NSD2 degradation agent and application thereof

Abstract

The invention discloses an NSD2 degradation agent and application thereof. The NSD2 degradation agent comprises a compound as shown in a formula I, a formula II or a formula III or pharmaceutically acceptable salts, stereoisomers, prodrugs, solvates and deuterated compounds of the compound as shown in the formula I, the formula II or the formula III. According to the compounds shown in the formula I, the formula II or the formula III or pharmaceutically acceptable salts, stereoisomers, prodrugs, solvates and deuterated compounds of the compounds, SET structural domain ligands are combined with primary amine degradation ligands, adamantane or VHL E3 ligase ligands, and the structures shown in the formula I, the formula II or the formula III or the pharmaceutically acceptable salts, the stereoisomers, the prodrugs, the solvates and the deuterated compounds are formed. According to the compound, FBXO22 or VHL E3 ligase can be recruited to achieve degradation of NSD2, or degradation is achieved through an adamantane hydrophobic label, two subtypes of NSD2, namely a long subtype and RE-IIBP, can be degraded at the same time, and an excellent anti-tumor effect is achieved. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the field of targeted protein degradation technology, and specifically relates to a class of NSD2 degradation agents and uses thereof. Background Art

[0002] Nuclear receptor-binding SET domain protein (NSD) is a type of histone lysine methyltransferase. The SET domain is its catalytic domain. It belongs to the SETD2-Like family in the SET superfamily. There are three subtypes: NSD1, 2, and 3, which are involved in the dimethylation of lysine 36 of histone H3 (H3K36me2). The NSD2 subtype is also known as MMSET (MM, multiple myeloma refers to multiple myeloma) and WHSC1 (Wolf-Hirschhorn syndrome candidate 1). Due to alternative splicing, NSD2 has three expression products: long, short and RE-IIBP. Its conserved region is mainly composed of PWWP1, PWWP2 domains (proline-tryptophan-tryptophan-proline-rich structures), multiple PHD zinc finger structures, SET domains with catalytic functions, pre-SET (AWS), post-SET domains, HMG box structures and cysteine-histidine-rich C5HCH domains. RE-IIBP contains the SET domain but not the PWWP1 domain, and the long type contains the SET domain and the PWWP1 domain. NSD2 regulates the expression of multiple genes through epigenetic modification and plays an important physiological function. The absence or inactivation of NSD2 can lead to developmental abnormalities and disrupt the immune system, and is associated with the onset of congenital diseases such as Rauch-Steindl syndrome and Wolf-Hirschhorn syndrome. The overexpression and abnormal enhancement of NSD2 activity are closely related to the onset of various malignant blood diseases and malignant tumors (J Med Chem, 64 (2021) 14913-14929). In summary, NSD2 is an important target for cancer treatment.

[0003] The existing small molecule regulators targeting NSD2 are mainly divided into three categories: inhibitors acting on the SET domain, inhibitors acting on the PWWP1 domain, and degraders acting on the PWWP1 domain. Among them, NSD2 SET domain inhibitors and NSD2 PWWP1 domain inhibitors can inhibit the methylation catalytic activity of NSD2 protein for histone H3K36 site by occupying the SET domain and PWWP1 domain of NSD2 protein. However, the mechanism of action of inhibitors determines that they cannot regulate other non-enzyme functions of NSD2 and abnormal methylation of other histone sites. In addition, it is difficult for inhibitors to effectively intervene in NSD2 E1099K gain-of-function mutants in malignant tumors, while degraders can degrade such mutants, which has advantages in tumor therapeutics over inhibitors. Degraders based on PROTAC (proteolysis-targeting chimeras, protein degradation targeting consortium) technology can use the ubiquitin-protease system to degrade NSD2, thereby regulating the non-enzyme function of NSD2.

[0004] The three isoforms of NSD2, long, short and RE-IIBP, have complex non-enzymatic functions. For example, RE-IIBP can promote the progression of malignant tumors by interacting with the SMN splicing complex (PLoS One, 9 (2014) e99493), and can produce abnormal methylation at sites such as H3K4, H3K27, and H3K79, thereby promoting tumor proliferation and migration (Sci Rep, 5 (2015) 12485). These non-enzymatic functions of NSD2 proteins cannot be intervened by NSD2 inhibitors, but can be regulated by NSD2 degraders. However, since RE-IIBP does not contain the PWWP1 domain, existing degraders that act on the PWWP1 domain cannot degrade RE-IIBP. Such degraders reported in the literature only degrade the long and short isoforms of NSD2. At present, there is no degrader that can simultaneously degrade the RE-IIBP isoforms of NSD2. Therefore, existing technical means cannot effectively intervene in the non-enzymatic function of the NSD2 isoform RE-IIBP to promote cancer. Summary of the invention

[0005] The present invention relates to a class of NSD2 degraders based on SET domain ligands, which can effectively degrade RE-IIBP and long isomers of NSD2, downregulate H3K36 methylation levels, and inhibit the proliferation of malignant tumor cells. The present invention has good application prospects in treating cancer, diseases caused by abnormal NSD2, and other diseases mediated by SET family proteins.

[0006] The object of the present invention is to provide a degradation agent that can simultaneously and efficiently degrade the long-type and RE-IIBP-type isomers of NSD2.

[0007] The first aspect of the present invention is:

[0008] Provided is a compound or a pharmaceutically acceptable salt, stereoisomer, prodrug, solvate and deuterated compound thereof.

[0009] A second aspect of the present invention is:

[0010] Provided is a NSD2 degrader.

[0011] The third aspect of the present invention is:

[0012] Provided is a drug for treating diseases mediated by SET domain proteins.

