CN116535400B

CN116535400B - Azaspiro compounds and uses thereof

Info

Publication number: CN116535400B
Application number: CN202310463934.8A
Authority: CN
Inventors: 肖贻崧; 谷晓辉; 赖焜民; 李铭曦
Original assignee: Shanghai Pailong Biotechnology Co ltd
Current assignee: Shanghai Pailong Biotechnology Co ltd
Priority date: 2023-04-26
Filing date: 2023-04-26
Publication date: 2024-08-16
Anticipated expiration: 2043-04-26
Also published as: CN116535400A

Abstract

本发明提供了一类氮杂螺环化合物或其药学上可接受的盐、立体异构体、同位素异构体、前药、水合物或溶剂合物，此类化合物能够调节KIF18A蛋白，而且具有良好的PARP7抑制活性，可以用于制备预防或治疗癌症的药物。The present invention provides a class of azaspiro compounds or pharmaceutically acceptable salts, stereoisomers, isotope isomers, prodrugs, hydrates or solvates thereof. Such compounds can regulate KIF18A protein and have good PARP7 inhibitory activity, and can be used to prepare drugs for preventing or treating cancer.

Description

Azaspiro compounds and uses thereof

Technical Field

The invention belongs to the field of medicines, and relates to an azaspiro compound with KIF18A kinesin inhibitory activity and PARP7 inhibitory activity and application thereof in treating cancers.

Background

KIF18A is a member of the kinesin-8 family of kinesins and can move in the positive direction in a cell by virtue of energy released by hydrolyzing ATP, with microtubules as a rail. Meanwhile, the KIF18A is positioned at the tail end of the positive electrode of the microtubule, can regulate and control the dynamic instability of the microtubule, and plays an activity similar to that of microtubule depolymerizing enzyme. In the mitosis process, the KIF18A can regulate and control the dynamics of spindle microtubules and the amplitude of chromosomes, and has a key effect on timely finishing whole lines of mitosis-stage chromosomes, maintaining stable genomes and smoothly finishing mitosis.

The KIF18A gene belongs to the kinesin-8 subfamily and is a positive-end directed motor. KIF18A is thought to influence the dynamics of the positive end of the centromere microtubules to control correct chromosomal location and spindle tension. Depletion of human KIF18A results in longer spindles in HeLa cervical cancer cells, increased chromosomal oscillations in metaphase (chromosomeoscillation) and activation of mitotic spindle assembly checkpoints (MIMayr et al, currentBiology [ contemporary biology ]17, 488-98, 2007). KIF18A is considered a viable target for cancer treatment. KIF18A is overexpressed in various types of cancers, including, but not limited to, colon, breast, lung, pancreas, prostate, bladder, head, neck, cervical and ovarian cancers. Furthermore, in cancer cell lines, gene deletions or knockouts or KIF18A inhibition affect mitotic spindle devices. In particular, inhibition of KIF18A has been found to induce mitotic cell arrest, a known weakness that can promote mitotic cell death by apoptosis, mitotic catastrophe, or multiphase driven lethality or death following mitotic slippage in the interphase. Thus, researchers are interested in finding inhibitors of the KIF18A protein. Inhibition of KIF18AATP enzyme activity is therefore a promising approach to develop new anticancer agents.

Poly ADP-ribose polymerase (PARPs) is an enzyme (S.Vyas,M.Chesarone-Cataldo,T.Todorova,Y.H.Huang,P.Chang,Asystematic analysis ofthePARPprotein familyidentifies newfunctions critical forcellphysiology,Nat.Common.,4,2240(2013)). having 17 family members that catalyzes the transfer of poly or single ADP to their target substrate proteins, whose functions include gene expression, protein degradation, and biological functions of some of the various cellular stress (M.S.Cohen,P.Chang,Insights intothebiogenesis,function,andregulationofADP-ribosylation,Nat.Chem.Biol.,14,236-243(2018)). enzymes, like PARP1 and PARP2, are well understood, but the biological functions of other enzymes are not well understood at present. There are 17 members of this family, 4 of which (PARP 1, PARP2, PARP5A and PARP 5B) are capable of synthesizing the PAR chain. Most other enzymes in the family can only build a single ADP ribose (ADP-ribose) unit and are therefore classified as mono (ADP-ribosyl) ases (MARs), i.e.single ADP ribosylases. The viability of tumor cells under stress is a fundamental mechanism of tumorigenesis and is an emerging therapeutic approach. PARP1, a member of the PARP family, has been shown to be a potent cancer target. Whether caused by genetic mutation or DNA damage associated with cytotoxic chemotherapy, thereby causing cellular stress. There are six approved drugs clinically, and several others are in advanced stages of development (A.Ohmoto,S.Yachida,Current status of poly(ADP-ribose)polymerase inhibitors and future directions,Onco.Targets Ther.,10,5195-5208(2017).

Aryl Hydrocarbon Receptors (AHR) are coordinated activating transcription factors (S.Feng,Z.Cao,X.Wang,Role ofaryl hydrocarbon receptor in cancer,Biochim.Biophys.Acta,1836,197-210(2013);and B.Stockinger,P.Di Meglio,M.Gialitakis,J.H.Duarte,The aryl hydrocarbon receptor:multitasking in the immune system,Annu.Rev.Immunol.,32,403-432(2014)).AHR involved in regulating a variety of cellular functions, including pro-inflammatory reactions and biological metabolism, that can be activated by a number of ligands, including endogenous tryptophan produced by metabolism, such as kynurenine, and activation of certain polycyclic aromatic hydrocarbons (C.A.Opitz et al.,An endogenous tumour-promoting ligand ofthe human aryl hydrocarbon receptor,Nature,478,197-203(2011)).AHR, to induce target gene expression, including genes involved in metabolism, such as cytochrome P4501A1 and P4501B1. Activation also resulted in an increase in the AHR target gene TCDD induced poly (ADP-ribose) polymerase (TIPARP). Like PARP7, act as a down regulator of certain AHR transcription targets (L.MacPherson et al.,Aryl hydrocarbon receptor repressor and TIPARP(ARTD14)use similar,but also distinct mechanisms to repress aryl hydrocarbon receptor signaling,Int.J.Mot.Sci.,15,7939-7957(2014).

PARP7 is a gene regulated by AHR and is an important member of the PARP family. PARP7 is only able to transfer one single ADP-ribose (MAR), belonging to monoPARP. The PARP catalytic domain of PARP7 contains a zinc finger motif that confers DNA binding, and a WWE domain that mediates protein interactions (Ma, Q et al, biochemistry (biochem.), 289,499-506,2001). Its mediated single ADP ribosylation is a reversible post-translational modification that involves a variety of important biological processes, such as immune cell function, transcriptional regulation, protein expression, and DNA repair. PARP7 is part of the negative feedback loop that regulates AHR activity, which can regulate immune function, inflammation and stem differentiation, and play a role in cancer. PARP7 has been shown to be overactive in tumors and plays a key role in cancer cell survival. More importantly, many cancer cells rely on PARP7 to achieve intrinsic cell survival, and studies have shown that PARP7 can enable cancer cells to "harbor" outside the immune system; inhibition of PARP7 is effective in inhibiting cancer cell growth, restoring interferon signaling, and inhibiting the "brake" of the innate and adaptive immune mechanisms. In several cancer models, PARP7 inhibitors exhibit durable tumor growth inhibition, potent antiproliferative activity, and interferon signaling restoration.

In addition, PARP7 may also be regulated by other transcription factors and signaling pathways, including androgen receptor (E.C.Bolton etal.,Cell-and gene-specific regulation ofprimary target genes by the androgen receptor,Genes.Dev.,21,2005-2017(2007), platelet-derived growth factor (J.Schmahl,C.S.Raymond,P.Soriano,PDGF signaling specificity is mediated through multiple immediate early genes,Nat.Genet.,39,52-60(2007)) and hypoxia inducible factor 1(N.Hao et al.,Xenobiotics and loss ofcell adhesion drive distinct transcriptional outcomes by aryl hydrocarbon receptor signaling,Mot.Pharmacol.,82,1082-1093(2012)).PARP7, have a variety of cellular functions. In the context of the AHR signal, PARP7 as a negative feedback mechanism to regulate expression (L.MacPherson et al.,Aryl hydro-carbon receptor repressor and TIPARP(ARTD14)use similar,but also distinct mechanisms to repress aryl hydrocarbon receptor signaling,Int.J.Mot.Sci.,15,7939-7957(2014),and L.MacPherson et al.,2,3,7,8-Tetrachlorodibenzo-p-dioxin poly(ADP-ribose)polymerase(TIPARP,ARTD14)is a mono-ADP-ribosyltransferase and repressor ofaryl hydro-carbon receptor transactivation,Nucleic.Acids Res.,41,1604-1621(2013)).PARP7 of P4501Al and P4501B1 is also described as an ADP-ribosyl liver X receptor, which results in modulation (C.Bindesboll et al.,TCDD-inducible poly-ADP-ribose polymerase(TIPARP/PARP7)mono-ADP-ribosylates and co-activates liver X receptors.Biochem.J.473,899-910(2016)). of its transcriptional activity during viral infection, PARP7 can bind to Sindbis virus (SINV) to promote viral RNA degradation ((T.Kozaki et al.,Mitochondrial damage elicits a TCDD-inducible poly(ADP-ribose)polymerase-mediated antiviral response,Proc.Natl.Acad.Sci.USA,114,2681-2686(2017)). also in the case of viral infection, AHR-induced PARP7 can interact with TBK1, TBK1 being a major kinase activated during initiation of pathogen-associated molecular pattern pathways, resulting in activation (T.Yamada et al.,Constitutive aryl hydrocarbon receptor signaling constrains Type I interferon-mediated antiviral innate defense,Nat.Immunol.,17,687-604(2016)).PARP7 of type I interferon response and antiviral immunity having activity to nuclear the TBK1 ADP, inactivating TBK1, thereby achieving inhibition of type I interferon response.

