A class of PI3K / HDAC dual inhibitors and their applications

Abstract

The present invention belongs to the field of medicinal chemistry, and specifically relates to a class of PI3K / HDAC dual inhibitors and their applications. The PI3K / HDAC dual inhibitors are compounds represented by general formulas (I)-(IV) and their pharmaceutically acceptable salts, deuterated compounds or optical isomers, which have dual inhibitory effects on PI3K and HDAC, have better therapeutic effects than PI3K inhibitors, and can be used to prepare anti-tumor, anti-inflammatory and autoimmune disease drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to a class of PI3K / HDAC dual inhibitors and their applications. Background Art

[0002] The over-activation of the PI3K / Akt / mTOR signaling pathway is one of the most common carcinogenic factors. The key kinase PI3K of this pathway is divided into three classes according to its structure and function. Among them, the abnormal regulation of class I PI3K is closely related to cancer. Because the four subtypes (PI3Kα, β, γ, δ) of class I PI3K have different functions, to avoid side effects caused by off-target effects, subtype-selective PI3K inhibitors have become the current research and development trend. The PI3Kδ inhibitors Idelalisib, Duvelisib, and Umbralisib have now been approved for the treatment of B-cell lymphoma - due to their ability to selectively inhibit PI3Kδ, or only inhibit PI3Kδ and γ, compared with class I PI3K inhibitors, the off-target effect is significantly reduced.

[0003] In addition to being a therapeutic target for hematological tumors, recent studies have shown that PI3Kδ is abnormally activated in solid tumors such as triple-negative breast cancer (TNBC), colon cancer, and liver cancer. For example, Guney Eskiler et al. found that PI3Kδ is overexpressed in the TNBC cell line MDA-MB-231, and the expression level is closely related to the malignancy and disease progression of the tumor. Targeting PI3Kδ can produce an anti-proliferative effect on tumor cells with overexpression of this enzyme by interfering with intracellular signal transduction pathways. Research has shown that PI3Kδ is a protein necessary for maintaining the functions of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) - the infiltration of these two immunosuppressive cells in the tumor microenvironment can lead to the inactivation of cytotoxic T lymphocytes (CTLs), thereby mediating tumor immune escape. Targeting PI3Kδ can reduce the recruitment of both in the tumor microenvironment, and then activate the anti-tumor immunity mediated by CD8 + CTLs. PI3Kδ inhibitors can provide effective therapies for PI3Kδ-overexpressing solid tumors through a dual mechanism of interfering with internal tumor signal pathways and remodeling the tumor microenvironment to activate anti-tumor immunity.

[0004] However, inhibiting PI3Kδ alone may lead to cross-activation of PI3K-related signal bypass; and there are multiple pathways in the tumor microenvironment that can mediate tumor immune escape. Intervening in PI3Kδ alone is likely to induce drug resistance. HDAC can induce the occurrence and development of tumors by affecting epigenetic processes and is an effective therapeutic target for malignant tumors. Among the multiple subtypes of HDAC, the HDAC6 subtype has a special structure and function. Its overexpression can lead to the activation of PI3K-related signal bypasses such as Ras and EGFR, promote carcinogenic transformation, as well as the growth, proliferation, and angiogenesis of tumor cells, and enhance the invasiveness and metastasis of tumor cells. Recent studies have shown that selective HDAC6 inhibitors inhibit tumor growth by activating the tumor suppressor protein PTEN (the activation of PTEN can block PI3K signal transduction). Therefore, simultaneously targeting PI3Kδ / HDAC6 is expected to obtain a synergistic effect in interfering with intracellular signal transduction in cancer cells and can weaken the drug resistance caused by PI3K-related signal bypasses. Selectively inhibiting HDAC6 can also downregulate the expression of the programmed death-ligand 1 (PD-L1) in tumor cells, thereby exerting an immunotherapeutic effect. Given that existing studies have shown that the downregulation of PD-L1 levels can synergistically activate tumor immunity with the inhibition of the functions of Tregs and MDSCs, single-molecule dual-target inhibition of PI3Kδ / HDAC6 is expected to obtain a synergistic effect both in interfering with intracellular signal pathways in cancer cells and in activating tumor immunity.

[0005] Currently, the clinical trials of the PI3K / HDAC dual-target inhibitor CUDC-907 for the treatment of multiple myeloma and diffuse large B-cell lymphoma are underway. However, due to its lack of selectivity for PI3K and HDAC subtypes and poor safety, there is an urgent need to seek PI3K / HDAC dual inhibitors with subtype selectivity to intervene in the functions of PI3Kδ and HDAC6, obtain the synergistic effect as described above, and address the drug resistance problem.

[0006] Both PI3Kδ and HDAC6 are also potential targets for the treatment of inflammation and autoimmune diseases. Simultaneously targeting PI3Kδ / HDAC6 is expected to provide a new option for the treatment of inflammation and autoimmune diseases. Therefore, it is an urgent problem to be solved to propose a dual inhibitor that can simultaneously target PI3Kδ and HDAC6. Summary of the Invention

[0007] In view of the deficiencies of existing PI3Kδ inhibitors in anti-tumor efficacy and the problem of easy drug resistance, the present invention provides a class of dual inhibitors that can simultaneously target PI3Kδ and HDAC6. The molecular structure of the inhibitors has the structural units required for inhibiting PI3Kδ and HDAC6. Multiple activity tests have confirmed that the compounds in the present invention have both PI3Kδ and HDAC6 inhibitory activities, as well as significant anti-tumor activities. At the same time, some compounds of the present invention have selectivity for inhibiting PI3Kδ and HDAC6. Existing inhibitors that act on both PI3K and HDAC targets, such as the clinical investigational drug CDUC-907, lack subtype selectivity and have poor safety. Therefore, the corresponding compounds in the present invention are beneficial to improving the safety of existing PI3K / HDAC dual-target inhibitors.

[0008] On the one hand, the present invention provides a class of PI3K / HDAC dual inhibitors, and the PI3K / HDAC dual inhibitors are compounds represented by the following general formulas (I)-(IV) and their pharmaceutically acceptable salts, deuterated compounds or optical isomers:

[0009]

[0010] In the general formulas (I)-(IV), R1 is hydrogen, halogen, cyano, C1-6 alkyl or C2-6 unsaturated aliphatic hydrocarbon group, and R1 is substituted at any position of the benzene ring;

[0011] In general formula (II), R2 is methyl, ethyl, trifluoromethyl or cyclopropyl;

[0012] In general formula (IV), R3 is hydrogen, C1-6 alkyl, C3-8 cycloalkyl, C2-6 unsaturated aliphatic hydrocarbon group or C3-8 unsaturated cycloalkyl;

[0013] In the general formulas (I)-(IV), ring A is selected from a monocyclic heteroaryl group or a fused bicyclic heteroaryl group substituted by 1-4 R4s, where R4 is selected from one of hydrogen, halogen, cyano, C1-6 alkyl, NR5R6, OR5, SO2NR5R6, NR5SO2R6, NR5CONR6R7, NR5COOR6, NR5COR6, COOR5, CONR5R6; R5, R6, and R7 are each independently selected from hydrogen, C1-6 alkyl, C3-8 cycloalkyl or C2-6 unsaturated aliphatic hydrocarbon group;

[0014] In the general formulas (I) and (III), ring B is R8 is hydrogen or hydroxyl, and R9 is hydrogen or C1-6 alkyl;

[0015] In the general formulas (I)-(IV), ring C is a C6-14 aryl or a C5-14 heteroaryl. In addition to being substituted by the structural units on both sides shown in the general formulas (I)-(IV), ring C may also be substituted by at least one R 10 wherein R 10 is selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, carbamoyl, trifluoromethyl, trifluoromethoxy, C1-6 alkyl, C1-6 alkoxy, C2-6 unsaturated aliphatic hydrocarbon group, NR 11 R 12 、NR 11 OR 12 、NR 11 NR 12 R 13 、 SO2NR 11 R 12 、NR 11 SO2R 12 、NR 11 CONR 12 R 13 、NR 11 COOR 12 、NR 11 COR 12 、 CONR 11 R 12 ; R 11 、R 12 、R 13 are each independently selected from the group consisting of hydrogen, C1-6 alkyl, C3-8 cycloalkyl, and C2-6 unsaturated aliphatic hydrocarbon group;

[0016] In the general formula (I), X is n1 = 1-10, and R 14 is hydrogen, C1-6 alkyl, or C3-8 cycloalkyl;

[0017] In the general formulas (II) and (III), Y is (CH2)n2, where n2 = 1-10;

[0018] In the general formula (IV), Z is n3 = 1-10, R 15 is C1-6 alkyl, R 16 is hydrogen, C1-6 alkyl, C3-8 cycloalkyl, or n4 = 1-4.

[0019] Furthermore, when ring A is substituted by two or more R4, two or more of the R4 may be the same or different.

[0020] Furthermore, when ring C is substituted by two or more R 10 wherein two or more of the R 10They may be the same or different from each other.

[0021] Further, when the ring C does not exist, the hydroxamic acid group in the general formulas (I)-(IV) is directly connected to X, Y or Z.

[0022] Further, the PI3K / HDAC dual inhibitor is selected from the following compounds and their pharmaceutically acceptable salts, deuterated compounds or optical isomers:

[0023]

[0024]

[0025]

[0026]

[0027]

[0028] Further, the PI3K / HDAC dual inhibitor is selective for PI3Kδ and HDAC6, and is a compound shown in the general formulas (II), (III) or (IV) and their pharmaceutically acceptable salts, deuterated compounds or optical isomers, wherein R1, R2, R3, ring A, ring B, ring C, Y, Z are as defined above.

[0029] The class of PI3K / HDAC dual inhibitors can be used in the preparation of drugs for treating anti-tumor, inflammation and autoimmune diseases, wherein the tumors include solid tumors and hematological tumors.

[0030] On the other hand, the present invention also provides a PI3K / HDAC dual inhibitor composition, which includes a PI3K / HDAC dual inhibitor and at least one pharmaceutical carrier or excipient.

[0031] Further, the PI3K / HDAC dual inhibitor composition can be used in the preparation of drugs for treating anti-tumor, inflammation and autoimmune diseases.

[0032] Further, the PI3K / HDAC dual inhibitor composition further includes at least one other therapeutic agent, and the dosage form of the PI3K / HDAC dual inhibitor composition is any clinically or pharmaceutically acceptable dosage form.

[0033] The PI3K / HDAC dual inhibitor composition can be used in the preparation of drugs for treating anti-tumor, inflammation and autoimmune diseases.