[0013] Specifically, the technical solution adopted according to the first aspect of the present invention is:

[0014] A compound having a structure shown in Formula I, II, or III, or a pharmaceutically acceptable salt, stereoisomer, prodrug, solvate, or deuterated compound thereof:

[0015]

[0016] Among them, each R 1 independently selected from hydrogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl or hydroxy halide C 1-6 alkyl;

[0017] Each R 2 independently selected from hydrogen, halogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl or C 1-6 Alkoxy;

[0018] The linking group in formula I, II, and III is -(L 1 ) n -(L 2 ) n -(L 3 ) n -(L 4 ) n -(L 5 ) n -; among them, L 1 , L 2 , L 3 , L 4 , L 5 Each is independently selected from: -CH2-, -O-, -NH-, -CH2CH2O-, piperazine or 4-amino-piperidine; and each n is independently selected from an integer of 0-20.

[0019] According to the embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0020] The compound of the present invention or its pharmaceutically acceptable salt, stereoisomer, prodrug, solvate and deuterated compound is formed by combining the SET domain ligand with the primary amine degradation ligand, adamantane or VHL E3 ligase ligand to form the structure shown. The compound of the present invention can recruit FBXO22 or VHL E3 ligase to achieve degradation of NSD2, or achieve degradation through the adamantane hydrophobic tag, and can simultaneously degrade two subtypes of NSD2: long type and RE-IIBP. Among them, the adamantane hydrophobic tag can induce heat shock protein-mediated degradation of NSD2; the VHL ligand can recruit the VHL E3 ubiquitin ligase to achieve degradation of NSD2, and NSD2 is an important pathogenic protein that promotes the proliferation of multiple myeloma; at the same time, the primary amine compound of the present invention has good NSD2 degradation agent activity because the fatty primary amine has the function of recruiting FBXO22 E3 ligase.

[0021] Unlike previous NSD2 inhibitors that inhibit enzyme function by occupying catalytic sites or H3 binding sites, the compounds of the present invention, when used as NSD2 degraders, recruit E3 ligases through E3 ligands, induce ubiquitination degradation of target proteins, directly reduce the content of NSD2 and / or isomers, and achieve dual regulation of its enzymatic function and non-enzymatic function, and therefore have potential anti-tumor advantages.

[0022] In particular, the isoform RE-IIBP expressed by alternative splicing of NSD2 has a unique non-enzymatic function and is closely related to tumorigenesis. The preferred embodiment of the present invention can simultaneously achieve the degradation of NSD2-RE-IIBP and the long form of NSD2, which has not been reported in the existing literature and is significantly innovative. The preferred embodiment of the present invention, the NSD2 degrader, has a more potent inhibitory activity against multiple myeloma cell lines under the same experimental conditions compared with known inhibitors, and has less toxicity against non-tumor normal cell lines, and has significant therapeutic advantages.

[0023] The primary amine compounds of the present invention have good NSD2 degradation agent activity. Preferably, the primary amine compounds with S configuration have better effects. More preferably, the ND-311B with S configuration and 11 carbon chain length has the best activity. Too short chain length and too long chain length are not conducive to degradation activity.

[0024] In addition, the degradation agent activity and SET binding affinity of the R-configuration enantiomer ND-311A (enantiomer of ND-311B) of the present invention are relatively weak, which indicates that the degradation of NSD2 depends on the binding ability of the S-configuration SET ligand. The degradation activity of the short aliphatic chain compound ND-(303-309) with an S-configuration SET ligand is relatively weak, which indicates that the degradation of NSD2 requires the SET ligand and the aliphatic primary amine ligand of appropriate length to work together.

[0025] Examples ND-311P and ND-311M are tertiary amine and amide structures derived from ND-311B, and their degradation activity is relatively weak, which indicates that the NSD2 degradation induced by the preferred examples is primary amine-dependent.

[0026] According to one embodiment of the present invention, the compound of the present invention is a class of compounds based on SET domain ligands.

[0027] According to one embodiment of the present invention, the compound of the present invention or its pharmaceutically acceptable salt, stereoisomer, prodrug, solvate and deuterated compound is a NSD2 degrading agent.

[0028] According to one embodiment of the present invention, the pharmaceutically acceptable salt is: a salt of a suitable compound of the present invention and an organic acid, an organic base, an inorganic acid, or an inorganic base, such as: hydrochloric acid, trifluoroacetic acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, lactic acid, fumaric acid, maleic acid, methanesulfonic acid, nitric acid, hydrobromic acid, tartaric acid, ethanolamine, diethanolamine, N-ethylethanolamine, N-methylethanolamine, triethanolamine, diethylaminoethanol, 2-amino-2-methyl-n-propanol, dimethylaminoisopropanol, 2-amino-2-methylpropane dial, triisopropanolamine, ethylenediamine, hexamethylenediamine, morpholine, piperidine, piperazine, cyclohexylamine, tributylamine, dodecylamine, dimethyldodecylamine; triethylaminebenzylamine, dibenzylamine, N-methylpiperazine, 4-methylcyclohexylamine, N-methylmorpholine, methylamine, ethylamine, alkaline earth metals, alkali metals, aluminum, and transition metal salts. These salts can be prepared from the compounds represented by formula I, II, and III by known salt-forming methods.

[0029] According to one embodiment of the present invention, the carbon number of the long-chain fatty amine connected to the linking group in Formula I is 7 to 17. When the carbon number is less than 7 or greater than 17, the compound will have no activity or poor activity.

[0030] According to one embodiment of the present invention, the R 1 Selected from R 1 Selected from hydrogen or

[0031] According to one embodiment of the present invention, the R 2 Selected from hydrogen, fluorine or C1-6 Alkoxy.

[0032] According to one embodiment of the present invention, the linking group is selected from one of the following structures:

[0033] The first linking group, The second linking group, The third linking group, The fourth linking group, The fifth connecting group, The sixth connecting group, The seventh connecting group, The eighth connecting group, The ninth connecting group, A tenth connecting group.