Based on the results of these viral infections, we can assume that cancer cells can use abnormally expressed and or activated PARP7 as a mechanism to evade the host immune system by T cell mediated anti-tumor immunity by inhibiting type I interferon. In fact, in a recent study PARP7 was identified as a possible tumor factor (D.Pan et al.,A major chromatin regulator determines resistance of tumor cells to Tcell-mediated killing,Science,359,770-775(2018)). that inhibits T cell activation in a mouse malignant cell line, PARP7 gene knockout increased proliferation and activation of co-cultured T cells suggesting that PARP7 inhibition may be a viable strategy to activate T cell mediated tumor killing.

Inhibition of KIF18A protein activity and inhibition of PARP7 activity are two different antitumor mechanisms, and can be used for development of antitumor drugs. The application surprisingly discovers an azaspiro compound, has the inhibition effect on KIF18A and PARP7, and has good anti-tumor drug development prospect.

Disclosure of Invention

The invention provides an azaspiro compound, a preparation method and application thereof.

In a first aspect of the invention there is provided a compound having the structure of formula (I) or a pharmaceutically acceptable salt, stereoisomer, isotopic isomer, prodrug, hydrate or solvate thereof:

wherein,

X ¹ is selected from CR ¹ or N; x ² is selected from CR ² or N; x ³ is selected from CR ³ or N;

R ¹、R²、R³ is each independently selected from hydrogen, halogen, -CN, -NO ₂、C₁-C₆ alkyl, C ₃-C₆ cycloalkyl, halogenated C ₁-C₆ alkyl, Hydroxy C ₁-C₆ alkyl 、-OR^a、-SR^a、-S(O)₂R^a、-S(O)R^aR^b、-NR^aR^b、-P(O)R^aR^b、-C(O)R^a、 or-C (O) NR ^aR^b; wherein R ^a、R^b is each independently selected from hydrogen, OH, halogen, C ₁-C₆ alkyl, C ₃-C₆ cycloalkyl, halogenated C ₁-C₆ alkyl, Hydroxy C ₁-C₆ alkyl, C ₁-C₆ alkoxy, -C (O) R ^c、-S(O)R^cR^d, or-S (O) ₂R^c;R^c、R^d are each independently selected from hydrogen, -OH, C ₁-C₆ alkyl, C ₃-C₆ cycloalkyl, hydroxy C ₁-C₆ alkyl, C ₁-C₆ alkoxy, or C ₁-C₆ acyl;

Or R ¹ and R ², or R ² and R ³, together with the atoms to which they are attached, form a 5-to 6-membered saturated or unsaturated ring; the 5-6 membered saturated or unsaturated ring contains 0-2 heteroatoms selected from O, S, N, the 5-6 membered saturated or unsaturated ring being optionally substituted with 0-2 substituents selected from the group consisting of: halogen, -CN, -NO ₂、C₁-C₆ alkyl, C ₃-C₆ cycloalkyl, haloC ₁-C₆ alkyl, hydroxyC ₁-C₆ alkyl 、-OR^a、-SR^a、-S(O)₂R^a、-S(O)R^aR^b、-NR^aR^b、-P(O)R^aR^b、-C(O)R^a、 or-C (O) NR ^aR^b; wherein R ^a、R^b is as defined above;

R ⁴、R⁵ and each R ⁶ are each independently selected from hydrogen, halogen, -CN, -NO ₂、-OH、C₁-C₆ alkyl, C ₃-C₆ cycloalkyl, halogenated C ₁-C₆ alkyl, hydroxyC ₁-C₆ alkyl, or C ₁-C₆ alkoxy;

m is selected from integers from 0 to 5.

In a preferred embodiment, formula (I) is formula (II):

Wherein X ¹ is CR ¹;X² is CR ²;X³ is CR ³;R¹、R²、R³、R⁴、R⁵、R⁶ as defined above.

In another preferred embodiment, the formula (I) is formula (III):

Wherein X ₁、X₂、X₃ is as defined above.

In another preferred embodiment, the structural fragment (III-1) of formula (III) is selected from one of the structural fragments represented by formula (III-2), (III-3), (III-4), (III-5) or (III-6)

Wherein R ⁷ is selected from halogen, -CN, -NO ₂、C₁-C₆ alkyl, C ₃-C₆ cycloalkyl, halogenated C ₁-C₆ alkyl, Hydroxy C ₁-C₆ alkyl 、-OR^a、-SR^a、-S(O)₂R^a、-S(O)R^aR^b、-NR^aR^b、-P(O)R^aR^b、-C(O)R^a、 or-C (O) NR ^aR^b; wherein R ^a、R^b is each independently selected from hydrogen, OH, halogen, C ₁-C₆ alkyl, C ₃-C₆ cycloalkyl, halogenated C ₁-C₆ alkyl, Hydroxy C ₁-C₆ alkyl, C ₁-C₆ alkoxy, -C (O) R ^c、-S(O)R^cR^d or-S (O) ₂R^c;R^c、R^d are each independently selected from hydrogen, -OH, C ₁-C₆ alkyl, C ₃-C₆ cycloalkyl, hydroxyc ₁-C₆ alkyl, C ₁-C₆ alkoxy or C ₁-C₆ acyl;

Y, Z are each independently selected from N, CR ⁷;R⁷ as defined above;

M is selected from O, S, NR ^c、CR^cR^d;R^c、R^d each independently selected from hydrogen, -OH, C ₁-C₆ alkyl, C ₃-C₆ cycloalkyl, hydroxy C ₁-C₆ alkyl, C ₁-C₆ alkoxy or C ₁-C₆ acyl.

In another preferred embodiment, the structural fragment (III-1) of formula (III) is selected from one of the structural fragments represented by formula (III-3), (III-4), (III-5), (III-6) or (III-7):

Wherein M is O; y, Z are each independently selected from N or CR ⁷;

R ⁷ is selected from -CN、-NO₂、-OR^a、-SR^a、-S(O)₂R^a、-S(O)R^aR^b、-NR^aR^b、-P(O)R^aR^b、-C(O)R^a、 or-C (O) NR ^aR^b;R^a、R^b each independently represents hydrogen, OH, C ₁-C₆ alkyl, C ₁-C₆ alkoxy, -C (O) R ^c、-S(O)R^cR^d or-S (O) ₂R^c;R^c、R^d each independently is selected from hydrogen, -OH, C ₁-C₆ alkyl, hydroxyC ₁-C₆ alkyl, C ₁-C₆ alkoxy or C ₁-C₆ acyl;

More preferably, R ⁷ is selected from-NR ^aR^b、-P(O)R^aR^b、-C(O)R^a, or-C (O) NR ^aR^b;R^a、R^b each independently represents hydrogen, OH, C ₁-C₆ alkyl, C ₁-C₆ alkoxy, -C (O) R ^c、-S(O)R^cR^d, or-S (O) ₂R^c;R^c、R^d each independently is selected from hydrogen, -OH, C ₁-C₆ alkyl, hydroxyC ₁-C₆ alkyl, C ₁-C₆ alkoxy, or C ₁-C₆ acyl.

In another preferred embodiment, the compound of formula (I) is selected from the group consisting of:

In a second aspect of the invention, there is provided the use of a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt, stereoisomer, isotopic isomer, prodrug, hydrate or solvate thereof, as an inhibitor of KIF18A kinesin and an inhibitor of PARP7, for the manufacture of a medicament for the prophylaxis or treatment of a disease associated with KIF18A or PARP7 activity or expression. These diseases include cancer.

