CN120383600A - A Menin-MLL protein inhibitor, its pharmaceutical composition and use - Google Patents

Abstract

The present invention provides compounds represented by Formula I, pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof, as well as pharmaceutical compositions containing the same, and their use in preparing drugs for preventing, alleviating or treating diseases related to the interaction with the Menin-MLL protein.

Description

Menin-MLL protein inhibitor, pharmaceutical composition and application thereof

Technical Field

The invention relates to a Menin-MLL protein inhibitor, a pharmaceutical composition containing the same, and application of the same in preparing medicines for preventing or treating related diseases caused by interaction with the Menin-MLL protein.

Background

MLL (Mixed Lineage Leukemia) protein is a histone methyltransferase, also known as KMT2A (Histone-lysine N-METHYLTRANSFERASE A). MLL rearrangement (MLL-r) leukemia results from translocation of the 11q23 chromosomal locus containing the gene encoding KMT2A, which is currently known to produce more than 60 oncogenic fusion proteins-fused from the amino terminus of MLL with a variety of different proteins-with the MLL-AF4/9 fusion mutation being the most malignant. MLL-r leukemia accounts for 5% -10% of adult acute leukemia and 70% of infant acute leukemia, and the current treatment schemes are limited, most of the treatment schemes are chemotherapy drugs, and the prognosis is poor and the recurrence is very easy.

Menin is a protein mainly located in the nucleus, is encoded by multiple endocrine tumor type I (Multiple Endocrine Neoplasia Type, MEN 1) genes, is an important cofactor of oncogenic MLL-r fusion proteins, and has high affinity with MLL-r proteins. After binding of the Menin to the MLL-r protein, a chromatin modifying enzyme such as Dot1L or pTEFb complex is recruited, resulting in enhanced transcription of genes including HOXA, MEIS1, etc., whose abnormal expression impedes differentiation and promotes proliferation of hematopoietic cells. In vitro and in vivo experiments have shown that the Menin inhibitors disrupt the interaction of Menin with MLL-r and specifically lead to growth inhibition and apoptosis of leukemia cells harboring MLL-r mutations (CANCER CELL, 660-673). The study also found that the Menin inhibitors were also effective against leukemia with NPM1 gene mutations, which account for approximately 20-30% of the cases of acute myelogenous leukemia (Science 367, 586-590).

A number of Menin inhibitors are currently being developed for clinical phase 1/2 studies in patients with relapsed/refractory acute leukemia carrying MLL-r or NPM1 mutations, such as KO-539 of SNDX-5613,Kura Oncology from Syndax, and DS-1594b from Japanese first Co (Daichi Sankyo Group). Of 60 patients who could be evaluated in one clinical trial, AUGMENT-101, 53 had responded to the drug, however, after the second treatment cycle, some had developed resistance to SNDX-5613. These drug resistant patients were found to have undergone a mutation in the MEN1 gene resulting in amino acid changes in the Menin proteins M3271, M327V, G331R, G331D, T349M and S160C. These amino acid point mutations located within the binding pocket of the Menin drug interfere with the binding of the drug molecule to the target protein, thereby reducing the affinity of the drug, and importantly, the affinity of these mutant Menin proteins for KMT2A peptides is not greatly affected by structural changes. Studies have shown that the sensitivity of the proteins of the M327I and T349M point mutations to reported inhibitors is significantly reduced, thus obtaining significant selection advantages (Nature 615, 913-919). Therefore, it is important to develop inhibitors that bind to both the Menin wild type and the muteins, particularly the M327I and T349M point muteins, which would bring about a cure hope for this part of drug resistant patients.

Furthermore, too high a level of expression of Menin may result in a blocked proliferation of beta cells, resulting in a relatively insufficient insulin secretion. A Menin-MLL inhibitor has been shown to enhance beta cell proliferation, thereby providing potential for use in the field of diabetes. There are currently 1 compound BMF-219 in the form of a phase 2 clinical study of type II diabetes.

In conclusion, the Menin-MLL interaction inhibitor has good application prospect as a drug research and development, and has good clinical requirements for developing the Menin-MLL interaction inhibitor.

Disclosure of Invention

The present invention provides a compound of formula I, a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof:

in formula I:

m=0, 1 or 2;n =0, 1,2 or 3.

R1 is selected from hydrogen, hydroxy, halogen, cyano, C1-C3 alkyl, C1-C3 alkoxy or C1-C3 haloalkyl.

In some embodiments, R1 is selected from hydrogen, fluoro, chloro, bromo, iodo, cyano, hydroxy, amino, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, chloromethyl, trifluoromethyl, or perfluoroethyl. In some embodiments, R1 is hydrogen or methyl.

R2 and R3 are each independently selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, R _x CO-, sulfonyl or phosphonyl, wherein R _x is alkyl or alkenyl.

In some embodiments, rx is C1-C3 alkyl, and in some specific embodiments Rx is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, rx is C2-C4 alkenyl, in some specific examples Rx is vinyl, 1-propenyl, or 2-propenyl.

In some embodiments, R2 is hydrogen.

In some embodiments, R3 is hydrogen.

In some embodiments, R2 is hydrogen and R3 is methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, chloromethyl, trifluoromethyl, perfluoroethyl, acetyl, acryloyl, dimethylphosphonyl, methanesulfonyl, or ethanesulfonyl.

R4 and R5 are respectively and independently selected from hydrogen, hydroxyl, halogen, C1-C3 alkyl, C1-C3 alkoxy or C1-C3 halogenated alkyl.

In some embodiments, R4 is hydrogen.

In some embodiments, R5 is hydrogen.

In some embodiments, R4 is hydrogen and R5 is selected from hydroxy, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 haloalkyl.

In some embodiments, R4, R5 are independently selected from hydrogen, hydroxy, fluoro, chloro, bromo, iodo, cyano, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, chloromethyl, trifluoromethyl, or perfluoroethyl.

R6 is selected from substituted or unsubstituted 5-7 membered aryl, 5-7 membered heteroaryl, 9-16 membered saturated or partially unsaturated cycloalkyl, 9-16 membered saturated or partially unsaturated heterocyclyl, 9-16 membered fused aryl, 9-16 membered fused heteroaryl.

In some embodiments, R6 is selected from substituted or unsubstituted 5-7 membered aryl, 5-7 membered heteroaryl, 9-12 membered saturated or partially unsaturated cycloalkyl, 9-12 membered saturated or partially unsaturated heterocyclyl, 9-12 membered fused aryl, 9-12 membered fused heteroaryl, wherein the heterocyclyl or heteroaryl optionally contains 1-3 heteroatoms selected from N, O, S in the backbone atoms.

In some embodiments, R6 may be monocyclic or polycyclic.

In some embodiments, R6 is a 9-12 membered fused bicyclic ring.

In some embodiments, R6 is a 9-12 membered fused heterocyclyl or a 9-12 membered fused heteroaryl.

In some embodiments, R6 may be selected from the group consisting of substituted or unsubstituted cyclic groups such as furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, phenyl, pyridyl, pyrimidinyl, pyrazinyl, 1,2, 3-triazinyl, 1,2, 4-triazinyl, 1,3, 5-triazinyl, indolyl, benzimidazolyl, benzofuranyl, benzothiazolyl, benzisothiazole, benzoxazolyl, benzothienyl, indazolyl, pyrrolo [1,2-a ] pyridine, imidazo [1,5-a ] pyridine, pyrazolo [1,5-a ] pyridine, isoindolin-1-onyl, 3, 4-dihydroisoquinolin-1 (2H) -onyl, naphthyl, quinolinyl, isoquinolinyl, naphthyridinyl, benzotriazinyl, benzometa triazinyl, benzopyranyl, tetrahydronaphthyridin-1, 3, 2-a ] naphthyridin-1, 3, 4-H-yl, and hexahydro-1, 4-oxazinyl.

In some embodiments, R6 is optionally substituted at any possible position with one or more groups selected from oxygen, hydroxy, amino, carboxy, cyano, halo, phosphono, sulfonyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, R _a CO-, C2-C4 alkenyl, C3-C6 cycloalkyl, 3-6 heterocycloalkyl, 5-7 membered aryl, or 5-7 membered heteroaryl, wherein R _a represents alkyl or alkenyl.

In some embodiments, R _a is C1-C5 alkyl, and in some embodiments, R _a is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl. In some embodiments, R _a is C2-C4 alkenyl, and in some specific examples R _a is vinyl, 1-propenyl, or 2-propenyl.

In some embodiments, R6 is selected from the following substituted or unsubstituted groups:

In some embodiments, R6 is optionally substituted at any possible position with one or more groups selected from oxygen, hydroxy, amino, carboxy, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, methoxy, ethoxy, chloromethyl, trifluoromethyl, 2-trifluoroethyl, perfluoroethyl, formyl, acetyl, acryloyl, methylphosphonyl, dimethylphosphonyl, methanesulfonyl, ethanesulfonyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, triazole, tetrazole, phenyl, pyridyl.

In some embodiments, the present invention provides a compound of formula II, a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof:

In formula II, n=0, 1,2 or 3.

The values of R1, R2, R3, R4, R5 and R6 are as described above.

In some embodiments, the invention provides a compound of formula III, a pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof:

In formula III, n=0, 1,2 or 3.

The values of R1, R2, R3, R4, R5 and R6 are as described above.

The present invention provides the following compounds, pharmaceutically acceptable salts, hydrates, isomers, prodrugs, or mixtures thereof:

The invention also provides a pharmaceutical composition comprising any of the foregoing compounds, pharmaceutically acceptable salts, hydrates, isomers, prodrugs, or mixtures thereof, and pharmaceutically acceptable excipients and/or carriers.

The invention also provides a pharmaceutical composition comprising any of the compounds of formula (I), pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof, for use in the preparation of a medicament for preventing, alleviating or treating a disease associated with the interaction of a Menin-MLL protein.

In the present invention, the related diseases caused by the interaction with the Menin-MLL protein include malignant tumors, diabetes mellitus, or complications related to the diseases. Wherein the malignant tumor comprises hematological tumor, lymphoma, and solid tumor.

