A heterocyclic compound and its preparation method and use

Abstract

The present invention provides a heterocyclic compound, its preparation method, and its use, belonging to the field of biopharmaceutical technology. The structure of the heterocyclic compound of the present invention is shown in Formula I. The compound of the present invention has excellent inhibitory activity against both PI3K and HDAC targets, and can be used as an effective dual-target inhibitor. The compound of the present invention can be used to prepare drugs for the prevention and / or treatment of diseases related to PI3K and / or HDAC, and has good application prospects. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a heterocyclic compound and a preparation method and use thereof. Background Art

[0002] Malignant tumors are one of the world's most serious public health problems. Although the emergence of new therapeutic drugs and treatments in recent years has significantly improved the quality of life for cancer patients, overall, the current treatment status of malignant tumors is far from satisfactory, and more efforts are urgently needed to find new anti-tumor drugs.

[0003] Chemotherapy has long played a vital role in cancer treatment due to its broad applicability and thorough elimination of tumor cells. Traditional chemotherapeutic agents, such as cytotoxic drugs, were the cornerstone of cancer therapy for decades, but their significant side effects have gradually faded from the mainstream. Targeted tumor drugs, such as kinase inhibitors, have revolutionized the treatment of malignant tumors over the past 20 years due to their rapid efficacy and minimal side effects. However, these drugs often only suppress tumor growth for a relatively short period of time due to the rapid development of drug-resistant mutations. The recent emergence of epigenetic target drugs, with their advantages of sustained efficacy and slow development of drug resistance, has offset the shortcomings of traditional kinase inhibitors and revolutionized the treatment of malignant tumors. However, these inhibitors also suffer from limitations such as limited indications and slow onset of efficacy. Given the complementary advantages of kinase inhibitors and epigenetic targets, small molecule compounds that simultaneously target both kinase and epigenetic targets hold promise as a new class of highly effective anti-cancer drugs.

[0004] Abnormal activation or amplification of PI3K is closely associated with the development and progression of hematological malignancies. Inhibitors targeting this protein could be used to treat hematological malignancies. Numerous PI3K inhibitors have been reported, but their clinical indications are limited, and their therapeutic efficacy remains far from satisfactory due to disease recurrence and drug resistance.

[0005] Histone deacetylases (HDACs) are a class of proteases that play a crucial role in chromatin structural modification and gene expression regulation. These proteins catalyze the deacetylation of histones, tightening the binding of DNA to the histone octamer, thereby inhibiting gene transcription, particularly tumor suppressor genes. Overexpression or aberrant activation of these proteins is closely associated with the development and progression of various tumors. Consequently, several small molecule inhibitors targeting these proteins, such as Vorinostat, Romidepsin, and Belinostat, have been approved for the treatment of cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and multiple myeloma (MM). Furthermore, a large number of HDAC inhibitors are currently in various stages of clinical development for the treatment of various hematologic malignancies and solid tumors. However, the efficacy of current HDAC inhibitors in cancer patients remains unsatisfactory, with most patients achieving only a few months of remission and prolonged survival with these drugs.

[0006] Single-target PI3K and HDAC inhibitors have limitations due to insufficient efficacy and the rapid development of drug-resistant mutations. Studies have shown that simultaneous inhibition of PI3K and HDAC can synergistically inhibit tumor growth and provide a wider therapeutic range than single inhibitors by improving efficacy and limiting drug resistance. Several dual-target PI3K / HDAC inhibitors have been reported (DOI: 10.1021 / acs.jmedchem.9b00390), such as CUDC-907, which inhibits cancer cell proliferation through a synergistic effect and exhibits superior efficacy compared to single-target inhibitors. However, these inhibitors still fall short of clinical needs. The development of more inhibitors that can simultaneously inhibit PI3K and HDAC is urgently needed, and this invention is therefore proposed. Summary of the Invention

[0007] The present invention aims to provide a heterocyclic compound, a preparation method, and uses thereof. The heterocyclic compound of the present invention can be used as a novel dual-target inhibitor, capable of simultaneously inhibiting kinase targets and epigenetic targets, and can be used to prepare highly effective anti-tumor drugs.

[0008] The present invention provides a compound represented by formula I, a salt thereof, a stereoisomer thereof or a solvate thereof:

[0009]

[0010]

[0011] in,

[0012] R1 is LR a R b CONHR c ,

[0013] L is selected from none, C1-C6 alkylene, carbonyl;

[0014] R a Select from none, C1~C 10 Alkylene, 6- to 10-membered aryl which is unsubstituted or substituted by one or more R's, 5- to 10-membered heteroaryl which is unsubstituted or substituted by one or more R's;

[0015] R b Select from none, C2~C 10 alkenylene;

[0016] R c A 6- to 10-membered aryl group selected from hydroxyl, unsubstituted or substituted by one or more R's;

[0017] And L, R a 、R b Not at the same time is nothing;

[0018] R2, R3, R4, R5, R6, R7, R8, and R9 are independently selected from hydrogen, or two substituents on the same carbon atom form ═O, and at most one of R2, R3, R4, R5, R6, R7, R8, and R9 forms ═O;

[0019] R 10 is selected from C1-C6 alkyl groups which are unsubstituted or substituted by one or more R's;

[0020] R 11 is selected from a cyclic group which is unsubstituted or substituted with one or more R's;

[0021] R' is independently selected from hydroxy, halogen, cyano, amino, C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, and C2-C6 alkynyl.

[0022] Furthermore, the compound is as shown in Formula II:

[0023]

[0024] in,

[0025] R1' is selected from C1 to C6 alkyl;

[0026] R1 is LR a R b CONHR c ,

[0027] L is selected from none, C1-C6 alkylene, carbonyl;

[0028] R a Select from none, C1~C 10Alkylene, 6- to 10-membered aryl which is unsubstituted or substituted by one or more R's, 5- to 10-membered heteroaryl which is unsubstituted or substituted by one or more R's;

[0029] R b Select from none, C2~C 10 alkenylene;

[0030] R c A 6- to 10-membered aryl group selected from hydroxyl, unsubstituted or substituted by one or more R's;

[0031] And L, R a 、R b Not at the same time is nothing;

[0032] R2, R3, R4, R5, R6, R7, R8, and R9 are independently selected from hydrogen, or two substituents on the same carbon atom form ═O, and at most one of R2, R3, R4, R5, R6, R7, R8, and R9 forms ═O;

[0033] R 10 is selected from C1-C6 alkyl groups which are unsubstituted or substituted by one or more R's;

[0034] R' is independently selected from hydroxy, halogen, cyano, amino, C1-C6 alkyl, C2-C6 alkenyl, C1-C6 alkoxy, and C2-C6 alkynyl.

[0035] Further, R1' is selected from C1 to C6 alkyl;

[0036] Selected from

[0037] R1 is LR a R b CONHR c ,

[0038] L is selected from none, C1-C6 alkylene, carbonyl;

[0039] R a Select from none, C1~C 10 Alkylene, 6- to 10-membered aryl which is unsubstituted or substituted by one or more R's, 5- to 10-membered heteroaryl which is unsubstituted or substituted by one or more R's;

[0040] R b Select from none, C2~C 10 alkenylene;

[0041] R c A 6- to 10-membered aryl group selected from hydroxyl, unsubstituted or substituted by one or more R's;

[0042] And L, R a 、R b Not at the same time is nothing;

[0043] R 10 is selected from C1-C6 alkyl groups which are unsubstituted or substituted by one or more R's;

[0044] R' is independently selected from amino, C1-C6 alkyl, and C2-C6 alkenyl.