[0034] The dosage of the compound of the present invention is 1 mg - 1000 mg per day, and this range may also be deviated from according to the severity of the condition or the type of dosage form. Unless otherwise defined, all the scientific and technical terms used in the present invention have the same meaning as generally understood by those skilled in the art to which the claimed subject matter pertains.

[0035] Among them, "halogen" refers to fluorine, chlorine, bromine, and iodine;

[0036] "C1-6 alkyl" refers to an alkyl group having 1 to 6 carbon atoms;

[0037] "C2-6 unsaturated aliphatic hydrocarbon group" refers to a straight-chain or branched-chain alkenyl, alkynyl, or enynyl group having 2 to 6 carbon atoms and containing a double bond or a triple bond. Specific examples of the unsaturated aliphatic hydrocarbon group include, but are not limited to, vinyl, 1-propenyl, 2-propenyl, ethynyl, etc.

[0038] "C3-8 cycloalkyl" refers to a cycloalkyl group having 3 to 8 carbon atoms, and specific examples include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, spiro[2.4]heptane, spiro[3.3]octane, spiro[3.4]octane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, etc.;

[0039] "C3-8 unsaturated alicyclic group" refers to an alicyclic group having 3 to 8 carbon atoms and containing a double bond or a triple bond. Specific examples of the unsaturated alicyclic group include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclooctene, cyclopentadiene, cyclohexadiene, cycloheptadiene, cyclooctadiene, benzene, cycloheptatriene, etc.;

[0040] "C6-14 aryl" refers to a fully carbon monocyclic or fused polycyclic group having 6 to 14 carbon atoms and having a completely conjugated π electron system, and specific examples include, but are not limited to, benzene ring, naphthalene ring, anthracene ring;

[0041] "C5-14 heteroaryl" refers to a non-fully carbon monocyclic or fused polycyclic group having 5 to 14 ring atoms and having a completely conjugated π electron system, and specific examples include, but are not limited to, pyridine, imidazole, thiophene, furan, thiazole, purine, indole, azaindole;

[0042] The compound of the present invention or its pharmaceutically acceptable salt or deuterated compound has the same efficacy, and the pharmaceutically acceptable salt herein refers to salts of general formula (I), (II), (III), or (IV), including alkali metal salts, alkaline earth metal salts, other metal salts, inorganic base salts, organic base salts, inorganic acid salts, organic acid salts, lower alkanesulfonates, arylsulfonates, and amino acid salts.

[0043] The "pharmaceutical carrier" refers to a conventional pharmaceutical carrier in the pharmaceutical field, including conventional diluents, excipients (such as water, etc.), fillers (such as starch, etc.), binders (such as cellulose derivatives, gelatin, etc.), wetting agents (such as glycerol, etc.), disintegrants (such as agar, calcium carbonate, etc.), absorption promoters (such as quaternary ammonium compounds, etc.), surfactants (such as cetyl alcohol, etc.), adsorption carriers (such as kaolin and saponite, etc.), lubricants (such as talc powder, etc.). When necessary, flavoring agents, sweetening agents, etc. can also be added.

[0044] The "other therapeutic agents" refer to therapeutic agents that can be formulated with the PI3K / HDAC dual inhibitor, including but not limited to mitotic inhibitors (such as vinblastine, vindesine), tubulin depolymerizing inhibitors (such as paclitaxel), biologic alkylating agents (such as cyclophosphamide), antimetabolites (such as 5-fluorouracil, tegafur, methotrexate), antitumor antibiotics (such as doxorubicin, mitomycin), enzymes (such as asparaginase), topoisomerase inhibitors (such as etoposide and camptothecin), biologic response modifiers (such as interferon), proteasome inhibitors (such as bortezomib).

[0045] The "any pharmaceutically acceptable dosage form" is suitable for administration by any appropriate route, such as oral (including buccal or sublingual administration), rectal administration, nasal administration, topical administration (including buccal, sublingual, transdermal or inhalation administration), vaginal administration or parenteral administration (including subcutaneous injection, intramuscular injection, intravenous injection or intradermal injection) routes. These preparations can be prepared by any method known in the pharmaceutical field. For example, by mixing the active ingredient with a carrier or excipient.

[0046] The "solid tumor or hematological tumor" includes but not limited to breast cancer, sarcoma, lung cancer, prostate cancer, colon cancer, rectal cancer, kidney cancer, pancreatic cancer, neuroblastoma, glioma, head cancer, cervical cancer, thyroid cancer, liver cancer, ovarian cancer, uterine cancer, endometrial cancer, gastric cancer, bladder cancer, gastrointestinal stromal tumor, nasopharyngeal cancer, leukemia, lymphoma, multiple myeloma.

[0047] Advantages of the present invention:

[0048] The PI3K / HDAC dual inhibitors proposed by the present invention have been confirmed through multiple experiments to possess dual inhibitory activities against PI3Kδ and HDAC6. Most of these compounds can strongly inhibit PI3Kδ, HDAC1, or / and HDAC6. Some compounds, while strongly inhibiting PI3Kδ, HDAC1, or / and HDAC6, exhibit significant anti-tumor cell proliferation activity. Additionally, some compounds, while strongly inhibiting PI3Kδ and HDAC6, demonstrate excellent subtype selectivity for PI3Kδ and HDAC6. Pharmacodynamic experiments indicate that the compounds involved in the present invention have the prospect of being developed into novel anti-tumor, anti-inflammatory, and autoimmune disease drugs.

[0049] Other features and advantages of the present invention will be described in the following specification, and will, in part, be obvious from the specification, or can be learned by practicing the present invention. The objectives and other advantages of the present invention can be achieved and obtained by the structures pointed out in the specification, claims, and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0051] Figure 1 Shows the results of protein immunoblot analysis of the effects of compound 28 in Example 22 of the present invention on the expression levels of STAT3 and P-STAT3 (Y705);

[0052] Figure 2 Shows the results of protein immunoblot analysis of the effects of compound 28 in Example 22 of the present invention on the expression levels of AKT, P-AKT (S473), and PD-L1;

[0053] Figure 3 Shows a quantitative analysis bar graph of the regulatory effects of compound 28 in Example 22 of the present invention on different proteins in T47D cells; wherein, A is the quantitative analysis bar graph of the regulatory effect of compound 28 on P-STAT3 (Y705) in T47D cells, B is the quantitative analysis bar graph of the regulatory effect of compound 28 on P-AKT (S473) in T47D cells, and C is the quantitative analysis bar graph of the regulatory effect of compound 28 on PD-L1 in T47D cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0055] The PI3K / HDAC dual inhibitor in the present invention is prepared through four routes. Taking compound 3, compound 17, compound 28, and compound 36 as examples respectively, the following describes the four synthetic routes:

[0056] Route 1:

[0057]

[0058] In Route 1, the reactants and reaction conditions involved are as follows:

[0059] The reactant a is ammonium chloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), N,N-diisopropylethylamine (DIPEA), dimethyl sulfoxide (DMSO); the reaction condition is room temperature;

[0060] The reactant b is (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), DIPEA, N,N-dimethylformamide (DMF); the reaction condition is nitrogen protection and 90°C;

[0061] The reactant c is sodium hydroxide (NaOH), absolute ethanol (EtOH), 80°C;

[0062] The reactant d is methyl 4-(aminomethyl)benzoate hydrochloride, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBop), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), absolute acetonitrile; the reaction condition is 25°C;

[0063] The reactant e is trifluoroacetic acid (TFA), dichloromethane (DCM), 0°C - room temperature;

[0064] The reactant f is 2,4-diamino-6-chloropyrimidine-5-carbonitrile, anhydrous potassium fluoride (KF), DIPEA; the reaction condition is nitrogen protection and 90°C;

[0065] The reactant g is hydroxylamine (NH2OH), NaOH, tetrahydrofuran (THF), methanol (MeOH); reaction conditions: 0 °C - room temperature.

[0066] Route 2:

[0067]

[0068] In Route 2, the reactants and reaction conditions involved are:

[0069] The reactant a is methyl 4-(aminomethyl)benzoate hydrochloride, EDCI, HOBT, triethylamine (TEA), DCM; reaction conditions: room temperature;

[0070] The reactant b is ① thionyl chloride (SOCl2), anhydrous DMF,; reaction conditions: under nitrogen protection, 0 °C - 80 °C; ② (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid, TEA, anhydrous DCM, under nitrogen protection; reaction conditions: 0 °C - room temperature;

[0071] The reactant c is zinc powder, glacial acetic acid; reaction conditions: under nitrogen protection, 50 °C;

[0072] The reactant d is TFA, DCM; reaction conditions: 0 °C - room temperature;

[0073] The reactant e is 6-chloro-9H-purine, DIPEA, tert-butanol (t-BuOH); reaction conditions: under nitrogen protection, 80 °C;

[0074] The reactant f is NH2OH, NaOH, THF, MeOH; reaction conditions: 0 °C - room temperature.

[0075] Route 3:

[0076]

[0077] In Route 3, the reactants and reaction conditions involved are:

[0078] The reactant a is methyl 5-(bromomethyl)thiophene-2-carboxylate, potassium carbonate (K2CO3), DMF; reaction conditions: 50 °C;

[0079] The reactant b is TFA, DCM; reaction conditions: 0 °C - room temperature;

[0080] The reactant c is 2,4-diamino-6-chloropyrimidine-5-carbonitrile, anhydrous KF, DIPEA; reaction conditions: under nitrogen protection, 90 °C;

[0081] The reactant d is NH2OH, NaOH, THF, MeOH; reaction conditions: 0 °C - room temperature.

[0082] Route 4:

[0083]

[0084] In Route 4, the reactants and reaction conditions involved are as follows:

[0085] The reactant a is tert-butyldimethylchlorosilane (TBSCl), imidazole, DMF; reaction conditions: under nitrogen protection, room temperature;

[0086] The reactant b is ammonium chloride, EDCI, HOBT, DIPEA, DMSO; reaction conditions: room temperature;

[0087] The reactant c is (2S,4R)-1-(tert-butoxycarbonyl)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-2-carboxylic acid (Intermediate 62), HATU, DIPEA, DMF; reaction conditions: under nitrogen protection, 90 °C;

[0088] The reactant d is NaOH, EtOH; reaction conditions: 80 °C;

[0089] The reactant e is methyl iodide, K2CO3, DMF; reaction conditions: 50 °C;

[0090] The reactant f is tetrabutylammonium fluoride (TBAF), THF; reaction conditions: under nitrogen protection, room temperature;

[0091] The reactant g is ① N,N'-carbonyldiimidazole (CDI), TEA, toluene; reaction conditions: under nitrogen protection, 55 °C; ② methyl 4-(aminomethyl)benzoate hydrochloride; reaction conditions: TEA, DMF, room temperature;

[0092] The reactant h is TFA, DCM; reaction conditions: 0 °C - room temperature;

[0093] The reactant i is 2,4-diamino-6-chloropyrimidine-5-carbonitrile, anhydrous KF, DIPEA; reaction conditions: under nitrogen protection, 90 °C;

[0094] The reactant j is NH2OH, NaOH, THF, MeOH; reaction conditions: 0 °C - room temperature.