Hematological neoplasms include leukemias and myelomas, including, but not limited to, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute monocytic leukemia, chronic monocytic leukemia, childhood leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, mixed leukemia, hairy cell leukemia, precursor T-cell lymphocytic leukemia, large particle lymphocytic leukemia, meningeal leukemia, myelodysplastic syndrome, myeloproliferative disorders, myeloproliferative neoplasia, plasmacytoma, and multiple myeloma.

Lymphomas include, but are not limited to, cutaneous T-cell lymphomas, AIDS-related lymphomas, hodgkin's lymphomas, non-hodgkin's lymphomas, or malignant lymphomas.

Solid tumors include, but are not limited to, pancreatic, colon, rectal, liver, gastric, glioblastoma, lung, breast, and prostate cancers.

Related complications include, but are not limited to, leukemia meningitis.

Experiments prove that the Menin-MLL protein inhibitor has excellent in-vitro enzyme inhibition activity and cell proliferation inhibition activity, and particularly has excellent activity of inhibiting the interaction of the Menin mutant protein and the MLL protein compared with the existing compounds, thereby having good resistance drug-resistance prospect.

The invention is supported by Sichuan province scientific and technological plan.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Definition and general description:

indicating the position of attachment of the group to other structures.

Unless otherwise indicated, "substituted" means that a hydrogen atom in a molecule is replaced by a different atom or group.

"Oxygen substitution" means that the oxygen atom forms a "c=o" structure with the immediately adjacent carbon atom.

"Alkyl" refers to a saturated hydrocarbon group consisting of only carbon and hydrogen atoms, with single bonds between carbon and carbon, and between hydrocarbon, and the alkyl group may be straight or branched. Representative branched alkyl groups have one, two or three branches. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) and hexyl.

"Heteroatom" refers to a non-carbon atom in a carbon chain or backbone atom. Typical heteroatoms include, but are not limited to N, O and S, and the like.

"Yuan" means the number of skeleton atoms constituting a ring. Typical 5-membered rings include, for example, cyclopentyl, pyrrole, tetrahydropyrrole, imidazole, thiazole, furan, tetrahydrofuran, thiophene, and the like, and typical 6-membered rings include, for example, cyclohexyl, piperidine, piperazine, pyridine, pyran, pyrazine, thiopyran, pyridazine, pyrimidine, benzene, and the like. The ring containing hetero atoms in the skeleton atom is a heterocyclic ring, the aromatic group containing hetero atoms is heteroaryl, and the non-aromatic group containing hetero atoms is heterocycloalkyl.

"Cycloalkyl" refers to a non-aromatic cyclic group wherein the backbone atoms are all carbon atoms, and includes saturated or unsaturated monocyclic, bicyclic or polycyclic ring systems, such as fused 2, 3, 4 rings or spiro rings, and the like, particularly cyclic alkyl and alkenyl groups. Typical examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, and the like. Also included in the definition of "cycloalkyl" are cyclic groups formed by the fusion of a non-aromatic ring with an aromatic ring, such as benzo or pyrido derivative groups of cyclopentane, cyclopentene, cyclohexane, and the like, with specific examples being tetrahydronaphthalene.

"Heterocyclyl" and "heterocyclyl" have the same meaning and refer to a saturated or unsaturated monocyclic, bicyclic or polycyclic ring system (e.g., 2, 3 or 4 fused rings or spiro rings, etc.) containing one or more heteroatoms in the backbone atom. Wherein the heteroatoms are selected from N, O and S, and the remaining ring atoms are carbon. Examples of heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, piperazinyl, tetrahydropyranyl, or morpholinyl. The definition of "heterocycloalkyl" also includes cyclic groups formed by the fusion of the aforementioned cycloalkyl ring or heterocycloalkyl ring with an aromatic ring, wherein the aforementioned heteroatoms are included or excluded from the aromatic ring, and specific examples include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, or benzoγ -pyronyl.

"Aryl" refers to a monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic hydrocarbon group. Typical aryl groups include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, and the like.

"Heteroaryl" refers to a monocyclic or polycyclic (e.g., having 2,3, or 4 fused rings) aromatic heterocyclic moiety having one or more heteroatom ring members selected from N, S and O. Typical heteroaryl groups include, but are not limited to, pyridyl, indolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, benzothienyl, benzopyranyl, benzothiopyranyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, oxadiazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, and the like.

"Alkenyl" refers to an unsaturated hydrocarbon group having one or more-c=c- (carbon-carbon double bonds). Exemplary alkenyl groups include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, and the like.

"Halogen" means fluorine, chlorine, bromine or iodine.

"Cyano" refers to-CN.

"Haloalkyl" means an alkyl group substituted with one or more halogen atoms, wherein alkyl has the meaning described herein. Haloalkyl includes, but is not limited to, monohaloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl, and the like, such as chloromethyl, dichloromethyl, difluoromethyl, dibromomethyl, trifluoromethyl, 2-trifluoroethyl, perfluoroethyl, 2-trifluoro-1, 1-dichloroethyl, and the like.

"Alkoxy" refers to an-O-alkyl group, wherein alkyl has the meaning described herein.

"Amino" alone or in combination with other terms refers to a group of formula-NH 2.

"Hydroxy" refers to-OH.

"Sulfonyl" refers toA group represented by formula (I), wherein Rb represents an alkyl or aryl group as defined in the definition of the present invention. Exemplary sulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, phenylsulfonyl, p-toluenesulfonyl.

"Phosphonoyl" meansA group represented by Rc, wherein Rc represents a hydroxyl group or an alkyl group or an aryl group as defined in the definition of the invention, and Rd represents an alkyl group or an aryl group as defined in the definition of the invention. Exemplary phosphoryl groups include, but are not limited to, methylphosphono, dimethylphosphono, diethylphosphono.

In this context, the term "Ca-Cb" is used to refer to a radical having a-b carbon atoms (b is greater than a and both are integers) in that moiety unless otherwise indicated. For example, C _1-C₃ represents a moiety having 1 to 3 carbon atoms, such as 1 carbon atom, 2 carbon atoms, or 3 carbon atoms, in its modified portion.

"P-q membered (hetero) cyclic hydrocarbyl" refers to the number of carbon atoms having p-q (q is greater than p and both are integers) and heteroatoms involved in the ring in the moiety modified by the term. For example, a 3-6 membered heterocycloalkyl group means that it has 3, 4, 5 or 6 atoms in its modified ring structure, including carbon atoms and at least one heteroatom.

"Y-z membered (heteroaryl) group" refers to the total number of carbon atoms having y-z (z is greater than y and both are integers) and heteroatoms involved in ring formation in the moiety modified by the term. For example, a 5-7 membered aryl group means that it has 5,6 or 7 carbon atoms in its modified aryl structure, and a 5-7 membered heteroaryl group means that it has 5,6 or 7 atoms in its modified heteroaryl structure, including carbon atoms and at least one heteroatom.

"Optionally" means that the event or circumstance described subsequently can be freely chosen or not chosen.

"Hydrate" refers to an aggregate of one or more water molecules of the compounds of the present invention, including hemihydrate, monohydrate, dihydrate, trihydrate, and the like.

"Isomers" means that when a compound of the present invention contains one or more asymmetric centers, it may exist as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. The compounds of the invention may have asymmetric centers and thus result in the presence of two optical isomers. The scope of the present invention includes all possible optical isomers and mixtures thereof. If the compounds of the present invention contain olefinic double bonds, the scope of the present invention includes cis-isomers and trans-isomers unless specified otherwise. The compounds of the invention may exist in tautomeric (one of the functional group isomers) forms which have different points of attachment of hydrogen through one or more double bond shifts, for example, the keto and his enol forms are keto-enol tautomers. Each tautomer and mixtures thereof are within the scope of the present invention. Enantiomers of all compounds. Diastereomers, racemates, meso, cis-trans isomers, tautomers, geometric isomers, epimers, mixtures thereof, and the like are within the scope of the present invention.

"Prodrug" refers to a derivative compound that is capable of providing a compound of the invention directly or indirectly upon administration to a subject. Particularly preferred derivative compounds or prodrugs are compounds that, when administered to an individual, may increase the bioavailability of the compounds of the invention (e.g., are more readily absorbed into the blood) or promote delivery of the parent compound to the site of action (e.g., the lymphatic system). All prodrug forms of the compounds of the invention are within the scope of the invention unless otherwise indicated, and various prodrug forms are known in the art, see for example T.Higuchi,V.Stella,Pro-drugs as Novel Drug Delivery Systems[J],American Chemical Society,Vol.14,1975. furthermore, the invention also embraces the compounds of the invention containing a protecting group. During any process for preparing the compounds of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules of interest, thereby forming a chemically protected form of the compounds of the present invention. This can be achieved by conventional protecting groups, for example those described in T.W.Greene, P.G.M.Wuts, protective Groups in Organic Synthesis [ M ], john Wiley & Sons, 2006. These protecting groups may be removed at a suitable subsequent stage using methods known in the art.

"Pharmaceutical composition" refers to a formulation of a compound of the invention with a medium commonly accepted in the art for delivery of biologically active compounds to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote the administration of organisms, facilitate the absorption of active ingredients and further exert biological activity.

By "pharmaceutically acceptable" is meant a material such as a carrier, diluent, or excipient, etc., which does not affect the biological activity or properties of the compounds of the present invention, and which is relatively non-toxic, i.e., the material can be administered to an individual without causing an adverse biological reaction or interacting in an adverse manner with any of the components contained in the composition. For example, "excipients" include, but are not limited to, any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavoring agents, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are approved by the relevant government regulatory agency as acceptable for human or livestock use.

"Pharmaceutically acceptable salts" include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

The invention also provides a synthesis method of the compound, which is mainly a preparation method reported in chemical literature or a related synthesis method by taking a commercial chemical reagent as a starting material.