[0045] Furthermore, the compound is as shown in Formula III, Formula IV, and Formula V:

[0046]

[0047] in,

[0048] R1' is selected from C1 to C3 alkyl;

[0049] R 10 Selected from C1-C3 alkyl;

[0050] R1 is LR a R b CONHR c ,

[0051] L is selected from none, C1-C3 alkylene, carbonyl;

[0052] R a Selected from none, C1-C8 alkylene, phenyl;

[0053] R b Selected from none, C2-C3 alkenylene;

[0054] And L, R a 、R b Not at the same time is nothing;

[0055] R c is selected from hydroxy, phenyl which is unsubstituted or substituted by one or more R';

[0056] R' is each independently selected from amino;

[0057] Preferably, R1 is Furthermore, the compound is one of the following compounds:

[0058]

[0059]

[0060] The present invention also provides use of the above-mentioned compound, its salt, its stereoisomer or its solvate in the preparation of PI3K inhibitors and / or HDAC inhibitors.

[0061] Furthermore, the PI3K inhibitor is a PI3Kδ inhibitor, and the HDAC inhibitor is an HDAC6 inhibitor.

[0062] The present invention also provides use of the above-mentioned compound, its salt, its stereoisomer or its solvate in the preparation of a drug for preventing and / or treating diseases associated with PI3K and / or HDAC.

[0063] Furthermore, the disease associated with PI3K and / or HDAC is cancer, and the cancer is preferably lymphoma.

[0064] The present invention also provides a pharmaceutical composition, which is a preparation prepared by using the above-mentioned compound, its salt, its stereoisomer or its solvate as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0065] The compounds and derivatives provided herein can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0066] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined herein, they should be given the meaning that a person skilled in the art would give them based on the disclosure and context.

[0067] "Substitution" refers to the replacement of a hydrogen atom in a molecule by another different atom or molecule.

[0068] The minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C a ~C b Alkyl refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C1-C6 alkyl" refers to an alkyl group containing 1, 2, 3, 4, 5, or 6 carbon atoms. For example, C 1-6 Alkyl refers to straight or branched chain alkyl groups containing 1, 2, 3, 4, 5 or 6 carbon atoms, and so on.

[0069] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of carbon atoms. For example, C1-C6 alkyl refers to an alkyl group having from 1 to 6 carbon atoms, i.e., 1, 2, 3, 4, 5, or 6 carbon atoms. Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl, among others.

[0070] "Alkenyl" refers to an aliphatic hydrocarbon group having at least one carbon-carbon double bond. The alkenyl group may be straight-chain or branched.

[0071] "Alkynyl" refers to an aliphatic hydrocarbon group having at least one carbon-carbon triple bond. The alkynyl group may be straight-chain or branched.

[0072] "Aryl" refers to an all-carbon monocyclic group with a conjugated π electron system, such as phenyl. The aryl group does not contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent moiety must be on a carbon atom on the ring with a conjugated π electron system.

[0073] "Heteroaryl" or "heteroaromatic ring" refers to a heteroaromatic group containing one or more heteroatoms. The heteroatoms referred to herein include, but are not limited to, oxygen, sulfur, and nitrogen. Examples include furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like.

[0074] 5- to 10-membered heteroaryl refers to heteroaromatic groups containing 5, 6, 7, 8, 9 or 10 ring atoms, and so on.

[0075] "Parallel rings" refer to polycyclic rings in which two rings share two adjacent ring atoms.

[0076] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0077] The present invention provides a heterocyclic compound with excellent inhibitory activity against both PI3K and HDAC targets, making it an effective dual-target inhibitor, thereby effectively inhibiting tumor cell proliferation. This dual-target inhibitor can complement the advantages of the two targets, exerting a synergistic anti-tumor effect, with a wider range of indications, better efficacy, and reduced drug resistance.

[0078] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0079] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 is the H NMR spectrum of compound Z1.

[0081] Figure 2 This is the C NMR spectrum of compound Z1.

[0082] Figure 3 is the H NMR spectrum of compound Z2.

[0083] Figure 4 This is the C-NMR spectrum of compound Z2.

[0084] Figure 5 This is the H NMR spectrum of compound Z3.

[0085] Figure 6 This is the C NMR spectrum of compound Z3.

[0086] Figure 7 This is the H NMR spectrum of compound Z4.

[0087] Figure 8 This is the C NMR spectrum of compound Z4.

[0088] Figure 9 This is the H NMR spectrum of compound Z5.

[0089] Figure 10 This is the C NMR spectrum of compound Z5.

[0090] Figure 11 This is the H NMR spectrum of compound Z6.

[0091] Figure 12 This is the C NMR spectrum of compound Z6.

[0092] Figure 13 This is the H NMR spectrum of compound Z8.

[0093] Figure 14 This is the C NMR spectrum of compound Z8.

[0094] Figure 15 This is the H NMR spectrum of compound Z9.

[0095] Figure 16 This is the C NMR spectrum of compound Z9.

[0096] Figure 17 This is the H NMR spectrum of compound Z10.

[0097] Figure 18 This is the C NMR spectrum of compound Z10. DETAILED DESCRIPTION

[0098] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercial products.

[0099] The "room temperature" or "normal temperature" referred to in the present invention is 25±10°C.

[0100] Example 1

[0101] (E)-3-(4-(4-(2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)methylphenyl)-N-hydroxyacrylamide (Compound Z1)

[0102] Step 1: Preparation of tert-butyl 4-((2-chloro-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazine-1-carboxylate (Intermediate 1)

[0103]

[0104] Boc piperazinone (4.15 g, 16.6 mmol) was added to THF (10 ml), stirred and dissolved at ℃, and then t-BuOK (3.5 g, 31.13 mmol) was added. The mixture was stirred at 0℃ for 10 min, and then the raw material 4-(2-chloro-8-(chloromethyl)-9-methyl-9H-purin-6-yl)morpholine (5 g, 16.6 mmol) was added. The mixture was stirred at 0℃ for 20 min, and then stirred at room temperature for 1 h. The reaction progress was monitored by TLC. After the raw material was completely converted, water was added to precipitate the product, which was filtered and the filter cake was spin-dried to obtain 4.9 g of the product with a yield of 63.45%.

[0105] Step 2: Preparation of tert-butyl 4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazine-1-carboxylate (Intermediate 2)

[0106]

[0107] Intermediate 1 (4.3 g, 9.24 mmol), raw material 2-ethyl-1H-benzo[d]imidazole (1.35 g, 9.24 mmol), Pd2(dba)3 (0.96 g, 0.92 mmol), Xpos (0.88 g, 1.85 mmol), and CsCO3 (6 g, 18.48 mmol) were added to 1,4-dioxane (50 ml), and the mixture was purged with nitrogen three times under stirring. The reaction was stirred at 110°C under nitrogen protection for 3 h. The reaction progress was monitored by TLC. The raw material was completely converted, and the mixture was extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 4.5 g of the product with a yield of 85%.

[0108] Step 3: Preparation of 1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one hydrochloride (Intermediate 3)

[0109]

[0110] To intermediate 2 (4.5 g, 7.82 mmol) was added dioxane hydrochloride solution (50 ml), and the reaction was stirred at room temperature for 2 h. The reaction progress was monitored by TLC until Boc was completely removed. DCM was added to the reaction solution, and the mixture was sonicated. The liquid was poured out and washed twice with DCM. MeOH was then added to dissolve the solid portion, and the product was concentrated under reduced pressure to obtain 3.12 g of the product with a yield of 78%.

