Substituted polycyclic compound

Abstract

A substituted polycyclic compound having biological inhibitory activity represented by formula (II), a preparation method therefor, and a use thereof. The use comprises a use of the compound of formula (II) in the preparation of a drug for treating MLL-related diseases. In the preparation process, the compound is obtained by means of a series of reactions such as substitution, cyclization, reduction, and deprotection.

Description

Substituted polycyclic compounds

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202311648598.0 filed on December 4, 2023, entitled “Substituted Polycyclic Compounds”, Chinese Patent Application No. 202410472871.7 filed on April 18, 2024, entitled “Substituted Polycyclic Compounds”, Chinese Patent Application No. 202410895093.2 filed on July 4, 2024, entitled “Substituted Polycyclic Compounds”, and Chinese Patent Application No. 202411230078.2 filed on September 3, 2024, the entire disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention generally relates to novel substituted heterocyclic compounds having Menin-MLL interaction inhibitory activity, methods for their preparation, and pharmaceutical compositions thereof, and also relates to the use of such compounds and pharmaceutical compositions thereof in treating diseases that benefit from inhibition of Menin-MLL interaction, such as the treatment of acute leukemia. Background Art

[0004] The development of human acute leukemia is associated with genetic and epigenetic alterations (Figueroa 2010; Rodríguez 2011). Approximately 5% to 10% of children and adults with acute leukemia develop a chromosomal translocation in the 11q23 region of chromosomes, resulting in rearrangement of KMT2A (MLL1) (Bill 2020). MLL1 rearrangement (MLL-r) triggers an aggressive form of leukemia with a 5-year survival rate of only 35% (Dimartino 1999; Marschalek 2011). This translocation occurs in up to 80% of infants with acute lymphoblastic leukemia (Brown 2013). MLL-r leukemia generally carries a poor prognosis, especially in infants with acute lymphoblastic leukemia, and effective and less toxic treatments are still lacking.

[0005] MLL1 is a histone lysine-N methyltransferase responsible for H3K4 methylation and is essential for the expression of the HOX family of hematopoietic cell differentiation regulators. MLL1 rearrangement to form MLL1 fusion proteins leads to transcriptional aberrations, driving the development of acute leukemia (Meyer 2018). MLL1 fusion proteins regulate target gene expression through interactions with chromatin-associated protein complexes. Menin, a key MLL partner protein in this complex, plays a crucial role in MLL fusion protein-driven target gene expression, regulating the expression of MEIS1 and HOX family proteins (Chen 2006). Menin has a surface binding pocket for MLL1, allowing it to bind with high affinity to a conserved sequence at the N-terminus of MLL1. This interaction is crucial for the transcriptional regulation of target genes by MLL1 fusion proteins (Yokoyama 2005; Caslini 2007). Therefore, modulating the Menin-MLL interaction is an important target for small molecule therapies for MLL-r leukemia. Blocking the interaction between Menin and MLL1 fusion protein using small molecule inhibitors has been shown to be a potential therapeutic option for treating MLL-r leukemia.

[0006] Currently, no Menin-MLL inhibitors have been approved for marketing, but six inhibitors have recently entered clinical trials abroad. Syndax's inhibitor SNDX-5613 (revumenib) first entered a Phase I / II clinical trial (NCT04065399) on August 22, 2019, targeting acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), mixed lineage acute leukemia (MLAL), mixed phenotype acute leukemia (MPAL), and unidentified lineage acute leukemia (ALAL) with MLL rearrangements or NPM1 mutations. Kura's inhibitor KO-539 began a Phase I / II clinical trial on August 26, 2019 (NCT04067336). Janssen began a Phase I clinical trial of JNJ-75276617 on March 21, 2021 (NCT04065399). Daiichi Sankyo's inhibitor DS-1594 began a Phase I clinical trial on March 25, 2021. Biomea's inhibitor BMF-219 began a Phase I clinical trial on January 24, 2022. Sumitomo's inhibitor DSP-5336 began a Phase I clinical trial on February 28, 2022. Yehui Pharmaceutical's BN104 began a Phase I clinical trial on September 4, 2023. Bayer, Agios, the University of Michigan, the University of Pennsylvania, and others also have patent portfolios.

[0007] Recently, according to the results of the AUGMENT-101 (NCT04065399) study, patients developed acquired resistance after receiving treatment with the Menin inhibitor revumenib (Perner 2023). Second-generation sequencing of bone marrow samples from resistant patients revealed somatic mutations in Menin. The mutation sites included M327V or M327I, T349M, G331R, and S160T, with a mutation frequency of 5.9% to 28.2%. The PDX model showed that when mice were continuously treated with the Menin inhibitor VTP-50469, 68% of the relapsed samples had mutations in Menin, including MEN1. M327V , MEN1 M327I , MEN1 G331R , MEN1 G331D , MEN1 T349M and MEN1 S160C , nearly identical to the patient's mutation site. Crystal structures revealed that the three most frequently mutated residues, M327, G331, and T349, are located near W346, impairing hydrogen bonding between revumenib and the indole NH group of W346. This reduces revumenib's binding activity to the Menin protein, but does not affect the Menin-MLL binding surface. Cellular and in vitro binding experiments also demonstrated a significant reduction in revumenib binding activity to the mutant Menin protein, thereby preventing revumenib-mediated regulation of leukemia gene expression by the Menin-MLL1 protein complex.

[0008] Therefore, developing a new generation of Menin inhibitors targeting Menin resistance mutations will effectively overcome the problem of drug resistance recurrence in patients, which is a huge clinical need. We use computer-aided drug design to discover novel compounds and, through structural modification, we obtain the desired new compounds with strong efficacy and good pharmaceutical properties against Menin resistance mutations. Summary of the Invention

[0009] The present invention provides a compound represented by formula (II) or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof,

[0010] in,

[0011] Ring A is a 5-8 membered nitrogen-containing heterocyclic ring or nitrogen-containing heteroaromatic ring, X is independently C or N,

[0012] R 1a H, -C(=O)-NR 2a R 2b 、 Phenyl, or 5-6 membered heteroaryl, said heteroaryl containing 1 oxygen atom and / or 1 sulfur atom and / or 1-3 nitrogen atoms, and optionally containing a carbonyl group, and said phenyl and heteroaryl are optionally substituted by 3-6 membered cycloalkyl or C 1-4 Alkyl substitution,

[0013] R 2a and R 2b Each independently is H or C 1-4 Alkyl, which may be optionally substituted with -OH or -NH2,

[0014] R 1b is F or Cl,

[0015] R2 is selected from H, halogen, C 1-4 Alkyl, -OC 1-4 Alkyl and -NR 3a R 3b ,

[0016] R 3a and R 3b Each independently is H or C 1-4 alkyl,

[0017] X1 and X2 are each independently N or CH,

[0018] X3 is N or R 10 ,

[0019] R 10 H, halogen, C 1-6 Alkyl, or -OC 1-6 alkyl,

[0020] L1 is -CH2-, -CH2-CH2- or -CH2-CH2-CH2-,

[0021] L2 and L3 are each independently -CH2- or -CH2-CH2-,

[0022] R4 is C 1-6 Alkyl, 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl,

[0023] R3 is -C 1-6 Alkylene-NR 4a R 4b 、-C 1-6 Alkylene-C(O)-NR 5a R 5b 、-C 1-6 Alkylene-OH, or -C 1-6 Alkylene-NR7-C(O)-OC 1-4 Alkylene-OC(O)-C1-4 Alkyl, wherein the alkyl or alkylene group may be optionally substituted with halogen, -CN, -OH or -OC l-4 Alkyl substitution,

[0024] R 4a and R 4b Each independently selected from H, C 1-6 Alkyl, -C(O)-C 1-4 Alkyl, -C(O)-OC 1-4 Alkyl and -C(O)-NR 8a R 8b , the C 1-6 The alkyl group may be optionally substituted with halogen, -CN, -OH, -S(O)2-C 1-4 Alkyl, -OC 1-4 Alkyl, -C(O)-NR 6a R 6b or -NR 6c -C(O)-C 1-4 Alkyl substitution,

[0025] R 5a 、R 5b 、R 6a 、R 6b 、R 6c , R7, R 8a and R 8b Each independently selected from H and C 1-6 alkyl.