[0095] The preparation routes of other compounds are similar to the above. Among them, Compounds 1, 2, and 4 are prepared according to Route 1; Compounds 5-9, 12-16, 18, and 19 are prepared according to Route 2; Compounds 10, 11, 20-27, 29-31 are prepared according to Route 3; Compounds 32-35, 37-44 are prepared according to Route 4.

[0096] Example 1

[0097] (S)-4-((5-Chloro-2-(5-(2,6-diamino-5-cyanopyrimidin-4-yl)-5-azaspiro[2.4]heptan-6-yl)quinazolin-4-yl)amino)methyl)-N-hydroxybenzamide (Compound 3) Synthesis

[0098] (1) Synthesis of 2-Amino-6-chlorobenzamide (Intermediate 46):

[0099] Add 2-Amino-6-chlorobenzoic acid (30.0 g, 175 mmol, 1.0 eq), EDCI (50.1 g, 262 mmol, 1.5 eq), HOBT (35.4 g, 262 mmol, 1.5 eq) and DMSO (100 mL) into a reaction flask, stir at room temperature for 1 h, then add DIPEA (61.0 mL, 1.05 mol, 4.0 eq), ammonium chloride (30.4 g, 0.569 mol, 3.25 eq), and continue to stir at room temperature for 12 h. After the reaction is completed, add water to quench, wash successively with water and saturated NaCl solution, extract with ethyl acetate (EA), combine the organic phases, dry with anhydrous Na2SO4, concentrate under reduced pressure, and obtain 25.8 g of a white foam-like solid by silica gel column chromatography, which is Intermediate 46.

[0100] (2) Synthesis of tert-Butyl (S)-6-((2-carbamoyl-3-chlorophenyl)carbamoyl)-5-azaspiro[2.4]heptane-5-carboxylate (Intermediate 47):

[0101] Add Intermediate 46 (25.0 g, 147 mmol, 1.0 eq), (S)-5-(tert-Butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid (40.0 g, 220 mmol, 1.5 eq), HATU (111 g, 293 mmol, 2.0 eq), DIPEA (76.0 mL, 494 mmol, 3.0 eq) into a reaction flask, then add DMF (100 mL) to dissolve, protect with nitrogen, and react at 90 °C for 10 h. After the reaction is completed, add water to quench, wash successively with water and saturated NaCl solution, extract with EA, combine the organic phases, dry with anhydrous Na2SO4, concentrate under reduced pressure, and obtain 48.8 g of a white oil by silica gel column chromatography, which is Intermediate 47.

[0102] (3) Synthesis of tert-Butyl (S)-6-(5-Chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate (Intermediate 48):

[0103] Intermediate 47 (48.0 g, 122 mmol, 1.0 eq) was added to a reaction flask, dissolved in EtOH (40.0 mL), and then 10 M NaOH solution (5.00 mL, 488 mmol, 4.0 eq) was added. The reaction was carried out at 80 °C for 5 h. After the reaction was completed, the pH of the system was adjusted to neutral with glacial acetic acid, washed successively with water and saturated NaCl solution, extracted with EA, the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain 42.8 g of a crude product as a white foamy solid, namely Intermediate 48.

[0104] (4) Synthesis of tert-butyl (S)-6-(5-chloro-4-((4-(methoxycarbonyl)benzyl)amino)quinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate (Intermediate 49):

[0105] Intermediate 48 (1.00 g, 2.68 mmol, 1.0 eq), methyl 4-(aminomethyl)benzoate hydrochloride (804 mg, 4.00 mmol, 1.5 eq), PyBop (2.29 g, 4.40 mmol, 1.6 eq), and DBU (1.20 mL, 8.00 mmol, 3.0 eq) were added to a reaction flask, and then dissolved in anhydrous acetonitrile (20.0 mL). The reaction was carried out at 25 °C for 4 h. After the reaction was completed, the acetonitrile was removed by rotary evaporation, washed successively with water and saturated NaCl solution, extracted with DCM, the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 907 mg of a light yellow foamy solid, namely Intermediate 49.

[0106] (5) Synthesis of methyl (S)-4-(((5-chloro-2-(5-azaspiro[2.4]heptan-6-yl)quinazolin-4-yl)amino)methyl)benzoate (Intermediate 50):

[0107] Intermediate 49 (600 mg, 1.15 mmol, 1.0 eq) was added to a reaction flask and dissolved in DCM (6.00 mL). TFA (1.50 mL) was added at 0 °C in an ice bath, and then the system was transferred to room temperature for reaction for 3 h. After the reaction was completed, it was neutralized with NaHCO3, and then washed successively with water and saturated NaCl solution, extracted with DCM, the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 460 mg of a light yellow foamy solid, namely Intermediate 50.

[0108] (6) Synthesis of methyl (S)-4-((5-chloro-2-(5-(2,6-diamino-5-cyanopyrimidin-4-yl)-5-azaspiro[2.4]heptan-6-yl)quinazolin-4-yl)amino)methyl)benzoate (Intermediate 51):

[0109] Add intermediate 50 (400 mg, 0.948 mmol, 1.0 eq), 2,4-diamino-6-chloropyrimidine-5-carbonitrile (161 mg, 0.948 mmol, 1.0 eq), anhydrous KF (113 mg, 1.90 mmol, 2.0 eq), DIPEA (890 μL, 5.22 mmol, 5.5 eq) and DMSO (5.00 mL) into a reaction flask. Under nitrogen protection, react at 90 °C for 12 h. After the reaction is completed, add an appropriate amount of water. A solid precipitates out. Filter by suction, wash the filter cake with water, dry it. The crude product is purified by silica gel column chromatography to obtain 503 mg of a white solid, which is intermediate 51.

[0110] (7) Synthesis of (S)-4-((5-chloro-2-(5-(2,6-diamino-5-cyanopyrimidin-4-yl)-5-azaspiro[2.4]heptan-6-yl)quinazolin-4-yl)amino)methyl)-N-hydroxybenzamide (Compound 3):

[0111] Add NH2OH (2.50 mL) and NaOH (144 mg, 3.60 mmol, 8.0 eq) into a reaction flask. React at 0 °C in an ice bath for 0.5 h. Then add a solution of intermediate 51 (250 mg, 0.450 mmol, 1.0 eq) in THF—MeOH (V:V = 1:1) (5.00 mL). Transfer the reaction system to room temperature and react for 4 h. After the reaction is completed, adjust the pH of the system to neutral with glacial acetic acid. Rotate to remove THF and CH3OH. Add an appropriate amount of water. Filter by suction, wash the filter cake with a small amount of water, dry it. The crude product is purified by silica gel column chromatography to obtain 220 mg of a light white solid.

[0112] Perform nuclear magnetic resonance and electrospray mass spectrometry (ESI-MS) detection on the light white solid. The nuclear magnetic resonance results are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 8.96 (s, 1H), 8.58 (t, J = 5.2 Hz, 1H), 7.66 (t, J = 8.4 Hz, 3H), 7.59 (d, J = 7.2 Hz, 1H), 7.52 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 6.35 (s, 2H), 6.10 (s, 2H), 5.52 (d, J = 6.4 Hz, 1H), 4.73 (d, J = 5.9 Hz, 2H), 3.74 (d, J = 10.0 Hz, 1H), 3.56 (s, 1H), 2.33 (s, 1H), 1.67 (s, 1H), 0.51~0.37 (m, 2H), 0.08~0.11 (m, 2H); ESI-MS: m / z = 557 [M + H] + 。

[0113] Example 2

[0114] Synthesis of (S)-4-((5-chloro-2-(1-(2,6-diamino-5-cyanopyrimidin-4-yl)pyrrolidin-2-yl)quinazolin-4-yl)amino)-N-hydroxybenzamide (Compound 2).

[0115] Compound 2 was prepared with reference to Example 1. Only in step (2), (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid was replaced by (tert-butoxycarbonyl)-L-proline, and then the same method was used for hydroxylamine cleavage to obtain an off-white solid.

[0116] The off-white solid was subjected to nuclear magnetic resonance and ESI-MS detection. The nuclear magnetic resonance results were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.10(s,1H),8.95(s,1H),8.55(t,J=5.2Hz,1H),7.67(d,J=7.6Hz,3H),7.62(t,J=11.2Hz,1H),7.52(d,J=7.2Hz,1H),7.38(d, J=7.6Hz,2H),6.33(s,2H),6.03(s,2H),5.40~5.33(m,1H),4.82~4.69(m, 2H),3.82(s,1H),3.73(s,1H),2.22~2.13(m,1H),2.03~1.95(m,1H),1.81~1.73(m,1H),1.62~1.52(m,1H); ESI-MS:m / z=531[M+H] + 。

[0117] Example 3

[0118] Synthesis of (S)-4-(2-((5-chloro-2-(5-(2,6-diamino-5-cyanopyrimidin-4-yl)-5-azaspiro[2.4]heptan-6-yl)quinazolin-4-yl)amino)-N-hydroxybenzamide (Compound 4).

[0119] Compound 4 was prepared with reference to Example 1. Only in step (4), methyl 4-(aminomethyl)benzoate hydrochloride was replaced by methyl 4-(aminoethyl)benzoate hydrochloride, and then the same method was used for hydroxylamine cleavage to obtain an off-white solid.

[0120] The off-white solid was subjected to nuclear magnetic resonance and ESI-MS detection. The nuclear magnetic resonance results were as follows: 11H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 8.97 (s, 1H), 7.98 (t, J = 5.2 Hz, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.65 - 7.57 (m, 2H), 7.46 (dd, J = 7.2, 1.6 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 6.35 (s, 2H), 6.11 (s, 2H), 5.59 (s, 1H), 3.92 (d, J = 10.8 Hz, 1H), 3.78 (s, 2H), 3.69 (td, J = 13.0, 7.2 Hz, 1H), 2.90 - 2.80 (m, 2H), 2.53 (s, 1H), 1.92 (d, J = 6.8 Hz, 1H), 0.67 - 0.52 (m, 2H), 0.48 - 0.37 (m, 1H), 0.27 - 0.18 (m, 1H); ESI-MS: m / z = 571 [M + H] + 。

[0121] Example 4

[0122] Synthesis of (S)-4-((2-(5-(9H-Purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-5-fluoro-4-oxoquinazolin-3(4H)-yl)methyl)-N-hydroxybenzamide (Compound 17).