[0111] Step 4: Preparation of 1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (Intermediate 4)

[0112]

[0113] To intermediate 3 (3.12 g, 6.1 mmol) was added saturated potassium carbonate solution (30 ml), and the mixture was shaken. The mixture was then extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Dissolved in DCM, the sample was mixed with silica gel, and loaded onto a column. 2.5 g of the product was extracted with DCM:MeOH (100:1 to 10:1) for an 85% yield.

[0114] Step 5: Preparation of (E)-methyl 3-(4-(4-(2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)methylphenyl)acrylate (Intermediate 5)

[0115]

[0116] Intermediate 4 (500 mg, 1.05 mmol) was dissolved in acetonitrile solution (10 ml), and methyl 4-bromomethylcinnamate (268 mg, 1.05 mmol) and potassium carbonate (435 mg, 3.15 mmol) were added. The mixture was heated with stirring at 85° C. for 2 h. The reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was dried, extracted with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 654 mg of the product with a yield of 86%.

[0117] Step 6: Preparation of (E)-3-(4-(4-(2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)methylphenyl)acrylic acid (Intermediate 6)

[0118]

[0119] Intermediate 5 (650 mg, 1 mmol) was dissolved in a mixture of THF (8 ml), MeOH (8 ml), and H₂O (2 ml). LiOH·H₂O (210 mg, 5 mmol) was then added and stirred at 70°C for 1 h. The reaction progress was monitored by TLC. Upon completion, the solvent was removed from the reaction solution by spin drying, and the THF and MeOH were removed. A small amount of water was then added, and the solution was acidified by adding HCl with stirring until a precipitate formed. The pH test indicated a weak acidity. The solid portion was then washed three times with ethyl acetate (EA), dissolved in DCM and MeOH, and concentrated under reduced pressure to yield 495 mg of the product in a 78% yield.

[0120] Step 7: Preparation of (E)-3-(4-((4-(2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)methyl)phenyl)-N-(((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (Intermediate 7)

[0121]

[0122] Intermediate 6 (200 mg, 0.31 mmol) was dissolved in DMF (5 ml), and HATU (178 g, 0.47 mmol) and DIEA (60 mg, 0.47 mmol) were added, followed by the addition of raw material O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (36 mg, 0.31 mmol). The mixture was stirred at room temperature for 30 min. After the reaction was complete, a small amount of water was added, and the mixture was extracted twice with EA, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 315 mg of a crude product. After plate purification, 180 mg of the product was obtained with a yield of 79%.

[0123] Step 8: Preparation of Compound Z1

[0124]

[0125] To intermediate 7 (62 mg, 0.084 mmol) was added 6 mol / L HCl aqueous solution (4 ml), stirred at room temperature, and reacted for 30 min. The reaction progress was monitored by TLC. After the reaction was complete, sodium bicarbonate solution was used for alkali adjustment. When a white solid precipitated and pH test paper showed alkaline, the reaction solution was filtered, and the filter cake was dissolved in DCM and MeOH, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 38.7 mg of the product with a yield of 70%.

[0126] Characterization data: 1H NMR (600MHz, DMSO-d6) δ8.27–8.18(m,1H),7.94–7.84(m,1H),7.78–7.55(m,6H),7.46(d,J=15.8Hz,1H),6.62(d,J=15.8Hz,1H),5.11(s,2 H), 4.91–4.62 (m, 2H), 4.48 (s, 2H), 4.04 (s, 2H), 3.78 (d, J = 9.2Hz, 9H), 3.67 (d, J = 2.1Hz, 2H), 3.52 (d, J = 7.5Hz, 4H), 1.48 (t, J = 7.4Hz, 3H).

[0127] HRMS (ESI+) m / z theoretical value: C 34 H 38 N 10 O4[M+H]+651.3150, actual value: 651.3160.

[0128] Example 2

[0129] (E)-N-(2-aminophenyl)-3-(4-(4-(2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)phenyl)acrylamide (Compound Z2)

[0130] Step 1: Preparation of (E)-tert-butyl(2-(3-(4-(4-(2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)methyl)phenyl)acrylamido)phenyl)carbamate (Intermediate 8)

[0131]

[0132] Intermediate 6 (0.24 mmol, 150 mg) was dissolved in DMF, and HATU (0.36 mmol, 136.8 mg) and DIEA (0.36 mmol, 46 mg) were added, and finally the raw material amine (0.24 mmol, 49.5 mg) was added. The mixture was stirred at room temperature and reacted for 30 min. The reaction progress was monitored by TLC. After the reaction was complete, a small amount of water was added to the reaction solution, and the mixture was extracted with EA, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 185 mg of the product with a yield of 93.4%.

[0133] Step 2: Preparation of compound Z2

[0134]

[0135] HCl dioxane (10 ml) was added to raw material A (150 mg, 0.18 mmol), and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC. After the reaction was complete, the solid portion was washed three times with DCM, and then MeOH was added to dissolve the solid portion. The mixture was concentrated under reduced pressure to obtain 112 mg of the product with a yield of 82%.

[0136] Characterization data: 1 H NMR (600MHz, DMSO-d6) δ11.02 (s, 1H), 8.23 (dq, J = 6.9, 4.2Hz, 1H), 7.90 (dd ,J=6.1,3.1Hz,1H),7.83–7.59(m,8H),7.54(d,J=7.9Hz,1H),7.42(t,J=7.7 Hz,1H),7.31(t,J=7.6Hz,1H),7.12(d,J=15.8Hz,1H),5.41–4.70(m,7H),4. 49 (s, 6H), 4.33–3.83 (m, 10H), 3.51 (d, J = 7.4Hz, 2H), 1.47 (t, J = 7.4Hz, 3H).

[0137] HRMS (ESI+) m / z theoretical value: C 40 H 43 N 11 O3[M+H]+726.3623, actual value: 726.3571.

[0138] Example 3

[0139] 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-N-hydroxy-6-oxohexanamide (Compound Z3)

[0140] Step 1: Preparation of methyl 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-6-oxohexanoate (Intermediate 9)

[0141]

[0142] Intermediate 4 (200 mg, 0.42 mmol) was dissolved in DMF (5 ml), and HATU (240 mg, 0.63 mmol) and DIEA (81 mg, 0.63 mmol) were added, followed by the addition of raw material 6-methoxy-6-oxohexanoic acid (67 mg, 0.42 mmol). The mixture was stirred at room temperature for 30 min. After the reaction was complete, a small amount of water was added, and the mixture was extracted twice with EA, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 332 mg of crude product. After plate purification, 186 mg of the product was obtained with a yield of 72%.

[0143] Step 2: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-6-oxohexanoic acid (Intermediate 10)

[0144]

[0145] NaOH (60 mg, 1.5 mmol) was dissolved in water (2 ml) and methanol (4 ml), and intermediate 9 (186 mg, 0.3 mmol) was added. The mixture was stirred at 70°C for 2 h. The reaction progress was monitored by TLC. After the reaction was complete, the pH was adjusted to 5 with 1.5-2.5 N hydrochloric acid. The reaction solution was spin-dried and directly used in the next step.

[0146] Step 3: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-6-oxo-N-((tetrahydro-2H-pyran-2-yl)oxy)hexanamide (Intermediate 11)

[0147]

[0148] Intermediate 10 (180 mg, 0.3 mmol) was dissolved in THF (4 ml), and HATU (170 mg, 0.45 mmol) and DIEA (58 mg, 0.45 mmol) were added and dissolved by ultrasonication. Then, the raw material O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (35 mg, 0.3 mmol) was added and the reaction was stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction was complete, a small amount of water was added, and the mixture was extracted twice with EA, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 192 mg of crude product, which was purified by plate climbing to obtain 115 mg of the final product with a yield of 55%.