In some preferred embodiments, the cancer comprises: multiple myeloma, B-cell lymphoma, T-cell lymphoma, acute and chronic myeloid leukemia, acute and chronic lymphoid leukemia, monocytic leukemia, splenomegaly, eosinophilia syndrome, fibrosarcoma, salivary gland carcinoma, liver cancer, rectal cancer, bladder cancer, throat cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, stomach cancer, and the like.

In a third aspect of the present invention there is provided a pharmaceutical composition comprising a compound of the first aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, isotopic isomer, prodrug, hydrate or solvate thereof, and a pharmaceutically acceptable carrier.

In some cases, pharmaceutically acceptable carriers include, but are not limited to, diluents, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants. The pharmaceutical composition can be prepared into liquid preparation forms such as tablets, capsules, powder, granules, troches, suppositories, oral liquid or sterile parenteral suspension and injection forms such as large or small volume injection and freeze-dried powder.

In some cases, the uracil-like PARP7 inhibitors may also be used in combination with other known drugs.

Detailed Description

The methods and techniques of the present invention are generally performed according to conventional methods known in the art, unless otherwise indicated. Nomenclature that and the laboratory procedures and techniques associated with the biology, pharmacy, medicine, and chemistry described herein are those well known and commonly employed in the art.

Definition of the definition

The following terms, as used in this specification and claims, have the meanings discussed below, unless otherwise indicated.

"Alkyl" refers to a saturated aliphatic hydrocarbon group or linking arm, preferably straight and branched chain groups of 1 to 6 carbon atoms.

"Cycloalkyl" is an all-carbon monocyclic structure in which the ring may contain one or more double bonds, but none of such rings has a complete conjugated pi-electron system. "C ₁-C₆ cycloalkyl" refers to a cyclic group consisting of 3 to 6 carbon atoms, examples being, but not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexanediene, and the like.

"Alkoxy" or "alkyloxy" means alkyl-O-, -C ₁-C₆ alkoxy (or alkyloxy) "includes C ₁、C₂、C₃、C₄、C₅、C₆ alkoxy. Examples of alkoxy groups are, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.

"Acyl" refers generally to alkyl-C (O) - (special cases such as formyl, referring to H-C (O) -), "C ₁-C₆ acyl" includes C ₁、C₂、C₃、C₄、C₅、C₆ acyl, examples of acyl are, but are not limited to, formyl, acetyl, propionyl, and butyryl (e.g., n-butyryl and isobutyryl), and the like.

"Substituted" means that at least one hydrogen atom is replaced with a non-hydrogen group, provided that the normal valence is maintained and that the substitution results in a stable compound.

Unless otherwise defined, the definition of substituents of the invention are independent of each other and are not related to each other, e.g., for R ^a (or R ^b) in the substituents, they are independent of each other in the definition of the different substituents. Specifically, when one definition is selected for R ^a (or R ^b) in one substituent, it does not mean that the R ^a (or R ^b) has the same definition in the other substituents. More specifically, for example (by way of non-exhaustive list) for NR ^aR^b, when the definition of R ^a (or R ^b) is selected from hydrogen, it does not mean that R ^a (or R ^b) in-C (O) -NR ^aR^b is necessarily hydrogen.

"Halo" or "halogen" includes fluoro, chloro, bromo and iodo. "haloalkyl" includes branched or straight-chain saturated aliphatic hydrocarbon groups having the indicated number of carbon atoms and substituted with 1 or more halogens. 1 or more halogens may each be independently selected from fluorine, chlorine, bromine, and iodine. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl.

In the present disclosure, the expression C _x-C_y is used when referring to some substituents, which means that the number of carbon atoms in the substituent may be x to y. For example, C ₁-C₆ represents that the group contains 1,2, 3, 4, 5, or 6 carbon atoms, and C ₃-C₆ represents that the group contains 3, 4, 5, or 6 carbon atoms.

In the present disclosure, the expression "x-y membered ring" is used when referring to a cyclic group (cycloalkyl and heterocycloalkyl), which means that the number of ring atoms of the group may be x to y. For example, a 3-6 membered ring means that the cyclic group may be a 3, 4, 5 or 6 membered ring, the number of ring atoms of which may be 3, 4, 5 or 6 said ring atoms may be carbon atoms or heteroatoms, for example heteroatoms selected from N, O and S. When the ring is a heterocyclic ring, the heterocyclic ring may contain 1,2 or more ring heteroatoms, for example heteroatoms selected from N, O and S.

In the present invention, when any variable occurs more than once in any composition or formula of a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-3R, then the group may optionally be substituted with up to three R groups, and R is independently selected at each occurrence from the definition of R. Furthermore, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

By "pharmaceutically acceptable salt" is meant an organic or inorganic salt of the active molecule that is toxicologically compatible. Exemplary salts include, but are not limited to: sulfate, citrate, acetate, oxalate, chloride bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, tartrate, ascorbate, succinate. Maleate, gentisate, fumarate, gluconate, glucuronate, formate, benzoate, glutamate, methanesulfonic acid "mesylate", ethanesulfonate, benzenesulfonate, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions. If the active molecule is a base, the pharmaceutically acceptable salt may be prepared by conventional chemical methods by treating the free base with an acid. Such acids include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, and the like, or organic acids such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, uronic acid (such as glucuronic acid or galacturonic acid), alpha hydroxy acid, citric acid, tartaric acid, amino acids (such as aspartic acid, glutamic acid), aromatic acids (such as benzoic acid or cinnamic acid), sulfonic acids (such as p-toluenesulfonic acid or ethanesulfonic acid), and the like. If the active molecule is an acid, the desired pharmaceutically acceptable salt may be prepared by suitable methods with inorganic or organic bases such as ammonia, amines, alkali or alkaline earth hydroxides, and the like. Examples of suitable salts include, but are not limited to, amino acid salts (e.g., glycine and arginine), ammonium salts, primary amine salts, secondary amine salts, tertiary amine salts, cyclic amines (e.g., piperidine, morpholine, and piperazine), sodium salts, calcium salts, potassium salts, magnesium salts, manganese salts, iron salts, copper salts, zinc salts, aluminum salts, and lithium salts.

An "effective amount" refers to an amount of a drug or pharmaceutical agent (i.e., a compound of the invention) that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount of: such amounts result in improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or disorder, as compared to a corresponding subject not receiving such amounts. An effective amount may be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or route of administration. The term also includes within its scope an effective amount to enhance normal physiological function.

The term "treatment" includes its broad meaning, covering therapeutic treatment and/or prophylactic treatment of a subject. In particular, the "treatment" includes any treatment that results in alleviation, inhibition, elimination, and amelioration and/or prevention of a condition, disease, disorder, etc., such as alleviation, reduction, regulation, amelioration, elimination, prevention, or amelioration of a symptom thereof. The therapeutic treatment includes alleviation, inhibition, or amelioration of the symptoms or conditions of the disease; inhibit the occurrence of complications; improving underlying metabolic syndrome; inhibiting the occurrence of a disease or condition, such as controlling the progression of a disease or condition; alleviating a disease or symptom; causing the disease or symptom to subside; alleviating complications caused by diseases or symptoms, or treating signs caused by diseases or symptoms. The prophylactic treatment includes prior treatment to prevent, block or delay, slow the occurrence or progression of, or attenuate the severity of a disease or disorder.

The term "cancer", as used herein, refers to an abnormal growth of cells that is not controllable and is capable of metastasis (transmission) under certain conditions. Cancers of this type include, but are not limited to, solid tumors (e.g., bladder, intestine, brain, chest, uterus, heart, kidney, lung, lymphoid tissue (lymphoma), ovaries, pancreas or other endocrine organs (e.g., thyroid), prostate, skin (melanoma), or hematological tumors (e.g., nonleukemia), particularly such as multiple myeloma, B-cell lymphoma, T-cell lymphoma, acute and chronic myeloid leukemia, acute and chronic lymphoid leukemia, monocytic leukemia, splenomegaly, eosinophilic leukocytosis syndrome, fibrosarcoma, salivary gland carcinoma, liver cancer, rectal cancer, bladder cancer, throat cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, stomach cancer, and the like.

The term "subject," "subject," or "patient" includes mammals and non-mammals. Mammals include, but are not limited to, mammals: humans, non-human primates such as gorillas, apes, and monkeys; agricultural animals such as cattle, horses, goats, sheep, pigs; domestic animals such as rabbits and dogs; laboratory animals include rodents such as rats, mice, guinea pigs, and the like. Non-mammalian animals include, but are not limited to, birds, fish, and the like.

As used herein, a compound or pharmaceutical composition, when administered, results in an improvement in a disease, condition, or condition, particularly an improvement in severity, delay in onset, slow progression, or decrease in duration of the condition. Whether stationary or temporary, continuous or intermittent, may be due to or associated with administration.