[0026] In another aspect, the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof,

[0027] in,

[0028] Ring A is a 5-8 membered nitrogen-containing heterocyclic ring or nitrogen-containing heteroaromatic ring, X is independently C or N,

[0029] R 1a -C(=O)-NR 2a R 2b 、 or a 5-6 membered heteroaryl group, wherein the heteroaryl group contains 1-3 nitrogen atoms and optionally contains a carbonyl group, and the phenyl group and the heteroaryl group are optionally replaced by a 3-6 membered cycloalkyl group or a C 1-4 Alkyl substitution,

[0030] R 2a and R 2b Each independently is H or C 1-4 Alkyl, which may be optionally substituted with -OH or -NH2,

[0031] R 1b is F or Cl,

[0032] R2 is selected from H, halogen, C 1-4 Alkyl, -OC 1-4 Alkyl and -NR 3a R 3b ,

[0033] R 3a and R 3b Each independently is H or C 1-4 alkyl,

[0034] X1 and X2 are each independently N or CH,

[0035] L1 is -CH2-, -CH2-CH2- or -CH2-CH2-CH2-,

[0036] L2 and L3 are each independently -CH2- or -CH2-CH2-,

[0037] R4 is C 1-6 Alkyl, 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl,

[0038] R3 is -C 1-6 Alkylene-NR 4a R 4b 、-C 1-6 Alkylene-C(O)-NR 5a R 5b 、-C 1-6 Alkylene-OH, or -C 1-6 Alkylene-NR7-C(O)-OC 1-4 Alkylene-OC(O)-C 1-4 Alkyl, wherein the alkyl or alkylene group may be optionally substituted with halogen, -CN, -OH or -OC l-4 Alkyl substitution,

[0039] R 4a and R 4b Each independently selected from H, C 1-6 Alkyl, -C(O)-C 1-4 Alkyl, -C(O)-OC 1-4 Alkyl and -C(O)-NR 8a R 8b , the C 1-6 The alkyl group may be optionally substituted with halogen, -CN, -OH, -S(O)2-C 1-4 Alkyl, -OC 1-4 Alkyl, -C(O)-NR 6a R6b or -NR 6c -C(O)-C 1-4 Alkyl substitution,

[0040] R 5a 、R 5b 、R 6a 、R 6b 、R 6c , R7, R 8a and R 8b Each independently selected from H and C 1-6 alkyl.

[0041] In some embodiments, for or Preferably or

[0042] In some embodiments, R 1a -C(=O)-NR 2a R 2b , R 2a and R 2b Each independently is H or C 1-4 alkyl.

[0043] In some embodiments, R 1a for

[0044] In some embodiments, R 1b For F.

[0045] In some embodiments, R4 is isopropyl.

[0046] In some embodiments, R2 is H or C 1-4 Alkyl, preferably H.

[0047] In some embodiments, R3 is -C 1-6 Alkylene-NR 4a R 4b , R 4a and R 4b Each independently selected from C 1-6 Alkyl, the C 1-6 The alkyl group may be optionally replaced by -OC 1-4 Alkyl substitution.

[0048] In some embodiments, R3 is -C 1-6 Alkylene-N(C 1-6 alkyl)-C 1-6 Alkylene-OC 1-4alkyl.

[0049] In some embodiments, R3 is

[0050] In some embodiments, the present invention provides the following compounds or pharmaceutically acceptable salts, solvates, polymorphs, tautomers, metabolites or prodrugs thereof:

[0051] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph, or isomer thereof, and optionally comprising a pharmaceutically acceptable carrier.

[0052] In another aspect, the present invention provides a method for treating a disease associated with MLL activity, comprising administering to a subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, or a pharmaceutical composition of the present invention; in some embodiments, the disease associated with MLL activity is cancer, preferably acute leukemia (including MLL acute leukemia, MLL partial tandem duplication acute leukemia, NPM mutation acute leukemia, MOZ acute leukemia, NUP98 acute leukemia and CALM acute leukemia), chronic lymphocytic leukemia, chronic myeloid leukemia, myelodysplasia syndrome, polycythemia vera, malignant lymphoma (including B-cell lymphoma), myeloma (including multiple myeloma), brain tumor, head and neck cancer, esophageal cancer, thyroid cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, gallbladder and bile duct cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, colon cancer, rectal cancer, anal cancer, chorioepithelialoma, endometrial cancer, cervical cancer, ovarian cancer, bladder cancer, urothelial cancer, kidney cancer, renal cell carcinoma, prostate cancer, testicular tumor, testicular germ cell tumor, ovarian germ cell tumor, Wilms' tumor, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, soft tissue sarcoma, or skin cancer.

[0053] The compound of the present invention or its pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug, or the pharmaceutical composition of the present invention, is used in combination with at least one different agent, wherein the different agent is at least one agent selected from antitumor alkylating agents, antitumor antibiotics, plant-derived antitumor drugs, antitumor platinum coordination compounds, antitumor camptothecin derivatives, antitumor tyrosine kinase inhibitors, antitumor serine / threonine kinase inhibitors, antitumor phospholipid kinase inhibitors, antitumor monoclonal antibodies, interferons, biological response modifiers, hormone preparations, angiogenesis inhibitors, immune checkpoint inhibitors, epigenetic-related molecule inhibitors, protein post-translational modification inhibitors, proteasome inhibitors and other antitumor drugs.

[0054] In another aspect, the present invention provides a use of a compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, or a pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease associated with MLL activity; in some embodiments, the disease associated with MLL activity is cancer, preferably acute leukemia (including MLL acute leukemia, MLL partial tandem duplication acute leukemia, NPM mutation acute leukemia, MOZ acute leukemia, NUP98 acute leukemia and CALM acute leukemia), chronic lymphocytic leukemia, chronic myeloid leukemia, myelodysplastic syndrome , polycythemia vera, malignant lymphoma (including B-cell lymphoma), myeloma (including multiple myeloma), brain tumor, head and neck cancer, esophageal cancer, thyroid cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, gallbladder and bile duct cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, colon cancer, rectal cancer, anal cancer, chorioepithelialoma, endometrial cancer, cervical cancer, ovarian cancer, bladder cancer, urothelial cancer, kidney cancer, renal cell carcinoma, prostate cancer, testicular tumor, testicular germ cell tumor, ovarian germ cell tumor, Wilms' tumor, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, soft tissue sarcoma, or skin cancer.

[0055] Detailed Description of the Invention

[0056] Exemplary embodiments utilizing the principles of the present invention are set forth in the following detailed description of the invention. The features and advantages of the present invention may be better understood by reference to the following summary of the invention.

[0057] It should be understood that the scope of protection of various aspects of the present invention is determined by the claims, and methods and structures within the scope of these claims and their equivalents are all within the scope of the claims.

[0058] Unless otherwise defined, all technical and scientific terms herein have the same meanings as commonly understood by those skilled in the art to which the subject matter of the claims pertains. Unless otherwise indicated, all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

[0059] It should be understood that the foregoing brief description and the following detailed description are exemplary and explanatory, rather than limiting, of any subject matter of the present invention. Unless otherwise specifically stated, the use of the singular also includes the plural. Unless otherwise specified, the use of "or" and "alternatively" means "and / or." In addition, the use of the term "include," as well as other forms such as "comprises," "includes," and "comprising" are not limiting.

[0060] Some chemical terms

[0061] The terms "optional," "optional," or "optionally" mean that the subsequently described event or circumstance may or may not occur, and the description includes both the occurrence of the event or circumstance and the non-occurrence of the event or circumstance. For example, "optionally substituted alkyl" means "unsubstituted alkyl" or "substituted alkyl." Furthermore, an optionally substituted group may be unsubstituted (e.g., -CH2CH3), fully substituted (e.g., -CF2CF3), monosubstituted (e.g., -CH2CH2F), or any hierarchy between monosubstituted and fully substituted (e.g., -CH2CHF2, -CF2CH3, -CFHCHF2, etc.). It will be understood by those skilled in the art that for any group containing one or more substituents, no substitution or substitution pattern that would be sterically impossible and / or incomposable to synthesize will be introduced.

[0062] Unless otherwise indicated, conventional methods within the technical scope of the art, such as mass spectrometry, nuclear magnetic resonance, high performance liquid chromatography, infrared and ultraviolet / visible spectroscopy and pharmacological methods, are adopted. Unless specifically defined, the relevant terms and experimental procedures and techniques of analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry herein are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and in the treatment of patients. For example, the manufacturer's instructions for use of the test kit can be utilized, or reactions and purification can be carried out according to methods well known in the art or the description of the present invention. Conventionally, the above-mentioned techniques and methods can be implemented according to conventional methods well known in the art, based on the description in the multiple summaries and more specific documents cited and discussed in this specification. In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural moieties and compounds.

[0063] When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents that would result if the formula were written from right to left. For example, -CH2O- is equivalent to -OCH2-.

[0064] As used herein, the terms "group" and "chemical group" refer to a specific part or functional group of a molecule. A chemical group is often considered to be a chemical entity embedded in or attached to a molecule.

[0065] Some chemical groups named herein may be abbreviated to indicate the total number of carbon atoms. For example, C1-C6 alkyl describes an alkyl group, as defined below, having a total of 1 to 6 carbon atoms. The total number of carbon atoms indicated in the abbreviated notation does not include carbon atoms in possible substituents.

[0066] The term "halogen," "halo," or "halide" refers to bromine, chlorine, fluorine, or iodine.

[0067] As used herein, the terms "aromatic," "aromatic ring," "aromatic," "aromatic," and "aromatic ring" refer to a planar ring or ring moiety having a delocalized electron conjugate system containing 4n+2 electrons, where n is an integer. Aromatic rings can be formed by 5, 6, 7, 8, 9, or more atoms. Aromatic compounds can be optionally substituted and can be monocyclic or polycyclic with fused rings. The term aromatic compound includes all carbon rings (e.g., benzene rings) and rings containing one or more heteroatoms (e.g., pyridine).