[0123] (1) Synthesis of Methyl 4-((2-fluoro-6-nitrobenzamido)methyl)benzoate (Intermediate 53):

[0124] 2-Fluoro-6-nitrobenzoic acid (10.0 g, 54.0 mmol, 1.0 eq), EDCI (12.4 g, 64.8 mmol, 1.2 eq), HOBT (8.80 g, 64.8 mmol, 1.2 eq) and DCM (80.0 mL) were successively added to a reaction flask and stirred at room temperature for 1 h. Then TEA (22.5 mL, 162.1 mmol, 3.0 eq) and methyl 4-(aminomethyl)benzoate hydrochloride (19.2 g, 59.4 mmol, 1.1 eq) were added, and stirring was continued at room temperature. After TLC showed that the reaction was complete, the mixture was washed successively with saturated NaHCO3 solution, water, and saturated NaCl solution, extracted with DCM, the organic phases were combined, dried over anhydrous Na2SO4, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 16.8 g of a white solid, which is Intermediate 53.

[0125] (2) Synthesis of tert-Butyl (S)-6-((2-fluoro-6-nitrobenzoyl)(4-(methoxycarbonyl)benzyl)carbamoyl)-5-azaspiro[2.4]heptane-5-carboxylate (Intermediate 54):

[0126] In reaction flask ①, add intermediate 53 (1.00 g, 3.01 mmol, 1.0 eq), SOCl2 (15.0 mL) and anhydrous DMF (2 drops). After cooling thoroughly in an ice bath at 0 °C and protecting with nitrogen sufficiently, place it in an oil bath at 80 °C and react for 6 h. After the reaction is completed, quickly rotary evaporate SOCl2 and store it sealed for later use;

[0127] In reaction flask ②, add (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid (800 mg, 3.31 mmol, 1.1 eq), TEA (1.30 mL, 9.03 mmol, 3.0 eq) and anhydrous DCM (8.00 mL). After cooling thoroughly in an ice bath at 0 °C, replace the system with nitrogen sufficiently and react at 0 °C in the ice bath for 2 h;

[0128] Dissolve the crude product after the post-treatment of ① with anhydrous DCM (6.00 mL) and transfer it to reaction flask ②, then transfer it to room temperature and react for 10 h. After the reaction is completed, wash it successively with saturated NaHCO3 solution, water, saturated NaCl solution, extract with DCM, combine the organic phases, dry with anhydrous Na2SO4, concentrate under reduced pressure, and obtain 710 mg of off-white solid by silica gel column chromatography, namely intermediate 54.

[0129] (3) Synthesis of tert-butyl (S)-6-(5-fluoro-3-(4-(methoxycarbonyl)benzyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate (intermediate 55):

[0130] In the reaction flask, add intermediate 54 (700 mg, 1.26 mmol, 1.0 eq), zinc powder (1.60 g, 25.2 mmol, 20.0 eq) and glacial acetic acid (8.00 mL). After cooling thoroughly in an ice bath at 0 °C and protecting the system with nitrogen, place it in an oil bath at 50 °C and react for 6 h. After the reaction is completed, filter by suction, wash the filter cake with an appropriate amount of DCM, then neutralize it with NaHCO3 solution, wash it successively with water and saturated NaCl solution, extract with DCM, combine the organic phases, dry with anhydrous Na2SO4, concentrate under reduced pressure, and obtain 610 mg of white foamy solid by silica gel column chromatography, namely intermediate 55.

[0131] (4) Synthesis of methyl (S)-4-((5-fluoro-4-oxo-2-(5-azaspiro[2.4]heptan-6-yl)quinazolin-3(4H)-yl)methyl)benzoate (intermediate 56):

[0132] Add intermediate 55 (600 mg, 1.18 mmol, 1.0 eq) to a reaction flask, dissolve it in DCM (6.00 mL), add TFA (1.5 mL) under an ice bath at 0 °C, and then transfer the system to room temperature for reaction for 3 h. After the reaction is completed, neutralize it with NaHCO3, wash it successively with water and saturated NaCl solution, extract it with DCM, combine the organic phases, dry it over anhydrous Na2SO4, concentrate it under reduced pressure, and obtain 480 mg of a light yellow foamy solid by silica gel column chromatography, which is intermediate 56.

[0133] (5) Synthesis of (S)-4-((2-(5-(9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-5-fluoro-4-oxoquinazolin-3(4H)-yl)methyl)benzoate (intermediate 57):

[0134] Add intermediate 56 (150 mg, 0.368 mmol, 1.0 eq), 6-chloro-9H-purine (68.0 mg, 0.442 mmol, 1.2 eq), DIPEA (195 μL, 1.10 mmol, 3.0 eq) and tert-butanol (2.00 mL) to a reaction flask, protect it with nitrogen, and react at 80 °C for 12 h. After the reaction is completed, spin off the solvent, dilute it with water, extract it with EA, combine the organic phases, dry it over anhydrous Na2SO4, concentrate it under reduced pressure, and obtain 182 mg of a light white solid by silica gel column chromatography, which is intermediate 57.

[0135] (6) Synthesis of (S)-4-((2-(5-(9H-purin-6-yl)-5-azaspiro[2.4]heptan-6-yl)-5-fluoro-4-oxoquinazolin-3(4H)-yl)methyl)-N-hydroxybenzamide (compound 17):

[0136] Compound 17 refers to step (7) of Example 1, only replacing intermediate 51 with intermediate 57 to obtain an off-white solid.

[0137] Perform nuclear magnetic resonance and ESI-MS detection on the off-white solid. The nuclear magnetic resonance results are as follows: 11H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 11.21 (s, 1H), 9.01 (s, 1H), 8.27 - 8.00 (m, 2H), 7.81 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.68 (dd, J = 13.6, 6.0 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.28 - 7.17 (m, 2H), 5.66 - 5.57 (m, 1H), 5.52 (dd, J = 7.6, 3.6 Hz, 1H), 5.47 - 5.37 (m, 1H), 4.37 (d, J = 10.8 Hz, 1H), 4.23 (d, J = 10.8 Hz, 1H), 2.37 - 2.25 (m, 1H), 2.00 - 1.90 (m, 1H), 0.76 - 0.62 (m, 2H), 0.58 - 0.49 (m, 2H); ESI-MS: m / z = 527 [M+H] + 。

[0138] Example 5

[0139] Synthesis of (S)-4-((2-(1-((9H-purin-6-yl)amino)propyl)-5-fluoro-4-oxoquinazolin-3(4H)-yl)methyl)-N-hydroxybenzamide (Compound 6):

[0140] Compound 6 was prepared with reference to Example 4, except that in step (2), (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid was replaced with 2-((tert-butoxycarbonyl)amino)butyric acid, and then the same method was used for hydroxylamine cleavage to obtain an off-white solid.

[0141] The off-white solid was subjected to nuclear magnetic resonance and ESI-MS detection. The nuclear magnetic resonance results were as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 12.98 (s, 1H), 11.19 (s, 1H), 8.99 (s, 1H), 8.32 - 8.02 (m, 2H), 7.93 - 7.75 (m, 2H), 7.71 (d, J = 6.8 Hz, 2H), 7.46 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 6.3 Hz, 2H), 7.29 (s, 1H), 5.66 - 5.46 (m, 2H), 5.32 (s, 1H), 1.97 - 1.72 (m, 2H), 0.83 - 0.71 (s, 3H); ESI-MS: m / z = 489 [M+H] + 。

[0142] Example 6

[0143] Synthesis of (S)-5-((5-chloro-2-(1-((2,6-diamino-5-cyanopyrimidin-4-yl)amino)propyl)-4-oxoquinazolin-3(4H)-yl)methyl)-N-hydroxythiophene-2-carboxamide (Compound 8).

[0144] Compound 8 was prepared with reference to Example 4, except that 2-fluoro-6-nitrobenzoic acid was replaced with 2-chloro-6-nitrobenzoic acid and methyl 4-(aminomethyl)benzoate hydrochloride was replaced with methyl 5-(aminomethyl)thiophene-2-carboxylate hydrochloride in step (1), and (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid was replaced with 2-((tert-butoxycarbonyl)amino)butyric acid in step (2). In addition, in step (5), the corresponding intermediate was prepared by the method described in step (6) of Example 1, and then the same method was used for hydroxylamine cleavage to obtain an off-white solid.

[0145] The off-white solid was subjected to NMR and ESI-MS detection. The NMR results were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.19(s,1H),9.09(s,1H),7.75(t,J=7.6Hz,1H), 7.58(d,J=8.0Hz,2H),7.47(s,1H),7.32(d,J=7.6Hz,1H),6.90(d,J=7.6Hz,1H),6.64(s,2H),6.46(s,2H),5.65(d,J=16.4Hz,1H),5.52(d,J=16.0Hz,1H), 5.31(dd,J=13.2,7.6Hz,1H),1.87~1.75(m,1H),1.70~1.60(m,1H),0.76(t,J=7.2Hz,3H); ESI-MS:m / z=526[M+H] + .

[0146] Example 7

[0147] Synthesis of (S)-5-((5-chloro-2-(5-(2,6-diamino-5-cyanopyrimidin-4-yl)-5-azaspiro[2.4]heptan-6-yl)-4-oxoquinazolin-3(4H)-yl)methyl)-N-hydroxythiophene-2-carboxamide (Compound 28).

[0148] (1) Synthesis of tert-butyl (S)-6-(5-chloro-3-((5-(methoxycarbonyl)thiophen-2-yl)methyl)-4-oxo-3,4-dihydroquinazolin-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate (Intermediate 58):

[0149] In a reaction flask, add intermediate 48 (1.00 g, 2.67 mmol, 1.0 eq), methyl 5-(bromomethyl)thiophene-2-carboxylate (686 mg, 2.93 mmol, 1.1 eq), K2CO3 (440 mg, 3.20 mmol, 1.2 eq), and then add DMF (20.0 mL) to dissolve. React at 50 °C for 5 h. After the reaction is completed, add water to quench, wash successively with water and saturated NaCl solution, extract with EA, combine the organic phases, dry over anhydrous Na2SO4, concentrate under reduced pressure, and obtain 550 mg of a white solid by silica gel column chromatography, which is intermediate 58.