Description of the abbreviations

Boc represents t-butyl carbonate;

TsOH represents 4-methylbenzenesulfonic acid;

IPA represents isopropanol;

TEA represents triethylamine;

DCM represents dichloromethane;

DBU represents 1, 8-diazabicyclo [5,4,0] undecene-7;

THF represents tetrahydrofuran;

TMEDA represents N, N' -tetramethyl ethylenediamine;

TMSCl represents trimethylchlorosilane;

EDCl represents 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;

HOBT represents 1-hydroxybenzotriazole;

DIPEA represents N, N-diisopropylethylamine;

XPhos represents 2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl;

XPhosPdG3 denotes methanesulfonic acid (2-dicyclohexylphosphino-2 ',4',6 '-tri-isopropyl-1, 1' -biphenyl) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II);

KOAc represents potassium acetate;

dppf represents 1,1' -bis (diphenylphosphine) ferrocene;

Fmoc represents 9-fluorenylmethyl formate;

EA represents ethyl acetate;

MeI represents methyl iodide;

DCC means N, N' -dicyclohexylcarbodiimide;

PPh3 represents triphenylphosphine.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Preparation example 1

7-Bromo-1- (2, 2-trifluoroethyl) -1H-indole-2-carbonitrile

The synthetic route is as follows:

Step 1 preparation of 7-bromo-1H-indole-2-carboxamide

7-Bromo-1H-indole-2-carboxylic acid (3 g) was dissolved in 40mL of tetrahydrofuran, and N, N' -carbonyldiimidazole (3.04 g) was added thereto, followed by reaction at room temperature for 1.5 hours. Ammonia (5.78 mL) was added and the reaction was continued for 2h. TLC and LCMS were used to monitor completion of the reaction, concentrated under reduced pressure and purified by column chromatography to give the title compound (3.0 g).

MS(ESI)m/z 239.0(M+H)⁺

Step 2 preparation of 7-bromo-1H-indole-2-carbonitrile

7-Bromo-1H-indole-2-carboxamide (3 g) was dissolved in 40mL of toluene, phosphorus oxychloride (5.86 mL) was added, nitrogen was replaced three times, and reacted at 120℃for 2H. TLC monitoring the completion of the reaction under reduced pressure concentrating and purifying by column chromatography gave the title compound (2.63 g).

¹H NMR(400MHz,DMSO-d₆)δ12.62(s,1H),7.71(d,J＝8.0Hz,1H),7.59(d,J＝7.5Hz,1H),7.51(d,J＝2.0Hz,1H),7.11(t,J＝7.8Hz,1H).

Step 3 preparation of 7-bromo-1- (2, 2-trifluoroethyl) -1H-indole-2-carbonitrile

7-Bromo-1H-indole-2-carbonitrile (600 mg) was dissolved in 15mL of N, N-dimethylformamide, and potassium carbonate (1.13 g), 2-trifluoroethyl trifluoromethane sulfonate (0.79 mL) was added and reacted at 80℃for 10 hours. TLC was used to monitor the reaction, water was added thereto, extraction was performed 2 times with ethyl acetate, washing was performed with saturated brine, and column chromatography was performed to purify the title compound (800 mg).

¹H NMR(400MHz,DMSO-d₆)δ7.82(dd,J＝8.0,1.0Hz,1H),7.78(s,1H),7.74(dd,J＝7.6,1.1Hz,1H),7.21(t,J＝7.8Hz,1H),5.65(q,J＝8.6Hz,2H).

Using 7-bromo-1H-indole-2-carbonitrile (the title compound of step 2 of preparation 1) as a substrate, with various commercially available halogenated reagents, reference was made to step 3 of preparation to give various intermediates, as shown in Table 1.

Table 1 intermediates

Preparation example 2 preparation of methyl (S) -3- ((tert-Butoxycarbonyl) amino) -4-iodobutyrate

Step 1 preparation of 1- (2, 5-Dioxopyrrolidin-1-yl) 4-methyl (t-butoxycarbonyl) -L-aspartic acid

(S) -2- ((tert-Butoxycarbonyl) amino) -4-methoxy-4-oxobutanoic acid was dissolved in ethyl acetate (400 mL), 1-hydroxypyrrolidine-2, 5-dione (15.4 g) was added under ice-water bath, an ethyl acetate solution (100 mL) of N, N' -dicyclohexylcarbodiimide (26.3 g) was added dropwise, and after the addition was completed, the reaction was allowed to stand at room temperature overnight, and TLC was monitored to complete the reaction. The mixture was filtered, and the filtrate was washed with a saturated sodium hydrogencarbonate solution, a saturated brine, dried over anhydrous sodium sulfate and concentrated to give a crude title compound (41.8 g).

MS(ESI)m/z(M+H)⁺=345.1。

Step 2 preparation of (S) -methyl 3- ((tert-butoxycarbonyl) amino) -4-hydroxybutyrate.

Sodium borohydride (7.4 g) was added to a mixed solvent of tetrahydrofuran (300 mL) and water (40 mL) under ice-water bath conditions, a tetrahydrofuran solution (100 mL) of 1- (2, 5-dioxopyrrolidin-1-yl) 4-methyl (t-butoxycarbonyl) -L-aspartic acid (41.8 g) was added dropwise at this temperature, and after completion of the dropwise addition, the reaction was continued for half an hour under ice-water bath, TLC was used to monitor the disappearance of the starting material, quench the reaction by adding saturated ammonium chloride solution, extraction with ethyl acetate, merging the organic phases, drying over anhydrous sodium sulfate, and column chromatography was performed after concentration to purify the crude title compound (17.3 g).

MS(ESI)m/z(M+Na)⁺=256.1

Step 3 preparation of (S) -3- ((tert-Butoxycarbonyl) amino) -4-iodobutanoic acid methyl ester

Triphenylphosphine (19.5 g), imidazole (5.1 g) and iodine (18.9 g) were mixed in dry dichloromethane (300 mL), a solution of (S) -3- ((tert-butoxycarbonyl) amino) -4-hydroxybutyric acid methyl ester ((17.3 g) in dichloromethane (100 mL) was added dropwise under nitrogen protection, the reaction was allowed to proceed to room temperature for 3h, TLC was monitored to complete the consumption of the starting material, a part of triphenylphosphine oxide was removed by filtration, the filtrate was quenched with a sodium thiosulfate solution until the system was colorless, extracted with dichloromethane, the organic phase was combined, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (15.9 g).

MS(ESI)m/z(M+Na)⁺=366.1。

Some of the intermediates used in the present invention are shown in Table 2, which are all commercially available.

Table 2 intermediates

Example 1

(R) -2- ((5- (5- (3-amino-4-phenylbutyryl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

The synthetic route is as follows:

step 1 preparation of 3,4,5, 6-Tetrahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylic acid tert-butyl ester p-toluenesulfonate

Di-tert-butyl 4, 6-dihydropyrrolo [3,4-C ] pyrrole-2, 5 (1H, 3H) -dicarboxylate (1.7 g) was dissolved in isopropanol (30 mL), p-toluenesulfonic acid (1.04 g) was added and reacted at 55℃overnight. LCMS monitored reaction was complete. Cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate and dried to give the title compound (1.65 g).

MS(ESI)m/z(M+H)⁺=211.1。

Step 2 preparation of tert-butyl 5- (3, 6-dichloro-1, 2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate.

Tert-butyl 3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate p-toluenesulfonate (1.64 g) was added to dichloromethane (30 mL), triethylamine (1.30 g) was added dropwise, and then trichloro-1, 2, 4-triazine (0.95 g) was added in portions to react at room temperature for 2 hours. LCMS monitored completion of the reaction, quenched with water, extracted three times with dichloromethane, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (1.5 g).

MS(ESI)m/z(M+H)⁺=358.1

Step 3 preparation of tert-butyl 5- (3-chloro-6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate

Tert-butyl 5- (3, 6-dichloro-1, 2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-C ] pyrrole-2 (1H) -carboxylate (1.5 g) was added to tetrahydrofuran (20 mL), DBU (0.96 g) was added, and N-ethyl-5-fluoro-2-hydroxy-N-isopropylbenzamide (1.04 g) was reacted overnight at 55 ℃. LCMS monitored completion, stopped, cooled to room temperature, quenched with water, extracted with ethyl acetate, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (1.75 g).

MS(ESI)m/z(M+H)⁺=547.1。

Step 4 preparation of tert-butyl 5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate

Tert-butyl 5- (3-chloro-6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate (1.75 g) was added to tetrahydrofuran (30 mL), sodium borohydride (0.61 g) was added in portions, and tetramethyl ethylenediamine (0.74 g), 1' -bis (diphenylphosphine) ferrocene palladium (II) dichloride (0.23 g) was added, and the reaction was allowed to react overnight at room temperature under nitrogen. LCMS monitored completion of the reaction, quenched with water, extracted with ethyl acetate, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (1.27 g).

MS(ESI)m/z(M+H)⁺=513.1。

Step 5 preparation of N-ethyl-5-fluoro-N-isopropyl-2- ((5- (3, 4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) benzamide

Tert-butyl 5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate (1.27 g) was added to trifluoroethanol (20 mL), trimethylchlorosilane (0.8 mL) was added and the mixture was reacted at room temperature for 2H. LCMS monitored completion of the reaction, solvent was distilled off under reduced pressure, the residue was taken up in saturated sodium bicarbonate solution to basicity, extracted three times with dichloromethane/methanol mixed solvent, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness to afford the title compound (1 g).

MS(ESI)[M+H]⁺=413.1。

Step 6 preparation of (R) - (4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxo-1-phenylbutan-2-yl) carbamic acid tert-butyl ester

(R) -3- ((tert-Butoxycarbonyl) amino) -4-phenylbutyric acid (36 mg) was added to N, N-dimethylformamide (2 mL), N-diisopropylethylamine (45 mg), 1-hydroxybenzotriazole (30 mg), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (40 mg) were added in this order, and after half an hour of reaction at room temperature, N-ethyl-5-fluoro-N-isopropyl-2- ((5- (3, 4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yloxy) benzamide (50 mg) was added, and the reaction was carried out at room temperature overnight. LCMS monitored completion of the reaction, quenched with water, extracted with ethyl acetate, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (50 mg).