[0068] As used herein, the term "heteroatom" or "hetero" refers to atoms other than carbon and hydrogen, either alone or as part of another component. Heteroatoms are independently selected from the group consisting of oxygen, nitrogen, sulfur, phosphorus, silicon, selenium, and tin, but are not limited to these atoms. In embodiments where two or more heteroatoms are present, the two or more heteroatoms may be the same as one another, or some or all of the two or more heteroatoms may be different from one another.

[0069]

[0046] The term "fused" or "fused ring," as used herein, alone or in combination, refers to a cyclic structure in which two or more rings share one or more bonds.

[0070]

[00146] The term "spiro" or "spirocycle," as used herein, alone or in combination, refers to a cyclic structure in which two or more rings share one or more atoms.

[0071] The term "alkyl" as used herein alone or as part of another component (e.g., monoalkylamino) refers to an optionally substituted straight-chain or optionally substituted branched monovalent saturated hydrocarbon group having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms, connected to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, n-octyl, n-nonyl, n-decyl, and the like.

[0072] As used herein, the term "alkylene" refers to an optionally substituted straight-chain or optionally substituted branched divalent saturated hydrocarbon group having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms.

[0073] The term "aryl" refers to an all-carbon monocyclic or fused ring having a completely conjugated π electron system having 6-14 carbon atoms, preferably 6-12 carbon atoms, and most preferably 6 carbon atoms. Aryl can be unsubstituted or substituted with one or more substituents, examples of which include but are not limited to alkyl, alkyloxy, aryl, aralkyl, amino, halogen, hydroxyl, sulfonyl, sulfinyl, phosphoryl, and heteroalicyclic groups. Non-limiting examples of unsubstituted aryl include but are not limited to phenyl, naphthyl, and anthracenyl.

[0074] The term "heteroaryl" refers to a monocyclic or fused ring of 5-12 ring atoms, having 5, 6, 7, 8, 9, 10, 11 or 12 ring atoms, of which 1, 2, 3 or 4 are selected from N, O, S, with the remaining ring atoms being C, and having a completely conjugated π-electron system. Heteroaryl groups may be unsubstituted or substituted, with substituents including, but not limited to, alkyl, alkyloxy, aryl, aralkyl, amino, halogen, hydroxy, cyano, nitro, carbonyl and heteroalicyclic groups. Non-limiting examples of unsubstituted heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl and triazinyl.

[0075] The term "cycloalkyl," as used herein, alone or in combination, refers to a stable, monovalent, non-aromatic, monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, which may include fused, spiro, or bridged ring systems, containing 3-15 carbon atoms, preferably 3-10 carbon atoms, more preferably 3-8 carbon atoms, which may be saturated or unsaturated, and which is attached to the rest of the molecule by a single bond. Non-limiting examples of "cycloalkyl" include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0076] The terms "heterocyclyl," "heterocycloalkyl," and "heterocycle," as used herein, alone or as part of another component, refer to a stable 3-18 membered monovalent non-aromatic ring comprising 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, a heterocyclyl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may contain fused, spirocyclic, or bridged ring systems. The nitrogen, carbon, or sulfur atoms in the heterocyclyl group may be optionally oxidized, the nitrogen atom may be optionally quaternized, and the heterocyclyl group may be partially or fully saturated. The heterocyclyl group may be attached to the rest of the molecule via a single bond through a carbon atom or heteroatom in the ring. A heterocyclyl group containing a fused ring may contain one or more aromatic or heteroaromatic rings, as long as the atoms attached to the rest of the molecule are non-aromatic ring atoms. For the purposes of the present application, the heterocyclic group is preferably a stable 4-11 membered monovalent non-aromatic monocyclic or bicyclic ring containing 1-3 heteroatoms selected from nitrogen, oxygen and sulfur, more preferably a stable 4-8 membered monovalent non-aromatic monocyclic ring containing 1-3 heteroatoms selected from nitrogen, oxygen and sulfur. Non-limiting examples of heterocyclic groups include azepanyl, azetidinyl, decahydroisoquinolinyl, dihydrofuranyl, dihydroindolinyl, dioxolane, 1,1-dioxo-thiomorpholinyl, imidazolidinyl, imidazolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazinyl, piperazinyl, piperidinyl, 4-piperidonyl, pyranyl, pyrazolidinyl, pyrrolidinyl, quinolizinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydropyranyl, and the like.

[0077] The term "polymorph" or "polymorphism" as used herein refers to compounds of the present invention having multiple crystal lattice morphologies. Some compounds of the present invention may have more than one crystal form, and the present invention encompasses all polymorphic forms or mixtures thereof.

[0078] Intermediate compounds of the compounds of the present invention and polymorphs thereof are also within the scope of the present invention.

[0079] Unless otherwise specified, compounds of the present invention containing olefinic double bonds include both E and Z isomers.

[0080] The compounds of the present invention include compounds having one or more isotopic substitutions, and references to specific elements include within their scope all isotopes of that element. For example, references to hydrogen include within their scope 1 H. 2 H(D) and 3 H(T). Similarly, references to carbon and oxygen include within their scope 12 C. 13 C and 14 C and 16 O and 18 O.

[0081] It should be understood that the compounds of the present invention may contain asymmetric centers. These asymmetric centers can independently be in the R or S configuration. Some compounds of the present invention may also exhibit cis-trans isomerism, which is apparent to those skilled in the art. It should be understood that the compounds of the present invention include their individual geometric isomers and stereoisomers and mixtures thereof, including racemic mixtures. These isomers can be separated from their mixtures by implementing or modifying known methods, such as chromatography and recrystallization techniques, or they can be prepared separately from the appropriate isomers of their intermediates.

[0082] As used herein, the term "pharmaceutically acceptable salt" includes both acid-addition salts and base-addition salts.

[0083] "Pharmaceutically acceptable salts" refer to salts formed with inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, capric acid, hexanoic acid, carbonic acid, cinnamic acid, and citric acid, which retain the biological efficacy and properties of the free acid and are not biologically or otherwise undesirable. "Pharmaceutically acceptable base-added salts" refer to salts that retain the biological efficacy and properties of the free acid and are not biologically or otherwise undesirable. These salts are prepared by reacting the free acid with an inorganic or organic base. Salts formed by reaction with inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and manganese salts.

[0084] Organic bases for forming salts include, but are not limited to, primary amines, secondary amines, tertiary amines, cyclic amines, and the like, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, ethanolamine, dicyclohexylamine, ethylenediamine, purine, piperazine, piperidine, choline, and caffeine. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0085] Crystallization often produces solvates of the compounds of the invention. The term "solvate" as used herein refers to an association of one or more molecules of the compound of the invention with one or more solvent molecules.

[0086] The solvent may be water, in which case the solvate is a hydrate. Alternatively, it may be an organic solvent. Thus, the compounds of the present invention may exist as hydrates, including monohydrates, dihydrates, hemihydrates, trihydrates, tetrahydrates, and the like, as well as the corresponding solvated forms. The compounds of the present invention may be true solvates, but in other cases, the compounds of the present invention may simply accidentally retain water or a mixture of water and some other solvent. The compounds of the present invention may be reacted in a solvent or precipitated or crystallized in a solvent. Solvates of the compounds of the present invention are also encompassed within the scope of the present invention.

[0087] The term "pharmaceutical composition" as used herein refers to a preparation containing a compound of the present invention and a medium generally accepted in the art for delivering biologically active compounds to mammals (such as humans), including all pharmaceutically acceptable carriers.

[0088] The term "acceptable" with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.

[0089] As used herein, the term "pharmaceutically acceptable" refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention and is relatively non-toxic, that is, the substance can be administered to a subject without causing an adverse biological response or interacting in an adverse manner with any components contained in the composition.

[0090] "Pharmaceutically acceptable carrier" includes, but is not limited to, adjuvants, carriers, excipients, auxiliary agents, deodorants, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants and wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that have been approved by relevant government administrative departments for use in humans and domesticated animals.

[0091] As used herein, the terms "subject," "patient," "subject," or "individual" refer to individuals suffering from a disease, disorder, or condition, and include mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (e.g., chimpanzees and other apes and monkeys); livestock, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs. Examples of non-human mammals include, but are not limited to, birds and fish. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0092] The term "treatment" as used herein refers to the treatment of a disease or condition in a mammal, especially a human, including

[0093] (i) preventing the development of a disease or condition in a mammal, particularly a mammal that has been previously exposed to the disease or condition but has not yet been diagnosed with the disease or condition;

[0094] (ii) inhibiting the disease or condition, i.e., controlling its development;

[0095] (iii) alleviate the disease or condition, i.e., cause the disease or condition to regress;

[0096] (iv) Alleviate symptoms caused by a disease or condition.

[0097] As used herein, the terms "disease" and "disorder" are used interchangeably or may have different meanings because certain diseases or disorders do not yet have a known causative agent (and therefore the cause of the disease is unknown) and therefore cannot be considered diseases but rather are considered to be undesirable conditions or syndromes with more or less specific symptoms that have been confirmed by clinical researchers.

[0098] As used herein, the terms "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" refer to an amount of at least one agent or compound sufficient to provide some relief to some degree from one or more symptoms of the disease or condition being treated. This can result in a reduction and / or alleviation of signs, symptoms, or causes of disease, or any other desired change in a biological system. For example, a therapeutically effective amount is the amount of a composition comprising a compound disclosed herein that provides a clinically significant alleviation of symptoms. Techniques such as dose escalation studies can be used to determine the effective amount appropriate for any individual case.