[0150] (2) Synthesis of methyl (S)-5-((5-chloro-4-oxo-2-(5-azaspiro[2.4]heptan-6-yl)quinazolin-3(4H)-yl)methyl)thiophene-2-carboxylate (intermediate 59):

[0151] Intermediate 59 was prepared according to the procedure of step (5) of Reference Example 1, except that intermediate 49 was replaced with intermediate 58, to obtain 445 mg of an off-white foamy solid, which is intermediate 59.

[0152] (3) Synthesis of methyl (S)-5-((5-chloro-2-(5-(2,6-diamino-5-cyanopyrimidin-4-yl)-5-azaspiro[2.4]heptan-6-yl)-4-oxoquinazolin-3(4H)-yl)methyl)thiophene-2-carboxylate (intermediate 60):

[0153] Intermediate 60 was prepared according to the procedure of step (6) of Reference Example 1, except that intermediate 50 was replaced with intermediate 59, to obtain 500 mg of a white solid, which is intermediate 60.

[0154] (4) Synthesis of (S)-5-((5-chloro-2-(5-(2,6-diamino-5-cyanopyrimidin-4-yl)-5-azaspiro[2.4]heptan-6-yl)-4-oxoquinazolin-3(4H)-yl)methyl)-N-hydroxythiophene-2-carboxamide (Compound 28):

[0155] Compound 28 was prepared according to the procedure of step (7) of Example 1, except that intermediate 51 was replaced with intermediate 60, to obtain an off-white solid.

[0156] The off-white solid was subjected to nuclear magnetic resonance and ESI-MS detection. The nuclear magnetic resonance results were as follows: 11H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 9.10 (s, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.51 (t, J = 8.0 Hz, 2H), 7.46 (s, 1H), 7.29 (d, J = 3.6 Hz, 1H), 6.57 (s, 2H), 6.15 (s, 2H), 5.75 (s, 1H), 5.61 (d, J = 16.8 Hz, 2H), 4.00 (s, 1H), 3.82 (d, J = 9.6 Hz, 1H), 1.80 (s, 1H), 1.67 (s, 1H), 0.65 (t, J = 11.2 Hz, 2H), 0.50 - 0.37 (m, 2H); ESI-MS: m / z = 564 [M + H] + 。

[0157] Example 8

[0158] Synthesis of (S)-5-((5-chloro-2-(1-(2,6-diamino-5-cyanopyrimidin-4-yl)pyrrolidin-2-yl)-4-oxoquinazolin-3(4H)-yl)methyl)-N-hydroxy-thiophene-2-carboxamide (Compound 11).

[0159] (S)-tert-Butyl 2-(5-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidine-1-carboxylate was prepared according to Intermediate 48 of Reference Example 1, except that in step (2), (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid was replaced with (tert-butoxycarbonyl)-L-proline.

[0160] Compound 11 was prepared according to Reference Example 7, except that in step (1), Intermediate 48 was replaced with (S)-tert-Butyl 2-(5-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidine-1-carboxylate, and then a similar method was used for hydroxylamine cleavage to obtain an off-white solid.

[0161] The off-white solid was detected by nuclear magnetic resonance and ESI-MS. The nuclear magnetic resonance results were as follows: 11H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 9.12 (s, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.50 (dd, J = 18.0, 8.0 Hz, 3H), 7.32 (d, J = 3.4 Hz, 1H), 6.53 (s, 2H), 6.09 (s, 2H), 5.79 (s, 1H), 5.59 (d, J = 16.0 Hz, 1H), 5.47 (s, 1H), 4.02 (s, 1H), 3.92 (s, 1H), 2.25 - 2.11 (m, 1H), 1.97 - 1.86 (m, 2H), 1.64 - 1.52 (m, 1H); ESI-MS: m / z = 538 [M+H] + 。

[0162] Example 9

[0163] Synthesis of (S)-4-((5-chloro-2-(5-((2,6-diamino-5-cyanopyrimidin-4-yl)-5-azaspiro[2.4]heptan-6-yl)-4-oxoquinazolin-3(4H)-yl)methyl)-N-hydroxybenzamide (Compound 25).

[0164] Compound 25 was prepared with reference to Example 7, except that in step (1), the latter methyl 5-(bromomethyl)thiophene-2-carboxylate was replaced with methyl 4-(chloromethyl)benzoate, and a similar method was used for hydroxylamine cleavage to obtain an off-white solid.

[0165] The off-white solid was detected by nuclear magnetic resonance and ESI-MS. The nuclear magnetic resonance results were as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 11.21 (s, 1H), 9.01 (s, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.68 (t, J = 8.4 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 6.57 (s, 2H), 6.08 (s, 2H), 5.78 (d, J = 15.2 Hz, 1H), 5.43 (d, J = 16.8 Hz, 1H), 5.32 (s, 1H), 3.94 (s, 1H), 3.76 (d, J = 9.6 Hz, 1H), 1.63 (s, 1H), 1.47 (s, 1H), 0.70 - 0.54 (m, 2H), 0.48 - 0.38 (m, 1H), 0.31 - 0.23 (m, 1H); ESI-MS: m / z = 558 [M+H] + 。

[0166] Example 10

[0167] (S)-5-((5-Chloro-2-(5-(2,6-diamino-5-cyanopyrimidin-4-yl)-5-azaspiro[2.4]heptan-6-yl)-4-oxoquinazolin-3(4H)-yl)methyl)-N-hydroxyisoxazole-3-carboxamide (Compound 30) Synthesis.

[0168] Compound 30 was prepared with reference to Example 7. Only in step (1), the latter methyl 5-(bromomethyl)thiophene-2-carboxylate was replaced with methyl 5-(bromomethyl)isoxazole-3-carboxylate. Using the same method, it was prepared by hydroxylamine hydrolysis to obtain an off-white solid.

[0169] The off-white solid was detected by nuclear magnetic resonance and ESI-MS. The nuclear magnetic resonance results were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.92 (s, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.53 (d, J = 2.8 Hz, 1H), 7.51 (d, J = 3.2 Hz, 1H), 6.87 (s, 1H), 6.56 (s, 2H), 6.16 (s, 2H), 5.69 (s, 2H), 5.52 (s, 1H), 3.97 (s, 1H), 3.87 (d, J = 9.2 Hz, 1H), 2.04~1.94 (m, 1H), 1.83 (s, 1H), 0.72~0.62 (m, 2H), 0.55~0.46 (m, 2H); ESI-MS: m / z = 549 [M+H] + 。

[0170] Example 11

[0171] (3R,5S)-5-(5-Chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-(2,6-diamino-5-cyanopyrimidin-4-yl)pyrrolidin-3-yl (4-(hydroxyamino)phenyl)carboxylate (Compound 36) Synthesis.

[0172] (1) Synthesis of (2S,4R)-1-(tert-butoxycarbonyl)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-2-carboxylic acid (Intermediate 62):

[0173] (2S,4R)-1-(tert-Butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (20.0 g, 86.5 mmol, 1.0 eq), TBSCl (31.3 g, 208 mmol, 2.4 eq), and imidazole (29.4 g, 432 mmol, 5.0 eq) were added to a reaction flask, followed by the addition of DMF (70.0 mL) to dissolve. Under nitrogen protection, the reaction was carried out at room temperature for 18 h. After the reaction was completed, it was washed successively with saturated NH4Cl solution, water, and saturated NaCl solution, extracted with EA, the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain 15.2 g of a crude white solid, which was Intermediate 62.

[0174] (2) Synthesis of tert-butyl (2S,4R)-4-((tert-butyldimethylsilyl)oxy)-2-((2-carbamoyl-3-chlorophenyl)carbamoyl)pyrrolidine-1-carboxylate (Intermediate 63):

[0175] Intermediate 63 was prepared with reference to (2) in Example 1, only replacing (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid with Intermediate 62 to obtain Intermediate 63.

[0176] (3) Synthesis of tert-butyl (2S,4R)-4-((tert-butyldimethylsilyl)oxy)-2-(5-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidine-1-carboxylate (Intermediate 64):

[0177] Intermediate 64 was prepared with reference to (3) in Example 1, only replacing Intermediate 47 with Intermediate 63 to obtain Intermediate 64.

[0178] (4) Synthesis of tert-butyl (2S,4R)-4-((tert-butyldimethylsilyl)oxy)-2-(5-chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidine-1-carboxylate (Intermediate 65):

[0179] Intermediate 65 was prepared with reference to (1) in Example 7, only replacing Intermediate 48 with Intermediate 64 and replacing methyl 5-(bromomethyl)thiophene-2-carboxylate with methyl iodide to obtain Intermediate 65.

[0180] (5) Synthesis of tert-butyl (2S,4R)-2-(5-chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)-4-hydroxypyrrolidine-1-carboxylate (Intermediate 66):

[0181] Add intermediate 65 (1.00 g, 2.02 mmol, 1.0 eq), TBAF in 1M THF (10.0 mL) and THF (20.0 mL) to a reaction flask. After nitrogen protection, react at room temperature for 6 h. After the reaction is completed, rotary evaporate the solvent, wash successively with water and saturated NaCl solution, extract with EA, combine the organic phases, dry over anhydrous Na2SO4, concentrate under reduced pressure, and obtain 750 mg of a light yellow foamy solid by silica gel column chromatography, namely intermediate 66.

[0182] (6) Synthesis of tert-butyl (2S,4R)-2-(5-chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)-4-(((4-(methoxycarbonyl)benzyl)carbamoyl)oxy)pyrrolidine-1-carboxylate (intermediate 67):

[0183] ① Add intermediate 66 (700 mg, 1.84 mmol, 1.0 eq), CDI (1.20 g, 7.37 mmol, 4.0 eq), and TEA (640 μL, 4.61 mmol, 2.5 eq) to a reaction flask, dissolve in toluene (6.00 mL). After nitrogen protection, react at 55 °C for 4 h. After the reaction is completed, quench with saturated NaHCO3 solution, wash successively with water and saturated NaCl solution, extract with EA, combine the organic phases, dry over anhydrous Na2SO4, concentrate under reduced pressure, and dry the crude product for use; ② Add the crude product prepared in ① (880 mg, 1.86 mmol, 1.0 eq), methyl 4-(aminomethyl)benzoate hydrochloride (560 mg, 1.86 mmol, 1.0 eq), TEA (850 μL, 7.43 mmol, 4.0 eq) and DMF (4.00 mL) to a reaction flask and dissolve. Then react at room temperature for 12 h. After the reaction is completed, quench with water, precipitate a solid, filter the cake, wash with water, dry, and obtain 997 mg of a light yellow foamy solid by silica gel column chromatography, namely intermediate 67.