MS(ESI)[M+H]⁺=674.3。

Step 6 preparation of (R) -2- ((5- (5- (3-amino-4-phenylbutyryl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

Tert-butyl (R) - (4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxo-1-phenylbutan-2-yl) carbamate (50 mg) was added to trifluoroethanol (3 mL), trimethylchlorosilane (0.1 mL) was added, and the reaction was carried out at room temperature for 2 hours. LCMS monitored completion of reaction, solvent was distilled off under reduced pressure, the residue was adjusted to weakly basic with saturated sodium bicarbonate solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and purified by pre-HPLC (preparative high performance liquid chromatography, the same applies hereinafter) to give the title compound (23.73 mg).

MS(ESI)[M+H]⁺=574.3。

¹H NMR(400MHz,DMSO-d₆)δ8.56(d,J＝2.0Hz,1H),7.49-7.44(td,J＝8.9,4.7Hz,1H),7.40-7.34(m,2H),7.31-7.27(t,J＝7.5Hz,2H),7.22-7.18(m,3H),4.78(m,2H),4.37-4.25(m,4H),4.19-4.10(m,2H),3.70-3.63(p,J＝6.6Hz,1H),3.34(m,1H),3.07-3.00(m,1H),2.72-2.67(dd,J＝13.1,5.9Hz,1H),2.61-2.54(m,1H),2.24-2.21(m,2H),1.56(br,2H),1.09-1.03(m,3H),1.03-0.89(m,4H),0.72(m,3H).

The compounds of examples 2-21 were obtained by reference to the synthetic route with steps 6-7 of example 1 using N-ethyl-5-fluoro-N-isopropyl-2- ((5- (3, 4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) benzamide (i.e. the title compound of step 5 of example 1) as starting material using the intermediates and commercially available reagents as shown in table 2 and the specific structure and characterization of the compounds are shown in table 3.

TABLE 3 examples 2-21

Example 22

(R) -3- (2-amino-4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoic acid

The synthetic route is as follows:

Step 1 preparation of (R) -methyl 3- (2- ((tert-Butoxycarbonyl) amino) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoate

Tert-butyl (R) - (1- (3-bromophenyl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-C ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate (80 mg, synthesized in accordance with example 18) was added to methanol (2 mL), triethylamine (30 uL) and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex (9 mg) were added in sequence, after the system was replaced with carbon monoxide, the reaction was monitored for completion of the reaction at 110℃for two days by LCMS, the solvent was distilled off under reduced pressure, and the title compound (30 mg) was purified by column chromatography.

MS(ESI)m/z[M+H]⁺=732.3。

Step 2 preparation of (R) -methyl 3- (2-amino-4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoate

Methyl (R) -3- (2- ((tert-butoxycarbonyl) amino) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoate (30 mg) was added to trifluoroethanol (3 mL), trimethylchlorosilane (0.1 mL) was added, and the mixture was reacted at room temperature for 2 hours. LCMS monitored completion of the reaction, solvent was distilled off under reduced pressure, the residue was adjusted to weak base with saturated sodium bicarbonate solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (15 mg).

MS(ESI)m/z[M+H]⁺=632.3。

Step 3 preparation of (R) -3- (2-amino-4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoic acid

Methyl (R) -3- (2-amino-4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoate (15 mg) was added to tetrahydrofuran (1 mL), a mixed solvent of methanol (0.3 mL) and water (0.3 mL), and lithium hydroxide monohydrate (6 mg) was added to react at room temperature for 2 hours. LCMS was monitored to completion and the pH of the system was adjusted to 2-3 with 2M dilute hydrochloric acid, the solvent was distilled off under reduced pressure and the residue was purified by pre-HPLC to give the title compound (2.41 mg)

MS(ESI)m/z[M+H]⁺=618.3。

¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),7.80(s,1H),7.77-7.76(d,J＝6.7Hz,1H),7.49-7.44(m,1H),7.40-7.33(m,4H),4.77(m,2H),4.36-4.28(m,4H),4.17-4.12(m,2H),3.70-3.63(m,2H),3.06(m,1H),2.83-2.73(m,2H),2.39-2.37(m,2H),1.09-1.03(m,4H),0.99-0.89(m,3H),0.72(m,3H)

Example 23

(R) -2- (2-amino-4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoic acid

The synthetic route is as follows:

Step 1 preparation of (R) -2- (2- ((tert-Butoxycarbonyl) amino) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrole [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoic acid

Methyl (R) -2- (2- ((tert-butoxycarbonyl) amino) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoate (80 mg, synthesized according to the procedure of example 22 using the procedure of example 19, was added to a mixed solvent of tetrahydrofuran (1 mL), methanol (0.3 mL) and water (0.3 mL) and lithium hydroxide monohydrate (14 mg) was added, followed by reaction at room temperature for 2 hours. LCMS monitored disappearance of starting material, pH of system adjusted to 2-3 with 2M dilute hydrochloric acid, extraction with ethyl acetate, combined organic phases, dried over anhydrous sodium sulfate, concentrated and residue purified by pre-TLC to give the title compound (15 mg).

MS(ESI)m/z[M+H]⁺=718.3。

Step 2 preparation of (R) -2- (2-amino-4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoic acid

(R) -2- (2- ((tert-Butoxycarbonyl) amino) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrole [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutyl) benzoic acid (15 mg) was added to trifluoroethanol (3 mL), trimethylchlorosilane (0.1 mL) was added, and after 2 hours of reaction at room temperature. LCMS monitored completion of the reaction and solvent was distilled off under reduced pressure, after the residue was made slightly alkaline with saturated sodium bicarbonate solution, the solvent was distilled off under reduced pressure and the residue was purified by pre-HPLC to give the title compound (1.77 mg).

MS(ESI)m/z[M+H]⁺=618.3。

¹H NMR(400MHz,DMSO-d₆)δ8.98(br,1H),8.57(s,1H),7.51-7.45(m,2H),7.41-7.35(m,2H),7.29-7.18(m,3H),4.82(m,2H),4.41-4.36(m,4H),4.21-4.16(m,2H),3.70-3.61(m,2H),3.06-2.94(m,3H),2.82-2.77(dd,J＝17.1,4.6Hz,1H),2.68-2.60(m,1H),1.10-1.03(m,4H),1.00-0.87(m,3H),0.79(m,3H).

Example 24

(R) -2- ((5- (5- (3-amino-4- (2-cyanophenyl) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

The synthetic route is as follows:

Step 1 preparation of (R) - (1- (2-cyanophenyl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamic acid tert-butyl ester

Tert-butyl (R) - (1- (2-bromophenyl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-C ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate (120 mg, see example 19), potassium hexacyanoferrate trihydrate (48 mg), 2- (dicyclohexylphosphorus) -2',4',6 '-tri-isopropyl-1, 1' -biphenyl (8 mg), methanesulfonic acid (2-dicyclohexylphosphino-2 ',4',6 '-tri-isopropyl-1, 1' -biphenyl) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II) (7 mg), potassium acetate (32 mg), 1, 4-dioxane (1.5 mL) and water (0.4 mL) were mixed solvent and the reaction was sealed at 120 ℃ under nitrogen protection for 3H, and the product was monitored. Cooled to room temperature, water was added, extraction was performed with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated, followed by column chromatography purification to give the title compound (30 mg).

MS(ESI)m/z[M+H]⁺=699.3。

Step 2 preparation of (R) -2- ((5- (5- (3-amino-4- (2-cyanophenyl) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

Tert-butyl (R) - (1- (2-cyanophenyl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate (30 mg) was added to trifluoroethanol (3 mL), trimethylchlorosilane (0.1 mL) was added, and the mixture was reacted at room temperature for 2 hours. LCMS monitored completion of the reaction and solvent was distilled off under reduced pressure, after the residue was made slightly alkaline with saturated sodium bicarbonate solution, the solvent was distilled off under reduced pressure and the residue was purified by pre-HPLC to give the title compound (4.04 mg).

MS(ESI)m/z[M+H]⁺=599.3。

¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),7.78-7.76(d,J＝7.7Hz,1H),7.66-7.62(t,J＝7.6Hz,1H),7.52-7.45(m,2H),7.43-7.34(m,3H),4.79(s,2H),4.38-4.29(m,4H),4.20-4.13(m,2H),3.70-3.63(p,J＝6.6Hz,1H),3.42-3.38(m,1H),3.07-3.02(m,1H),2.95-2.90(dd,J＝13.4,5.7Hz,1H),2.85-2.76(ddd,J＝13.1,7.8,3.4Hz,1H),2.36-2.34(d,J＝6.8Hz,2H),1.72(br,2H),1.10-1.03(m,4H),0.99-0.90(m,3H),0.72(m,3H).

Example 25

(R) -2- ((5- (5- (3-amino-4- (3- (dimethylphosphoryl) phenyl) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

The synthetic route is as follows:

Step 1 preparation of methyl (R) -4- (3-bromophenyl) -3- ((tert-butoxycarbonyl) amino) butyrate

(R) -4- (3-bromophenyl) -3- ((t-butoxycarbonyl) amino) butyric acid (200 mg) was dissolved in N, N-dimethylformamide, potassium hydrogencarbonate (84 mg), methyl iodide (55 uL) was added at room temperature, the reaction was allowed to proceed at room temperature for 4h, LCMS was monitored for completion of the reaction, quenched with water, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound (220 mg).

MS(ESI)m/z[M+H]⁺=372.1。

Step 2 preparation of (R) -3- ((tert-Butoxycarbonyl) amino) -4- (3- (dimethylphosphoryl) phenyl) butanoic acid

Methyl (R) -4- (3-bromophenyl) -3- ((t-butoxycarbonyl) amino) butyrate (130 mg), dimethylphosphine oxide (82 mg), palladium acetate (8 mg), 1' -bis (diphenylphosphine) ferrocene (40 mg), N, N-diisopropylethylamine (0.15 mL) were mixed in DMF (2 mL), nitrogen blanket, 120℃for 8h, and LCMS monitored reaction was complete. Cooling to room temperature, adding water for quenching, extracting with ethyl acetate, combining organic phases, drying with anhydrous sodium sulfate, and concentrating to obtain a crude product of (R) -3- ((tert-butoxycarbonyl) amino) -4- (3- (dimethylphosphoryl) phenyl) methyl butyrate.