[0149] Step 4: Preparation of compound Z3

[0150]

[0151] Intermediate 11 (115 mg, 0.16 mmol) was dissolved in methanol (4 ml), and 6N aqueous hydrochloric acid solution (0.2 ml) was added. The mixture was stirred at room temperature for 30 min and the reaction progress was monitored by TLC. After the reaction was complete, the pH was adjusted to 8 with sodium bicarbonate solution. The reaction solution was spin-dried, filtered, and purified by plate climbing to obtain 65 mg of the final product with a yield of 64%.

[0152] Characterization data: 1 H NMR(600MHz,DMSO-d6)δ10.37(s,1H),8.68(s,1H),8.10–7.94(m,1H),7.70 –7.59(m,1H),7.26(tt,J=7.3,5.6Hz,2H),4.88(s,2H),4.17(d,J=59.5Hz,6 H),3.79–3.67(m,8H),3.47(s,3H),3.26(q,J=7.4Hz,2H),2.34(dt,J=14.9, 6.9Hz, 2H), 1.96 (t, J = 7.0Hz, 2H), 1.55–1.44 (m, 4H), 1.33 (t, J = 7.4Hz, 3H).

[0153] HRMS (ESI+) m / z theoretical value: C 30 H 38 N 10 O5[M+H]+619.3099, actual value: 619.3083.

[0154] Example 4

[0155] 8-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-N-hydroxy-8-oxooctamide (Compound Z4)

[0156] Step 1: Preparation of methyl 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-6-oxooctanoate (Intermediate 12)

[0157]

[0158] Intermediate 4 (200 mg, 0.42 mmol) was dissolved in DMF (5 ml), and HATU (240 mg, 0.63 mmol) and DIEA (81 mg, 0.63 mmol) were added, followed by the addition of raw material 6-methoxy-6-oxooctanoic acid (80 mg, 0.42 mmol). The mixture was stirred at room temperature for 30 min. After the reaction was complete, a small amount of water was added, and the mixture was extracted twice with EA, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 340 mg of a crude product. After plate purification, 173 mg of the product was obtained with a yield of 64%.

[0159] Step 2: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-6-oxooctanoic acid (Intermediate 13)

[0160]

[0161] NaOH (54 mg, 1.35 mmol) was dissolved in water (2 ml) and methanol (4 ml), and intermediate 12 (173 mg, 0.27 mmol) was added. The mixture was stirred at 70°C for 2 h. The reaction progress was monitored by TLC. After the reaction was complete, the pH was adjusted to 5 with 1.5-2.5 N hydrochloric acid. The reaction solution was spin-dried and directly used in the next step.

[0162] Step 3: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-6-oxo-N-((tetrahydro-2H-pyran-2-yl)oxy)octanamide (Intermediate 14)

[0163]

[0164] Intermediate 13 (170 mg, 0.27 mmol) was dissolved in THF (4 ml), and HATU (154 mg, 0.41 mmol) and DIEA (104 mg, 0.81 mmol) were added and dissolved by ultrasonication. Then, the raw material O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (32 mg, 0.27 mmol) was added and the reaction was stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction was complete, a small amount of water was added, and the mixture was extracted twice with EA, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 165 mg of a crude product. The final product was purified by plate climbing to obtain 85 mg with a yield of 43%.

[0165] Step 4: Preparation of compound Z4

[0166]

[0167] Intermediate 14 (85 mg, 0.12 mmol) was dissolved in methanol (4 ml), and 6N aqueous hydrochloric acid solution (0.2 ml) was added. The mixture was stirred at room temperature for 30 min and the reaction progress was monitored by TLC. After the reaction was complete, the pH was adjusted to 8 with sodium bicarbonate solution. The reaction solution was spin-dried, filtered, and purified by plate climbing to obtain 36 mg of the final product with a yield of 46%.

[0168] Characterization data: 1 H NMR(600MHz,DMSO-d6)δ10.34(s,1H),8.66(s,1H),8.01(d,J=7.2Hz,1H),7.64 (d,J=7.1Hz,1H),7.26(q,J=6.5Hz,2H),4.88(s,2H),4.49–3.98(m,6H),3.84–3 .66(m,8H),3.51–3.39(m,3H),3.21(dd,J=45.2,6.1Hz,2H),2.33(dt,J=15.2,7 .3Hz,2H),1.93(t,J=7.6Hz,2H),1.48(q,J=8.5,7.8Hz,3H),1.39–1.18(m,8H).

[0169] HRMS (ESI+) m / z theoretical value: C 32 H 43 N 10 O5[M+H]+647.3412, actual value: 647.3400.

[0170] Example 5

[0171] 10-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-N-hydroxy-10-oxodecanamide (Compound Z5)

[0172] Step 1: Preparation of methyl 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-6-oxodecanoate (Intermediate 15)

[0173]

[0174] Intermediate 4 (200 mg, 0.42 mmol) was dissolved in DMF (5 ml), and HATU (240 mg, 0.63 mmol) and DIEA (81 mg, 0.63 mmol) were added, followed by the addition of raw material 6-methoxy-6-oxodecanoic acid (91 mg, 0.42 mmol). The mixture was stirred at room temperature for 30 min. After the reaction was complete, a small amount of water was added, and the mixture was extracted twice with EA, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 280 mg of a crude product. After plate purification, 130 mg of the product was obtained with a yield of 46%.

[0175] Step 2: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-6-oxodecanoic acid (Intermediate 16)

[0176]

[0177] NaOH (40 mg, 0.97 mmol) was dissolved in water (2 ml) and methanol (4 ml), and intermediate 15 (130 mg, 0.2 mmol) was added. The mixture was stirred at 70°C for 2 h. The reaction progress was monitored by TLC. After the reaction was complete, the pH was adjusted to 5 with 1.5-2.5 N hydrochloric acid. The reaction solution was spin-dried and directly used in the next step.

[0178] Step 3: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)-6-oxo-N-((tetrahydro-2H-pyran-2-yl)oxy)decanamide (Intermediate 17)

[0179]

[0180] Intermediate 16 (127 mg, 0.19 mmol) was dissolved in THF (4 ml), and HATU (110 mg, 0.29 mmol) and DIEA (74 mg, 0.58 mmol) were added and dissolved by ultrasonication. Raw material B (23 mg, 0.19 mmol) was added and the reaction was stirred at room temperature for 30 min. The reaction progress was monitored by TLC. After the reaction was complete, a small amount of water was added, and the mixture was extracted twice with EA, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by plate climbing to obtain 122 mg of the final product with a yield of 84%.

[0181] Step 4: Preparation of compound Z5

[0182]

[0183] Intermediate 17 (122 mg, 0.16 mmol) was dissolved in methanol (8 ml), and 6N aqueous hydrochloric acid solution (2 ml) was added. The mixture was stirred at room temperature for 30 min and the reaction progress was monitored by TLC. After the reaction was complete, the pH was adjusted to 8 with sodium bicarbonate solution. The reaction solution was spin-dried and filtered to obtain 102 mg of the product with a yield of 94%.