Route of administration: suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, auditory canal, nasal, and topical. Further, by way of example only, parenteral administration includes intramuscular, subcutaneous, intravenous, intramedullary, ventricular, intraperitoneal, intralymphatic, and intranasal.

By "pharmaceutically acceptable carrier" is meant a pharmaceutical substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate or zinc stearate or stearic acid), or solvent encapsulating material, which involves carrying or transporting the subject compound from one organ or body part to another organ or body part. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient.

Pharmaceutically acceptable carriers are according to a number of factors within the purview of one skilled in the art. These factors include, but are not limited to: the type and nature of the active agent formulated; a subject to whom the active agent-containing composition is to be administered; the intended route of administration of the composition; and targeted therapeutic indications. Pharmaceutically acceptable carriers include aqueous and nonaqueous liquid media and various solid and semi-solid dosage forms.

The carrier may include a number of different ingredients and additives in addition to the active agent, which other ingredients are included in the formulation for a variety of reasons known to those skilled in the art, such as stabilizing the active agent, binder, etc. For a description of suitable pharmaceutically acceptable carriers and factors involved in carrier selection, see a number of readily available sources, e.g Allen L.V.Jr.et al.Remington:The Science and Practice of Pharmacy(2Volumes),22nd Edition(2012),Pharmaceutical Press.

The scope of the present invention includes pharmaceutical compositions (alone or in combination with pharmaceutically acceptable carriers) comprising a therapeutically effective amount of at least one compound of the present invention as an active ingredient. Optionally, the compounds of the present invention may be used alone, in combination with other compounds of the present invention, or in combination with one or more other therapeutic agents (e.g., anticancer agents or other pharmaceutically active substances).

The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features disclosed in this specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention.

The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

NMR measurements were performed using BrukerAVANCE-400 nuclear magnetic instruments. The measurement solvent is noted in the spectrum analysis.

The MS is determined by Agilent 1200-G1956A/1200-6110A/1200-6140A/1260-6125B/Prime-6125B/1260-6120 liquid chromatography-mass spectrometry, SHIMADZU 20A-2010/20A-2020 liquid chromatography-mass spectrometry and WATERS ACQ-QDA liquid chromatography-mass spectrometry.

HPLC analysis was performed using a SHIMADZU 20A high performance liquid chromatograph.

SFC analysis and determination uses Waters UPCC with PDADetector and QDa Detector ultra-high performance co-phase chromatograph, waters UPC ² with PDA detector ultra-high performance co-phase chromatograph, agilent 1260with DAD detector high performance liquid chromatograph, shimadzu LC-20AB with PDA detector high performance liquid chromatograph, shimadzu LC-20AD with PDAdetector high performance liquid chromatograph.

Preparative HPLC separation using Shimadzu LC-20AP pump,Shimadzu LH-40Liquid Handler,Shimadzu SPD-20A Detector,Gilson GX-281Liquid Handler,Gilson 322pump,Gilson 156UV Detector preparative chromatograph.

SFC separation and use The Berger MG II、MG III,Sepiatec's Prep SFC 100system,Waters Prep80Q SFC SYSTEM、Prep 150AP SFC SYSTEM、Prep 200SFC SYSTEM、Prep 350SFC SYSTEM.

Flash column chromatography separation using Biotage IsoleraOne flash preparative chromatography.

The thin layer chromatography silica gel plate uses GF254 acrylic acid adhesive silica gel plate of Anhui good ministerial silicon source material company, the specification of the silica gel plate used by the Thin Layer Chromatography (TLC) is 0.25mm, and the specification of the thin layer chromatography separation and purification product is 0.5mm.

A pressurized hydrogenation reaction hydrogenation bottle and a hydrogen steel bottle.

The microwave reaction uses Biotage Initiator + microwave synthesizer.

Glove box the glove box was custom made using deluxe DELLIX.

The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention. Various changes and modifications to the specific embodiments of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

Example 1: synthesis of intermediates

1.1 Intermediate A1:2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-amine

The first step: to a solution of 2, 4-dichloro-6-methylpyrimidine (3.00 g,18.40 mmol) in dimethyl sulfoxide (30 mL) at 25℃was added aqueous ammonia (2.5 mL,18.04 mmol), and the reaction was stirred at 25℃for 16 hours. The reaction was extracted with ethyl acetate (50 ml×3), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the dried yellow oil was concentrated and purified by slurrying (ethyl acetate: petroleum ether=1:1, 10 mL) to give 2-chloro-6-methylpyrimidin-4-amine as a white solid (2.00 g, yield 75%). LCMS (ESI) [ m+h ] ⁺ =144.1.

And a second step of: to a solution of 2-chloro-6-methylpyrimidin-4-amine (7.50 g,41.79 mmol), 4-difluoropiperidine hydrochloride (12.00 g,76.13 mmol) in N-methylpyrrolidone (75 mL) was added N, N-diisopropylethylamine (25 mL) at 25 ℃. The reaction was stirred at 160℃for 6 hours, then the reaction mixture was concentrated to dryness and purified by flash column chromatography (silica gel, 0-10% gradient methanol/dichloromethane) to give crude yellow oil. The crude product was dissolved in ethyl acetate (50 mL) and washed with water (50 mL. Times.2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to give a pale yellow solid, which was stirred in petroleum ether (30 mL) for 30 minutes. The white precipitate was filtered to give 2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-amine (intermediate A1,3.00g, yield 38%). LCMS (ESI) [ m+h ] ⁺ = 229.1.

1.2 Intermediate A2: 4-bromo-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -2- (6-azaspiro [2.5] oct-6-yl) benzamide

The first step: 4-bromo-2-fluorobenzoic acid (20.00 g,90.41 mmol), sodium carbonate (14.52 g,135.61 mmol) was dissolved in N, N-dimethylformamide (200 mL). Benzyl bromide (17.18 g,99.45 mmol) was added to the solution at 30 ℃. The reaction mixture was stirred at 30℃for 15 hours. The reaction mixture was quenched with water (400 mL) and extracted with methyl tert-butyl ether (3X 300 mL). The combined organic extracts were washed with brine (400 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give benzyl 4-bromo-2-fluorobenzoate (29.00 g) as a crude white solid. LCMS (ESI) [ m+h ] ⁺ =308.

And a second step of: to a solution of benzyl 4-bromo-2-fluorobenzoate (20.00 g,59.79 mmol) in dimethyl sulfoxide (120 mL) was added sodium carbonate (21.82 g,203.8 mmol), followed by 6-azaspiro [2.5] octane hydrochloride (13.38 g,89.69 mmol), and the reaction mixture was stirred at 100℃for 15 hours. The reaction mixture was quenched with water (200 mL) and extracted with methyl tert-butyl ether (3X 200 mL). The combined organic extracts were washed with brine (3X 200 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give benzyl 4-bromo-2- (6-azaspiro [2.5] oct-6-yl) benzoate (23.00 g, yield: 89%) as a red oil. LCMS (ESI) [ m+h ] ⁺ =402.1.

And a third step of: to a solution of benzyl 4-bromo-2- (6-azaspiro [2.5] oct-6-yl) benzoate (7.00 g,16.24 mmol) in methanol/water (60 mL/15 mL) was added lithium hydroxide monohydrate (1.15 g,48.72 mmol), and the mixture was stirred at 35℃for 15 hours. The reaction mixture was concentrated, the residue was dissolved in water (50 mL), the aqueous phase was washed with ethyl acetate (3×70 mL), then the aqueous phase was adjusted to ph=5 with aqueous hydrogen chloride (1 m aq,20 mL), and extracted with ethyl acetate (3×50 mL), the organic layer was washed with saturated aqueous sodium chloride (100 mL), dried over anhydrous sodium sulfate and concentrated to give 2- (6-azaspiro [2.5] oct-6-yl) -4-bromobenzoic acid (4.00 g, yield: 73%) as a red solid. LCMS (ESI) [ m+h ] ⁺ = 311.7

Fourth step: to a solution of 4-bromo-2- (6-azaspiro [2.5] oct-6-yl) benzoic acid (1000 mg,3.22 mmol) in dichloromethane (10 mL), N, N-dimethylformamide (0.2 mL) was added thionyl chloride (1.10 g,9.67 mmol), and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was dried by spinning, toluene (10 mL) was added, concentrated, and the procedure was repeated 3 times. 2- (4, 4-Difluoropiperidin-1-yl) -6-methylpyrimidin-4-amine (730 mg,3.22 mmol) and potassium tert-butoxide (1096 mg,9.67 mmol) were added to tetrahydrofuran (10 mL), the crude product of the above step was dissolved in tetrahydrofuran (20 mL), and the mixture was slowly added dropwise to the reaction solution, followed by stirring at room temperature for 4 hours. The reaction solution was filtered off with suction, concentrated and flash column chromatographed (silica gel, 0-25% gradient of ethyl acetate/petroleum ether) to give 4-bromo-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -2- (6-azaspiro [2.5] oct-6-yl) benzamide (intermediate A2, 600mg, yield 35%) as a pale yellow solid.