[0184] (7) Synthesis of methyl 4-((((((3R,5S)-5-(5-chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)pyrrolidin-3-yl)oxy)carbonyl)amino)methyl)benzoate (intermediate 68):

[0185] Intermediate 68 was prepared with reference to step (5) of Reference Example 1, only replacing intermediate 49 with intermediate 67 to obtain intermediate 68.

[0186] (8) Synthesis of 4-((((((3R,5S)-5-(5-chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-(2,6-diamino-5-cyanopyrimidin-4-yl)pyrrolidin-3-yl)oxy)amino)methyl)benzoate (Intermediate 69):

[0187] Intermediate 69 was prepared according to Step (6) of Reference Example 1, with only Intermediate 50 replaced by Intermediate 68 to obtain Intermediate 69.

[0188] (9) Synthesis of (3R,5S)-5-(5-chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-(2,6-diamino-5-cyanopyrimidin-4-yl)pyrrolidin-3-yl (4-(hydroxyamino)phenyl)benzoate (Compound 36):

[0189] Compound 36 was prepared according to Step (7) of Reference Example 1, with only Intermediate 51 replaced by Intermediate 69. A white solid was obtained.

[0190] The white solid was subjected to NMR and ESI-MS analysis. The NMR results were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 8.99 (s, 1H), 7.94 (t, J = 5.6 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.64 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 6.56 (s, 2H), 6.28 (s, 2H), 5.69~5.22 (m, 2H), 4.37~4.13 (m, 3H), 4.04 (d, J = 8.8 Hz, 1H), 3.65 (s, 3H), 2.70~2.53 (m, 1H), 2.43~2.23 (m, 1H); ESI-MS: m / z = 605 [M + H] + 。

[0191] Example 12

[0192] Synthesis of 4-((((3R,5S)-5-(5-chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-(2,6-diamino-5-cyano-pyrimidin-4-yl)pyrrolidin-3-yl)oxy)-N-hydroxybenzamide (Compound 32).

[0193] Compound 32 Refer to Example 11, only step (6) is different: Add intermediate 66 (700 mg, 1.84 mmol, 1.0 eq) and sodium hydride (177 mg, 7.37 mmol, 4.0 eq) into a reaction flask. Add DMF (6.00 mL) to dissolve under an ice bath, then protect with nitrogen and react at room temperature for 1 h. Then add methyl 4-(chloromethyl)benzoate ((1.36 g, 7.37 mmol, 4.0 eq) and continue to react at room temperature for 3 h. After the reaction is completed, add water to terminate the reaction, extract with EA, combine the organic phases, dry with anhydrous Na2SO4, concentrate under reduced pressure, and obtain 770 mg of a light yellow foamy solid through silica gel column chromatography. Then, use the same method to prepare a white solid through hydroxylamine hydrolysis.

[0194] Perform nuclear magnetic resonance and ESI-MS detection on the said white solid. The nuclear magnetic resonance results are as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 9.03 (s, 1H), 7.73 (d, J = 8.0 Hz, 2H), 7.63 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 7.2 Hz, 3H), 6.56 (s, 2H), 6.26 (s, 2H), 5.49 (s, 1H), 4.65~4.46 (m, 3H), 4.18 (s, 1H), 4.08~4.01 (m, 1H), 3.66 (s, 3H), 2.63~2.54 (m, 1H), 2.31~2.14 (m, 1H); ESI-MS: m / z = 562 [M + H] + 。

[0195] Example 13

[0196] (3R,5S)-5-(5-Chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-(2,6-diamino-5-cyanopyrimidin-4-yl)pyrrolidin-3-yl ((5-(hydroxyamino)thiophen-2-yl)methyl)carbamate (Compound 37) synthesis.

[0197] Compound 37 Refer to Example 11, only in step (6), replace methyl 4-(aminomethyl)benzoate hydrochloride with methyl 2-(aminomethyl)thiophene-5-carboxylate hydrochloride, and then use the same method to prepare a white solid through hydroxylamine hydrolysis.

[0198] Perform nuclear magnetic resonance and ESI-MS detection on the said white solid. The nuclear magnetic resonance results are as follows: 11H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 9.08 (s, 1H), 8.05 (t, J = 4.8 Hz, 1H), 7.64 (t, J = 6.4 Hz, 1H), 7.48 (d, J = 6.4 Hz, 1H), 7.43 (d, J = 6.4 Hz, 2H), 6.94 (s, 1H), 6.57 (s, 2H), 6.28 (s, 2H), 5.59 - 5.34 (m, 2H), 4.40 - 4.24 (m, 3H), 4.02 (s, 1H), 3.65 (s, 3H), 2.67 - 2.56 (m, 1H), 2.40 - 2.25 (m, 1H); ESI-MS: m / z = 611 [M+H] + 。

[0199] Example 14

[0200] Synthesis of (3R,5S)-5-(5-chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-(2,6-diamino-5-cyanopyrimidin-4-yl)pyrrolidin-3-yl (4-hydroxycarbamoyl)phenethyl)carbamate (Compound 38).

[0201] Compound 38 was prepared with reference to Example 11, except that methyl 4-(aminomethyl)benzoate hydrochloride was replaced by methyl 4-(aminoethyl)benzoate hydrochloride in step (6), and then the same method was used for hydroxylamine cleavage to obtain an off-white solid.

[0202] The off-white solid was subjected to nuclear magnetic resonance and ESI-MS detection. The nuclear magnetic resonance results were as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 1H), 9.00 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.43 (t, J = 7.2 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 6.60 (s, 2H), 6.28 (s, 2H), 5.53 - 5.22 (m, 2H), 4.26 (s, 1H), 3.98 (s, 1H), 3.65 (s, 3H), 3.22 (dd, J = 12.7, 6.8 Hz, 2H), 2.75 (t, J = 7.2 Hz, 2H), 2.71 - 2.64 (m, 1H), 2.35 - 2.21 (m, 1H); ESI-MS: m / z = 619 [M+H] + 。

[0203] Example 15

[0204] (3R,5S)-5-(5-Chloro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1-(2,6-diamino-5-cyanopyrimidin-4-yl)pyrrolidin-3-yl (4-hydroxycarbamoyl)(methyl)aminobenzoate (Compound 39) synthesis.

[0205] Compound 39 was prepared with reference to Example 11. Only in step (6), methyl 4-(aminomethyl)benzoate hydrochloride was replaced with methyl 4-((methylamino)methyl)benzoate hydrochloride, and then the same method was used for hydroxylamine cleavage to obtain an off-white solid.

[0206] The off-white solid was subjected to nuclear magnetic resonance and ESI-MS detection. The nuclear magnetic resonance results were as follows: 1 H NMR(400MHz,DMSO-d6)δ11.16(s,1H),9.01(s,1H),7.71(t,J=6.8Hz,2H), 7.64(t,J=6.4Hz,1H),7.48(d,J=6.4Hz,1H),7.43(d,J=6.4Hz,1H),7.29(dd, J=12.2,6.4Hz,2H),6.57(s,2H),6.28(s,2H),5.60(s,1H),5.39(s,1H),4.47(m,2H),4.28(s,1H),4.09(dd,J=8.4,4.2Hz,1H),3.62(d,J=23.6Hz,3H),2.83(s, 3H),2.71~2.55(m,1H),2.44~2.25(m,1H); ESI-MS:m / z=619[M+H] + 。

[0207] Example 16

[0208] (3R,5S)-5-(5-Chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-1-(2,6-diamino-5-cyanopyrimidin-4-yl)pyrrolidin-3-yl (4-(hydroxycarbamoyl)benzyl) formate (Compound 42) synthesis.

[0209] Compound 42 was prepared with reference to Example 11. Only in step (4), methyl iodide was replaced with chlorobenzene, and then the same method was used for hydroxylamine cleavage to obtain an off-white solid.

[0210] The off-white solid was subjected to nuclear magnetic resonance and ESI-MS detection. The nuclear magnetic resonance results were as follows: 11H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 9.00 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.77 (t, J = 6.0 Hz, 1H), 7.73 - 7.66 (m, 3H), 7.63 - 7.58 (m, 1H), 7.57 - 7.74 (m, 2H), 7.52 (d, J = 2.4 Hz, 1H), 7.51 - 7.46 (m, 2H), 7.21 (d, J = 8.0 Hz, 2H), 6.58 (s, 2H), 6.25 (s, 2H), 5.24 (s, 1H), 4.80 (s, 1H), 4.21 - 4.06 (m, 3H), 3.97 (s, 1H), 2.35 - 2.27 (m, 1H), 1.95 - 1.86 (m, 1H); ESI-MS: m / z = 667 [M + H] + 。

[0211] Example 17

[0212] Synthesis of (3S,5S)-5-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-1-(2,6-diamino-5-cyanopyrimidin-4-yl)pyrrolidin-3-yl (4-(hydroxycarbamoyl)benzyl) formate (Compound 43).

[0213] Compound 43 was prepared with reference to Example 11, except that in step (1), (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid was replaced with (2S,4S)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid, and in step (4), methyl iodide was replaced with chlorobenzene. Then, the same method was used for hydroxylamine cleavage to obtain an off-white solid.

[0214] The off-white solid was subjected to nuclear magnetic resonance and ESI-MS detection. The nuclear magnetic resonance results were as follows: 11H NMR (400 MHz, DMSO-d6) δ 11.15 (s, 1H), 8.98 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.72 (s, 1H), 7.68 (dd, J = 8.4, 3.2 Hz, 3H), 7.63 - 7.51 (m, 5H), 7.42 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 6.55 (s, 2H), 6.26 (s, 2H), 5.00 - 4.91 (m, 1H), 4.77 (s, 1H), 4.36 (dd, J = 10.4, 7.2 Hz, 1H), 4.18 (d, J = 5.6 Hz, 2H), 3.84 (s, 1H), 2.25 - 2.14 (m, 1H), 2.03 - 1.94 (m, 1H); ESI-MS: m / z = 667 [M+H] + 。

[0215] Example 18: PI3K and HDAC inhibitory activities

[0216] In this example, the marketed PI3Kδ inhibitor Idelalisib was used as the positive control to determine the PI3Kδ inhibitory activity by ADP-Glo Assay. In addition, the marketed broad-spectrum HDAC inhibitor SAHA (succinylanilide hydroxamic acid) was used as the positive control to evaluate the HDAC6 and HDAC1 enzyme inhibitory activities of the compounds of the present invention by Fluorescent-based HDAC Activity Assay. Other compounds of the present invention have similar beneficial effects to the compounds listed below, but this should not be construed as the compounds of the present invention having only the following beneficial effects.