[0184] Characterization data: 1 H NMR(600MHz,DMSO-d6)δ10.36(d,J=6.0Hz,1H),8.66(s,1H),8.13–7.86(m,1H), 7.78–7.53(m,1H),7.36–7.16(m,2H),4.88(s,2H),4.57–3.96(m,6H),3.79–3.69 (m,8H),3.46(t,J=5.1Hz,1H),3.26(t,J=7.4Hz,2H),2.38–2.26(m,2H),1.93(t, J=7.3Hz,2H),1.50–1.43(m,4H),1.33(t,J=7.4Hz,3H),1.24(d,J=11.8Hz,10H).

[0185] Example 6

[0186] (E)-3-(4-(4-(2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-1-yl)methylphenyl)-N-hydroxyacrylamide (Compound Z6)

[0187] Step 1: Preparation of tert-butyl 4-((2-chloro-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazine-1-carboxylate (Intermediate 18)

[0188] Boc-piperazine (1.233 g, 6.619 mmol) was dissolved in acetonitrile and potassium carbonate (2.740 g, 19.85 mmol). After stirring for 5 minutes, 4-(2-chloro-8-(chloromethyl)-9-methyl-9H-purin-6-yl)morpholine (2 g, 6.619 mmol) was added and the reaction was stirred at room temperature for 4 hours. After sampling, water was added, and the mixture was extracted with EA and plated. After the reaction was substantially complete, water was added to produce solid precipitation. The reaction mixture was filtered to obtain a filter cake. The filter cake was dissolved in a mixed solvent of DCM and MeOH, dried over anhydrous sodium sulfate, and spin-dried to obtain 1.29 g of the product.

[0189] Step 2: Preparation of tert-butyl 4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazine-1-carboxylate (Intermediate 19)

[0190]

[0191] Intermediate 18 (1.500 g, 3.319 mmol) was dissolved in dioxane, and the starting materials 2-ethyl-1H-benzo[d]imidazole (485 mg, 3.319 mmol), Pd(dba) (343 mg, 0.332 mmol), XPhos (316 mg, 0.664 mmol), and CsCO (2.163 g, 6.638 mmol) were added in that order. The reaction mixture was refluxed at 110°C under nitrogen for 5 hours until the starting materials were fully converted. The reaction mixture was extracted with EA and water. The organic solution above was plated, indicating near-complete conversion of the starting materials. EA, water extraction, and column chromatography yielded 1.293 g of the product, a 69% yield.

[0192] Step 3: Preparation of 1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazine hydrochloride (Intermediate 20)

[0193]

[0194] Intermediate 19 (2.708 g, 4.82 mmol) was added to a mixed solvent of HCl and 1,4-dioxane and stirred at room temperature for 20 minutes. The developing solvent was DCM:MeOH = 5:1. The starting materials reacted substantially completely, with a single product spot. Dichloromethane and methanol were added to separate the solids. The reaction solution was spin-dried and then dichloromethane was added again to precipitate the solid. The dichloromethane layer was removed and spin-dried again to yield 1.859 g of the solid product, a yield of 77%.

[0195] Step 4: Preparation of (E)-methyl 3-(4-(4-(2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)methylphenyl)acrylate (Intermediate 21)

[0196]

[0197] Intermediate 20 (200 mg, 0.402 mmol) was dissolved in acetonitrile (5 ml), and methyl 4-bromomethylcinnamate (102.45 mg, 0.402 mmol) and potassium carbonate (166.50 mg, 1.205 mmol) were added. The mixture was stirred and refluxed at 85°C. After sampling, DCM was added to the plate using a developing solvent of DCM:MeOH = 10:1. The raw materials were essentially reacted. The solvent was dried by spin-drying, and the mixture was extracted three times with water and EA. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and then spin-dried. The product (100 mg) was obtained by scraping with a plate, with a yield of 40%.

[0198] Step 5: Preparation of (E)-3-(4-(4-(2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-oxopiperazin-1-yl)methylphenyl)acrylic acid (Intermediate 22)

[0199]

[0200] Intermediate 21 (100 mg, 0.157 mmol) and LiOH monohydrate (19.77 mg, 0.472 mmol) were dissolved in a mixed solvent (THF:MeOH:H2O = 4:4:1) and stirred at reflux at 70°C for 2 hours. After sampling, EA was added and the developing solvent was DCM:MeOH = 10:1. The raw materials were essentially reacted. The acetonitrile and methanol were dried to a water-only level. Hydrochloric acid was added while stirring to acidify the mixture. A white precipitate precipitated and easily adhered to the wall of the flask. The solid product was isolated, dissolved in dichloromethane and methanol, and then vortexed until semi-dry. It was then washed with ethyl acetate. Filtered with suction, the filter cake was dried to obtain a solid product, 80 mg, with a yield of 82%.

[0201] Step 6: Preparation of (E)-3-(4-((4-(2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazin-1-yl)methyl)phenyl)-N-(((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (Intermediate 23)

[0202]

[0203] The raw material O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (18.84 mg, 0.16 mmol) was dissolved in DMF, and intermediate 22 (100 mg, 0.16 mmol), HATU (91.74 mg, 0.242 mmol), and DIEA (31.18 mg, 0.242 mmol) were added and reacted at room temperature for 3 hours. A sample was taken and extracted with water and EA using a developing solvent of DCM:MeOH = 10:1. The raw material was essentially reacted. Water and EA were added to extract, and DMF was removed as much as possible. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and dried by spin drying. The product was scraped to obtain 46 mg, with a yield of 40%.

[0204] Step 7: Preparation of Compound Z6

[0205]

[0206] Intermediate 23 (40 mg, 0.055 mmol) was dissolved in 6 mol / L aqueous hydrochloric acid. The reaction was allowed to proceed for half an hour, and the reaction was monitored by a microplate reader. After sampling, extraction with DCM was performed, and the developing solvent was a 5:1 ratio of DCM:MeOH. The reaction was essentially complete. Sodium hydroxide solution was added to adjust the base, and a solid precipitated. The reaction mixture was filtered, and the filter cake was dried to obtain 29.4 mg of the solid, a yield of 83%.

[0207] Representation structure: 1 H NMR (400MHz, DMSO-d6) δ8.02(d,J=6.9Hz,1H),7.63(d,J=6.6Hz,1H),7.46(dd,J=28.2,11.1Hz,3H),7.35–7.12(m,4H),6.49(d,J=15.5 Hz, 1H), 4.24 (s, 4H), 3.78 (dd, J = 13.3, 8.7Hz, 10H), 3.46 (s, 2H), 3.26 (q, J = 7.4Hz, 2H), 2.44 (d, J = 41.3Hz, 8H), 1.33 (t, J = 7.3Hz, 3H).

[0208] HRMS (ESI+) m / z theoretical value: C 34 H 40 N 10 O3[M+H]+637.3357, actual value: 637.3346.

[0209] Example 7

[0210] 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-1-yl)-N-hydroxy-6-oxohexanamide (Compound Z8)

[0211] Step 1: Preparation of methyl 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazin-1-yl)-6-oxohexanoate (Intermediate 24)

[0212]

[0213] The raw material 6-methoxy-6-oxohexanoic acid (62.66 mg, 0.391 mmol) was dissolved in DMF, and HATU (223.14 mg, 0.587 mmol) and DIEA (75.84 mg, 0.587 mmol) were added in sequence. After stirring for 5 minutes, intermediate 20 (200 mg, 0.391 mmol) was added and reacted for about two hours. After sampling, EA and water were added for extraction. The organic layer was spot-blotted with a developing solvent of DCM:MeOH = 10:1. The raw materials were basically reacted. Appropriate amount of water and EA were added to the reaction solution for extraction, and the organic phase was collected three times. The product content in the water and organic phase was monitored by spotting. Anhydrous sodium sulfate was added for drying, spin-drying, and scraping to obtain 100 mg of product, with a yield of 42%.