The crude product was added to a mixed solvent of tetrahydrofuran (2 mL), methanol (0.6 mL) and water (0.6 mL), and lithium hydroxide monohydrate (40 mg) was added thereto, followed by reaction at room temperature for 2 hours. LCMS monitors the disappearance of starting material, extracts the system three times with ethyl acetate, discards the organic phase, adjusts the pH of the system to 2-3 with 2M dilute hydrochloric acid, extracts with dichloromethane/methanol mixed solvent, combines the organic phases, washes with saturated saline, dries over anhydrous sodium sulfate, and concentrates to give crude title compound (80 mg)

MS(ESI)m/z[M-H]^--=354.1。

Step 3 preparation of tert-butyl (R) - (1- (3- (dimethylphosphoryl) phenyl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrole [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate

(R) -3- ((tert-Butoxycarbonyl) amino) -4- (3- (dimethylphosphoryl) phenyl) butanoic acid (80 mg) was added to N, N-dimethylformamide (2 mL), N-diisopropylethylamine (0.07 mL), 1-hydroxybenzotriazole (35 mg), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (50 mg) and, after half an hour at room temperature, N-ethyl-5-fluoro-N-isopropyl-2- ((5- (3, 4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) benzamide (70 mg) was added and reacted overnight at room temperature. LCMS monitored completion of the reaction, quenched with water, extracted with ethyl acetate, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column to give the title compound (45 mg).

MS(ESI)m/z[M+H]⁺=750.3。

Step 4 preparation of (R) -2- ((5- (5- (3-amino-4- (3- (dimethylphosphoryl) phenyl) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

Tert-butyl (R) - (1- (3- (dimethylphosphoryl) phenyl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrole [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate (45 mg) was added to trifluoroethanol (3 mL), trimethylchlorosilane (0.1 mL) was added, and the mixture was reacted at room temperature for 2 hours. LCMS monitored complete reaction, solvent was distilled off under reduced pressure, the residue was taken up in saturated sodium bicarbonate solution to weakly basic, solvent was distilled off under reduced pressure, and the residue was purified by pre-HPLC to give the title compound (26.94 mg)

MS(ESI)m/z[M+H]⁺=650.3。

¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),7.63-7.57(m,2H),7.49-7.34(m,5H),4.79(m,2H),4.38-4.30(m,4H),4.19-4.14(m,2H),3.70-3.63(p,J＝6.7Hz,1H),3.09-3.01(m,2H),2.81-2.76(dd,J＝13.3,5.8Hz,1H),2.69-2.63(q,J＝6.6Hz,1H),2.38-2.25(m,2H),1.65(s,3H),1.62(s,3H),1.09-0.99(m,4H),0.97-0.89(m,3H),0.74-0.70(m,3H).

The compound of example 26 was obtained by reference to the synthetic route with example 25 using the intermediate (R) -4- (2-bromophenyl) -3- ((t-butoxycarbonyl) amino) butanoic acid as starting material and using commercially available reagents, as specifically shown in table 4.

Table 4 example 26

Example 27

(R) -2- ((5- (5- (3-amino-4- (1-methyl-1H-indol-3-yl) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

The synthetic route is as follows:

step 1 preparation of (R) - (4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1- (1-methyl-1H-indol-3-yl) -4-oxobutan-2-yl) carbamic acid tert-butyl ester

Tert-butyl (R) - (4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1- (1H-indol-3-yl) -4-oxobutan-2-yl) carbamate (40 mg, see example 6) was added to tetrahydrofuran (2 mL), sodium hydroxide (34 mg) was added under ice water bath, methyl iodide (16 mg) was added later, and the reaction was allowed to warm to room temperature overnight. TLC monitoring the disappearance of starting material, water quenching, extraction with ethyl acetate, combining organic phases, drying over anhydrous sodium sulfate, concentration and purification by pre-TLC gave the title compound (15 mg). .

MS(ESI)m/z[M+H]⁺=727.3。

Step 2 preparation of (R) -2- ((5- (5- (3-amino-4- (1-methyl-1H-indol-3-yl) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

Tert-butyl (R) - (4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1- (1-methyl-1H-indol-3-yl) -4-oxobutan-2-yl) carbamate (15 mg) was added to trifluoroethanol (3 mL), trimethylchlorosilane (0.1 mL) was added, and the mixture was reacted at room temperature for 2 hours. LCMS monitored completion of the reaction, solvent was distilled off under reduced pressure, the residue was adjusted to weak base with saturated sodium bicarbonate solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and purified by pre-HPLC to give the title compound (4.79 mg).

MS(ESI)m/z[M+H]⁺=627.3。

¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),7.58-7.56(d,J＝7.9Hz,1H),7.49-7.44(dd,J＝8.8,4.7Hz,1H),7.40-7.34(m,3H),7.14-7.10(m,2H),7.02-6.98(t,J＝7.4Hz,1H),4.78(m,2H),4.37-4.30(m,3H),4.21-4.13(m,3H),3.74(s,3H),3.70-3.63(p,J＝6.6Hz,1H),3.41-3.37(m,1H),3.05(m,1H),2.82-2.78(dd,J＝14.0,5.8Hz,1H),2.69-2.63(m,1H),2.40-2.35(dd,J＝15.5,4.5Hz,1H),2.28-2.23(dd,J＝15.5,8.0Hz,1H),1.65(br,2H),1.09-1.03(m,4H),0.99-0.89(m,3H),0.72(m,3H).

Example 28

(R) -2- ((5- (5- (3-amino-4- (1-cyclopropyl-1H-indol-3-yl) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

The synthetic route is as follows:

Step 1 preparation of tert-butyl (R) - (1- (1-cyclopropyl-1H-indol-3-yl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate

Tert-butyl (R) - (4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-C ] pyrrol-2 (1H) -yl) -1- (1H-indol-3-yl) -4-oxobutan-2-yl) carbamate (80 mg, see example 6), copper acetate (44 mg), cyclopropylboronic acid (19 mg), 4-dimethylaminopyridine (27 mg) were mixed in toluene and reacted for 3H under nitrogen protection at 110℃with LCMS monitoring as the main product with starting material remaining, copper acetate (44 mg), cyclopropylboronic acid (19 mg) being fed and the reaction continued overnight. LCMS monitored completion of the reaction, cooled to room temperature, suction filtered through celite, and the filtrate concentrated and purified by column chromatography to give the title compound (35 mg).

MS(ESI)m/z[M+H]⁺=753.3。

Step 2 preparation of (R) -2- ((5- (5- (3-amino-4- (1-cyclopropyl-1H-indol-3-yl) butyryl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

Tert-butyl (R) - (1- (1-cyclopropyl-1H-indol-3-yl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate (35 mg) was added to trifluoroethanol (3 mL), trimethylchlorosilane (0.1 mL) was added, and the mixture was reacted at room temperature for 2 hours. LCMS monitored completion of the reaction, solvent was distilled off under reduced pressure, the residue was adjusted to weak base with saturated sodium bicarbonate solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and purified by pre-HPLC to give the title compound (6.32 mg).

MS(ESI)m/z[M+H]⁺=653.3。

¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),7.58-7.56(d,J＝7.9Hz,1H),7.51-7.45(m,2H),7.40-7.34(m,2H),7.16-7.12(m,2H),7.04-7.01(t,J＝7.4Hz,1H),4.78(m,2H),4.37-4.06(m,7H),3.70-3.63(p,J＝6.6Hz,1H),3.06-3.01(s,1H),2.80-2.75(dd,J＝14.0,5.8Hz,1H),2.67-2.60(m,1H),2.40-2.35(dd,J＝15.5,4.6Hz,1H),2.28-2.22(dd,J＝15.5,7.9Hz,1H),1.75(br,2H),1.10-0.89(m,12H),0.72(m,3H).

Example 29

(R) -2- ((5- (5- (4- (benzo [ b ] thiophen-3-yl) -3- (methylamino) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

The synthetic route is as follows:

Step 1 preparation of tert-butyl (R) - (1- (benzothien-3-yl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrole [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) (methyl) carbamate

Tert-butyl (R) - (1- (benzothien-3-yl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate (60 mg, cf. Example 3) was added to dry tetrahydrofuran (2 mL), sodium hydride (6 mg, purity 60%) was added under ice-water bath, methyl iodide (24 mg) was added later, the reaction was warmed to room temperature for 2H, lcms was monitored to completion, quenched with saturated ammonium chloride solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and column chromatography was purified to give the title compound (40 mg).

MS(ESI)m/z[M+H]⁺=743.3。

Step 2 preparation of (R) -2- ((5- (5- (4- (benzo [ b ] thiophen-3-yl) -3- (methylamino) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

Tert-butyl (R) - (1- (benzothien-3-yl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrole [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) (methyl) carbamate (40 mg) was added to trifluoroethanol (3 mL), trimethylchlorosilane (0.1 mL) was added, and the mixture was reacted at room temperature for 2 hours. LCMS monitored completion of the reaction, solvent was distilled off under reduced pressure, the residue was adjusted to weak base with saturated sodium bicarbonate solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and purified by pre-HPLC to give the title compound (17.44 mg).

MS(ESI)m/z[M+H]⁺=644.3。

¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),7.97-7.91(m,2H),7.50-7.44(m,2H),7.42-7.33(m,4H),4.78-4.74(m,2H),4.36-4.33(m,2H),4.26-4.12(m,4H),3.70-3.63(dt,J＝13.1,6.4Hz,1H),3.26-3.19(t,J＝6.3Hz,1H),3.07-3.05(m,1H),3.01-2.89(m,2H),2.48-2.44(m,1H),2.31(s,3H),2.28-2.33(dd,J＝15.6,6.2Hz,1H),1.71(br,1H),1.09-1.03(m,4H),0.99-0.90(m,3H),0.72(m,3H).

Example 30

(R) -2- ((5- (3-amino-4- (4-hydroxyphenyl) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

The synthetic route is as follows:

Step 1 preparation of (9H-fluoren-9-yl) methyl (R) - (1- (4- (tert-butoxy) phenyl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate

(R) -3- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4- (4- (t-butoxy) phenyl) butanoic acid (60 mg) was added to N, N-dimethylformamide (2 mL), N-diisopropylethylamine (45 mg), 1-hydroxybenzotriazole (30 mg), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (40 mg) was added sequentially, and after half an hour of reaction at room temperature, N-ethyl-5-fluoro-N-isopropyl-2- ((5- (3, 4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) benzamide (50 mg) was added and reacted overnight at room temperature. LCMS monitored completion of the reaction, quenched with water, extracted with ethyl acetate, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (50 mg).