[0034] According to one embodiment of the present invention, the compound of formula I, formula II or formula III comprises:

[0035]

[0036] According to one embodiment of the present invention, the compound comprises:

[0037]

[0038]

[0039]

[0040] According to one embodiment of the present invention, the compound is:

[0041]

[0042] The compound of this structure is named as compound ND-311B in the present invention. The compound ND-311B is a further preferred compound in the embodiments of the present invention. ND-311B exhibits stronger multiple myeloma cell inhibitory activity than known SET inhibitors, and can significantly change the H3K36 methylation level in multiple myeloma and prostate cancer cells, while achieving synchronous degradation of RE-IIBP and long isomers, showing good application prospects in disease treatment.

[0043] According to one embodiment of the present invention, the present invention synthesizes some of the compounds through the following synthetic route, and the compounds are named as compounds ND-(101-117), ND-(202-210), ND-(303-312), ND-311M or ND-311P in the present invention:

[0044]

[0045] Specifically, the second aspect of the present invention relates to a NSD2 degradation agent:

[0046] The NSD2 degrading agent includes one of the compounds described above.

[0047] A pharmaceutical composition comprises the compound or its pharmaceutically acceptable salt, stereoisomer, prodrug, solvate and deuterated compound, and also comprises pharmaceutically acceptable excipients.

[0048] According to one embodiment of the present invention, the excipient is selected from: lactose, microcrystalline cellulose, methylcellulose, sodium hydroxymethylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, starch, dextrin, cellulose derivatives, povidone, gelatin, polyethylene glycol, polyvinyl alcohol, brown sugar, distilled water, ethanol, starch slurry, antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the subject's blood, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers and preservatives.

[0049] According to one embodiment of the present invention, the pharmaceutical composition is suitable for enteral administration or parenteral administration.

[0050] Another aspect of the present invention also provides a drug for treating diseases mediated by SET domain proteins, including the compound or its pharmaceutically acceptable salt, stereoisomer, prodrug, solvate and deuterated compound as described in the first embodiment. Since the application adopts all the technical solutions of the above-mentioned compound or its pharmaceutically acceptable salt, stereoisomer, prodrug, solvate and deuterated compound, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments.

[0051] Preferably, a method for preparing a drug for treating diseases mediated by nuclear receptor binding SET domain protein type 2 (NSD2) is also provided.

[0052] According to one embodiment of the present invention, the disease is cancer.

[0053] According to one embodiment of the present invention, the disease is selected from: multiple myeloma, leukemia, prostate cancer, lymphoma, lung cancer, breast cancer, gastric cancer, liver cancer, head and neck malignancies, pancreatic cancer, and ovarian cancer.

[0054] According to one embodiment of the present invention, the diseases mediated by the SET domain protein include at least one of multiple myeloma, myeloid leukemia, lymphocytic leukemia, granulocytic leukemia, prostate cancer, non-Hodgkin's lymphoma, lung cancer, breast cancer, gastric cancer, skin cancer, cervical cancer, liver cancer, head and neck malignancies, pancreatic cancer, and ovarian cancer.

[0055] According to one embodiment of the present invention, the cancer is selected from: lymphoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine tumor, carcinoid tumor, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, intestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, metastatic breast cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, myeloma, esophageal cancer, biliary tract tumors, head and neck cancer and hematological malignancies.

[0056] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0058] Figure 1 The diagram shows the synthesis steps, separation steps and absolute configuration identification of some intermediates in Route 1 in the embodiment.

[0059] Figure 2 The graphs are the assay diagrams of the NSD2 degradation activity and multiple myeloma cell proliferation inhibition activity of the compounds of Examples 1-17.

[0060] Figure 3 The graphs show the NSD2 degradation activity and multiple myeloma cell proliferation inhibition activity assays of the compounds of Examples 18-23.

[0061] Figure 4 The graphs show the changes in the contents of NSD2 and its long form, and RE-IIBP after treatment with the compounds of Examples 24-34, the changes in the levels of other SET family proteins NSD1 and NSD2, the changes in the methylation level at H3K36, and the SPR affinity test.

[0062] Figure 5The graphs are the test graphs of the proliferation inhibition activity of the compound of Example 25 and the control compound on KMS11 multiple myeloma cells and the test graphs of the cytotoxicity on human embryonic kidney cells 293T. DETAILED DESCRIPTION

[0063] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0064] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0065] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of the present invention.

[0066] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0067] In the examples, compounds ND-(101-117), ND-(202-210), ND-(303-312), ND-311M and ND-311P were synthesized by the following route 1:

[0068]

[0069] In route 1, the synthetic reagents and conditions are as follows:

[0070] (a) Reagents: 1,1-difluoro-2-iodoethane, LHMDS, THF; Conditions: first react at -78°C and then at room temperature; (b) Reagents: LiOH, methanol-water, Conditions: room temperature; (c) Reagents: DPPA, TEA, toluene, Conditions: reflux; (d) Reagents: (Boc)2O, DIPEA, DCM, Conditions: room temperature; (e) Reagents: H2, Pd(OH)2, methanol, Conditions: room temperature; (f) Reagents: 2-bromo-5-fluoroisonicotinic acid methyl ester, Pd(dppf)Cl2, Cs2CO3, 1,4-dioxane-water, Conditions: reflux in argon atmosphere; (g) Reagents: DIPEA, DMSO, Conditions: 120°C; (h) Reagents: LiCl, NaBH4, methanol-THF, Conditions: 0°C; (i) Reagents: SOCl2, DCM, Conditions: 0°C; (j) Reagents: N 6 -diBoc-adenosine, K2CO3, acetonitrile, condition: room temperature; (k) reagent: H2, Pd(OH)2 / C, AcOH, ethanol, condition: room temperature; (l) reagent: benzyl bromoacetate, K2CO3, acetonitrile, condition: room temperature; (m) reagent: H2, Pd(OH)2, ethanol, condition: room temperature; (n) reagent: HATU, DIPEA, DMF, condition: room temperature; (o) reagent: CF3COOH, DCM, condition: room temperature.