1.3 Intermediate A3: 4-nitro-2- (6-azaspiro [2.5] oct-6-yl) benzoyl chloride

The first step: a mixed solution containing 2-fluoro-4-nitrobenzoic acid (10.00 g,53.48 mmol), 6-azaspiro [2.5] octane hydrochloride (8.69 g,58.83 mmol), potassium carbonate (22.60 g,160.45 mmol) and dimethyl sulfoxide (50 mL) was stirred at 140℃for 12 hours. To the reaction solution was added dropwise a 2M aqueous hydrochloric acid solution to adjust the pH to about 5, followed by filtration and drying of the cake to give the compound 4-nitro-2- (6-azaspiro [2.5] octane-6-yl) benzoic acid (10.60 g) as a yellow solid.

And a second step of: a mixed solution of 4-nitro-2- (6-azaspiro [2.5] oct-6-yl) benzoic acid (500 mg,1.81 mmol) and thionyl chloride (5 mL) was stirred at 90℃for 1 hour. The reaction mixture was concentrated with methylene chloride to give 4-nitro-2- (6-azaspiro [2.5] oct-6-yl) benzoyl chloride (intermediate A3, 620 mg) as a yellow solid. LCMS (ESI) [ m+h ] ⁺ =291.1.

1.4 Intermediate A4: 2-sulfamylacetic acid ethyl ester

The first step: ethyl 2- (chlorosulfonyl) acetate (5.00 g,26.53 mmol) was added to dichloromethane (25 mL) and the reaction was cooled to 0deg.C. Hexamethyldisilazane (4.32 g,26.53 mmol) was slowly added dropwise to the reaction solution. The reaction solution was stirred at 25℃for 1 hour. The reaction solution was evaporated to dryness to give a residue. The residue was dissolved in ethanol (20 mL) at 0deg.C, and the reaction mixture was stirred at 25deg.C for 1 hour. The reaction solution was evaporated to dryness to give 2-sulfamylacetic acid ethyl ester as a white solid (intermediate A4,4.20g, yield 94%).

Example 2: synthesis of N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -4- (dimethylphosphoryl) -2- (6-azaspiro [2.5] oct-6-yl) benzamide (compound 1)

The first step: 4-bromo-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -2- (6-azaspiro [2.5] oct-6-yl) benzamide (100 mg,0.19 mmol), dimethylphosphine oxide (30 mg,0.38 mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (34 mg,0.058 mmol), palladium acetate (4 mg,0.019 mmol), cesium carbonate (127 mg,0.38 mmol) were added to 1, 4-dioxane (1 mL), nitrogen substitution, and reacted at 100℃for 16 hours. The reaction solution is filtered by suction, concentrated and subjected to flash column chromatography (silica gel, 0-10% gradient to obtain methanol/dichloromethane) to obtain crude products. Crude preparative chromatography (C18, 30-70% gradient acetonitrile/water) afforded N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -4- (dimethylphosphoryl) -2- (6-azaspiro [2.5] oct-6-yl) benzamide (compound 1,0.6mg, 0.54%) as a pale yellow solid .LCMS(ESI):[M+H]⁺＝518.0;¹H NMR(600MHz,CDCl3)δ13.43(s,1H),8.38(dd,J＝7.9,3.7Hz,1H),7.95(dd,J＝12.3,1.5Hz,1H),7.50(s,1H),7.45(ddd,J＝10.8,7.9,1.4Hz,1H),4.00(t,J＝5.8Hz,4H),3.14(t,J＝5.3Hz,4H),2.39(s,3H),2.00(td,J＝11.1,8.3,4.3Hz,4H),1.78(d,J＝13.0Hz,6H),1.56–1.20(m,4H),0.41(s,4H).

Example 3: synthesis of N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -4- (2-oxopropanamido) -2- (6-azaspiro [2.5] octyl-6-yl) benzamide (compound 2)

The first step: 4-Nitro-2- (6-azaspiro [2.5] oct-6-yl) benzoic acid (550 mg,0.99 mmol) was added to thionyl chloride (5 mL) and reacted at 90℃for 2 hours. The reaction solution was evaporated to dryness. 2- (4, 4-Difluoropiperidin-1-yl) -6-methylpyridin-4-amine (227 mg,0.99 mmol) and potassium tert-butoxide (338 mg,2.98 mmol) were added to tetrahydrofuran (5 mL) and stirred at room temperature for 15 minutes. The residue was evaporated to dryness in tetrahydrofuran (5 mL), and the mixture was slowly added dropwise to the reaction mixture, followed by stirring at room temperature for 4 hours. The reaction solution was filtered off with suction, concentrated and flash column chromatographed (silica gel, 10% -25% gradient ethyl acetate/petroleum ether) to give N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl) -4-nitro-2- (6-azaspiro [2.5] oct-6-yl) benzamide (200 mg, yield 28%) as a tan solid. LCMS (ESI) [ m+h ] ⁺ = 487.2.

And a second step of: n- (2- (4, 4-Dihaloperidol-1-yl) -6-methylpyrimidin-4-yl) -4-nitro-2- (6-azaspiro [2.5] octane-6-yl) benzamide (195 mg,0.28 mmol) was added to a solution of ethanol (5 mL), water (1 mL), ammonium chloride (76 mg,1.40 mmol), iron powder (79 mg,1.40 mmol) was added to the reaction solution, and the mixture was reacted at 90℃for 2 hours. The reaction solution was filtered by suction, the filtrate was poured into water (30 mL), extracted with ethyl acetate (30 mL. Times.3), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to give crude 4-amino-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -2- (6-azaspiro [2.5] oct-6-yl) benzamide (140 mg, yield 87%) as a yellow solid. LCMS (ESI) [ m+h ] ⁺ =457.2.

And a third step of: 4-amino-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -2- (6-azaspiro [2.5] oct-6-yl) benzamide (50 mg,0.11 mmol), N, N-diisopropylethylamine (29 mg,0.22 mmol), pyruvic acid (19 mg,0.22 mmol) were added to dichloromethane (2 mL), and 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (84 mg,0.22 mmol) was added to the reaction solution, which was stirred at room temperature for 3 hours. The reaction mixture was poured into water (30 mL), extracted with ethyl acetate (30 mL. Times.3), washed with saturated brine (30 mL. Times.3), the organic phases were combined, concentrated, and flash column chromatographed (silica gel, 10-25% gradient ethyl acetate/petroleum ether) to give crude product. Crude prep chromatography (C18, 50-85% gradient acetonitrile/water) afforded N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -4- (2-oxopropanamido) -2- (6-azaspiro [2.5] octyl-6-yl) benzamide (compound 2,1.4mg, 2.27%) as a white solid .LCMS(ESI):[M+H]⁺＝527.2;¹H NMR(600MHz,CDCl3)δ13.42(s,1H),8.90(s,1H),8.29(d,J＝8.6Hz,1H),8.01(d,J＝2.2Hz,1H),7.52(s,1H),7.34(dd,J＝8.6,2.1Hz,1H),4.02(t,J＝5.8Hz,4H),3.09(t,J＝5.3Hz,4H),2.60(s,3H),2.41(s,3H),2.00(dt,J＝18.8,6.2Hz,4H),1.45–1.19(m,4H),0.41(s,4H).

Example 4: synthesis of N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl) -3- (2-hydroxyethyl) sulfanyl) -5- (6-azaspiro [2.5] oct-6-yl) benzo [ d ] isoxazole-6-carboxamide (compound 3)

The first step: a mixed solution containing 4-bromo-2, 5-difluorobenzoic acid (4.90 g,20.68 mmol), 6-azaspiro [2.5] octane hydrochloride (3.40 g,22.74 mmol), potassium carbonate (8.70 g,62.03 mmol) and dimethyl sulfoxide (50 mL) was stirred at 130℃for 12 hours, the reaction solution was poured into water and filtered, and petroleum ether was used: the filter cake was washed with ethyl acetate=10:1 to give the compound 4-bromo-5-fluoro-2- (6-azaspiro [2.5] oct-6-yl) benzoic acid (3.40 g, yield: 50%) as a yellow solid. LCMS (ESI) [ m+h ] ⁺ =328.1.