[0214] Step 2: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazin-1-yl)-6-oxohexanoic acid (Intermediate 25)

[0215]

[0216] Intermediate 24 (100 mg, 0.165 mmol) and LiOH monohydrate (20.29 mg, 0.495 mmol) were dissolved in a mixed solvent (THF:MeOH:H2O = 4:4:1) and stirred at reflux at 70°C for 2 hours. After sampling, EA was added and the developing solvent was DCM:MeOH = 10:1. The raw materials were essentially reacted. The acetonitrile and methanol were dried to the point where only water remained. Hydrochloric acid was added while stirring to acidify the mixture. A white precipitate precipitated and easily adhered to the wall of the bottle. The solid product was separated, dissolved in dichloromethane and methanol, and then vortexed until semi-dry. It was then washed with ethyl acetate. Filtered with suction, the filter cake was dried to obtain a solid product, 70 mg, with a yield of 70%.

[0217] Step 3: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazin-1-yl)-6-oxo-N-((tetrahydro-2H-pyran-2-yl)oxy)hexanamide (Intermediate 26)

[0218]

[0219] The raw material O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (13.59 mg, 0.116 mmol) was dissolved in DMF, followed by the addition of HATU (66.18 mg, 0.174 mmol) and DIEA (22.49 mg, 0.174 mmol). After stirring for 5 minutes, the intermediate 25 (70 mg, 0.116 mmol) was added and allowed to react for approximately two hours. After sampling, the mixture was extracted with EA and water. The organic layer was stripped using a developing solvent of DCM:MeOH = 10:1. The raw materials were essentially reacted. The reaction solution was extracted with appropriate amounts of water and EA, and the extraction was repeated three times. The organic phase was collected. The product content in the aqueous and organic phases was monitored by stripping. The mixture was dried over anhydrous sodium sulfate, spin-dried, and scraped to obtain 46 mg of the product, with a yield of 57%.

[0220] Step 4: Preparation of Compound Z8

[0221]

[0222] Intermediate 26 (40 mg, 0.067 mmol) was dissolved in 6 mol / L aqueous hydrochloric acid. The reaction was allowed to proceed for half an hour, and the reaction was monitored by a microplate reader. After sampling, DCM extraction was performed, and the developing solvent was a 5:1 ratio of DCM:MeOH. The reaction was essentially complete. Sodium hydroxide solution was added to adjust the base, and a solid precipitated. The reaction mixture was filtered, and the filter cake was dried to obtain 30 mg of the solid, a yield of 75%.

[0223] Representation structure: 1 H NMR(600MHz,DMSO-d6)δ10.40(d,J=18.3Hz,1H),8.70(s,1H),8.02(d,J=7.6Hz,1H), 7.84–7.58(m,1H),7.26(q,J=6.8,6.2Hz,2H),4.53–4.00(m,3H),3.95–3.67(m,7H), 3.46(s,8H),3.27(q,J=7.4Hz,2H),2.44(dt,J=28.2,5.0Hz,3H),2.29(t,J=7.1Hz,2 H), 1.96 (t, J = 6.9Hz, 2H), 1.47 (ddt, J = 30.3, 15.6, 7.4Hz, 4H), 1.34 (t, J = 7.4Hz, 3H).

[0224] Example 8

[0225] 8-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-1-yl)-N-hydroxy-8-oxooctamide (Compound Z9)

[0226] Step 1: Preparation of methyl 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazin-1-yl)-6-oxooctanoate (Intermediate 27)

[0227]

[0228] The raw material 6-methoxy-6-oxooctanoic acid (132.40 mg, 0.704 mmol) was dissolved in DMF, followed by the addition of HATU (401.2 mg, 1.06 mmol) and DIEA (135.2 mg, 1.06 mmol). After stirring for 5 minutes, intermediate 20 (350 mg, 0.704 mmol) was added and allowed to react for about two hours. After sampling, EA and water were added for extraction. The organic layer was spot-blotted using a developing solvent of DCM:MeOH = 30:1. The raw materials were essentially reacted. Appropriate amounts of water and EA were added to the reaction solution for extraction, repeated three times, and the organic phase was collected. The product content in the water and organic phases was monitored by spotting. The product was dried over anhydrous sodium sulfate, spin-dried, and scraped to obtain 215 mg of the product, with a yield of 48%.

[0229] Step 2: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazin-1-yl)-6-oxooctanoic acid (Intermediate 28)

[0230]

[0231] Intermediate 27 (215 mg, 0.398 mmol) and NaOH (79.53 mg, 1.988 mmol) were dissolved in a mixed solvent (MeOH:H2O = 2:1) and stirred at reflux at 70°C for 2 hours. After sampling, EA was added and the developing solvent was DCM:MeOH = 10:1. The raw materials were essentially reacted. The acetonitrile and methanol were dried to a concentration of only water. Hydrochloric acid was added while stirring to adjust the pH to 5-6. The reaction solution was dried and used directly in the next step. The amount of other raw materials in the next step was calculated based on the theoretical yield.

[0232] Step 3: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazin-1-yl)-6-oxo-N-((tetrahydro-2H-pyran-2-yl)oxy)octanamide (Intermediate 29)

[0233]

[0234] The raw material O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (46.63 mg, 0.398 mmol) was dissolved in DMF, followed by the addition of HATU (227.00 mg, 0.597 mmol) and DIEA (154.31 mg, 0.597 mmol). After stirring for 5 minutes, intermediate 28 (245.70 mg, 0.398 mmol) was added and allowed to react for approximately two hours. After sampling, the mixture was extracted with EA and water. The organic layer was stripped using a developing solvent of DCM:MeOH = 10:1. The raw material was essentially reacted. The reaction mixture was extracted with appropriate amounts of water and EA, and the extraction was repeated three times. The organic phase was collected. The product content in the aqueous and organic phases was monitored by stripping. The mixture was dried over anhydrous sodium sulfate, spin-dried, and scraped to yield 130 mg of the product, a yield of 57%.

[0235] Step 4: Preparation of compound Z9

[0236]

[0237] Intermediate 29 (65 mg, 0.067 mmol) was dissolved in 6 mol / L aqueous hydrochloric acid. The reaction was allowed to proceed for half an hour, and the reaction was monitored by a microplate reader. After sampling, DCM extraction was performed, and the developing solvent was a 5:1 ratio of DCM:MeOH. The reaction was essentially complete. Sodium hydroxide solution was added to adjust the base, and a solid precipitated. The reaction mixture was filtered, and the filter cake was dried to obtain 30 mg of the solid, a yield of 63%.

[0238] Representation structure: 1H NMR(600MHz,DMSO-d6)δ10.37(s,1H),8.69(s,1H),8.19–7.90(m,1H),7.73–7.55(m,1H) ),7.26(dt,J=11.9,7.4Hz,2H),4.52–4.05(m,3H),3.90–3.71(m,7H),3.44(d,J=12.8Hz ,8H),3.27(q,J=7.4Hz,2H),2.44(dt,J=26.7,4.9Hz,3H),2.27(t,J=7.5Hz,2H),1.94( t,J=7.4Hz,2H),1.46(tt,J=12.7,6.3Hz,4H),1.34(t,J=7.4Hz,3H),1.30–1.18(m,4H).