[0071] In route 1, intermediates MN1-MN5 were synthesized by methods reported in the literature (WO2021028854). VHL or CRBN intermediates MV01-MV11 and MA03-MA11 were synthesized by similar steps reported previously (J Med Chem, 63 (2020) 7510-7528; Eur J Med Chem, 218 (2021) 113341). Other reagents and primary amine building blocks were purchased from reagent suppliers such as Bidex, Adamas, Anergy and Bailingwei.

[0072] In route 1, the synthesis steps of the intermediate 2-(3-(benzyloxy)-4-fluorophenyl)-5-fluoroisonicotinic acid methyl ester (M1) are as follows: 28.01g of 2-bromo-5-fluoroisonicotinic acid methyl ester and 30.05g of SM2, 1.50g of Pd(dppf)Cl2 and 78.30g of Cs2CO3 are dissolved in 250mL of 1,4-dioxane / water (4:1) solution. The reaction mixture is refluxed under Ar atmosphere for 2h and then filtered through diatomaceous earth. The filtrate is then concentrated under reduced pressure and the resulting mixture is further purified by flash chromatography (EA:PE=2-30%) to obtain 25.93g of M1 as a yellow solid with a yield of ~61%. The H NMR spectrum is: 1H NMR (500 MHz, Chloroform-d) δ 8.67–8.58 (m, 1H), 8.11 (d, J = 5.4 Hz, 1H), 7.81–7.71 (m, 1H), 7.54–7.45 (m, 3H), 7.44–7.38 (m, 2H), 7.38–7.29 (m, 1H), 7.24–7.14 (m, 1H), 5.24 (s, 2H), 4.01 (s, 3H). Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 20 H 16 F2NO3 356.1[M+H] + , the detection result is 356.1.

[0073] In route 1, the synthesis steps of the intermediate 2-(3-(benzyloxy)-4-fluorophenyl)-5-(3-(tert-butoxycarbonyl)amino)-3-(2,2-difluoroethyl)piperidin-1-yl)isonicotinic acid methyl ester (M2) are as follows: 13.51g M1 and 10.07g MN5 are dissolved in 25mL DMSO, and then 8mL DIPEA is added. The reaction mixture is stirred at 120°C for 12 hours. After cooling to room temperature, the reaction mixture is diluted with 100mL water and then extracted with EA (80mL×3). The mixed EA solution is washed with a saturated aqueous NaCl solution, dried over Mg2SO4, and concentrated under reduced pressure to obtain a crude product, which is further purified by flash chromatography (EA:PE=10-50%) to obtain 12.85g M2, a yellow oil, with a yield of ~56%. The hydrogen nuclear magnetic resonance spectrum is: 1 H NMR (600 MHz, Chloroform-d) δ 8.50 (s, 1H), 7.88 (s, 1H), 7.78–7.71 (m, 1H), 7.55–7.43 (m, 3H), 7.42–7.37 (m, 2H), 7.36–7.31 (m, 1H), 7.20–7.13 (m, 1H), 6.08–5.87 (m, 1H), 5.23 (s, 2H), 4.01 (s, 3H), 3.43–3.23 (m, 2H), 2.96–2.47 (m, 4H), 2.00–1.85 (m, 2H), 1.73–1.66 (m, 1H), 1.47 (s, 9H). Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 32 H 37 F3N3O5 600.3[M+H] + , the detection result is 600.5.

[0074] In route 1, the synthesis steps of the intermediate tert-butyl (1-(6-(3-benzyloxy)-4-fluorophenyl)-4-(hydroxymethyl)pyridin-3-yl)-3-(2,2-difluoroethyl)piperidin-3-yl)carbamate (M3) are as follows: 12.03 g of M2 was dissolved in 100 mL of methanol-THF solution (1:2), and then cooled to 0°C. 8.40 g of LiCl (10 equivalents) and 3.78 g of NaBH4 (5 equivalents) were slowly added. After stirring at 0°C for 1 hour, LC-MS monitoring showed that the reaction was complete. The reaction was quenched with 100 mL of water at 0°C. The resulting mixture was extracted with EA (100 mL×3). The mixed EA solution was washed with saturated NaCl aqueous solution, dried over Mg2SO4, and concentrated under reduced pressure to obtain a crude product, which was further purified by flash chromatography (EA:PE=20-50%) to obtain 7.01 g of M3 as a light yellow solid with a yield of ~61%. The H NMR spectrum is: 1 H NMR(600MHz,Chloroform-d)δ8.40(s,1H),7.79–7.71(m,1H),7.67(s,1H),7.51–7 .43(m,3H),7.42–7.37(m,2H),7.36–7.30(m,1H),7.19–7.11(m,1H),6.07–5.82(m ,1H),5.22(s,2H),4.86–4.73(m,2H),3.48–3.22(m,2H),3.12–3.05(m,1H),2.89–2.75(m,2H),2.55–2.16(m,2H),1.93–1.82(m,1H),1.79–1.62(m,3H),1.46(s,9H). Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 31 H 37 F3N3O4 572.3[M+H] + , the detection result is 572.4.