And a second step of: the reaction solution containing 4-bromo-5-fluoro-2- (6-azaspiro [2.5] oct-6-yl) benzoic acid (3.00 g,9.14 mmol) and thionyl chloride (10 mL) was stirred at 90℃for 1 hour, and the reaction solution was concentrated with methylene chloride to give the crude compound 4-bromo-5-fluoro-2- (6-azaspiro [2.5] oct-6-yl) benzoyl chloride (3.20 g) as a yellow solid. LCMS (ESI) [ m+h ] ⁺ = 342.1, (corresponding methyl ester).

And a third step of: a mixed solution containing 2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-amine (2.00 g,8.66 mmol), potassium t-butoxide (2.00 g,17.31 mmol) and tetrahydrofuran (20 mL) was stirred at room temperature for 30 minutes, then 4-bromo-5-fluoro-2- (6-azaspiro [2.5] oct-6-yl) benzoyl chloride (3.00 g,8.66 mmol) was dissolved in tetrahydrofuran and added dropwise to the reaction solution, stirring was continued at room temperature for 12 hours, the reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (50 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography (silica gel, 0-100% gradient ethyl acetate/petroleum ether) to give the compound 4-bromo-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl) -2, 5-difluorobenzamide (400 mg, 10% as a solid yield. LCMS (ESI) [ m+h ] ⁺ = 447.1.

Fourth step: a mixed solution containing N- [2- (4, 4-difluoropiperidinyl) -6-methylpyridin-4-yl ] (4-bromo-2, 5-difluorophenyl) carboxamide (300 mg,0.67 mmol), 6-azaspiro [2.5] octane hydrochloride (120 mg,0.80 mmol), potassium carbonate (284 mg,2.01 mmol) and dimethyl sulfoxide (5 mL) was stirred at 130℃for 12 hours, the reaction mixture was poured into water (20 mL), ethyl acetate (20 mL. Times.3) was extracted, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated after filtration to give the compound 4-bromo-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl) -5-fluoro-2- (6-azaspiro [2.5] oct-6-yl) benzamide (300 mg, 83%) as a yellow solid by column chromatography purification (silica gel, 0-100% gradient of ethyl acetate/petroleum ether). LCMS (ESI) [ m+h ] ⁺ = 538.3.

Fifth step: a mixed solution containing [2- (6-azaspiro [2.5] oct-6-yl) -4-bromo-5-fluorophenyl ] -N- [2- (4, 4-difluoropiperidinyl) -6-methylpyrimidin-4-yl ] carboxamide (300 mg,0.56 mmol), zinc cyanide (79 mg,0.67 mmol), tetrakis triphenylphosphine palladium (64 mg,0.056 mmol) and N, N-dimethylformamide (5 mL) was replaced with nitrogen for 3 minutes, the reaction solution was stirred at 120℃for 12 hours, the reaction mixture was poured into water (20 mL), ethyl acetate (20 mL. Times.3) was extracted, the organic phase was dried over anhydrous sodium sulfate, and after concentration by filtration, the ethyl acetate/petroleum ether was purified by column chromatography (silica gel, 0-100% gradient) to give the compound 4-cyano-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -5-fluoro-2- (6-aza [2.5] oct-6-yl) benzamide (220 mg) as a pale yellow solid in 81% yield. LCMS (ESI) [ m+h ] ⁺ = 485.3.

Sixth step: a mixed solution containing [2- (6-azaspiro [2.5] oct-6-yl) -4-cyano-5-fluorophenyl ] -N- [2- (4, 4-difluoropropiiperidin-6-yl ] carboxamide (200 mg,0.41 mmol), acetohydroxamic acid (38 mg,0.50 mmol), potassium tert-butoxide (94 mg,0.83 mmol) and N, N-dimethylformamide (5 mL) was stirred at room temperature for 12 hours, the reaction solution was poured into water (20 mL) and extracted with ethyl acetate (20 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, and after concentration by filtration was purified by column chromatography (silica gel, ethyl acetate/petroleum ether in a gradient of 0-100%) to give the compound 3-amino-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl) -5- (6-aza [2.5] oct-6-yl) benzo [ d ] isoxazol-6-carboxamide (160 mg, 77 mg) as a pale yellow solid in light yellow yield. LCMS (ESI) [ m+h ] ⁺ =498.3.

Seventh step: ethyl 2- (chlorosulfonyl) acetate (63 mg,0.33 mmol) was added dropwise to a mixed solution containing [ 3-amino-5- (6-azaspiro [2.5] oct-6-yl) benzo [ d ] isoxazol-6-yl ] -N- [2- (4, 4-difluoropiperidinyl) -6-methylpyrimidin-4-yl ] carboxamide (150 mg,0.30 mmol), N-diisopropylethylamine (80 mg,0.60 mmol) and dichloromethane (5 mL), stirred at room temperature for 30 minutes, the reaction solution was poured into water (20 mL) and extracted with dichloromethane (20 mL. Times.3), and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give the crude product 2- { (5- (6-azaspiro [2.5] oct-6-yl) -6- { N- [2- (4, 4-difluoropiperidinyl) -6-methylpyrimidin-4-yl ] carbamoyl } benzo [ d ] isoxazol-3-yl) amino ] sulfonyl } ethyl acetate (120 mg) as a yellow solid in 61% yield. LCMS (ESI) [ m+h ] ⁺ = 648.4.

Eighth step: lithium aluminum hydride (17 mg,0.46 mmol) was added to a mixed solution containing ethyl 2- { (5- (6-azaspiro [2.5] oct-6-yl) -6- { N- [2- (4, 4-difluoropiperidinyl) -6-methylpyrimidin-4-yl ] carbamoyl } benzo [ d ] isoxazol-3-yl) amino ] sulfonyl } acetate (100 mg,0.15 mmol) and tetrahydrofuran (5 mL), stirred at room temperature for 30 minutes, a saturated aqueous ammonium chloride solution (20 mL) was added dropwise to the reaction solution, the reaction was quenched, then extracted with ethyl acetate (20 mL. Times.3), and the organic phase was dried over anhydrous sodium sulfate, concentrated by filtration, and purified by column chromatography (silica gel, ethyl acetate/petroleum ether in a gradient of 0-100%) to give the compound (5- (6-azaspiro [2.5] oct-6-yl) -3- { [ (2-hydroxyethyl) sulfonyl ] amino } benzo [ d ] isoxazol-6-yl) -N- [2- (4, 4-difluoropiperidinyl) -6-methylpyrimidine-yl (4 mg, 6%) as a yellow solid, the compound was obtained as a solid (4 mg, 4%) .LCMS(ESI):[M+H]⁺＝606.4;¹H NMR(400MHz,DMSO)δ14.05(s,1H),8.31(s,1H),8.23(s,1H),7.43(s,1H),4.97(s,2H),3.93(t,J＝5.6Hz,4H),3.83(t,J＝6.4Hz,2H),3.65(t,J＝5.6Hz,2H),2.96-3.14(m,5H),2.27-2.39(m,4H),2.12–1.89(m,6H),0.41(s,4H).

Example 5: synthesis of N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl) -3- (2-hydroxyethyl) sulfanyl) -7- (6-azaspiro [2.5] oct-6-yl) benzo [ d ] isoxazole-6-carboxamide (Compound 4)

The first step: a mixed solution containing 4-bromo-2, 3-difluorobenzoic acid (5.00 g,21.10 mmol), 6-azaspiro [2.5] octane hydrochloride (3.50 g,23.21 mmol), potassium carbonate (8.90 g,63.29 mmol) and dimethyl sulfoxide (50 mL) was stirred at 130℃for 12 hours, the reaction solution was poured into water and filtered, and the filter cake was washed with petroleum ether/ethyl acetate=10:1 to give the compound 4-bromo-3-fluoro-2- (6-azaspiro [2.5] octane-6-yl) benzoic acid (6.20 g) as a brown solid. LCMS (ESI) [ m+h ] ⁺ =328.1.

And a second step of: the reaction solution containing 4-bromo-3-fluoro-2- (6-azaspiro [2.5] oct-6-yl) benzoic acid (4.00 g,12.19 mmol) and thionyl chloride (20 mL) was stirred at 90℃for 1 hour, and the reaction solution was concentrated with methylene chloride to give the crude compound 4-bromo-3-fluoro-2- (6-azaspiro [2.5] oct-6-yl) benzoyl chloride (4.20 g, yield: 99%) as a yellow solid. LCMS (ESI) [ m+h ] ⁺ = 342.1, (corresponding methyl ester).

And a third step of: a mixed solution containing 2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-amine (3.20 g,13.85 mmol), potassium t-butoxide (3.90 g,34.62 mmol) and tetrahydrofuran (20 mL) was stirred at room temperature for 30 minutes, then 4-bromo-5-fluoro-2- (6-azaspiro [2.5] oct-6-yl) benzoyl chloride (4.00 g,11.54 mmol) was dissolved in tetrahydrofuran and added dropwise to the reaction solution, stirring was continued at room temperature for 30 minutes, the reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (50 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography (silica gel, 0-70% gradient ethyl acetate/petroleum ether) to give the compound 4-bromo-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -3-fluoro-2- (6-azaspiro [2.5] benzoyl amine (12 g) as a yellow solid in a yield of 0.75 g. LCMS (ESI) [ m+h ] ⁺ = 538.2.