[0099] As used herein, the terms "administer," "administer," "dosing," and the like refer to methods that enable a compound or composition to be delivered to the desired site of biological action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration, and rectal administration. In preferred embodiments, the compounds and compositions discussed herein are administered orally.

[0100] Preparation of the compounds of the present invention

[0101] The following specific examples are provided for the purpose of enabling those skilled in the art to more clearly understand and implement the present invention. They should not be considered as limiting the scope of the present invention, but are merely illustrative and representative of the present invention. It will be appreciated by those skilled in the art that there are other synthetic pathways for forming the compounds of the present invention, and the following are non-limiting examples.

[0102] All operations involving raw materials that are easily oxidized or hydrolyzed were carried out under nitrogen protection. Unless otherwise specified, the raw materials used in the present invention were purchased directly from the market and used directly without further purification.

[0103] Column chromatography used silica gel (200-300 mesh) produced by Qingdao Chemical Co., Ltd. Thin layer chromatography used prefabricated plates (silica gel 60PF) produced by E. Merck 254 Chiral compound separation and enantiomeric excess (ee) determination were performed using Agilent LC 1200 series (column: CHIRALPAKAD-H, mm, 5 μm, 30°C). Nuclear magnetic resonance chromatography (NMR) was determined using a Varian VNMRS-400 nuclear magnetic resonance instrument; liquid chromatography-mass spectrometry (LC / MS) was performed using a FINNIGAN Thermo LCQAdvantage MAX, Agilent LC 1200 series (column: Waters Symmetry C18, mm, 5 μm, 35 °C), using ESI (+) ion mode.

[0104] Experimental part

[0105] Intermediate 1: 6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-one

[0106] 6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-one was synthesized according to the same method as intermediate 162 in patent WO2021121327.

[0107] Intermediate 2: 2-(7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-9'(5'H)-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0108] Step 1: tert-Butyl 5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-1-carboxylate

[0109] Under nitrogen, 4-iodopyridin-3-amine (880 mg), tert-butyl 7-oxo-2-azaspiro[3.5]nonane-2-carboxylate (956 mg), palladium acetate (90 mg), and 1,4-diazabicyclo[2.2.2]octane (1.35 g) were added to N,N-dimethylformamide (10 mL) and heated to 120°C for 8 hours. The reaction mixture was cooled to room temperature, poured into water (50 mL), and extracted three times with ethyl acetate (20 mL). The extracts were combined, washed sequentially with water and saturated brine, and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product was separated by silica gel column chromatography (petroleum ether / ethyl acetate, 3:1) to obtain the title compound (790 mg). MS m / z [LC-MS]: 314.19 [M+1].

[0110] Step 2: 2-(1-(tert-Butyloxycarbonyl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-9'(5'H)-yl)-5-fluorobenzoic acid

[0111] Under nitrogen protection, tert-butyl 5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-1-carboxylate (628 mg), 5-fluoro-2-iodobenzoic acid (638 mg), (1S,2S)-N 1 ,N 2 1,2-Dimethylcyclohexane-1,2-diamine (28 mg), cuprous iodide (38 mg), and potassium phosphate (1.27 g) were added to 1,4-dioxane (10 mL) and heated to 100°C for 8 hours. The reaction solution was cooled to room temperature, poured into water (50 mL), and adjusted to pH 4-5 with 1M hydrochloric acid. The solid was filtered, dried, and separated by silica gel column chromatography (dichloromethane / methanol, 15:1) to obtain the title compound (500 mg). MS m / z [LC-MS]: 451.20 [M+1].

[0112] Step 3: tert-Butyl 9'-(2-(ethyl(isopropyl)formyl)-4-fluorophenyl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-1-carboxylate

[0113] 2-(1-(tert-Butyloxycarbonyl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-9'(5'H)-yl)-5-fluorobenzoic acid (450 mg), N-ethylpropan-2-amine (104 mg), diisopropylethylamine (387 mg), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (380 mg) were added to acetonitrile (5 mL) and stirred at room temperature for 2 hours. The solvent was then concentrated under reduced pressure. The residue was dissolved in dichloromethane (20 mL) and washed with water. The organic phase was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether / ethyl acetate, 2:1) to obtain the title compound (500 mg). MS m / z [LC-MS]: 521.29 [M+1].

[0114] Step 4: 2-(7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-9'(5'H)-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0115] Dissolve tert-butyl 9'-(2-(ethyl(isopropyl)formyl)-4-fluorophenyl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-1-carboxylate (480 mg) in dichloromethane (10 mL). Add trifluoroacetic acid (3 mL) and stir at room temperature for 2 hours. Adjust the pH to 12-13 with 10% sodium hydroxide solution. The organic phase is separated, and the aqueous phase is extracted with dichloromethane. The combined organic phases are washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield the title compound (380 mg), which is used directly in the next step. MS m / z [LC-MS]: 421.24 [M+1].

[0116] Intermediate 3: 2-(6',9'-dihydrospiro[azetidine-3,8'-pyrimido[5,4-b]indol]-5'(7'H)-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0117] Step 1: tert-Butyl 7-(pyrrolidin-1-yl)-2-azaspiro[3.5]non-6-ene-2-carboxylate

[0118] tert-Butyl 7-oxo-2-azaspiro[3.5]nonane-2-carboxylate (480 mg), tetrahydropyrrole (142 mg), Molecular sieves (1.0 g) were added to toluene (10 mL) and stirred at room temperature for 8 hours. The molecular sieves were filtered off and the solution was used directly in the next step. MS m / z [LC-MS]: 293.22 [M+1].

[0119] Step 2: tert-Butyl 6-(6-chloro-5-nitropyrimidin-4-yl)-7-oxo-2-azaspiro[3.5]nonane-2-carboxylate

[0120] A toluene solution of tert-butyl 7-(pyrrolidin-1-yl)-2-azaspiro[3.5]non-6-ene-2-carboxylate prepared in Step 1, 4,6-dichloro-5-nitropyrimidine (388 mg), and diisopropylethylamine (260 mg) were added to dichloromethane (10 mL) and stirred at room temperature for 8 hours. 1M hydrochloric acid (10 mL) and ethanol (10 mL) were then added and stirred for 2 hours. Dichloromethane (20 mL) was then added, and the organic phase was separated, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether / ethyl acetate, 3:1) to obtain the title compound (480 mg). MS m / z [LC-MS]: 397.13 [M+1].

[0121] Step 3: tert-Butyl 5',6',7',9'-tetrahydrospiro[azetidine-3,8'-pyrimido[5,4-b]indole]-1-carboxylate

[0122] tert-Butyl 6-(6-chloro-5-nitropyrimidin-4-yl)-7-oxo-2-azaspiro[3.5]nonane-2-carboxylate (397 mg) and palladium on carbon (10%, 40 mg) were added to ethyl acetate (10 mL). The mixture was evacuated and replaced with hydrogen three times. The reaction was allowed to proceed at room temperature for 4 hours. The palladium on carbon was removed by filtration, and the mixture was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether / ethyl acetate, 2:1) to obtain the title compound (220 mg). MS m / z [LC-MS]: 315.18 [M+1].

[0123] Step 4: tert-Butyl 5'-(2-(ethyl(isopropyl)formyl)-4-fluorophenyl)-5',6',7',9'-tetrahydrospiro[azetidine-3,8'-pyrimido[5,4-b]indole]-1-carboxylate

[0124] Under nitrogen, tert-butyl 5',6',7',9'-tetrahydrospiro[azetidine-3,8'-pyrimido[5,4-b]indole]-1-carboxylate (210 mg), N-ethyl-5-fluoro-2-iodo-N-isopropylbenzamide (335 mg), tris(dibenzylideneacetone)dipalladium (61 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (32 mg), and sodium tert-butoxide (128 mg) were added to toluene (10 mL) and heated to 100°C for 8 hours. The mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether / ethyl acetate, 2:1) to obtain the title compound (180 mg). MS m / z [LC-MS]: 522.29 [M+1].

[0125] Step 5: 2-(6',9'-dihydrospiro[azetidine-3,8'-pyrimido[5,4-b]indol]-5'(7'H)-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0126] Following the procedure in Step 4 of Intermediate 2, using tert-butyl 5'-(2-(ethyl(isopropyl)formyl)-4-fluorophenyl)-5',6',7',9'-tetrahydrospiro[azetidine-3,8'-pyrimido[5,4-b]indole]-1-carboxylate as starting material, the title compound was obtained and used directly in the next step. MS m / z [LC-MS]: 422.24 [M+1].

[0127] Intermediate 4: 2-(7',8'-dihydrospiro[azetidine-3,6'-[1,2,4]triazino[6,5-b]indol]-9'(5'H)-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0128] Step 1: tert-Butyl 6-(3,6-dichloro-1,2,4-triazin-5-yl)-7-oxo-2-azaspiro[3.5]nonane-2-carboxylate

[0129] Following the procedure of Step 2 in Intermediate 3, using 3,5,6-trichloro-1,2,4-triazine as starting material, the title compound (540 mg) was obtained. MS m / z [LC-MS]: 387.10 [M+1].