[0075] In route 1, the synthesis steps of the intermediate tert-butyl (9-((2-(3-(benzyloxy)-4-fluorophenyl)-5-(3-((tert-butoxycarbonyl)amino)-3-(2,2-difluoroethyl)piperidin-1-yl)pyridin-4-yl)methyl)-9H-purin-6-yl)(tert-butoxycarbonyl)carbamate (M4) are as follows: 6.29g of M3 was dissolved in 60mL of dry DCM, then cooled to 0°C, and then 2.5eq.3.27g of SOCl2 was added dropwise. After stirring at 0°C for 30min, TLC showed that the reaction was complete. The reaction was quenched with 100mL of Na2CO3 at 0°C. The resulting mixture was extracted with DCM (100mL×3). The combined DCM solution was washed with a saturated aqueous NaCl solution, dried over Mg2SO4, and concentrated under reduced pressure to obtain a crude chloro product, which was directly used in the next step. The chloro-substituted product 3.85g N 6 -diBoc-adenine, 3.05 g K2CO3 were dissolved in 50 mL acetonitrile. The reaction mixture was stirred at rt for 18 hours and then filtered through a celite pad. The filtrate was then concentrated under reduced pressure and the crude product was further purified by flash chromatography (C18 column, acetonitrile: water 0.1% FA = 10-100%) to obtain 5.18 g M4 as a white powder in a yield of 53% (2 steps). The H NMR spectrum is: 1 H NMR(500MHz,Chloroform-d)δ8.86(s,1H),8.51(s,1H),8.24(s,1H),8.01(s,1H),7.64–7.58(m,1H),7.51–7 .42(m,2H),7.41–7.35(m,2H),7.35–7.29(m,1H),7.22–7.13(m,2H),7.09–7.03(m,1H),6.14–5.88(m,1H),5 .77–5.40 (m, 2H), 5.17 (s, 2H), 3.68–3.47 (s, 2H), 3.05–2.97 (m, 2H), 2.90–2.82 (m, 4H), 2.72–2.58 (m, 1H), 2.38–2.28 (m, 2H), 2.09–1.94 (m, 1H), 1.83–1.72 (m, 1H), 1.70–1.64 (m, 1H), 1.60 (s, 9H), 1.44–1.34 (s, 18H). Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 46 H 56 F3N8O7 889.4[M+H] + , the detection result was 889.7.

[0076] In route 1, the synthesis steps of the intermediate tert-butyl (tert-butoxycarbonyl) (9-((5-(3-((tert-butoxycarbonyl)amino)-3-(2,2-difluoroethyl)piperidin-1-yl)-2-(4-fluoro-3-hydroxyphenyl)pyridin-4-yl)methyl)-9H-purin-6-yl)carbamate (M5) are as follows: 5.05g of M4 was dissolved in 50mL of ethanol, and then 0.50g of Pd(OH)2 / C and 1mL of CH3COOH were added. The reaction was stirred for 36 hours under H2 atmosphere. LC-MS monitoring showed that the reaction was complete, and the reaction mixture was filtered through a celite pad. The filtrate was then concentrated under reduced pressure; the crude product was further purified by flash chromatography (C18 column, acetonitrile: water 0.1% FA = 10-100%) to obtain 3.63g of M5 as a white powder with a yield of ~81%. The H NMR spectrum is: 1 H NMR(500MHz,Chloroform-d)δ8.87(s,1H),8.49(s,1H),8.25(s,1H),7.26–7.22( m,2H),7.07–7.02(m,1H),6.98(s,1H),6.12–5.87(m,1H),5.74–5.42(m,2H),3.6 8–3.54 (m, 2H), 3.08–2.97 (m, 2H), 2.92–2.85 (s, 2H), 2.72–2.55 (m, 1H), 2.40–2.28 (m, 2H), 2.07–1.92 (m, 1H), 1.86–1.63 (m, 4H), 1.48–1.42 (m, 18H), 1.36 (s, 9H). Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 39 H 50 F3N8O7 799.4[M+H] + , the detection result was 799.5.

[0077] In route 1, the synthesis steps of the intermediate benzyl 2-(5-(4-((6-(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)methyl)-5-(3-((tert-butoxycarbonyl)amino)-3-(2,2-difluoroethyl)piperidin-1-yl)pyridin-2-yl)-2-fluorophenoxy)acetate (M6) are as follows: 3.50 g of M5, 1.05 g of benzyl bromoacetate and 1.20 g of K2CO3 are mixed in 50 mL of acetonitrile. After stirring at rt for 6 hours, LC-MS monitoring showed that the reaction was complete, and the reaction mixture was filtered through a celite pad. The filtrate was then concentrated under reduced pressure, and the crude product was further purified by flash chromatography (C18 column, acetonitrile: water 0.1% FA = 10-100%) to obtain 2.07 g of M6 as a light yellow powder with a yield of 50%. The H NMR spectrum is: 1H NMR(500MHz,Chloroform-d)δ8.86(s,1H),8.48(s,1H),8.24(s,1H),8.02(s,1H),7.63–7.53(m,1H),7.40 –7.27(m,9H),7.25–7.17(m,2H),7.10–7.02(m,1H),6.15–5.85(m,1H),5.75–5.40(m,2H),5.26–5.18(m,4 H), 4.79 (s, 2H), 4.20 (d, J = 5.3 Hz, 1H), 3.67 (s, 1H), 3.55–3.45 (m, 1H), 3.06–2.97 (m, 2H), 2.87–2.80 (m, 2H), 2.71–2.60 (m, 1H), 2.40–2.27 (m, 3H), 2.03–1.74 (m, 2H), 1.70–1.63 (m, 1H), 1.58 (s, 18H), 1.43 (s, 9H). Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 48 H 58 F3N8O9 947.4[M+H] + , the detection result was 947.7.