MS(ESI)m/z[M+H]⁺=868.3。

Step 2 preparation of (9H-fluoren-9-yl) methyl (R) - (4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1- (4-hydroxyphenyl) -4-oxobutan-2-yl) carbamate

(9H-fluoren-9-yl) methyl (R) - (1- (4- (tert-butoxy) phenyl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate (50 mg) was added to ethyl acetate (3 mL), 4M hydrochloric acid (0.5 mL) was added, and the mixture was reacted overnight at room temperature. LCMS monitored completion of the reaction, saturated sodium bicarbonate solution was adjusted to about 4, extracted with ethyl acetate, the organic phases combined, dried over anhydrous sodium sulfate, and concentrated to give crude (50 mg).

MS(ESI)m/z[M+H]⁺=812.3。

Step 3 preparation of (R) -2- ((5- (5- (3-amino-4- (4-hydroxyphenyl) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

(9H-fluoren-9-yl) methyl (R) - (4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1- (4-hydroxyphenyl) -4-oxobutan-2-yl) carbamate (50 mg crude) was added to dichloromethane (3 mL), piperidine (0.5 mL) was added, and the mixture was reacted at room temperature for 4 hours. LCMS monitored completion of the reaction, water, dichloromethane extraction, combined organic phases, dried over anhydrous sodium sulfate, concentrated and purified by pre-HPLC to give the title compound (29.54 mg).

MS(ESI)m/z[M+H]⁺=590.3。

¹H NMR(400MHz,DMSO-d₆)δ9.10(br,1H),8.49(s,1H),7.42-7.38(mz,1H),7.33-7.27(m,2H),6.93-6.91(d,J＝7.9Hz,2H),6.62-6.60(d,J＝7.9Hz,2H),4.71(m,2H),4.30-4.06(m,6H),3.63-3.56(p,J＝6.6Hz,1H),3.17(m,1H),3.00-2.94(m,1H),2.51-2.44(m,1H),2.42-2.38(m,1H),2.27-2.09(m,2H),1.51(br,2H),1.03-0.96(m,4H),0.92-0.82(m,3H),0.65(m,3H).

The compounds of examples 31-32 were obtained by reference to the synthetic route with example 30 using N-ethyl-5-fluoro-N-isopropyl-2- ((5- (3, 4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) benzamide (i.e. the title compound of step 5 in example 1) as starting material using the intermediates shown in table 2 and commercially available reagents, as detailed in table 5.

TABLE 5 examples 31-32

Example 33

(R) -2- ((5- (5- (4- (3- (1H-tetrazol-5-yl) phenyl) -3-aminobutanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

The synthetic route is as follows:

Step 1 preparation of methyl (R) -4- (3-cyanophenyl) -3- ((tert-butoxycarbonyl) amino) butyrate

(R) -4- (3-cyanophenyl) -3- ((tert-Butoxycarbonyl) amino) butanoic acid (200 mg) was dissolved in N, N-dimethylformamide, potassium hydrogen carbonate (84 mg), methyl iodide (55 uL) was added at room temperature, the reaction was allowed to react at room temperature for 4h, LCMS was monitored for completion of the reaction, quenched with water, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the title compound (220 mg).

MS(ESI)m/z[M+H]⁺=319.1。

Step 2 preparation of methyl (R) -4- (3- (1H-tetrazol-5-yl) phenyl) -3- ((tert-butoxycarbonyl) amino) butyrate

Methyl (R) -4- (3-cyanophenyl) -3- ((tert-butoxycarbonyl) amino) butyrate (220 mg) was added to N, N-dimethylformamide (3 mL), ammonium chloride (85 mg), sodium azide (82 mg) was added at room temperature and reacted for two days at 115℃with LCMS monitoring small amounts of starting material remaining, mainly product. Cooling to room temperature, quenching with sodium hypochlorite solution, evaporating the solvent under reduced pressure, and purifying the residue by column chromatography to give the title compound (220 mg)

MS(ESI)m/z[M+H]⁺=362.1。

Step 3 preparation of (R) -4- (3- (1H-tetrazol-5-yl) phenyl) -3- ((t-butoxycarbonyl) amino) butanoic acid

Methyl (R) -4- (3- (1H-tetrazol-5-yl) phenyl) -3- ((t-butoxycarbonyl) amino) butyrate (100 mg) was added to a mixed solvent of tetrahydrofuran (2 mL), methanol (0.6 mL) and water (0.6 mL), and lithium hydroxide monohydrate (28 mg) was added to react at room temperature for 2 hours. LCMS monitored disappearance of starting material, pH of the system was adjusted to 7 with 2M dilute hydrochloric acid, and the solvent was distilled off under reduced pressure and concentrated to give crude title compound.

MS(ESI)m/z[M-H]^-=348.1。

Step 4 preparation of tert-butyl (R) - (1- (3- (1H-tetrazol-5-yl) phenyl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate

Crude (R) -4- (3- (1H-tetrazol-5-yl) phenyl) -3- ((tert-butoxycarbonyl) amino) butyric acid obtained in step 3 was added to N, N-dimethylformamide (2 mL), N-diisopropylethylamine (0.06 mL), 1-hydroxybenzotriazole (30 mg), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (40 mg) were added in this order, and after half an hour of reaction at room temperature, N-ethyl-5-fluoro-N-isopropyl-2- ((5- (3, 4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) benzamide (50 mg) was added and reacted overnight at room temperature. LCMS monitored completion of the reaction, quenched with water, extracted with ethyl acetate, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (25 mg).

MS(ESI)m/z[M+H]⁺=742.3。。

Step 5 preparation of (R) -2- ((5- (5- (4- (3- (1H-tetrazol-5-yl) phenyl) -3-aminobutanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

Tert-butyl (R) - (1- (3- (1H-tetrazol-5-yl) phenyl) -4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -4-oxobutan-2-yl) carbamate (25 mg) was added to trifluoroethanol (3 mL), trimethylchlorosilane (0.1 mL) was added, and the mixture was reacted at room temperature for 2 hours. LCMS monitored complete reaction, solvent was distilled off under reduced pressure, the residue was taken up in saturated sodium bicarbonate solution to weakly basic, solvent was distilled off under reduced pressure, and the residue was purified by pre-HPLC to give the title compound (4.5 mg)

MS(ESI)m/z[M+H]⁺=642.3。。

¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),7.90-7.87(m,2H),7.52-7.46(m,1H),7.40-7.33(m,3H),7.15-7.13(d,J＝7.5Hz,1H),4.76-4.72(m,2H),4.35(m,2H),4.25-4.15(m,4H),3.81-3.74(q,J＝6.7Hz,1H),3.69-3.63(p,J＝6.7Hz,1H),3.04-2.99(m,3H),2.92-2.87(m,1H),2.67-2.54(m,1H),1.09-1.02(m,4H),0.99-0.85(m,3H),0.71(m,3H).

Example 34

(R) -2- ((5- (5- (3-amino-4- (2-methyl-1-oxoisoindolin-4-yl) butanoyl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

The synthetic route is as follows:

step 1 preparation of methyl (R) -3- ((tert-Butoxycarbonyl) amino) -4- (2-methyl-1-oxoisoindolin-4-yl) butyrate

Zinc powder (171 mg) was added to dry N, N-dimethylformamide (4 mL), nitrogen was used to protect it, iodine (66 mg) was added at room temperature, after five minutes the elemental color of iodine disappeared, a solution of trimethylchlorosilane (50 uL) in DMF (0.5 mL) was added, the reaction was allowed to proceed for half an hour at room temperature, a solution of (S) -3- ((tert-butoxycarbonyl) amino) -4-iodobutanoic acid methyl ester (300 mg) in N, N-dimethylformamide (1 mL) was added dropwise, the reaction was allowed to proceed for five minutes at room temperature, 4-bromo-2-methylisoindoline-1-one (236 mg), 2-dicyclohexylphosphine-2 ',6' -dimethoxybiphenyl (36 mg), tris (dibenzylideneacetone) dipalladium (79 mg) was added, the reaction was allowed to proceed to 4h at 70℃with LCMS monitoring the product formation and the consumption of the raw materials was completed. Cooled to room temperature, quenched with saturated sodium bicarbonate solution, extracted with ethyl acetate, the combined organic phases dried over anhydrous sodium sulfate, and concentrated before purification by column chromatography to give the title compound (250 mg).

MS(ESI)m/z(M+Na)⁺=385.1。

Step 2 preparation of (R) -3- ((tert-Butoxycarbonyl) amino) -4- (2-methyl-1-oxoisoindolin-4-yl) butanoic acid

Methyl (R) -3- ((tert-butoxycarbonyl) amino) -4- (2-methyl-1-oxoisoindolin-4-yl) butyrate (250 mg) was added to a mixed solvent of tetrahydrofuran (3 mL), methanol (3 mL) and water (1 mL), lithium hydroxide monohydrate (60 mg) was added at room temperature, after 2h LCMS was monitored to be complete, the system was added with water and ethyl acetate to extract three times, the organic phase was discarded, the pH of the system was adjusted to 2 with 2M dilute hydrochloric acid, the dichloromethane/methanol mixed solvent to extract, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to give the title compound (120 mg crude product).

MS(ESI)m/z[M+H]⁺=349.1。

Step 3 preparation of (R) - (4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1- (2-methyl-1-oxoisoindol-4-yl) -4-oxobutan-2-yl) carbamic acid tert-butyl ester

Crude (R) -3- ((tert-butoxycarbonyl) amino) -4- (2-methyl-1-oxoisoindolin-4-yl) butanoic acid (120 mg) was added to N, N-dimethylformamide (2 mL), N-diisopropylethylamine (0.1 mL), 1-hydroxybenzotriazole (80 mg), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (100 mg) were added sequentially, and after half an hour of reaction at room temperature, N-ethyl-5-fluoro-N-isopropyl-2- ((5- (3, 4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) benzamide (90 mg) was added and reacted overnight at room temperature. LCMS monitored completion of the reaction, quenched with water, extracted with ethyl acetate, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (110 mg).