Fourth step: a mixed solution containing 4-bromo-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -3-fluoro-2- (6-azaspiro [2.5] oct-6-yl) benzamide (750 mg,1.39 mmol), zinc cyanide (200 mg,1.67 mmol), tetrakis triphenylphosphine palladium (164 mg,0.139 mmol) and N, N-dimethylformamide (10 mL) was replaced with nitrogen for 3 minutes, the reaction solution was stirred at 120℃for 12 hours, the reaction mixture was poured into water (30 mL), ethyl acetate (30 mL. Times.3) was extracted, the organic phase was dried over anhydrous sodium sulfate, and after concentration by filtration, ethyl acetate/petroleum ether was purified by column chromatography (silica gel, 0-100% gradient) to give 4-cyano-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -3-fluoro-2- (6-aza [2.5] oct-6-yl) benzamide (630 mg) as a yellow solid. LCMS (ESI) [ m+h ] ⁺ = 485.3.

Fifth step: a mixed solution containing acetohydroxamic acid (38 mg,0.50 mmol), potassium tert-butoxide (94 mg,0.83 mmol) and N, N-dimethylformamide (5 mL) was stirred at room temperature for 30 minutes, then 4-cyano-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -3-fluoro-2- (6-azaspiro [2.5] oct-6-yl) benzamide (200 mg,0.41 mmol) was added to the reaction flask and stirring was continued at room temperature for 12 hours, the reaction solution was poured into water (30 mL) and extracted with ethyl acetate (30 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, and after concentration by filtration was purified by column chromatography (silica gel, 0-100% gradient ethyl acetate/petroleum ether) to give the compound 3-amino-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -7- (6-aza [2.5] oct-6-yl) benzo [ 460-oxazazole ] d-6-carboxamide as a yellow solid (mg). LCMS (ESI) [ m+h ] ⁺ =498.3.

Sixth step: ethyl 2- (chlorosulfonyl) acetate (121 mg,0.64 mmol) was added dropwise to a mixed solution containing 3-amino-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -7- (6-azaspiro [2.5] oct-6-yl) benzo [ d ] isoxazole-6-carboxamide (320 mg,0.64 mmol), N-diisopropylethylamine (127 mg,0.97 mmol) and dichloromethane (5 mL), stirred at room temperature for 1 hour, the reaction solution was poured into water (20 mL) and extracted with dichloromethane (20 mL. Times.3), and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give the product 2- (N- (6- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl) carbamoyl) -7- (6-aza [2.5] oct-6-yl) benzo [ d ] isoxazol-3-yl) sulfamoyl) ethyl acetate (370 mg) in yield: 88%) as a yellow solid. LCMS (ESI) [ m+h ] ⁺ = 648.4.

Seventh step: lithium aluminum hydride (41 mg,1.16 mmol) was added to a mixed solution containing ethyl 2- (N- (6- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl) carbamoyl) -7- (6-azaspiro [2.5] oct-6-yl) sulfamoyl) acetate (250 mg,0.39 mmol) and tetrahydrofuran (3 mL), the reaction solution was stirred at room temperature for 30 minutes, poured into water (30 mL), then extracted with ethyl acetate (30 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, and the crude product obtained after concentration by filtration was purified by column chromatography (silica gel, 0-50% gradient of methanol/dichloromethane) was purified by HPLC (C18, 30-100% gradient of acetonitrile/pure water) to give the compound N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl) -3- (2-hydroxyethyl) sulfanilamide (6-azaspiro [ 2.5-oxa-6-azol-6-yl) m-1 mg, 01mg of the product: 0.4%) as a yellow solid .LCMS(ESI):[M+H]⁺＝606.3;¹H NMR(400MHz,DMSO)δ14.03(s,1H),11.74(s,1H),8.07-8.18(m,1H),7.92-8.01(m,1H),7.44(s,1H),3.88-4.01(m,4H),3.80-3.87(m,2H),3.64-3.74(m,2H),2.34(s,6H),1.92-2.11(m,6H),1.70-1.84(m,4H),1.20-1.30(m,2H),0.44(s,4H).

Example 6: synthesis of dimethyl (4- ((2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) carbamoyl) -3- (6-azaspiro [2.5] oct-6-yl) phenyl) phosphonate (compound 5) and methylhydrogen (4- ((2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) carbamoyl) -3- (6-azaspiro [2.5] oct-6-yl) phenyl) phosphonate (compound 6)

The first step: 4-bromo-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -2- (6-azaspiro [2.5] oct-6-yl) benzamide (200 mg,0.38 mmol), triethylamine (79 mg,0.77 mmol), dimethyl phosphite (88 mg,0.77 mmol), 1' -bis (diphenylphosphine) ferrocene (44 mg,0.077 mmol), palladium acetate (9 mg,0.038 mmol), and potassium carbonate (108 mg,0.77 mmol) were added to acetonitrile (2 mL) and after nitrogen substitution, reacted at 100℃for 16 hours. The reaction solution is filtered by suction, concentrated and purified by rapid column chromatography to obtain crude products. Chromatography of the residue (C18, 30-75% gradient of acetonitrile/water) afforded dimethyl (4- ((2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl) carbamoyl) -3- (6-azaspiro [2.5] oct-6-yl) phenyl) phosphonate (compound 5, 35mg, 16% yield) as a white solid .LCMS(ESI):[M+H]⁺＝550.3;¹H NMR(400MHz,Chloroform-d)δ13.35(s,1H),8.39(dd,J＝8.0,5.2Hz,1H),7.85(d,J＝14.4Hz,1H),7.74–7.64(m,1H),7.53(s,1H),4.03(s,4H),3.83(d,J＝11.1Hz,6H),3.14(t,J＝5.3Hz,4H),2.42(s,3H),2.09–1.42(m,8H),0.44(s,4H).

Preparation of the chromatograph (C18, 5-30% gradient acetonitrile/water) to give methyl hydrogen (4- ((2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) carbamoyl) -3- (6-azaspiro [2.5] oct-6-yl) phenyl) phosphonate (compound 6, 31mg, yield 15%) as a white solid .LCMS(ESI):[M+H]⁺＝536.0;¹H NMR(400MHz,Methanol-d4)δ8.23(dd,J＝7.9,4.1Hz,1H),7.91(dd,J＝13.1,1.3Hz,1H),7.71(ddd,J＝11.3,8.0,1.3Hz,1H),7.49(s,1H),4.02(t,J＝5.8Hz,4H),3.53(d,J＝10.9Hz,3H),3.33(q,J＝1.7Hz,4H),3.15(t,J＝5.3Hz,4H),2.39(s,3H),2.01(tt,J＝13.4,5.8Hz,4H),0.46(s,4H).

EXAMPLE 7 Synthesis of (2- (6-azaspiro [2.5] oct-6-yl) -4- { N- [ (2-hydroxyethyl) sulfonyl ] carbamoyl } phenyl) -N- [2- (4, 4-difluoropiperidinyl) -6-methylpyridin-4-yl ] carboxamide (Compound 7)

The first step: 4-bromo-N- (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl) -2- (6-azaspiro [2.5] oct-6-yl) benzamide (1.30 g,2.37 mmol) and [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (II) (319 mg,0.47 mmol) were added to methanol (26 mL), triethylamine (490 mg,4.7 mmol) was added, stirred magnetically, ice-water bath, reflux at 80℃and carbon monoxide protection, and reaction was carried out for 15 hours. The reaction was quenched with purified water (50 mL), extracted with dichloromethane (3×50 mL), the combined organic phases washed once with water (50 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to give the crude product, which was purified by flash column chromatography (silica gel, 0-50% gradient of ethyl acetate/petroleum ether) to give nitrogen (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyridin-4-yl-4-iodo-2- (6-azaspiro [2.5] oct-6-yl) benzamide (500 mg, 40% yield) as a pale yellow solid LCMS (ESI): [ m+h ] + = 499.9).

And a second step of: nitrogen (2- (4, 4-difluoropiperidin-1-yl) -6-methylpyrimidin-4-yl-4-iodo-2- (6-azaspiro [2.5] oct-6-yl) benzamide (500 mg,0.95 mmol) and lithium hydroxide monohydrate (144 mg,3.5 mmol) were magnetically stirred, nitrogen protected, heated to 40 ℃ and reacted for 5 hours, the reaction solution was quenched with water (10 mL), pH=5-6 was adjusted with 1N HCl, a large amount of solids was washed out, a filter cake was obtained by suction filtration, rinsed twice with petroleum ether, and dried to obtain 3- (6-azaspiro [2.5] oct-6-yl) -4- { N- [2- (4, 4-difluoropiperidin-4-yl ] carbamoyl } benzoic acid (450 mg, 97% yield): [ M+H ] ⁺ = 485.9 ] S (ESI).