[0078] In route 1, the synthesis steps of the intermediate 2-(5-(4-((6-(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)methyl)-5-(3-(tert-butoxycarbonyl)amino)-3-(2,2-difluoroethyl)piperidin-1-yl)pyridin-2-yl)-2-fluorophenoxy)acetic acid (M7) are as follows: 2.05g of M6 is dissolved in 50mL of ethanol, and then 0.50g of Pd(OH)2 / C and 1mL of CH3COOH are added. The reaction is stirred for 36 hours under H2 atmosphere. LC-MS monitoring shows that the reaction is complete, and the reaction mixture is filtered through a celite pad. The filtrate is then concentrated under reduced pressure; the crude product is further purified by flash chromatography (C18 column, acetonitrile: water 0.1% FA = 10-100%) to obtain 1.61g of M7 as a light yellow powder with a yield of ~87%. The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.85–8.81(m,2H),8.47(s,1H),7.98(s,1H),7.54–7. 50(m,1H),7.34(s,1H),7.25–7.20(m,1H),7.12–7.06(m,1H),7.02(s,1H),6.20–5 .94(m,1H),5.88–5.62(m,2H),4.65(s,2H),4.08–4.00(s,1H),3.65(s,1H),3.14–3.01(m,2H),2.97–2.87(m,2H),2.56–2.38(m,2H),2.37–2.24(m,2H),2.10–1.98(m 1H),1.76–1.56(m,3H),1.45–1.25(s,27H). Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 41 H 52 F3N8O9 857.4[M+H] + , the detection result is 857.6.

[0079] In route 1, a UV-absorbing benzoyl group was introduced into the chiral carbon of (-)-M4A to provide (-)-M4A-Bz for ECD-based absolute configuration determination.

[0080] In route 1, the chiral separation of the intermediate M4 was performed using a C3 IH chiral column and methanol-carbon dioxide SFC supercritical fluid chromatography to obtain two enantiomers, namely (-)-M4A with a specific rotation [α] D 20 -28.3 (c 0.7, EtOH) and (+)-M4B specific rotation [α] D 20 +26.0 (c 0.5, EtOH).

[0081] In route 1, the synthesis of the intermediate (-)-M4A-Bz for configuration identification: 30 mg (-)-M4A was dissolved in 2 mL DCM before adding 1 mL CF3COOH. After stirring at room temperature for 30 minutes, the reaction mixture was concentrated to dryness to obtain a crude amine product. The amine intermediate was then dissolved in 4 mL DMF, followed by the addition of 0.3 mL DIPEA and 5 mg DMAP. To the stirred solution, 12 mg benzoyl chloride was added at 40 ° C. After stirring at 40 ° C for 20 minutes, LC-MS monitoring showed that the reaction was complete. Concentrated under reduced pressure, the residue was further purified by flash chromatography (C18 column, acetonitrile: water 0.1% TFA = 10-100%) to obtain 5 mg (-)-M4A-Bz as a yellow semi-solid with a yield of ~20%. The hydrogen nuclear magnetic resonance spectrum is:1 H NMR(500MHz,Methanol-d4)δ8.73–8.53(m,3H),8.16–8.05(m,3H),7.75–7.65(m,3H),7.61–7.53(m, 2H),7.51–7.46(m,1H),7.43–7.39(m,2H),7.38–7.31(m,3H),7.30–7.25(m,2H),7.25–7.12(m,3H),6 .24–5.96(m,1H),5.84–5.75(m,2H),5.40–5.31(t,J=5.0Hz,1H),5.15(s,2H),3.99(d,J=11.9Hz,1H ),3.10–3.11(m,1H),2.71–2.59(m,2H),2.58–2.49(m,1H),2.22–1.92(m,5H),1.88–1.58(m,3H).[α] D 20 -32.0 (c 0.5, EtOH). HRMS (ESI) calculated for C 45 H 40 F3N8O3 + 797.3170[M+H] + , the detection result is 797.3168.

[0082] The synthesis steps of (-)-M4A-Bz in route 1 and the chiral separation steps of intermediate M4 are shown in FIG. Figure 1 As shown in A, the absolute configuration test diagram of (-)-M4A-Bz in route 1 was determined by ECD and calculation as shown in Figure 1 As shown in B.

[0083] In the examples, the final target compounds were synthesized by General Method A and General Method B (Method B for primary amine compounds).

[0084] Among them, the amide condensation process and subsequent deprotection process of ND-(V01-V11), ND-(C02-C11) and ND-L11P in the examples of the present invention are prepared using General Method A. Typical procedure: 30 mg (0.035 mmol) of M7, 5 equivalents of DIPEA and 1.0 equivalents of the corresponding amine intermediates (MV01-MV11, MC02-MC12, PL11) were mixed in 3-5 mL of CH3CN before adding HATU (1.1 equivalents). After LC-MS monitoring showed that the reaction was complete, the reaction was quenched with 0.5 mL of water and concentrated to dryness. The residue was then dissolved in 2 mL of CF3COOH to remove the Boc group. After LC-MS monitoring showed the reaction was completed, the mixture was concentrated and purified by flash chromatography (C18 column, acetonitrile: water: 0.1% TFA = 10-100%, in some cases using 0.1% formic acid aqueous solution) to give ND-(V01-V11), ND-(C02-C11) and ND-L11P as TFA salts or FA salts with yields of about 35% to 60% for 2 steps.

[0085] Wherein, adopt universal method B to prepare the amide condensation method and subsequent deprotection method of ND-(303-312) and ND-311M in the embodiment of the present invention.Typical step is slightly different from method A, adds excessive alkyl primary amine and solvent to avoid side reaction 30mg (0.035mmol) M7, 10 equivalents of DIPEA and 8.0 equivalents of corresponding alkyl primary amine intermediates (M303-M312, M311M) are mixed in 10mL acetonitrile.Add HATU (1.1 equivalents) again.After LC-MS monitoring display reaction is completed, use 0.5mL water quenching reaction, and concentrate to remove solvent.Then residue is dissolved in 3mL CF in COOH to remove Boc group. After LC-MS monitoring showed that the reaction was completed, the mixture was concentrated and purified by flash chromatography (C18 column, acetonitrile: water 0.1% TFA = 5-100%) to obtain the TFA salt of ND-(303-312) and ND-311M, with a total yield of about 30-45% for two steps. Racemic M7 was used to prepare ND-311, (+)-M7 was used to prepare ND-311B, ND-(303-309), ND-312, ND-311M and ND-311P, and (-)-M7 was used to prepare ND-311A.