MS(ESI)m/z[M+H]⁺=743.3。

Step 4 preparation of (R) -2- ((5- (5- (3-amino-4- (2-methyl-1-oxoisoindolin-4-yl) butyryl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

(R) - (4- (5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) -3,4,5, 6-tetrahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1- (2-methyl-1-oxoisoindol-4-yl) -4-oxobutan-2-yl) carbamic acid tert-butyl ester (110 mg) was added to trifluoroethanol (3 mL), trimethylchlorosilane (0.2 mL) was added, and the mixture was reacted at room temperature for 2 hours. LCMS monitored completion of the reaction and solvent was distilled off under reduced pressure, and the residue was adjusted to weakly basic with saturated sodium bicarbonate solution, concentrated and purified by pre-HPLC to give the title compound (56.13 mg).

MS(ESI)m/z[M+H]⁺=643.3。

¹H NMR(400MHz,DMSO-d₆)δ8.56(s,1H),7.52-7.35(m,6H),4.79(m,2H),4.48(s,2H),4.37-4.32(m,4H),4.19-4.13(m,2H),3.70-3.63(p,J＝6.7Hz,1H),3.39(m,1H),3.08(s,3H),3.01(m,1H),2.82-2.77(dd,J＝13.4,5.4Hz,1H),2.65-2.59(m,1H),2.41-2.29(m,2H),1.67(br,2H),1.10-1.03(m,4H),0.99-0.90(m,3H),0.74-0.70(m,3H).

The compounds of examples 35-64 were obtained by reference to the synthetic route with example 34 using (S) -methyl 3- ((tert-butoxycarbonyl) amino) -4-iodobutyrate as starting material and using commercially available or prepared halogenated reagents as reactants, as shown in Table 6.

TABLE 6 examples 35 to 64

Example 65

N-ethyl-2- ((5- ((((1 r,4 r) -4- (ethylsulfonamide) cyclohexyl) methyl) hexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -5-fluoro-N-isopropylbenzamide

Step 1 preparation of 5- (3, 6-dichloro-1, 2, 4-triazin-5-yl) hexahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylic acid tert-butyl ester

Hexahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylic acid tert-butyl ester (5 g) was added to dichloromethane (50 mL), triethylamine (7.24 g) was added dropwise, and then trichloro-1, 2, 4-triazine (4.4 g) was added in portions to react for 1H at room temperature. LCMS monitored completion of the reaction, quenched with water, extracted three times with dichloromethane, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (7.5 g).

MS(ESI)m/z(M+H)⁺=360.1。

Step 2 preparation of tert-butyl 5- (3-chloro-6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) hexahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate

Tert-butyl 5- (3, 6-dichloro-1, 2, 4-triazin-5-yl) hexahydropyrrolo [3,4-C ] pyrrole-2 (1H) -carboxylate (7.5 g) was added to tetrahydrofuran (50 mL), DBU (9.5 g) was added, and N-ethyl-5-fluoro-2-hydroxy-N-isopropylbenzamide (4.7 g) was reacted overnight at 50 ℃. LCMS monitored completion, stopped, cooled to room temperature, quenched with water, extracted with ethyl acetate, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (8.74 g).

MS(ESI)m/z(M+H)⁺=549.2。

Step 3 preparation of tert-butyl 5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) hexahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate

Tert-butyl 5- (3-chloro-6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) hexahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate (8.74 g) was added to tetrahydrofuran (80 mL), sodium borohydride (3.02 g) was added in portions, and tetramethyl ethylenediamine (3.70 g), 1' -bis (diphenylphosphine) ferrocene palladium (II) dichloride (0.58 g) was added, and the reaction was allowed to proceed overnight at room temperature under nitrogen. LCMS monitored completion of the reaction, quenched with water, extracted with ethyl acetate, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (5.12 g).

MS(ESI)m/z(M+H)⁺=515.2。

Step 4 preparation of N-ethyl-5-fluoro-2- ((5- (hexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-isopropylbenzamide

Tert-butyl 5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) hexahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carboxylate (5.12 g) was added to trifluoroethanol (20 mL), trimethylchlorosilane (3.8 mL) was added and reacted at room temperature for 2H. LCMS monitored completion of the reaction, solvent was distilled off under reduced pressure, the residue was taken up in saturated sodium bicarbonate solution to basicity, and the solvent was lyophilized followed by column chromatography purification to give the title compound (4.0 g).

MS(ESI)[M+H]⁺=415.3。

Step 5 preparation of tert-butyl ((1 r,4 r) -4- ((5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) hexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) methyl) cyclohexyl) carbamate

N-ethyl-5-fluoro-2- ((5- (hexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-isopropylbenzamide (0.27 g) was added to methanol (5 mL), tert-butyl ((1 r,4 r) 4-formylcyclohexyl) carbamate (0.1 g) was added, and the reaction was carried out at room temperature under nitrogen atmosphere for 3 hours with sodium cyanoborohydride (0.08 g). LCMS monitored completion of the reaction, water was added to the reaction after spinning dry the methanol solvent, dichloromethane extraction, combined organic phases, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (190 mg).

MS(ESI)[M+H]⁺=626.3。

Step 6 preparation of 2- ((5- (5- ((1 r,4 r) -4-aminocyclohexyl) methyl) hexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide

Tert-butyl ((1 r,4 r) -4- ((5- (6- (2- (ethyl (isopropyl) carbamoyl) -4-fluorophenoxy) -1,2, 4-triazin-5-yl) hexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) methyl) cyclohexyl) carbamate (190 mg) was added to trifluoroethanol (5 mL), trimethylchlorosilane (0.2 mL) was added, and after 2 hours at room temperature. LCMS monitored completion of the reaction, solvent was distilled off under reduced pressure, the residue was adjusted to weak base with saturated sodium bicarbonate solution, extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give the title compound (157 mg).

MS(ESI)[M+H]⁺=526.3。

Step 7 preparation of N-ethyl-2- ((5- ((((1 r,4 r) -4- (ethylsulfonamide) cyclohexyl) methyl) hexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -5-fluoro-N-isopropylbenzamide

2- ((5- (5- ((1 R,4 r) -4-aminocyclohexyl) methyl) hexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -1,2, 4-triazin-6-yl) oxy) -N-ethyl-5-fluoro-N-isopropylbenzamide (157 mg) was added to dichloromethane (5 mL), triethylamine (90 mg) was added dropwise, and ethyl-xanthic chloride (38 mg) was added thereto for reaction at room temperature for 1H. LCMS monitored completion of the reaction, quenched with water, extracted three times with dichloromethane, the combined organic phases dried over anhydrous sodium sulfate, concentrated and purified by pre-HPLC to give the title compound (60.77 mg).

MS(ESI)m/z(M+H)⁺=618.3。

¹H NMR(400MHz,DMSO-d₆)δ7.45-7.39(m,1H),7.38-7.34(m,1H),7.33(dt,J＝4.6,1.5Hz,1H),6.99(d,J＝7.7Hz,1H),4.14(s,1H),2.95(q,J＝7.4Hz,4H),2.89-2.81(m,2H),2.54(s,1H),2.42(d,J＝6.7Hz,2H),2.15(d,J＝7.2Hz,2H),1.86-1.80(m,2H),1.74(d,J＝13.0Hz,2H),1.34-1.02(m,12H),1.02-0.65(m,9H).

Experimental example 1 Menin-MLL protein interaction inhibitory Activity assay

(1) Test of inhibitory Activity of interaction between Menin and MLL proteins

IC ₅₀ values of test compounds inhibiting the interaction of the Menin-MLL proteins were measured using the method of Fluorescence Polarization.

The specific procedure was to three-fold dilutions of compound stock (10 mM, DMSO configuration) to ten concentrations of 10000.00, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52, 0.51nM in DMSO. 50nL of test compounds (10 gradient concentrations) and DMSO solutions without compound (negative control wells) were each taken by ECHO 665Series Acoustic Liquid Handlers (BECKMAN Inc.), transferred to 384 well plates and centrifuged at 1000rpm for use. mu.L of Menin (ICE Inc., cat No. E2208F-H15H) was pipetted into each assay well using I.DOT (DISPENDIX Inc.), and incubated for 10min at 25 ℃. mu.L of FITC-MLL4-43 (Genscrip Inc.) was pipetted using I.DOT into each assay well, centrifuged at 1000rpm and incubated for 60 minutes. FP signals were measured using a PHERASTAR FSX multifunctional microplate reader (BMG Labtech inc.) and the data processed.

Compound IC50 = (negative control well signal-compound well signal)/(negative control well signal-background signal) 100% (background signal is signal value detected in assay well containing only 10 μm SNDX-5613), log value of compound concentration is used as X-axis, percent Inhibition (Inhibition%) is used as Y-axis, and dose-response curve is fitted to obtain IC50 value of each compound inhibiting the interaction of the Menin-MLL protein. The experimental results are shown in table 7.

TABLE 7 IC ₅₀ values for compounds of the invention which inhibit the interaction of the Menin-MLL proteins

Note that in table 7, "-" indicates a temporary undetected. The numbers under the term "compound number" denote the compounds in the corresponding examples. SNDX5613 refers to N-ethyl-2- ((4- (7- (((1 r,4 r) -4- (ethylsulfonylamino) cyclohexyl) methyl) -2, 7-diazaspiro [3.5] non-2-yl) pyrimidin-5-yl) oxy) -5-fluoro-N-isopropylbenzamide (hereinafter, also referred to as "Shanghai Lai Biotechnology Co., ltd.) having the following structural formula:

The above experiments show that the compounds of the present invention have excellent activity in inhibiting the interaction of the Menin-MLL protein.

(2) Mutant Menin-M327I, menin-T349M and MLL protein interaction inhibition Activity assay

IC ₅₀ values of test compounds that inhibit the interaction of Menin-M327I, menin-T349M with MLL proteins were tested using the Fluorescence Polarization method.