[0130] Step 2: tert-Butyl 3'-chloro-9'-(2-(ethyl(isopropyl)formyl)-4-fluorophenyl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-[1,2,4]triazino[6,5-b]indole]-1-carboxylate

[0131] tert-Butyl 6-(3,6-dichloro-1,2,4-triazin-5-yl)-7-oxo-2-azaspiro[3.5]nonane-2-carboxylate (387 mg), 2-amino-N-ethyl-5-fluoro-N-isopropylbenzamide (224 mg), and acetic acid (12 mg) were added to toluene (10 mL) and heated to 80°C with stirring for 10 hours. The mixture was cooled to room temperature, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether / ethyl acetate, 2:1) to obtain the title compound (260 mg). MS m / z [LC-MS]: 557.24 [M+1].

[0132] Step 3: tert-Butyl 9'-(2-(ethyl(isopropyl)formyl)-4-fluorophenyl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-[1,2,4]triazino[6,5-b]indole]-1-carboxylate

[0133] Following the procedure in Step 3 of Intermediate 3, using tert-butyl 3'-chloro-9'-(2-(ethyl(isopropyl)formyl)-4-fluorophenyl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-[1,2,4]triazino[6,5-b]indole]-1-carboxylate as starting material, the title compound (220 mg) was obtained. MS m / z [LC-MS]: 523.28 [M+1].

[0134] Step 4: 2-(7',8'-dihydrospiro[azetidine-3,6'-[1,2,4]triazino[6,5-b]indol]-9'(5'H)-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0135] Following the procedure in Step 4 of Intermediate 2, using tert-butyl 9'-(2-(ethyl(isopropyl)formyl)-4-fluorophenyl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-[1,2,4]triazino[6,5-b]indole]-1-carboxylate as starting material, the title compound was obtained and used directly in the next step. MS m / z [LC-MS]: 423.23 [M+1].

[0136] Intermediate 5: N-ethyl-5-fluoro-N-isopropyl-2-(4'-methyl-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-9'(5'H)-yl)benzamide

[0137] Following the synthesis method of Intermediate 2, using 4-iodo-5-methylpyridin-3-amine as the starting material, the target compound was obtained and used directly in the next step. MS m / z [LC-MS]: 435.26 [M+1].

[0138] Intermediate 6: (R)-N 1 -(2-methoxyethyl)-N 1 ,5-Dimethylhexane-1,4-diamine hydrochloride

[0139] (R)-N 1 -(2-methoxyethyl)-N 1 ,5-Dimethylhexane-1,4-diamine hydrochloride was synthesized according to the same method as intermediate 231 in patent WO2022262796.

[0140] Intermediate 7: (R)-2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2-azaspiro[3.5]nonan-7-one

[0141] Step 1: (R)-4-(8,11-dioxa-2-azaspiro[3.2.4 7 .2 4 ]Tridec-2-yl)-N-(2-methoxyethyl)-N,5-dimethylhexane-1-amine

[0142] (1,4-Dioxaspiro[4.5]decane-8,8-diyl)dimethanol (2.02 g) and diisopropylethylamine (5.16 g) were added to dry acetonitrile (50 mL), cooled to -30°C, and trifluoromethanesulfonic anhydride (6.21 g) was added dropwise. The mixture was stirred at -30°C for 1 hour. Intermediate 5 (3.30 g) and diisopropylethylamine (5.16 g) were added, and the mixture was refluxed for 24 hours. The mixture was cooled to room temperature, concentrated under reduced pressure to remove the solvent, and saturated aqueous sodium bicarbonate (100 mL) was added. The mixture was extracted with ethyl acetate (50 mL x 3). The extract was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane / methanol, 15:1, supplemented with 2% concentrated aqueous ammonia) to obtain the title compound (2.40 g). MS m / z [LC-MS]: 369.31 [M+1].

[0143] Step 2: (R)-2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2-azaspiro[3.5]nonan-7-one

[0144] (R)-4-(8,11-dioxa-2-azaspiro[3.2.4 7 .2 4 1-Tridecane-2-yl)-N-(2-methoxyethyl)-N,5-dimethylhexane-1-amine (1.85 g) and p-toluenesulfonic acid (2.15 g) were added to an acetone-water mixture (3:1, 20 mL) and stirred at reflux for 16 hours. After cooling to room temperature, the pH was adjusted to 12 with 1M sodium hydroxide solution. The mixture was extracted with ethyl acetate (30 mL x 3). The extract was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and used directly in the next reaction. MS m / z [LC-MS]: 325.29 [M+1].

[0145] In the following table, intermediates 8 to 17, 21 to 35, and 40 to 44 were prepared by referring to the synthesis method of intermediate 2 using the corresponding starting materials.

[0146] Intermediate 18: N-ethyl-5-fluoro-N-isopropyl-2-(7-(1,2,3,6-tetrahydropyridin-4-yl)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)benzamide

[0147] Step 1: 2-(7-Bromo-5H-pyrrolo[3,2-d]pyrimidin-5-yl)-5-fluorobenzoic acid

[0148] Under nitrogen, 7-bromo-5H-pyrrolo[3,2-d]pyrimidine (594 mg), 5-fluoro-2-iodobenzoic acid (798 mg), copper powder (191 mg), and potassium carbonate (414 mg) were added to N,N-dimethylformamide (6 mL), heated to 100°C, and stirred for 8 hours. The reaction mixture was cooled to room temperature, filtered, and the filtrate was poured into water (50 mL) and adjusted to pH 3-4 with 1M hydrochloric acid. The mixture was filtered and the filter cake dried to obtain the crude target compound (950 mg), which was used directly in the next reaction. MS m / z [LC-MS]: 335.98 [M+1].

[0149] Step 2: 2-(7-Bromo-5H-pyrrolo[3,2-d]pyrimidin-5-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0150] Following the procedure in Step 3 of Intermediate 2, using 2-(7-bromo-5H-pyrrolo[3,2-d]pyrimidin-5-yl)-5-fluorobenzoic acid as the starting material, the title compound was obtained. MS m / z [LC-MS]: 405.07 [M+1].

[0151] Step 3: tert-Butyl 4-(5-(2-(ethyl(isopropyl)formyl)-4-fluorophenyl)-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3,6-dihydropyridine-1(2H)-carboxylate

[0152] Under nitrogen, 2-(7-bromo-5H-pyrrolo[3,2-d]pyrimidin-5-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide (404 mg), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (464 mg), Pd(dppf)Cl2 (73 mg), and potassium carbonate (414 mg) were added to a mixture of 1,4-dioxane and water (8:1, 10 mL). The mixture was heated to 100°C and stirred for 12 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to remove the solvent. The residue was added to ethyl acetate (30 mL), washed with water, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 2:1) to obtain the title compound (310 mg). MS m / z[LC-MS]:508.27[M+1].

[0153] Step 4: N-ethyl-5-fluoro-N-isopropyl-2-(7-(1,2,3,6-tetrahydropyridin-4-yl)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)benzamide

[0154] Following the procedure in Step 4 of Intermediate 2, the title compound was obtained using tert-butyl 4-(5-(2-(ethyl(isopropyl)formyl)-4-fluorophenyl)-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3,6-dihydropyridine-1(2H)-carboxylate as the starting material. MS m / z [LC-MS]: 408.22 [M+1].

[0155] In the following table, intermediates 19-20 and 36-39 were prepared by referring to the synthesis method of intermediate 18 using the corresponding starting materials.

[0156] Intermediate 45: 9'-(4-Fluorophenyl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]

[0157] Step 1: tert-Butyl 9'-(4-fluorophenyl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-1-carboxylate

[0158] Under nitrogen, tert-butyl 5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-1-carboxylate (628 mg), 4-fluoroiodobenzene (888 mg), copper powder (64 mg), and cesium carbonate (1.96 g) were added to 1,4-dioxane (10 mL) and heated to 100°C for 24 hours. The reaction mixture was cooled to room temperature and filtered. The filtrate was then dried and separated by silica gel column chromatography (dichloromethane / methanol, 40:1) to obtain the title compound (160 mg). MS m / z [LC-MS]: 408.21 [M+1].

[0159] Step 2: 9'-(4-Fluorophenyl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]

[0160] Dissolve tert-butyl 9'-(4-fluorophenyl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-1-carboxylate (160 mg) in dichloromethane (2 mL). Add trifluoroacetic acid (1 mL) and stir at room temperature for 2 hours. Adjust the pH to 12-13 with 10% sodium hydroxide solution. The organic phase is separated, and the aqueous phase is extracted with dichloromethane. The combined organic phases are washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield the title compound (110 mg), which is used directly in the next step. MS m / z [LC-MS]: 308.16 [M+1].

[0161] Intermediate 46: 9'-(5-fluoropyridin-2-yl)-5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]

[0162] Following the synthesis method of Intermediate 45, 2-bromo-5-fluoropyridine was used as the starting material to obtain the target compound, which was used directly in the next step. MS m / z [LC-MS]: 309.15 [M+1].