[0217] The test procedure for the PI3Kδ enzyme inhibitory activity is as follows: Prepare a DMSO solution of the test compound, and sequentially prepare a kinase buffer, a kinase solution, a substrate solution, and a kinase catalytic reaction system according to the operating instructions of the commercial kit, terminate the reaction as required, and add a detection reagent; Use a multi-functional microplate reader to read the chemiluminescence value, calculate the inhibition rate at each concentration, and the IC 50 Obtained by fitting with GraphPad Prism 5 software.

[0218] The test procedure for the inhibitory activity of HDAC6 enzyme is as follows: Prepare a DMSO solution of the compound to be tested, and sequentially prepare a buffer solution, an enzyme solution, and a corresponding Substrate / Trypsin mixed solution according to the kit instructions; Add compound solutions with gradient concentrations, enzyme solutions, and Substrate / Trypsin mixed solutions to a 384-well plate respectively to prepare a catalytic reaction system (set up no-compound control and no-enzyme control wells); After incubating at room temperature for a certain period of time, continuously read the fluorescence signal values using a Synergy microplate reader, and select the linear reaction segment to obtain the slope, and then calculate the inhibition rate at each concentration, IC 50 Fitted by GraphPad Prism 5 software.

[0219] The test method for the inhibitory activity of the compound on HDAC1 refers to the test method for HDAC6 inhibitory activity, and only the substrate of the corresponding catalytic reaction system needs to be replaced.

[0220] Table 1 Inhibitory activities of the compounds against PI3Kδ, HDAC6, and HDAC1 enzymes

[0221]

[0222]

[0223]

[0224] In Table 1: “++++” represents 0 - 10 nM; “+++” represents 10 - 100 nM; “++” represents 100 - 1000 nM; “+” represents 1000 - 10000 nM; “-” represents not determined.

[0225] From the enzyme inhibitory activity data in Table 1, it can be seen that most of the compounds in the present invention have significant dual enzyme inhibitory activities of PI3K / HDAC, and the IC 50 At the two-digit nanomolar or single-digit nanomolar level, its inhibitory activity against PI3K is equivalent to or better than that of Idelalisib. At the same time, its inhibitory activity against HDAC6 is equivalent to or better than that of SAHA. The literature shows that the IC 50 of the compound inhibiting HDAC1 50The difference between them can reflect the HDAC6 selectivity of the compound. Among the above compounds with significant PI3K / HDAC dual enzyme inhibitory activity, the inhibitory activity of some compounds against HDAC6 is better than that against HDAC1, such as Compound 5, 8, 9 conforming to General Formula (II), Compound 10 - 13, 15, 16, 18, 20 - 24, 26, 28, 31 conforming to General Formula (III), and Compound 32 - 34, 37 - 44 conforming to General Formula (IV).

[0226] Example 19: Antitumor cell proliferation activity

[0227] In this example, Idelalisib and SAHA were used as positive controls, and the CCK-8 method was adopted to evaluate the anti-proliferation activity of the compounds of the present invention with significant PI3K / HDAC dual enzyme inhibitory activity against colon cancer cell line KM-12, colon cancer cell line HCT116, and B-cell lymphoma SU-DHL-6. Other compounds of the present invention have similar beneficial effects to the compounds listed below, but this should not be construed as the compounds of the present invention only having the following beneficial effects.

[0228] The test steps for antitumor cell proliferation activity are as follows: Digest and collect tumor cells, inoculate them in a 96-well culture plate at a certain density, and place them in an incubator (37 °C, 5% CO2) overnight. Treat the cells with compound solutions at different concentrations. After the compound acts for 72 h, discard the culture medium, and then gently wash the cells 3 times with PBS. Subsequently, add a certain volume of culture medium and CCK-8 to each well of the culture plate, and continue to culture for a certain time. Finally, use a multifunctional microplate reader to measure the absorbance OD value at a wavelength of 570 nm, calculate the inhibition rate, and the IC 50 value is obtained by fitting with GraphPad Prism 5 software, and the results are shown in Table 2.

[0229] Table 2 Antitumor cell proliferation activity of compounds with significant P13K / HDAC dual enzyme inhibitory activity

[0230] Cpd. <![CDATA[T47D(IC 50 )]]> <![CDATA[KM-12(IC 50 )]]> <![CDATA[HCT116(IC 50 )]]> SU-DHL-6(IC;0) 5 ++++ +++ ++ ++++ 10 +++ +++ ++ +++ 11 +++ ++++ ++ +++ 22 ++++ +++ ++ ++++ 24 ++++ +++ +++ ++++ 28 ++++ ++++ +++ ++++ 32 ++++ +++ +++ ++++ 33 +++ +++ ++ ++ 37 ++++ +++ +++ +++ 38 ++++ ++++ ++ ++ 39 ++++ ++++ ++ ++++ 41 ++++ ++++ ++ ++++ 42 ++++ ++++ ++ ++++ 43 ++++ ++++ ++ ++++ Idelalisib +++ +++ ++ ++++ SAHA ++ ++ ++ +++

[0231] In Table 2: "++++" represents < 0.1 μM; "+++" represents 0.1 - 1.0 μM; "++" represents 1.0 - 10 μM.

[0232] As can be seen from the anti-proliferative activity data in Table 2, most of the compounds with significant dual PI3K / HDAC enzyme inhibitory activity also showed significant anti-proliferative activity against solid tumor cell lines T47D, KM-12, HCT116 and hematological tumor cell line SU-DHL-6. Among them, more than half of the compounds had better or equivalent anti-proliferative activity against the four cell lines than Idelalisib, and the vast majority of the compounds had better or equivalent anti-proliferative activity against the four cell lines than SAHA, showing good application prospects. Compound 5 conforming to general formula (II), compounds 10, 11, 22, 24, 28 conforming to general formula (III), and compounds 32, 33, 37, 38, 41 - 43 conforming to general formula (IV) had better or equivalent anti-proliferative activity against T47D, KM-12, HCT116 than Idelalisib and SAHA while being selective for HDAC6; among these compounds, compound 5 conforming to general formula (II), compounds 22, 24, 28 conforming to general formula (III), and compounds 32, 39, 41, 42, 43 conforming to general formula (IV) had better or equivalent anti-proliferative activity against SU-DHL-6 than Idelalisib and SAHA.

[0233] Example 20: PI3Kδ selectivity

[0234] Further evaluation of the enzyme inhibitory activities of compounds against PI3Kα, PI3Kβ, and PI3Kγ can reflect the inhibitory selectivity of the compounds for the PI3Kδ subtype. The following further elaborates on the selectivity for PI3Kδ through the inhibitory activity data of some compounds of the present invention with significant dual PI3K / HDAC enzyme inhibitory and anti-proliferative activities against PI3Kα, β, γ, and δ. It should not be understood that only the following compounds have PI3Kδ selectivity.

[0235] The test method for the inhibitory activities of compounds against other PI3K subtypes refers to the test method for PI3Kδ inhibitory activity, and only the substrate of the catalytic reaction system is changed when testing the corresponding enzyme inhibitory activity.

[0236] Table 3 PI3Kδ selectivity of compounds

[0237]

[0238]

[0239] In Table 3: “++++” represents 0 - 10 nM; “+++” represents 10 - 100 nM; “++” represents 100 - 1000 nM; “+” represents 1000 - 10000 nM.

[0240] As shown in Table 3, Compound 5 conforming to General Formula (II), Compounds 11, 22, 24, and 28 conforming to General Formula (III), and Compounds 32, 37, 39, 41, 42, and 43 conforming to General Formula (IV) have significant enzyme inhibitory activity against PI3K δ, while their inhibitory activities against PI3Kα, PI3Kβ, and PI3Kγ are relatively weak. Therefore, they are selective PI3Kδ inhibitors, and their selectivity is equivalent to or better than that of the marketed drug Idelalisib. Compound 10 conforming to General Formula (III) has significant enzyme inhibitory activity against PI3Kδ and PI3Kγ, while its inhibitory activities against PI3K α and PI3Kβ are relatively weak. Therefore, it is a PI3Kδ / γ inhibitor. The above results indicate that the compounds of the present invention have subtype selectivity in inhibiting PI3K, which is beneficial to avoiding the off-target effects caused by simultaneously inhibiting the four subtypes.

[0241] Example 21: HDAC6 Selectivity

[0242] The test of the enzyme inhibitory activities of HDAC1, HDAC2, HDAC3, HDAC6, HDAC8, HDAC10, and HDAC11 can reflect the selectivity of the compound for inhibiting the HDAC6 subtype. The following further elaborates the selectivity for HDAC6 through the inhibitory activity data of some compounds of the present invention with significant PI3K enzyme inhibitory activity, HDAC6 enzyme inhibitory activity, and anti-proliferative activity against HDAC1, 2, 3, 6, 8, 10, and 11. It should not be understood that only the following compounds of the present invention have HDAC6 selectivity. The IC of the compound for other subtypes 50 Compared with the IC for HDAC6 50 The larger the ratio, the better the HDAC6 selectivity of the compound.

[0243] The test method for the inhibitory activity of the compound against other HDAC subtypes refers to the test method for HDAC6 inhibitory activity, and only the substrate of the catalytic reaction system is changed when testing the corresponding enzyme inhibitory activity.

[0244] Table 4 HDAC6 Selectivity of Compounds

[0245]

[0246] In Table 4: “++++” represents >100; “+++” represents 50 - 100; “++” represents 10 - 50; “+” represents 5 - 10, and “-” represents less than 5.

[0247] As can be seen from Table 1 and Table 4, Compound 5 conforming to General Formula (II), Compounds 22 and 28 conforming to General Formula (III), and Compounds 32, 37, 39, 41, 42, and 43 conforming to General Formula (IV) significantly inhibit HDAC6 while having excellent HDAC6 selectivity, with selectivity superior to that of the HDAC6 inhibitor ACY-1215 under clinical research. The activities of the broad-spectrum HDACs inhibitor SAHA in inhibiting HDAC1, 2, 3, 8, and 10 subtypes are relatively close, lacking subtype selectivity. This indicates that Compounds 5, 22, 28, 32, 37, 39, 41, 42, and 43 are beneficial for reducing the toxicity caused by the inhibition of all or multiple HDAC subtypes while significantly inhibiting the HDAC6 subtype.