[0086] Example 1

[0087] A compound, named ND-111, has the following structure:

[0088]

[0089] The above compound (ND-111) is: (2S, 4R)-1-((2S)-2-(12-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)dodecylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide·TFA.

[0090] The above compound was prepared by general method A using M7 and MV11 to give ND-111 as a colorless solid (TFA salt) in 45% yield.

[0091] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.89(s,1H),8.53(s,1H),8.41(s,1H),8.29(s,1H),7.70(dd,J=12.6,2.1Hz,1H),7.60–7.5 4(m,1H),7.47–7.33(m,5H),7.10(t,J=8.6Hz,1H),6.51–6.19(m,1H),5.76–5.58(m,2H),5.00(q,J=7.0Hz,1H),4.61(d, J=6.5Hz,2H),4.58–4.54(m,1H),4.47–4.41(m,1H),3.92–3.70(m,2H),3.41–3.33(m,2H),3.25(t,J=7.1Hz,3H),3.12–2 .90(m,2H),2.51–2.43(m,5H),2.30–2.13(m,3H),2.05–1.93(m,4H),1.60-1.46(m,6H),1.34–1.18(m,16H),1.03(s,9H).

[0092] The carbon NMR spectrum is: 13 C NMR(125MHz,Methanol-d4)δ176.12,173.29,172.36,170.32,154.65(d,J C-F=98.4Hz),152.93,150.41,149.07,148.19,146.09,145.71,144.59,143.57,142.88,134. 21,133.42,131.54,130.53,127.67,127.50,123.99,120.50,120.12,116.81,115.86,115. 70,71.02,69.54,60.64,59.89,59.07,58.02,55.47,54.95,50.18,43.23,40.18,38.84,36.70,36.54,32.43,30.62,30.56,30.42,30.39,30.36,27.89,27.11,22.42,22.07,15.82.

[0093] 19 F NMR(471MHz, Methanol-d4)δ-76.92,-114.59,-134.77.

[0094] High resolution mass spectra: HRMS (ESI) calculated as C 61 H 81 F3N 13 O6S1180.6100[M+H] + , the detection result is 1180.6100.

[0095] Example 2

[0096] A compound, named ND-110, has the following structure:

[0097]

[0098] The above compound (ND-110) is: (2S, 4R)-1-((2S)-2-(11-(2-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)undecylamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0099] The above compound was prepared from M7 and MV10 by general method A as ND-110, a light yellow solid (TFA salt), with a yield of 53%.

[0100] The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.86(s,1H),8.47(s,1H),8.25–8.18(m,2H),7.60–7.54(m,1H),7.48–7.37(m,4H),7.36–7.31 (m,1H),7.29(s,1H),7.20–7.12(m,8.5Hz,1H),6.33–6.06(m,1H),5.70–5.58(m,2H),5.00(q,J=7.0Hz,1H),4.65–4.53(m,4 H),4.45–4.40(brs,1H),3.91–3.70(m,2H),3.25(t,J=6.9Hz,2H),3.05–2.84(m,4H),2.47(s,3H),2.29–2.14(m,4H),2.13 –1.99(m,2H),1.99–1.87(m,2H),1.81–1.66(m,3H),1.62–1.52(m,2H),1.52–1.41(m,4H),1.32–1.11(m,10H),1.03(s,9H).

[0101] High resolution mass spectra: HRMS (ESI) calculated as C 60 H 79 F3N 13 O6S1166.5944[M+H] + , the detection result is 1166.5940.

[0102] Example 3

[0103] A compound, named ND-109, has the following structure:

[0104]

[0105] The above compound (ND-109) is: (2S, 4R)-1-((2S)-2-(10-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)decanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0106] The above compound was prepared by general method A with M7 and MV09 as reactants to give ND-109 as a light yellow solid (TFA salt) with a yield of 46%.

[0107] The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.86(s,1H),8.47(s,1H),8.26–8.18(m,2H),7.61–7.53(m,1H),7.45–7.38(m,4H),7.38–7.31(m,1 H),7.29(s,1H),7.23–7.12(m,1H),6.20(tt,J=56.3,4.7Hz,1H),5.72–5.53(m,2H),4.99(q,J=7.0Hz,1H),4.66–4.52(m,4H),4. 45–4.40(brs,1H),3.94–3.66(m,2H),3.24(t,J=7Hz,2H),3.09–2.86(m,4H),2.47(s,3H),2.31–2.15(m,3H),2.14–2.01(m,2H), 1.99–1.89(m,2H),1.78–1.65(m,3H),1.58–1.52(m,2H),1.51–1.48(m,2H),1.48–1.42(m,2H),1.25–1.12(m,10H),1.03(s,9H).

[0108] High resolution mass spectra: HRMS (ESI) calculated as C 59 H 77 F3N 13 O6S1152.5787[M+H] + , the detection result is 1152.5782.

[0109] Example 4

[0110] A compound, named ND-108, has the following structure:

[0111]

[0112] The above compound (ND-108) is: (2S, 4R)-1-((2S)-2-(9-(2-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)nonanamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0113] The above compound was prepared by general method A with M7 and MV08 as reactants to give ND-108 as a light yellow solid (TFA salt) with a yield of 49%.