[0163] Example 1: N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-9'(5'H)-yl)benzamide

[0164] Intermediate 1 (80 mg), Intermediate 2 (84 mg), and an ethanolic solution of acetic acid (10%, 12 mg) were added to ethanol (1 mL) and stirred at room temperature for 1 hour. Sodium triacetoxyborohydride (170 mg) was added and stirred for 8 hours. The solvent was then concentrated under reduced pressure. The residue was added to dichloromethane, washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column chromatography (dichloromethane / methanol, 15:1, containing 1% concentrated aqueous ammonia) to obtain the title compound (36 mg). MS m / z [LC-MS]: 606.42 [M+1].

[0165] Example 2: N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-6',9'-dihydrospiro[azetidine-3,8'-pyrimido[5,4-b]indol]-5'(7'H)-yl)benzamide

[0166] The target compound was obtained by following the method of Example 1 using Intermediate 3 as a starting material. MS m / z [LC-MS]: 607.41 [M+1]. 1 H NMR (400MHz, CDCl3), δ=8.91-8.93(m,1H),8.25-8.28(m,1H),7.14-7.41(m,3H) ,2.10-3.74(m,24H),1.38-1.98(m,7H),0.63-1.18(m,12H),0.38-0.53(m,3H).

[0167] Example 3: N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-7',8'-dihydrospiro[azetidine-3,6'-[1,2,4]triazino[6,5-b]indol]-9'(5'H)-yl)benzamide

[0168] The target compound was obtained by following the method of Example 1 using Intermediate 4 as a starting material. MS m / z [LC-MS]: 608.41 [M+1]. 1 H NMR (400MHz, CDCl3), δ = 9.37 (s, 0.7H), 9.36 (s, 0.3H), 7.38-7.42 (m, 1H), 7.23-7.29 (m,1H),7.14-7.19(m,1H),3.84-3.98(m,1H),3.42-3.53(m,2H),2.93-3.36(m,10H), 2.72-2.84(m,1H),2.49-2.64(m,3H),1.97-2.40(m,7H),1.68-1.82(m,1H),1.42-1. 58(m,2H),1.15-1.38(m,4H),1.04-1.09(m,3H),0.78-0.92(m,7H),0.52-065(m,5H).

[0169] In the following table, Examples 4, 6-27, 33-34, 37-44, and 46-52 were prepared by referring to the synthesis method of Example 1 using corresponding raw materials.

[0170] Example 5: (R)-N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-9'(5'H)-yl)benzamide

[0171] Step 1: (R)-N-(2-methoxyethyl)-N,5-dimethyl-4-(5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-1-yl)hexan-1-amine

[0172] Following the procedure of Step 1 in Intermediate 2, the target compound was obtained using Intermediate 7 as the starting material. MS m / z [LC-MS]: 399.31 [M+1].

[0173] Step 2: (R)-5-Fluoro-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-9'(5'H)-yl)benzoic acid

[0174] Following the procedure in Step 2 of Intermediate 2, using (R)-N-(2-methoxyethyl)-N,5-dimethyl-4-(5',7',8',9'-tetrahydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-1-yl)hexan-1-amine as starting material, the title compound was obtained. MS m / z [LC-MS]: 537.32 [M+1].

[0175] Step 3: (R)-N-Ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-9'(5'H)-yl)benzamide

[0176] Following the procedure in Step 3 of Intermediate 2, using (R)-5-fluoro-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indol]-9'(5'H)-yl)benzoic acid as the starting material, the title compound was obtained. MS m / z [LC-MS]: 606.42 [M+1].

[0177] Example 28: (S)-N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-4'-methyl-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-9'(5'H)-yl)benzamide

[0178] Example 29: (R)-N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-4'-methyl-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-9'(5'H)-yl)benzamide

[0179] Example 4 was separated using an IC-5 chiral preparative column (5 μm, 10×250 mm) with an elution solvent of n-hexane / ethanol / diethylamine 75:25:0.1 at a flow rate of 4.7 mL / min to obtain (S)-N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-4'-methyl-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-9'(5'H)-yl)benzamide. R = 14.8 min. MS m / z [LC-MS]: 620.43 [M+1]. 1H NMR (400MHz, CD3OD), δ = 8.26 (s, 0.2H), 8.00 (s, 0.8H), 7.80-7.85 (m, 1H), 7.48-7.51 (m, 0.8H), 7.37-7.43 (m, 1.2H), 7.30-7 .35(m,1H),3.54-3.82(m,1H),3.49(t,J=5.6Hz,2H),3.12-3.41(m,9H),2.93-3.09(m,1H),2.73-2.90(m,1H),2.66(s,2.4H) ,2.65(s,0.6H),2.58(t,J=5.6Hz,2H),2.29-2.46(m,3H),2.26(s,3H),2.15-2.24(m,1H),1.80-2.08(m,3H),1.48-1.61(m,2 H),1.15-1.43(m,3H),0.98-1.05(m,3H),0.93(d,J=6.8Hz,3H),0.74-0.91(m,5H),0.67(t,J=7.2Hz,2H),0.34-0.38(m,2H). At the same time, (R)-N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-4'-methyl-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-9'(5'H)-yl)benzamide was obtained. R = 23.2 min. MS m / z [LC-MS]: 620.43 [M+1]. 1H NMR (400MHz, CD3OD), δ = 8.26 (s, 0.2H), 8.00 (s, 0.8H), 7.80-7.85 (m, 1H), 7.48-7.51 (m, 0.8H), 7.37-7.43 (m, 1.2H), 7.30-7 .35(m,1H),3.54-3.82(m,1H),3.49(t,J=5.6Hz,2H),3.12-3.41(m,9H),2.93-3.09(m,1H),2.73-2.90(m,1H),2.66(s,2.4H) ,2.65(s,0.6H),2.58(t,J=5.6Hz,2H),2.29-2.46(m,3H),2.26(s,3H),2.15-2.24(m,1H),1.80-2.08(m,3H),1.48-1.61(m,2 H),1.15-1.43(m,3H),0.98-1.05(m,3H),0.93(d,J=6.8Hz,3H),0.74-0.91(m,5H),0.67(t,J=7.2Hz,2H),0.34-0.38(m,2H).

[0180] Example 30: (S)-2-(4'-chloro-1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-9'(5'H)-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0181] Example 31: (R)-2-(4'-chloro-1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-9'(5'H)-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide

[0182] Example 7 was separated using an IC-5 chiral preparative column (5 μm, 10×250 mm) with an elution solvent of n-hexane / ethanol / diethylamine 75:25:0.1 at a flow rate of 4.7 mL / min to obtain (S)-2-(4'-chloro-1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-9'(5'H)-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide. R = 15.5 min. MS m / z [LC-MS]: 640.38 [M+1]. 1H NMR (400MHz, CDCl3), δ = 8.46 (s, 0.2H), 8.13 (s, 0.8H), 8.12 (s, 0.2H), 8.06 (s, 0.8H), 7.32-7.35 (m, 0.8H) ,7.14-7.28(m,2.2H),3.86-3.96(m,0.2H),3.53-3.66(m,0.8H),3.30-3.51(m,6H),2.98-3.28(m,6H),2. 72-2.84(m,1H),2.54(t,J=5.6Hz,2H),2.28-2.42(m,3H),2.24(s,3H),1.68-2.10(m,4H),1.43-1.56(m,2 H),1.12-1.36(m,3H),0.94-1.06(m,3.2H),0.81-0.92(m,6.2H),0.66-0.78(m,3H),0.31-0.50(m,2.6H). At the same time, (R)-2-(4'-chloro-1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexane-3-yl)-7',8'-dihydrospiro[azetidine-3,6'-pyrido[3,4-b]indole]-9'(5'H)-yl)-N-ethyl-5-fluoro-N-isopropylbenzamide was obtained. R = 24.1 min. MS m / z [LC-MS]: 640.38 [M+1]. 1 H NMR (400MHz, CD3OD), δ = 8.44 (s, 0.3H), 8.28 (s, 0.7H), 8.13 (s, 1H), 7.76-7.81 (m, 4H), 7.51-7 .64(m,1H),7.33-7.50(m,8H),4.01-4.44(m,4H),3.61-3.89(m,3H),2.88-3.60(m,13H),2.89( s,3H),2.48-2.76(m,1H),2.08-2.44(m,3H),1.80-2.06(m,2H),1.54-1.78(m,2H),1.06-1.13 (m,6.6H),0.99(d,J=6.8Hz,3H),0.74-0.86(m,1.6H),0.52-0.70(m,3H),0.34-0.48(m,0.8H).

[0183] Example 32: N-ethyl-5-fluoro-N-isopropyl-2-(3-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)piperidin-4-yl)-1H-pyrrolo[2,3-c]pyridin-1-yl)benzamide

[0184] Example 18 (30 mg) and palladium on carbon (10%, 6 mg) were added to methanol (3 mL). After replacing the air in the reactor with hydrogen, the mixture was stirred at room temperature under 1 atmosphere of hydrogen for 8 hours. The palladium on carbon was then removed by filtration through celite. The filtrate was concentrated under reduced pressure and separated by silica gel thin-layer chromatography (dichloromethane / methanol, 15:1, containing 1% concentrated aqueous ammonia) to yield the title compound (24 mg). MS m / z [LC-MS]: 594.42 [M+1]. 1 H NMR (400MHz, CDCl3), δ = 8.49 (s, 1H), 8.03 (s, 1H), 7.44-7.47 (m, 1H), 7.40 (s, 1H), 7.22-7.26 (m, 1H), 7.14-7.17 (m, 1H), 3.78-3.86 (m, 3H), 3.58 -3.69(m,2H),3.43-3.56(m,1H),3.30-3.42(m,5H),2.72-3.24(m,10H) ,2.68(s,3H),2.25-2.38(m,2H),1.45-2.10(m,7H),0.76-1.38(m,15H).