And a third step of: 3- (6-azaspiro [2.5] oct-6-yl) -4- { N- [2- (4, 4-difluoropiperidinyl) -6-methylpyridin-4-yl ] carbamoyl } benzoic acid (200 mg,0.39 mmol) was dissolved in dichloromethane (2 mL), overdry N, N-dimethylformamide (0.2 mL) was added, oxalyl chloride (100 mg,0.78 mmol) was added dropwise at zero degree, and the reaction was carried out at room temperature for 2 hours after completion of the addition. The reaction solution was dried by spin-drying at low temperature to give 3- (6-azaspiro [2.5] octane-6-yl) -4- { N- [2- (4, 4-difluoropiperidinyl) -6-methylpyrimidin-4-yl ] carbamoyl } benzoyl chloride (200 mg, crude product) which was directly fed to the next step.

Fourth step: 2- (Benzylmethoxy) ethanesulfonamide (170 mg,0.75 mmol), N, N-diisopropylethylamine (196 mg,1.51 mmol), 4-dimethylaminopyridine (4.6 mg,0.04 mmol) was dissolved in N, N-dimethylformamide (2 mL), and 3- (6-azaspiro [2.5] oct-6-yl) -4- { N- [2- (4, 4-difluoropiperidinyl) -6-methylpyrimidin-4-yl ] carbamoyl } benzoyl chloride (200 mg, crude product) was dissolved in dichloromethane (0.5 mL) and was added dropwise to the reaction mixture at room temperature for 16 hours. The reaction mixture was poured into saturated ammonium chloride (20 mL), extracted with ethyl acetate (20 ml×3), the combined organic phases were dried over saturated brine (20 ml×3), anhydrous sodium sulfate, filtered, and the filtrate was dried by spin-drying, and the crude product obtained was purified by column to give [2- (6-azaspiro [2.5] octane-6-yl) -4- (N- { [2- (benzyloxy) ethyl ] sulfonyl } carbamoyl) phenyl ] -N- [2- (4, 4-difluoropiperidinyl) -6-methylpyridin-4-yl ] carboxamide as a pale yellow solid (140 mg, yield 54%) LCMS (ESI): [ m+h ] + = 683.1.

Fifth step: [2- (6-azaspiro [2.5] oct-6-yl) -4- (N- { [2- (benzyloxy) ethyl ] sulfonyl } carbamoyl) phenyl ] -N- [2- (4, 4-difluoropiperidinyl) -6-methylpyridin-4-yl ] carboxamide (120 mg,0.17 mmol) was dissolved in methanol (2 mL), palladium hydroxide (5 mg) was added, and the mixture was reacted at room temperature under hydrogen balloon protection for 18 hours. TLC detection reaction was complete, filtration, rinsing of the filter cake with a small amount of methanol, and purification of the crude filtrate by spin-drying using a large plate gave (2- (6-azaspiro [2.5] octane-6-yl) -4- { N- [ (2-hydroxyethyl) sulfonyl ] carbamoyl } phenyl) -N- [2- (4, 4-difluoropiperidinyl) -6-methylpyrimidin-4-yl ] carboxamide as a white solid (compound 7,6.7mg, yield) 7％)LCMS(ESI):[M+Na]+＝593.0.¹H NMR(400MHz,DMSO)δ13.89(s,1H),8.10(d,J＝8.2Hz,1H),8.03(s,1H),7.86(s,1H),7.42(s,1H),4.61(d,J＝6.1Hz,1H),3.92(s,4H),3.66(dd,J＝13.3,6.9Hz,2H),3.28(s,2H),3.03(t,J＝5.2Hz,4H),2.33(s,3H),2.03–1.97(m,4H),1.24(m,4H),0.40(s,4H).

Example 8: enzyme activity detection experimental material: human KIF18A (amino acid sequence 1-417), available from Shanghai Weiya Biotechnology Co., ltd; ADP-Glo ^TM protein kinase kit was purchased from Promega, america; tubulin is available from Cytoskeleton, usa; 384 well assay plates and multifunctional microplate reader Envision were purchased from perkin elmer, usa.

Enzymatic activity assay: dissolving the compound powder into 10mM stock solution using DMSO; the compounds were diluted in a gradient in the microplate and brought to a final concentration of between 0 and 10. Mu.M. Then 2.5. Mu.L each of tubulin, compound, ATP, and KIF18A protein was added to the microplate in this order, and reacted at room temperature for 120 minutes. The final concentration of the enzyme reaction was 60. Mu.g/mL microtubule, 25. Mu.M ATP, and 2.5nM KIF18A protein. After the enzyme reaction, 10 mu lADP-GLO reagent was added to each well and incubated at room temperature for 30 minutes. Then, 20. Mu.l of a detection reagent was added to each well, and incubated at room temperature for 30 minutes under light-shielding conditions. Finally, chemiluminescent detection was performed using a perkin elmer Envision.

The inhibition data of the KIF18AATP enzymatic activity by the compounds of the present invention are detailed in Table 1.

TABLE 1 inhibition data of KIF18AATP enzymatic Activity of Compounds of the invention

Numbering of compounds	IC50 for enzyme Activity (nM)
		1	318
2	2359
		3	550
4	1136
		5	1203
6	846
		7	363

From the above results, it can be seen that the compounds of the present invention can significantly inhibit KIF18AATP enzyme activity.

Example 9: test of PARP7 enzyme inhibitory Activity

1. Enzymatic reaction

Enzyme reaction buffer: 20mM HEPES ph= 8,100mM NaCl,0.1%BSA,2mM DTT and 0.002% tween20;

Enzyme reagent: PARP7 FLAG-tag (BPS 80527) was diluted at 30nM into the enzyme reaction;

ProbeA: dilute to enzyme reaction buffer at 5nM concentration;

a compound: compounds were diluted to reaction buffer with DMSO at 4-fold dilution 8 from 10 mM;

The enzyme reagent, probe A and test compound were added to PERKINELMER OPTI-Plate in a ratio of 4:4:2, respectively, with a total volume of 10. Mu.l, and mixed well and reacted at room temperature for 1 hour, with a final PARP7 concentration of 12nM and a final Probe A concentration of 2nM.

2. Time resolved fluorescence detection

Lance Eu-W1024 streptavidine reagent diluted at 8nM into 1xLance buffer;

ULight-anti FLAG was diluted at 0.5nM into 1xLance buffer;

5. Mu.l each of LANCE Eu-W1024 streptavidine, ULight-anti-FLAG was added to the reaction plate, and reacted at room temperature for 1 hour, with final concentrations of LANCE Eu-W1024 streptavidine, ULight-anti-FLAG being 4nM and 0.25nM, respectively.

3. Reading the signal value by an Envision instrument; excitation wavelength 320nm emission wavelengths were 615nm and 665nm.

4. Data analysis

IC50 was calculated with GRAPHPAD PRISM software and the following nonlinear fitting formula was used to obtain the IC50 (half inhibition concentration) of the compound:

Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))

Log of compound concentration, Y inhibition (%inhibition)

The data of the inhibitory activity of the compounds of the present invention against PARP7 enzyme are detailed in table 2.

TABLE 2 inhibition of PARP7 enzyme by the compounds of the invention

Numbering of compounds	PARP7 enzyme inhibitory Activity IC ₅₀
		1	29nM
2	120nM
		3	82nM
4	102nM
		5	12nM
6	32nM
		7	65nM

As can be seen from Table 2, the compounds of the present invention have a strong PARP7 inhibitory activity.

The aza-spiro compound provided by the invention has double activities of inhibiting KIF18A and PARP7, can simultaneously utilize two different antitumor mechanisms of KIF18A protein inhibition and PARP7 inhibition, and has excellent antitumor drug development prospects.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

2. Use of a compound as claimed in claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prophylaxis or treatment of a disease associated with KIF18A or PARP7 activity or expression level.

3. The use according to claim 2, wherein the disease associated with KIF18A or PARP7 activity or expression level is cancer.

4. The use of claim 3, wherein the cancer is selected from the group consisting of: multiple myeloma, B-cell lymphoma, T-cell lymphoma, acute and chronic myeloid leukemia, acute and chronic lymphoid leukemia, monocytic leukemia, splenomegaly, eosinophilia syndrome, fibrosarcoma, salivary gland carcinoma, liver cancer, rectal cancer, bladder cancer, throat cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, stomach cancer.

5. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.