The specific procedure was to three-fold dilutions of compound stock (10 mM, DMSO configuration) to ten concentrations of 10000.00, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, 1.52, 0.51nM in DMSO. 50nL of test compounds (10 gradient concentrations) and DMSO solutions without compound (negative control wells) were each taken by ECHO 665Series Acoustic Liquid Handlers (BECKMAN Inc.), transferred to 384 well plates and centrifuged at 1000rpm for use. mu.L of both Menin-M327I (ICE Inc. Cat No. A130412011) and Menin-T349M (ICE Inc. Cat No. A130413011) were pipetted separately into each assay well using I.DOT (DISPENDIX Inc.), and incubated for 10min at 25 ℃. mu.L of FITC-MLL4-43 (Genscrip Inc.) was pipetted using I.DOT into each assay well, centrifuged at 1000rpm and incubated for 60 minutes. FP signals were measured using a PHERASTAR FSX multifunctional microplate reader (BMG Labtech inc.) and the data processed.

Compound IC ₅₀ = (negative control well signal-compound well signal)/(negative control well signal-background signal) 100% (background signal is signal value detected in assay well containing only 100 μm SNDX-5613), log of compound concentration is used as X-axis, percent Inhibition (Inhibition%) is used as Y-axis, and dose-response curve is fitted to obtain IC ₅₀ value of each compound inhibiting interaction of Menin-M327I, menin-T349M with MLL protein. The experimental results are shown in table 8.

TABLE 8 IC50 values for compounds of the invention inhibiting the interaction of a Menin mutein with an MLL protein

Note that in table 8, the numbers under the "compound numbers" indicate compounds in the corresponding examples.

The above experiments show that the compound has excellent activity of inhibiting the interaction between the Menin mutant protein and the MLL protein, thereby having good drug resistance prospect.

Experimental example 2 cell proliferation inhibition experiment

(1) Cell plating:

the cells were removed from the incubator and placed on a console, gently swirled, mixed, and counted using CounterStar.

Cells were diluted to the desired density with fresh complete medium. MV-4-11 (cell source is Nanjac Bai, cat# CBP 60522), RPMI1640 (HEPES-containing) (BOSTER, cat# PYG 0122) +10% FBS (GIBCO, cat# 10099-141C) +1% P/S (Hyclone, cat# SV 30010), plating density 1X 10≡4 cells/well, 100. Mu.L/well.

Mu.L of each of the above cell suspensions was individually pipetted into a 96-well plate using an electric row gun.

(2) Preparing a compound:

The compound stock was diluted from 10mM to 4000, 1200, 400, 120, 40, 12, 4, 1.2, 0. Mu.M in DMSO, with 0. Mu.M as control well.

After thorough mixing, 1.3. Mu.L was removed by an electric lance and added to 258.7. Mu.L of medium, at which time the dilution was 100-fold and the DMSO content was 0.5%.

After the cells were thoroughly mixed in the row gun mixing mode, two wells were prepared for each compound, and 100. Mu.L of the diluted compound of step 2 was added to each well, wherein the final concentrations of the compound were 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0. Mu.M, 200. Mu.L of medium was used in each well of the cell culture plate, and the DMSO content was 0.25%, wherein the wells containing only 0.25% DMSO (compound concentration: 0) were control wells.

Cells were returned to 37℃in a 5% CO2 incubator for continued culture and were examined after 3 days of treatment with the compound.

(3) CTG detection:

After incubation to time, cells were removed and a portion of the medium was aspirated so that 50. Mu.L of medium was left per well, and 50. Mu.L/well CTG reagent (cellcounting-Lite 2.0, vazyme, DD 1101-02) was added with a row gun.

Shaking and incubating for 15min at room temperature, and standing and balancing for 15min at room temperature.

And detecting by using a multifunctional enzyme-labeled instrument.

(4) Data analysis:

Cell viability was calculated, cell viability% = As/ac×100%. As: test wells (cell-containing medium, CTG, test compound), ac: control wells (cell-containing medium, CTG, no test compound).

The IC ₅₀ values of the Cell proliferation inhibitory activity of each compound were obtained by fitting a dose-response curve with the log of the compound concentration as the X-axis and the Cell viability as the Y-axis. The specific results are shown in Table 9.

TABLE 9 proliferation inhibitory Activity of the Compounds of the invention on MV-4-11 cells

In table 9, "-" indicates no content. The numbers under the term "compound number" denote the compounds in the corresponding examples. The test results show that the compound has excellent inhibitory activity on MV-4-11 cells.

Claims (15)

1. A compound of formula I, a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, In Formula 1: m = 0, 1 or 2; n = 0, 1, 2 or 3; R1 is selected from hydrogen, hydroxy, halogen, cyano, C1-C3 alkyl, C1-C3 alkoxy or C1-C3 haloalkyl; R2 and R3 are independently selected from hydrogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, RxCO-, sulfonyl or phosphonyl, wherein Rx is alkyl or alkenyl; R4 and R5 are independently selected from hydrogen, hydroxy, halogen, C1-C3 alkyl, C1-C3 alkoxy or C1-C3 haloalkyl; R6 is selected from substituted or unsubstituted 5-7 membered aryl, 5-7 membered heteroaryl, 9-16 membered saturated or partially unsaturated cycloalkyl, 9-16 membered saturated or partially unsaturated heterocyclic group, 9-16 membered fused aryl, 9-16 membered fused heteroaryl. 2. The compound according to claim 1, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that R1 is selected from fluorine, chlorine, bromine, iodine, cyano, hydroxyl, amino, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, chloromethyl, trifluoromethyl or perfluoroethyl. 3. The compound according to any one of claims 1 or 2, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that Rx is C1-C3 alkyl or C2-C4 alkenyl. 4. The compound according to claim 3, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that R2 is hydrogen, and R3 is selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, chloromethyl, trifluoromethyl, perfluoroethyl, acetyl, acryloyl, dimethylphosphonyl, methylsulfonyl or ethylsulfonyl. 5. The compound according to claim 1, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that R4 is hydrogen, and R5 is selected from hydrogen, hydroxyl, fluorine, chlorine, bromine, iodine, cyano, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, chloromethyl, trifluoromethyl or perfluoroethyl. 6. The compound according to claim 1, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that R6 is selected from substituted or unsubstituted 5-7 membered aryl, 5-7 membered heteroaryl, 9-12 membered saturated or partially unsaturated cycloalkyl, 9-12 membered saturated or partially unsaturated heterocyclyl, 9-12 membered fused aryl, 9-12 membered fused heteroaryl, wherein the backbone atoms of the heterocyclyl or heteroaryl optionally contain 1-3 heteroatoms selected from N, O, and S. 7. The compound according to claim 6, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that R6 can be selected from the following substituted or unsubstituted cyclic groups: furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl; phenyl, pyridyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl; indolyl, benzimidazolyl, benzofuranyl, benzothiazolyl, benzisothiazole, benzoxazolyl, benzisoxazole, benzothienyl, indazolyl, pyrrolo[1,2-a] Pyridine, imidazo[1,2-a]pyridine, imidazo[1,5-a]pyridine, pyrazolo[1,5-a]pyridine, isoindolin-1-one, 3,4-dihydroisoquinolin-1(2H)-one; naphthyl, quinolinyl, isoquinolinyl, naphthyridinyl, pyridadiazinyl, benzotriazinyl, benzotriazinyl, benzopyranyl, benzoγ-pyrone, tetrahydroisoquinolinyl, tetrahydronaphthyl, 3,4-dihydro-2H-1,4-benzoxazinyl, 2H-1,4-benzoxazin-3(4H)-one or 1,2,3,5,6,7-hexahydro-S-dipentacyclophenyl. 8. The compound according to claim 6 or 7, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that R6 is optionally substituted at any possible position by one or more groups selected from oxygen, hydroxyl, amino, carboxyl, cyano, halogen, phosphono, sulfonyl, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, RaCO-, C2-C4 alkenyl, C3-C6 cycloalkyl, 3-6 membered heterocycloalkyl, 5-7 membered aryl or 5-7 membered heteroaryl, wherein Ra is C1-C5 alkyl or Ra is C2-C4 alkenyl. 9. The compound according to any one of claims 1 to 8, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, characterized in that R6 is selected from the following substituted or unsubstituted groups: 10. The compound according to claim 9, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, wherein R6 is optionally substituted at any possible position by one or more groups selected from oxygen, hydroxyl, amino, carboxyl, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, methoxy, ethoxy, chloromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, perfluoroethyl, formyl, acetyl, acryloyl, methylphosphonyl, dimethylphosphonyl, methylsulfonyl, ethylsulfonyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, triazole, tetrazole, phenyl, or pyridyl. 11. The following compounds, their pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures thereof, 12. A pharmaceutical composition comprising the compound according to any one of claims 1 to 11, a pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, and a pharmaceutically acceptable excipient. 13. Use of the compound according to any one of claims 1 to 11, its pharmaceutically acceptable salt, hydrate, isomer, prodrug or mixture thereof, or the pharmaceutical composition according to claim 12, for preparing a medicament for preventing, alleviating or treating diseases related to the interaction with Menin-MLL protein. 14. The use according to claim 13, characterized in that the diseases caused by the interaction with Menin-MLL protein include malignant tumors, diabetes or complications related to the diseases, wherein the malignant tumors include hematological tumors, lymphomas, and solid tumors. 15. The use according to claim 14, characterized in that the blood tumors include leukemias and myelomas, including but not limited to acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute monocytic leukemia, chronic monocytic leukemia, childhood leukemia, acute myeloid leukemia, chronic myeloid leukemia, mixed lineage leukemia, hairy cell leukemia, precursor T-cell lymphocytic leukemia, large granular lymphocytic leukemia, meningeal leukemia, myelodysplastic syndrome, myeloproliferative disease, myeloproliferative neoplasia, plasma cell tumor, multiple myeloma; Lymphoma includes, but is not limited to, cutaneous T-cell lymphoma, lymphoma, AIDS-related lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or malignant lymphoma; Solid tumors include but are not limited to pancreatic cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, glioblastoma, lung cancer, breast cancer, and prostate cancer; Related complications include, but are not limited to, leukemic meningitis.