[0185] Example 35: (S)-N-Ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-4'-methyl-5',7'-dihydro-8'H-spiro[azetidin-3,6'-cyclopenta[4,5]pyrrolo[2,3-c]pyridine]-8'-yl)benzamide

[0186] Example 36: (R)-N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-4'-methyl-5',7'-dihydro-8'H-spiro[azetidin-3,6'-cyclopenta[4,5]pyrrolo[2,3-c]pyridin-8'-yl)benzamide

[0187] Example 21 was separated using an IC-5 chiral preparative column (5 μm, 10×250 mm) with an elution solvent of n-hexane / ethanol / diethylamine (75:25:0.1) at a flow rate of 4.7 mL / min to obtain (S)-N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-4′-methyl-5′,7′-dihydro-8′H-spiro[azetidin-3,6′-cyclopenta[4,5]pyrrolo[2,3-c]pyridin-8′-yl)benzamide. R=14.3 minutes. MS m / z [LC-MS]: 606.42 [M+1]. (R)-N-ethyl-5-fluoro-N-isopropyl-2-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-4'-methyl-5',7'-dihydro-8'H-spiro[azetidin-3,6'-cyclopenta[4,5]pyrrolo[2,3-c]pyridin-8'-yl)benzamide was also obtained. R = 22.5 min. MS m / z [LC-MS]: 606.42 [M+1].

[0188] Example 45: N-ethyl-5-fluoro-N-isopropyl-2-(7-(1-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)piperidin-4-yl)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)benzamide

[0189] The target compound was obtained by referring to the method of Example 32 using Example 16 as the starting material. MS m / z [LC-MS]: 595.41 [M+1]. 1 H NMR (400MHz, CDCl3), δ = 9.01 (s, 1H), 8.73 (s, 0.8H), 8.71 (s, 0.2H), 7.50 (s, 0.2H), 7.49 (s, 0.8H), 7.40- 7.45(m,1H),7.17-7.28(m,2H),4.20-4.30(m,0.2H),3.39-3.53(m,2.8H),3.35(s,2.4H),3.33(s,0.6H), 2.78-3.02(m,3H),2.54-2.67(m,3.8H),2.37-2.44(m,2.2H),2.29(s,2.4H),2.28(s,0.6H),2.02-2.14(m ,3H),1.40-1.85(m,6H),1.18-1.38(m,2H),1.02-1.14(m,1H),0.78-0.96(m,13H),0.28(d,J=6.8Hz,2H).

[0190] Biological testing

[0191] Biochemical assay for the inhibition of wild-type Menin binding to MLL by compounds

[0192] Wild-type Menin binds with high affinity to the conserved sequence at the N-terminus of MLL. Compounds compete with the MLL N-terminus for Menin binding. FITC-MLL(4-43) (synthesized by GenScript) contains the conserved sequence for MLL-Menin binding. We developed a method to detect Menin-MLL binding activity using a Fluorescence Polarization Binding Assay (FP Assay) and used it to detect compounds that inhibit binding activity. The specific method is as follows:

[0193] Compounds were serially diluted five-fold starting at 1 mM in 100% DMSO (8 concentrations total). 2 μl of each concentration was added to 48 μl of reaction buffer (50 mM NaCl, 50 mM Tris (pH 7.5), 0.05% Tween-20, 1 mM DTT) and mixed thoroughly to create the 4* compounds (final concentrations: 10,000, 2,000, 400, 80, 16, 3.2, 0.64, and 0 nM). 2*Menin and 4*FITC-MLL (4-43) were prepared in reaction buffer to a final concentration of 5 nM. 5 μl of each of the 4* compounds was added to a 384-well plate (OptiPlate-384, purchased from PerkinElmer). 10 μl of 2*Menin was added and centrifuged. 5 μl of FITC-MLL (4-43) was then added and the reaction initiated by centrifugation. The reaction was incubated at 23°C in the dark for 1 hour. After the reaction, the signal value was read on EnVison2104 Multilabel Reader (purchased from PerkinElmer) (excitation wavelength 480 nm / emission wavelength 535 nm). The data were processed using the data analysis software GraphPad Prism to obtain the IC value of the compound. 50 value

[0194] Biochemical assay for the inhibition of the binding of mutant Menin to MLL by compounds

[0195] Will Menin mut The DNA sequence was constructed into the pET28a vector by enzyme digestion and ligation, and the recombinant protein was expressed in BL21 Escherichia coli system and purified by Ni-NTA to obtain Menin. mut Menin can bind to the conserved sequence at the N-terminus of MLL with high affinity. The compound competes with the N-terminus of MLL and then binds to Menin. FITC-MLL -4-43(Synthesized by GenScript) contains the conserved sequence for MLL and Menin binding. We used FITC-MLL to bind to the conserved sequence of MLL and Menin through the Fluorescence Polarization Binding Assay (FPAssay). -4-43 with Menin mut After binding, the molecular weight increases and the fluorescence becomes stronger to detect the effect of the compound on Menin mut Inhibition of binding to MLL.

[0196] The specific method is as follows:

[0197] Dissolve the compound in DMSO to a final concentration of 10 mM. Dilute the 10 mM compound 10-fold with DMSO, then perform a 5-fold serial dilution for a total of 8 concentrations. Take 2 μl of each concentration and add it to 48 μl of reaction buffer (50 mM NaCl, 50 mM Tris, pH 7.5, 0.05% Tween-20, 1 mM DTT) and mix well. Prepare 4x compound (final concentrations of 10,000, 2,000, 400, 80, 16, 3.2, 0.64, 0 nM) for later use. Prepare 2x Menin in reaction buffer. mut , final concentration 5 nM, 4x FITC-MLL -4-43 , the final concentration is 2nM. Take 5μl of 4x compound and add it to 384-well plate (OptiPlate-384, purchased from PerkinElmer), add 10μl of 2x Menin, centrifuge, and then add 5μl of FITC-MLL -4-43 The reaction was initiated by brief centrifugation and incubated at 23°C in the dark for 1 hour. After completion of the reaction, the signal was read on an EnVison 2104 Multilabel Reader (PerkinElmer) (excitation wavelength 480 nm / emission wavelength 535 nm). The inhibitory effect of each compound was determined at eight concentrations. Data were processed using GraphPad Prism software to calculate the half-inhibitory concentration (IC50) for the binding of menin to MLL.

[0198] Note: x refers to multiplication, indicating multiples.

[0199] Table 1. Inhibitory activity of the example compounds on wild-type and mutant Menin-MLL protein binding

[0200] Table 1 shows the inhibitory activity IC of the compounds of the present invention on the wild-type and mutant Menin-MLL protein binding. 50The data show that the compounds provided by the present invention have good wild-type and mutant Menin-MLL protein binding inhibitory activity.

[0201] Determination of the inhibitory activity of compounds on MV-4-11 cells expressing wild-type Menin protein:

[0202] MV-4-11 cells are human acute myeloid leukemia cells that contain MLL-AF4 fusion protein. In this experiment, different concentrations of compounds were incubated with MV-4-11 cells and the cells were treated with Promega The detection reagents were used to establish a screening method for MV-4-11 cell proliferation inhibition.

[0203] MV-4-11 cells were cultured in 1640 medium (Biological Industries, Catalog No. 01-100-1ACS) supplemented with 10% fetal bovine serum (Biological Industries, Catalog No. 04-001-1ACS) at 37°C in 5% CO2. The cells were subcultured 2-3 times per week. Cells were seeded at 5000 cells / well in 96-well cell culture plates. 195 μL / well, and cultured at 37°C, 5% CO2. After 24 hours, the compound was diluted 3 times with 100% DMSO starting from 10 mM and mixed (a total of 10 concentrations), and then 4 μL of the compound at each concentration was added to 96 μL of RPMI-1640 culture medium for dilution and mixing. 5 μL of each diluted compound was added to the plated cell suspension, and the compound and cells were co-incubated in a cell culture incubator for 72 hours (3 days). Then 35 μL of CellTiter-Blue (Promega, product number G8082) reagent was added and reacted for 4 hours at 37°C, 5% CO2. The chemiluminescence value was read on a BMG Clariostar Microplate Reader, and the data was processed using GraphPad Prism software to calculate the IC value of the compound for cell proliferation inhibition. 50 value.

[0204] Note: * refers to multiplication, indicating multiples.

[0205] Determination of the inhibitory activity of compounds on MOLM13 cells expressing mutant Menin protein:

[0206] Will Menin M327IThe DNA sequence was constructed into the lentiviral vector pCDHL-CMV-3xFlag-puroR by enzyme digestion and ligation, and co-transfected with psPAX2 / pMD2G into 293T cells. After 48 hours, the virus was harvested and infected into MOLM13 cells. Pooling and monoclonal screening were performed by puromycin, and finally a stable expression of Menin was obtained. M327I Stable cell lines for recombinant protein production.