[0239] Example 9

[0240] 10-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-1-yl)-N-hydroxy-10-oxodecanamide (Compound Z10)

[0241] Step 1: Preparation of methyl 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazin-1-yl)-6-oxodecanoate (Intermediate 30)

[0242]

[0243] The raw material 6-methoxy-6-oxodecanoic acid (84.51 mg, 0.391 mmol) was dissolved in DMF, followed by the addition of HATU (223.14 mg, 0.587 mmol) and DIEA (75.84 mg, 0.587 mmol). After stirring for 5 minutes, intermediate 20 (200 mg, 0.391 mmol) was added and allowed to react for about two hours. After sampling, EA and water were added for extraction. The organic layer was spot-blotted using a developing solvent of DCM:MeOH = 30:1. The raw materials were essentially reacted. Appropriate amounts of water and EA were added to the reaction solution for extraction, repeated three times, and the organic phase was collected. The product content in the water and organic phases was monitored by spotting. The product was dried over anhydrous sodium sulfate, spin-dried, and scraped to obtain 115 mg of the product, with a yield of 40%.

[0244] Step 2: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazin-1-yl)-6-oxodecanoic acid (Intermediate 31)

[0245]

[0246] Intermediate 30 (115 mg, 0.174 mmol) and NaOH (34.8 mg, 0.87 mmol) were dissolved in a mixed solvent (MeOH:H2O = 2:1) and stirred at reflux at 70°C for 2 hours. After sampling, EA was added and the developing solvent was DCM:MeOH = 10:1. The raw materials were essentially reacted. The acetonitrile and methanol were dried to a concentration of only water. Hydrochloric acid was added while stirring to adjust the pH to 5-6. The reaction solution was dried and used directly in the next step. The amount of other raw materials in the next step was calculated based on the theoretical yield.

[0247] Step 3: Preparation of 6-(4-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)-3-piperazin-1-yl)-6-oxo-N-((tetrahydro-2H-pyran-2-yl)oxy)decanamide (Intermediate 32)

[0248]

[0249] The raw material O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (20.38 mg, 0.174 mmol) was dissolved in DMF, followed by the addition of HATU (99.24 mg, 0.261 mmol) and DIEA (33.73 mg, 0.261 mmol). After stirring for 5 minutes, intermediate 31 (115 mg, 0.174 mmol) was added and allowed to react for approximately two hours. After sampling, the mixture was extracted with EA and water. The organic layer was spot-blotted using a developing solvent of DCM:MeOH = 10:1. The raw material was essentially reacted. The reaction mixture was extracted with appropriate amounts of water and EA, repeated three times, and the organic phase was collected. The product content in the aqueous and organic phases was monitored by spotting. The mixture was dried over anhydrous sodium sulfate, spin-dried, and scraped to yield 50 mg of the product, a yield of 35%.

[0250] Step 3: Preparation of compound Z10

[0251]

[0252] Intermediate 32 (50 mg, 0.067 mmol) was dissolved in 6 mol / L aqueous hydrochloric acid. The reaction was allowed to proceed for half an hour, and the reaction was monitored by a microplate reader. After sampling, extraction with DCM was performed, and the developing solvent was a 5:1 ratio of DCM to MeOH. The reaction was essentially complete. Sodium hydroxide solution was added to adjust the base, and a solid precipitated. The reaction mixture was filtered, and the filter cake was dried to obtain 25 mg of the solid, a 50% yield.

[0253] Representation structure: 1H NMR(600MHz,DMSO-d6)δ10.32(s,1H),8.65(s,1H),8.15–7.97(m,1H),7.69–7.56(m,1H),7. 26(h,J=7.7,7.0Hz,2H),4.44–4.10(m,3H),3.87–3.76(m,7H),3.44(dd,J=11.0,5.8Hz,8H) ,3.27(d,J=7.4Hz,2H),2.44(dt,J=26.5,5.0Hz,3H),2.27(q,J=8.9,8.2Hz,2H),1.93(t,J= 7.4Hz, 2H), 1.47 (q, J = 12.2, 9.7Hz, 4H), 1.34 (t, J = 7.4Hz, 3H), 1.24 (dd, J = 9.6, 4.9Hz, 8H).

[0254] The beneficial effects of the present invention are demonstrated below through specific experimental examples.

[0255] Experimental Example 1: Affinity test of compound and target protein

[0256] The inhibition rate and IC of the compounds on PI3K family protein PI3Kδ and HDAC family protein HDAC6 50 The assay was performed by Wise Chemical Research Co., Ltd. Positive control compounds PI103 and SAHA were used as controls.

[0257] 1.1 The inhibition rate of the compound on PI3Kδ was detected using ADP-Glo Luminescent Assay. The specific experimental method is as follows:

[0258] 1.1.1 Compound preparation

[0259] 1) Compound dilution

[0260] The initial concentration of the compound tested on the kinase is 1 μM, and it needs to be prepared to a 100-fold concentration, i.e. 100 μM.

[0261] Add 99 μL of 100% DMSO to the second well of a 384-well Echo plate, followed by 1 μL of a 10 mM compound solution to create a 100 μM compound solution. Add 30 μL of 100% DMSO to the remaining wells. Add 15 μL of compound from the second well to the third well, and continue with the three-fold dilutions for a total of eight concentrations. Use an automated micropipette (Precision PRC384U). Transfer 40 μL of 100% DMSO to two empty wells, designated as the Max and Min wells.

[0262] 2) Transfer compounds to the reaction plate

[0263] An Echo 650 was used to transfer 50 nl of compound into a 384-well assay plate.

[0264] 1.1.2 Prepare 1x kinase buffer

[0265] PI3Kδ 1x Kinase Buffer

[0266] 50 mM HEPES, pH 7.5

[0267] 3mM MgCl2

[0268] 1mM EGTA

[0269] 100mM NaCl

[0270] 0.03% CHAPS

[0271] 2mM DTT

[0272] 1.1.3 Kinase reaction and termination

[0273] 1) Add kinase to 1x kinase buffer to form a 2x kinase solution;

[0274] 2) Transfer 2.5 μL of the 2x kinase solution to each well of a 384-well plate. Add 1x kinase buffer to the negative control wells. Incubate at room temperature for 10 minutes.

[0275] 3) Add substrate and ATP to 1x kinase buffer to form a 2x substrate solution;

[0276] 4) Transfer 2.5 μL of 2x substrate solution to the reaction wells of a 384-well plate;

[0277] 5) Incubate at room temperature for 60 minutes;

[0278] 6) Transfer 5 μL of ADP-Glo reagent 1 to a 384-well plate to terminate the reaction;

[0279] 7) react at room temperature for 120 minutes;

[0280] 8) Transfer 10 μL of ADP-Glo reagent 2 to a 384-well plate;

[0281] 9) Leave at room temperature for 30 minutes.

[0282] 1.1.4 Data Reading

[0283] The RLU values were read on the Envision2104 Multilable Reader.

[0284] 1.1.5 Data Calculation

[0285] 1) Copy the RLU reading

[0286] 2) Convert the above data into inhibition percentage using the formula

[0287] Percent inhibition=(max-sample RLU) / (max-min)*100

[0288] "min" is the reading of the control well without enzyme reaction; "max" is the reading of the control well with DMSO added

[0289] 3) Data were imported into MS Excel and curve fitted using XLFit Excel add-in version 5.4.0.8. The fitting formula was: Y = Bottom + (Top - Bottom) / (1 + (IC50 / X)^HillSlope)

[0290] 1.2 The inhibition rate of the compound on HDAC6 was detected by Fluroscence assay. The specific experimental method is as follows:

[0291] 1.2.1 Prepare 1x buffer

[0292] Prepare 1x assay buffer (modified Tris buffer).