[0248] As can be seen from Table 3 and Table 4, Compound 5 conforming to General Formula (II), Compounds 22 and 28 conforming to General Formula (III), and Compounds 32, 37, 39, 41, 42, and 43 simultaneously have significant inhibitory activities against PI3Kδ and HDAC6, while their inhibitory activities against PI3Kα, PI3Kβ, PI3Kγ, HDAC1, HDAC2, HDAC3, HDAC8, HDAC10, and HDAC11 are relatively weak. Therefore, they are selective PI3Kδ / HDAC6 dual-target inhibitors. Their selective inhibition of PI3Kδ and HDAC6 is beneficial for avoiding the toxic and side effects caused by the simultaneous inhibition of other PI3K or HDAC subtypes.

[0249] Example 22: Tumor Immune Regulation Effect of the Compounds of the Present Invention

[0250] In this example, the Western blot method was used to evaluate the regulatory effects of the compounds of the present invention on the STAT3 signaling pathway related to tumor cell immunity and the expression of the immune checkpoint PD-L1. Other compounds of the present invention have similar beneficial effects to the compounds listed below, but this should not be construed as meaning that the compounds of the present invention only have the following beneficial effects.

[0251] Test procedure: When detecting the regulatory effect of a compound on the STAT3 signaling pathway, T47D cells in the logarithmic growth phase were digested and then blown into a single-cell suspension, seeded in a 6-well plate at a certain density, added with culture medium, and placed overnight in an incubator. Different concentrations of the test compound solution were added to each well for pretreatment overnight. Then, the cells were further treated with IL-6 (30 ng / mL) for 20 min (blank control group, not treated with the compound or IL-6; model group, treated only with IL-6). The cells were washed with PBS solution, lysed with RIPA lysis buffer, centrifuged, and the supernatant was collected. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and incubated with STAT3, P-STAT3 (Y705) antibodies and secondary antibodies respectively, and then developed. When detecting the regulatory effect of the compound on the PI3K signaling pathway and the expression of immune checkpoint PD-L1, GAPDH was used as an internal reference, and only the test compound was added to co-incubate with T47D cells overnight without adding IL-6. After incubation, the cells were treated using the same method as above, and immunoblot analysis was performed. The results are shown in Figure 1 , Figure 2 and Figure 3 , among which, Figure 1 is the result of protein immunoblot analysis of the effect of compound 28 on the expression levels of STAT3 and P-STAT3 (Y705), Figure 2 is the result of protein immunoblot analysis of the effect of compound 28 on the expression levels of AKT, P-AKT (S473), and PD-L1, Figure 3 in which A is a quantitative analysis histogram of the regulatory effect of compound 28 on P-STAT3 (Y705) in T47D cells, Figure 3 in which B is a quantitative analysis histogram of the regulatory effect of compound 28 on P-AKT (S473) in T47D cells, Figure 3 in which C is a quantitative analysis histogram of the regulatory effect of compound 28 on PD-L1 in T47D cells.

[0252] P-STAT3 (Y705) is an important biomarker of the STAT3 signaling pathway. The upregulation of its level reflects the activation of the STAT3 pathway, and the activation of this pathway can mediate tumor immune tolerance, thereby escaping immune system surveillance. The literature shows that the inhibition of the HDAC6 target can regulate the STAT3 pathway. Figure 1It was shown that treatment of tumor cells with IL-6 could lead to an upregulation of the level of P-STAT3 (Y705); Compound 28 conforming to General Formula (III) could significantly inhibit the upregulation of the expression level of P-STAT3 (Y705) induced by IL-6 even at a concentration as low as 10 nM, and showed a dose-dependence as a whole, suggesting that the compound could play a role in tumor immune regulation by interfering with the activation of the STAT3 signaling pathway. This result also revealed the inhibitory effect of the compound on the HDAC6 target at the cellular level. P-AKT (S473) is an important biomarker of the PI3K signaling pathway, and the upregulation of its level reflects the activation of this pathway; PD-L1 is an immune checkpoint on the surface of tumor cells, and through its interaction with PD-1 on the surface of immune cells, tumor cells can escape immune system surveillance. From Figure 2 It can be seen that Compound 28 could significantly downregulate the biomarker P-AKT (S473) of the PI3K signaling pathway even at a concentration as low as 30 nM; at the same time, at this concentration, the compound also significantly reduced the expression of PD-L1. The above effects showed a dose-dependence as a whole. To sum up, the results of biological experiments showed that Compound 28 conforming to General Formula (III) could strongly inhibit PI3Kδ and HDAC6, and showed excellent selectivity for PI3Kδ and HDAC6; this compound had significant anti-tumor cell proliferation activity at the cellular level, and could act on both the PI3K pathway and the HDAC6 target; it was worth noting that this compound could also interfere with the STAT3 pathway related to immune escape and downregulate the expression of PD-L1, so it had tumor immune therapy activity and could intervene in tumors through "multiple pathways and multiple factors", which was beneficial to enhancing the drug effect and delaying drug resistance.

[0253] The literature shows that inhibiting the PI3Kδ target can exert anti-inflammatory and anti-autoimmune disease effects by inhibiting the release of inflammatory factors in B cells and T cells (Bartok, Beatrix et al. PI3 Kinase δ Is a Key Regulator of Synoviocyte Function in Rheumatoid Arthritis. Am. J. Pathol. 2012, 180, 1906-1916; Randis1, Tara M., Role of PI3Kd and PI3 Kc in inflammatory arthritis and tissue localization of neutrophils. Eur. J. Immunol. 2008, 38, 1215-1224); inhibiting the HDAC6 target can obtain anti-inflammatory effects by regulating the expression levels of intracellular inflammatory factors such as IL-6 (Park, Jin Kyun et al. Inhibition of histone deacetylase 6 suppresses inflammatory responses and invasiveness of fibroblast-like-synoviocytes in inflammatory arthritis. Arthritis Res. Ther. 2021, 23, 177); it can also maintain immune homeostasis by enhancing the immunosuppressive ability of Foxp3+ regulatory T cells, thereby slowing down or reversing the onset of autoimmune diseases (Akimova, Tatiana et al. Histone / protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs. Clin. Immunol. 2010, 136, 348-363; Kalin, Jay H et al. Second-generation histone deacetylase 6 inhibitors enhance the immunosuppressive effects of Foxp3+ T-regulatory cell. J. Med. Chem. 2012, 55, 639-651). In view of the significant dual-target inhibition effect of the compounds of the present invention on PI3Kδ / HDAC6 and excellent subtype selectivity, therefore, the compounds of the present invention have good application prospects in anti-tumor, anti-inflammatory, and anti-autoimmune disease aspects.

[0254] Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of PI3K / HDAC dual inhibitors, characterized in that: The PI3K / HDAC dual inhibitor is a compound represented by the following general formulas (I)-(IV) and its pharmaceutically acceptable salts: In the general formulas (I)-(IV), R1 is hydrogen, halogen, cyano, C1-6 alkyl or C2-6 unsaturated aliphatic hydrocarbon group, and R1 is substituted at any position on the benzene ring; In general formula (II), R2 is methyl, ethyl, trifluoromethyl or cyclopropyl; In general formula (IV), R3 is hydrogen, C1-6 alkyl, C3-8 cycloalkyl, C2-6 unsaturated aliphatic hydrocarbon group or C3-8 unsaturated cycloalkyl; In the general formulas (I)-(IV), ring A is selected from a monocyclic heteroaryl or a fused bicyclic heteroaryl substituted by 1-4 R4s, and the number of carbon atoms in ring A is C3-C9, where R4 is selected from one of hydrogen, halogen, cyano, C1-6 alkyl, NR5R6; R5 and R6 are each independently selected from hydrogen; In the general formulas (I) and (III), ring B is , R8 is hydrogen or hydroxyl, and R9 is hydrogen or C1-6 alkyl; In the general formulas (I)-(IV), ring C is a C6-14 aryl or a C5-14 heteroaryl. In addition to being substituted by the structural units on both sides shown in the general formulas (I)-(IV), ring C may also be substituted by at least one R 10 substitution, and the R 10 is selected from hydrogen; In the general formula (I), X is or , n1 = 1 to 3, R 14 is hydrogen; In the general formulas (II) and (III), Y is (CH2)n2, and n2 = 1-10; In the general formula (IV), Z is , , or , n3 = 1 - 3, R 15 is a C1-6 alkyl group, R 16 is hydrogen or a C1-6 alkyl group.

2. A class of PI3K / HDAC dual inhibitors according to claim 1, characterized in that: When ring A is substituted by two or more R4s, two or more of the R4s may be the same or different.

3. A class of PI3K / HDAC dual inhibitors according to claim 1, characterized in that: When the ring C is substituted by two or more Rs 10 the two or more Rs 10 may be the same or different from each other.

4. A class of PI3K / HDAC dual inhibitors according to claim 1, characterized in that: When ring C does not exist, the hydroxamic acid group in the general formulas (I)-(IV) is directly connected to X, Y or Z.

5. A class of PI3K / HDAC dual inhibitors as claimed in claim 1, characterized in that: The PI3K / HDAC dual inhibitor is selected from the following compounds and their pharmaceutically acceptable salts: 。 6. The class of PI3K / HDAC dual inhibitors according to claim 1, characterized in that: The PI3K / HDAC dual inhibitor is selective for PI3Kδ and HDAC6, and is a compound represented by general formula (II), (III) or (IV) and its pharmaceutically acceptable salt, wherein R1, R2, R3, ring A, ring B, ring C, Y, Z are as defined in claim 1.

7. Use of a class of PI3K / HDAC dual inhibitors as described in any one of claims 1-6 in the preparation of drugs for treating anti-tumor, inflammation, and autoimmune diseases, wherein, The tumor is selected from solid tumors and hematological tumors.

8. A PI3K / HDAC dual inhibitor composition, characterized in that: It includes the PI3K / HDAC dual inhibitor according to any one of claims 1-6, and also includes at least one pharmaceutical carrier or excipient.

9. Use of the PI3K / HDAC dual inhibitor composition according to claim 8 in the preparation of drugs for treating tumors, inflammation and autoimmune diseases.

10. The PI3K / HDAC dual inhibitor composition according to claim 8, characterized in that, It also includes at least one other therapeutic agent, and the dosage form of the PI3K / HDAC dual inhibitor composition is any clinically or pharmaceutically acceptable dosage form.

11. Use of the PI3K / HDAC dual inhibitor composition according to claim 10 in the preparation of drugs for treating tumors, inflammation and autoimmune diseases.

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