[0207] MOLM13 cells are human acute myeloid leukemia cells that contain MLL-AF9 fusion protein. M327I MOLM13 cells were incubated for 7 days, and viable cells were detected using Promega's Cell-Titer Glo.

[0208] MOLM13 cells were cultured in RPMI-1640 containing 20% ​​fetal bovine serum and seeded in 96-well cell culture plates at 500 cells / well the day before the addition of the compound. The volume of culture medium in each well was 195 μL. The compound was dissolved in DMSO to a final concentration of 10 mM. The 10 mM compound was diluted 3-fold with DMSO for a total of 10 concentrations. 2 μl of each concentration was added to 48 μl of RPMI-1640 culture medium for a 25-fold dilution, and added to the plated cell suspension as a 40x compound. After incubation in the incubator for 7 days, 35 μL of Cell-Titer Glo was added, and the cells were allowed to stand at room temperature for 10 minutes. The fluorescence signal was read on a CLARIO starPlus, and finally the IC of the compound for inhibition of cell proliferation was calculated using GraphPad Prism 5.0. 50 value.

[0209] Note: * refers to multiplication, indicating multiples.

[0210] Table 2: Proliferation inhibition activity of the compounds of the examples on MV-4-11 cells expressing wild-type Menin protein and MOLM13 cells expressing mutant Menin protein

[0211] Table 2 lists the IC values ​​of the compounds of the present invention against the proliferation inhibition of MV-4-11 cells expressing wild-type Menin protein and MOLM13 cells expressing mutant Menin protein. 50 The data show that the compounds provided by the present invention have good inhibitory activity on the proliferation of cells expressing wild-type and mutant Menin proteins.

[0212] Determination of pharmacokinetic data of compounds in SD rats

[0213] Male SD rats were obtained from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The rats were divided into groups of 3 in each group and orally gavaged with a suspension of the test sample (10 mg / kg, suspension was 0.5% MC + 0.1% SDS). The animals were fasted overnight before the experiment, and the fasting period was from 10 hours before administration to 4 hours after administration. Blood was collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. After anesthesia with isoflurane using a small animal anesthesia machine, 0.3 mL of whole blood was collected through the fundus venous plexus and placed in a heparin anticoagulant tube. The sample was centrifuged at 4°C and 4000 rpm for 5 minutes. The plasma was transferred to a centrifuge tube and stored at -80°C until analysis. The sample in the plasma was extracted using a protein precipitation method, and the extract was analyzed by LC / MS. The detection results of some compounds are shown in Table 3.

[0214] Table 3 Pharmacokinetic parameters of the compounds in the examples

[0215] Table 3 lists the pharmacokinetic data of the compounds of the present invention in SD rats, indicating that the compounds provided by the present invention have relatively good in vivo metabolism levels.

Claims

1. A compound of formula (II) or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, in, Ring A is a 5-8 membered nitrogen-containing heterocyclic ring or a nitrogen-containing heteroaromatic ring, and X is independently C or N, R 1a H, -C(=O)-NR 2a R 2b , phenyl, or a 5-6 membered heteroaryl, the heteroaryl containing 1 oxygen atom and / or 1 sulfur atom and / or 1-3 nitrogen atoms, and optionally containing a carbonyl group, and the phenyl and heteroaryl are optionally substituted by a 3-6 membered cycloalkyl or C 1-4 Alkyl substitution, R 2a and R 2b Each independently is H or C 1-4 Alkyl, which may be optionally substituted by -OH or -NH2, R 1b is F or Cl, R2 is selected from H, halogen, C 1-4 Alkyl, -OC 1-4 Alkyl and -NR 3a R 3b , R 3a and R 3b Each independently is H or C 1-4 alkyl, X1 and X2 are each independently N or CH, X3 is N or R 10 , R 10 H, halogen, C 1-6 Alkyl, or -OC 1-6 alkyl, L1 is -CH2-, -CH2-CH2- or -CH2-CH2-CH2-, L2 and L3 are each independently -CH2- or -CH2-CH2-, R4 is C 1-6 Alkyl, 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl, R3 is -C 1-6 Alkylene-NR 4a R 4b , -C 1-6 Alkylene-C(O)-NR 5a R 5b , -C 1-6 Alkylene -OH, or -C 1-6 Alkylene-NR7-C(O)-OC 1-4 Alkylene-OC(O)-C 1-4 Alkyl, wherein the alkyl or alkylene group may be optionally substituted with halogen, -CN, -OH or -OC l-4 Alkyl substitution, R 4a and R 4b Each independently selected from H, C 1-6 Alkyl, -C(O)-C 1-4 Alkyl, -C(O)-OC 1-4 Alkyl and -C(O)-NR 8a R 8b , the C 1-6 The alkyl group may be optionally substituted with halogen, -CN, -OH, -S(O)2-C 1-4 Alkyl, -OC 1-4 Alkyl, -C(O)-NR 6a R 6b or -NR 6c -C(O)-C 1-4 Alkyl substitution, R 5a , R 5b , R 6a , R 6b , R 6c , R7, R 8a and R 8b Each independently selected from H and C 1-6 alkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, which has a structure represented by formula (I), in, Ring A is a 5-8 membered nitrogen-containing heterocyclic ring or a nitrogen-containing heteroaromatic ring, and X is independently C or N, R 1a -C(=O)-NR 2a R 2b , or a 5-6 membered heteroaryl group, the heteroaryl group contains 1-3 nitrogen atoms and optionally contains a carbonyl group, and the heteroaryl group is optionally substituted by a 3-6 membered cycloalkyl group or a C 1-4 Alkyl substitution, R 2a and R 2b Each independently is H or C 1-4 Alkyl, which may be optionally substituted by -OH or -NH2, R 1b is F or Cl, R2 is selected from H, halogen, C 1-4 Alkyl, -OC 1-4 Alkyl and -NR 3a R 3b , R 3a and R 3b Each independently is H or C 1-4 alkyl, X1 and X2 are each independently N or CH, L1 is -CH2-, -CH2-CH2- or -CH2-CH2-CH2-, L2 and L3 are each independently -CH2- or -CH2-CH2-, R4 is C 1-6 Alkyl, 3-8 membered cycloalkyl or 3-8 membered heterocycloalkyl, R3 is -C 1-6 Alkylene-NR 4a R 4b , -C 1-6 Alkylene-C(O)-NR 5a R 5b , -C 1-6 Alkylene -OH, or -C 1-6 Alkylene-NR7-C(O)-OC 1-4 Alkylene-OC(O)-C 1-4 Alkyl, wherein the alkyl or alkylene group may be optionally substituted with halogen, -CN, -OH or -OC l-4 Alkyl substitution, R 4a and R 4b Each independently selected from H, C 1-6 Alkyl, -C(O)-C 1-4 Alkyl, -C(O)-OC 1-4 Alkyl and -C(O)-NR 8a R 8b , the C 1-6 The alkyl group may be optionally substituted with halogen, -CN, -OH, -S(O)2-C 1-4 Alkyl, -OC 1-4 Alkyl, -C(O)-NR 6a R 6b or -NR 6c -C(O)-C 1-4 Alkyl substitution, R 5a , R 5b , R 6a , R 6b , R 6c , R7, R 8a and R 8b Each independently selected from H and C 1-6 alkyl.

3. The compound according to claim 2 or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, wherein for or 4. The compound according to claim 2 or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, wherein for or 5. The compound according to claim 2, or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, wherein R 1a -C(=O)-NR 2a R 2b , R 2a and R 2b Each independently is H or C 1-4 Alkyl, R 1b is F, and R4 is isopropyl.

6. The compound according to claim 2, or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, wherein R2 is H or C 1-4 alkyl.

7. The compound according to claim 2, or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, wherein R3 is -C 1-6 Alkylene-N(C 1-6 Alkyl)-C 1-6 Alkylene-OC 1-4 alkyl.

8. The following compounds or their pharmaceutically acceptable salts, solvates, polymorphs, tautomers, metabolites or prodrugs 9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, and optionally comprising a pharmaceutically acceptable carrier.

10. Use of the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite or prodrug thereof, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating a disease associated with MLL activity.

11. The use according to claim 10, wherein the disease associated with MLL activity is cancer, preferably acute leukemia (including MLL acute leukemia, MLL partial tandem duplication acute leukemia, NPM mutation acute leukemia, MOZ acute leukemia, NUP98 acute leukemia and CALM acute leukemia), chronic lymphocytic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, polycythemia vera, malignant lymphoma (including B cell lymphoma), myeloma (including multiple myeloma) , brain tumor, head and neck cancer, esophageal cancer, thyroid cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, stomach cancer, gallbladder and bile duct cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, colon cancer, rectal cancer, anal cancer, chorioepithelialoma, endometrial cancer, cervical cancer, ovarian cancer, bladder cancer, urothelial cancer, kidney cancer, renal cell carcinoma, prostate cancer, testicular tumor, testicular germ cell tumor, ovarian germ cell tumor, Wilms' tumor, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's sarcoma, chondrosarcoma, soft tissue sarcoma, or skin cancer.

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