[0293] 1.2.2 Compound serial dilution:

[0294] Compounds were transferred to assay plates by Echo and diluted three-fold in 100% DMSO.

[0295] 1.2.3 Prepare enzyme solution in 1x assay buffer.

[0296] 1.2.4 Prepare the matrix solution:

[0297] Prepare substrate solution by adding trypsin and Ac peptide substrate in 1x assay buffer.

[0298] 1.2.5 Transfer 15 μL of enzyme solution to the assay plate, or 15 μL of 1x assay buffer for the low control, and incubate at room temperature for 15 minutes.

[0299] 1.2.6 Add 10 μL of substrate solution to each well to start the reaction.

[0300] 1.2.7 Kinetically read the plate on Envision with an excitation wavelength of 355 nm and an emission wavelength of 460 nm.

[0301] 1.2.8 Curve Fitting

[0302] Fit the data in Excel using equation (1) to obtain inhibition values

[0303] Equation (1): Inh% = (maximum signal) / (maximum minimum) * 100

[0304] The data were fitted in XL Fit using equation (2) to obtain IC50 values.

[0305] Equation (2): Y = bottom + (top - bottom) / (1 + (IC50 / X) * hillside)

[0306] Y is the percentage of inhibition and X is the compound concentration.

[0307] The results of the inhibitory effects of the compounds of the present invention on PI3Kδ and HDAC6 activities are shown in Table 1 below. 50 Value ≤ 5nM; letter B indicates 5nM ≤ IC 50 Value ≤ 20nM; letter C indicates 20nM ≤ IC 50 Value ≤ 100nM; letter C indicates 100nM ≤ IC 50 Value ≤1000nM.

[0308] Table 1 Statistical results of the inhibitory effects of the compounds in the examples on PI3Kδ and HDAC6 activities

[0309]

[0310]

[0311] As can be seen from Table 1, the inhibitory activity against PI3Kδ of the compounds of the present invention is comparable to or superior to that of the positive control compound PI103 (a known single-target PI3Kδ inhibitor). The inhibitory activity against HDAC6 of most compounds of the present invention is comparable to that of the positive control compound SAHA (a known single-target HDAC6 inhibitor).

[0312] More importantly, most of the compounds of the present invention showed inhibitory effects on PI3Kδ and HDAC6 at nanomolar concentrations. Among them, the preferred compounds Z1, Z5, and Z9 had an IC of 50 The value is ≤5nM, and the IC 50 The values are between 5 nM and 20 nM, indicating that the compounds of the present invention can be used to prepare dual-target inhibitors that simultaneously inhibit PI3Kδ and HDAC6.

[0313] Experimental Example 2: Test of the Cell Proliferation Inhibitory Activity of Compounds

[0314] The half inhibitory concentration (IC50) of the compounds on various tumor cells was investigated by MTS assay and Cell Titer-Glo cell viability assay. 50 ).

[0315] The tumor cells used in the experiment were: Jeko-1 and CA-46 cells.

[0316] Experimental method: Collect cells in the logarithmic growth phase in a centrifuge tube, place the centrifuge tube in a centrifuge, set it to 1200r / min, and centrifuge for 3 minutes. Resuspend the cells in fresh culture medium, count the resuspended cells, and dilute them to a cell concentration of 8×10 4 ~1×10 5 Cells were seeded into 96-well plates at a density of 100 μL per well (8,000 to 10,000 cells per well) using a cell suspension of 100 μL per well. The plates were then placed in an incubator for incubation. The drug was diluted to an appropriate concentration (typically threefold) and treated with 100 μL per well. Three replicates were set up for each drug concentration. Cells were treated with fresh culture medium containing 0.1% DMSO as a negative control. The 96-well plates were then incubated in the incubator for the desired time (24 h, 48 h, 72 h, 96 h, 120 h), then removed from the incubator. 20 μL of MTS solution was added to each well and incubated at 37°C for 2 to 4 hours (2 h is optimal). After the test solution and the color developer formed a colored solution, the absorbance at a wavelength of 492 nm was measured using a microplate reader. The absorbance (A) was proportional to the number of viable cells. The inhibitory rate of the drug on tumor cells was calculated according to the following formula: cell growth inhibition rate = (control group A495nm - experimental group A495nm) / control group A495nm × 100%. The relationship between cell growth inhibition rate and drug concentration was calculated using Graphpad Prism software, and the drug growth inhibition curve was fitted to calculate the half inhibitory concentration (IC 50 )value.

[0317] The proliferation inhibitory activity of the compounds of the present invention on non-Hodgkin's lymphoma cell lines CA-46 and JEKO-1 is shown in Table 2. Wherein, the letter A represents IC 50 Value ≤ 50nM; letter B indicates 50nM ≤ IC 50 Value ≤ 500nM; letter C indicates 500μM ≤ IC 50 Value ≤ 5 μM; letter D represents IC 50 Values >5 μM.

[0318] Table 2. Statistical results of the inhibitory activity of the compounds of the present invention on tumor cell proliferation

[0319]

[0320] According to the results in Table 2, the compounds of the present invention can effectively inhibit the proliferation of lymphoma cell lines, and the IC 50 The value can reach the nanomolar level.

[0321] In summary, the present invention provides a heterocyclic compound that exhibits excellent inhibitory activity against both PI3K and HDAC targets, making it a highly effective dual-target inhibitor. The compound of the present invention can be used to prepare medicaments for the prevention and / or treatment of diseases associated with PI3K and / or HDAC, and has promising application prospects.

Claims

1. A compound represented by formula II or a salt thereof: Formula II in, R1' is selected from C1~C3 alkyl; R 10 Selected from C1~C3 alkyl; R1 is LR a R b CONHR c , L is selected from none, C1~C3 alkylene, carbonyl; R a Selected from none, C1~C8 alkylene, phenyl; R b Selected from none, C2~C3 alkenylene; And L, R a 、R b Not at the same time is nothing; R c Selected from hydroxyl groups.

2. The compound or salt thereof according to claim 1, characterized in that: The R1 is 、 or .

3. The compound represented by formula III or its salt: Formula III in, R1' is selected from C1~C3 alkyl; R 10 Selected from C1~C3 alkyl; R1 is LR a R b CONHR c , L is a carbonyl group; R a Selected from C1~C8 alkylene; R b for nothing; R c Selected from hydroxyl groups.

4. The compound or salt thereof according to claim 3, characterized in that: The R1 is .

5. A compound or a salt thereof, characterized in that: The compound is one of the following compounds: 。 6. Use of the compound or salt thereof according to any one of claims 1 to 5 in the preparation of a PI3K inhibitor and / or an HDAC inhibitor.

7. The use according to claim 6, characterized in that: The PI3K inhibitor is a PI3Kδ inhibitor, and the HDAC inhibitor is an HDAC6 inhibitor.

8. Use of the compound or salt thereof according to any one of claims 1 to 5 in the preparation of a medicament for preventing and / or treating diseases associated with PI3K and / or HDAC.

9. The use according to claim 8, characterized in that: The disease associated with PI3K and / or HDAC is cancer.

10. The use according to claim 9, characterized in that: The cancer is lymphoma.

11. A pharmaceutical composition, characterized in that: The preparation is prepared by using the compound or salt thereof according to any one of claims 1 to 5 as an active ingredient and adding pharmaceutically acceptable excipients.

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