[0114] The H NMR spectrum is:1 H NMR(500MHz,Methanol-d4)δ8.86(s,1H),8.48(s,1H),8.26–8.20(m,2H),7.61–7.55(m,1H),7.47–7.38(m,4 H),7.36–7.31(m,2H),7.22–7.09(m,1H),6.37–6.09(m,1H),5.72–5.56(m,2H),4.99(q,J=6.9Hz,1H),4.67–4 .51(m,4H),4.44–4.40(brs,1H),3.93–3.66(m,2H),3.25(t,J=6.9Hz,2H),3.13–2.87(m,4H),2.47(s,3H),2. 27–2.09(m,2H),1.96–1.87(m,2H),1.83–1.70(brs,3H),1.49–1.41(m,2H),1.21–1.09(m,10H),1.03(s,9H).

[0115] High resolution mass spectra: HRMS (ESI) calculated as C 58 H 75 F3N 13 O6S1138.5631[M+H] + , the detection result is 1138.5646.

[0116] Example 5

[0117] A compound, named ND-107, has the following structure:

[0118]

[0119] The above compound (ND-107) is: (2S, 4R)-1-((2S)-2-(8-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)octanamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0120] The above compound was prepared by general method A with M7 and MV07 as reactants to give ND-107 as a light yellow solid (TFA salt) with a yield of 38%.

[0121] The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.86(s,1H),8.47(s,1H),8.24–8.18(m,2H),7.60–7.55(m,1H),7.46–7.36(m ,4H),7.37–7.29(m,1H),7.28(s,1H),7.20–7.14(m,1H),6.36–6.04(m,1H),5.71–5.54(m,2H),4.99(q,J=6 .9Hz,1H),4.66–4.50(m,4H),4.45–4.39(brs,1H),3.93–3.65(m,2H),3.24(t,J=6.9Hz,2H),3.06–2.86(m, 4H),2.47(s,3H),2.24–2.16(m,3H),2.01–1.64(m,5H),1.50–1.43(m,4H),1.29–1.13(m,6H),1.03(s,9H).

[0122] High resolution mass spectra: HRMS (ESI) calculated as C 57 H 73 F3N 13 O6S1124.5474[M+H] + , the detection result is 1124.5472.

[0123] Example 6

[0124] A compound, named ND-106, has the following structure:

[0125]

[0126] The above compound (ND-106) is: (2S, 4R)-1-((2S)-2-(7-(2-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)heptylamido)-3,3-dimethylbutyramido)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0127] The above compound was prepared by general method A with M7 and MV06 as reactants to give ND-106 as a light yellow solid (TFA salt) with a yield of 47%.

[0128] The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.86(s,1H),8.48(s,1H),8.25–8.18(m,2H),7.59–7.53(m,1H),7.47–7.36(m,4 H),7.35–7.31(m,1H),7.30–7.28(m,1H),7.20–7.13(m,1H),6.34–6.09(m,1H),5.74–5.56(m,2H),4.99(q,J =6.9Hz,1H),4.68–4.49(m,4H),4.45–4.39(brs,1H),3.94–3.70(m,2H),3.25(t,J=6.9Hz,2H),3.08–2.85(m ,4H),2.47(s,3H),2.25–2.12(m,3H),1.97–1.69(m,5H),1.53–1.41(m,4H),1.33–1.20(m,4H),1.03(s,9H).

[0129] High resolution mass spectra: HRMS (ESI) calculated as C 56 H 71 F3N 13 O6S1110.5318[M+H] + , the detection result is 1110.5331.

[0130] Example 7

[0131] A compound, named ND-105, has the following structure:

[0132]

[0133] The above compound (ND-105) is: (2S, 4R)-1-((2S)-2-(6-(2-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)hexanaphthylamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0134] The above compound was prepared by general method A with M7 and MV05 as reactants to give ND-105 as a light yellow solid (TFA salt) with a yield of 50%.

[0135] The H NMR spectrum is: 1H NMR(500MHz, Methanol-d4)δ8.91(s,1H),8.62–8.48(m,2H),8.39(s,1H),7.57(d,J=7.9Hz,1H),7.48–7.3 9(m,5H),7.37–7.31(m,1H),7.23–7.14(m,1H),6.54–6.23(m,1H),5.91–5.70(m,2H),5.15–4.96(m,1H),4 .68–4.49(m,4H),4.46–4.25(m,1H),3.95–3.56(m,6H),3.45–3.35(m,3H),3.24(t,J=6.4Hz,2H),3.18–2. 97(m,2H),2.57–2.45(m,5H),2.23–1.92(m,6H),1.55–1.44(m,6H),1.34–1.21(m,4H),1.07–0.94(m,9H).

[0136] High resolution mass spectra: HRMS (ESI) calculated as C 55 H 69 F3N 13 O6S1096.5161[M+H] + , the detection result is 1096.5175.

[0137] Example 8

[0138] A compound, named ND-104, has the following structure:

[0139]

[0140] The above compound (ND-104) is: (2S, 4R)-1-((2S)-2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)pentaminyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0141] The above compound was prepared from M7 and MV04 by general method A as a pale yellow solid (HCOOH salt) with a yield of 35%.

[0142] The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.87(s,1H),8.54(s,1H),8.34(s,1H),8.23(s,1H),8.11(t,J=5.8Hz,1H),7 .84–7.77(m,1H),7.65–7.59(m,1H),7.49–7.45(m,1H),7.45–7.37(m,4H),7.24–7.17(m,1H),6.46–6.22( m,1H),5.67–5.55(s,2H),4.99(t,J=7.0Hz,1H),4.66–4.52(m,4H),4.44–4.40(brs,1H),3.90–3.70(m,2 H),3.29–2.92(m,6H),2.47(s,3H),2.31–2.11(m,3H),2.01–1.87(m,3H),1.63–1.47(m,4H),1.02(s,9H).

[0143] High resolution mass spectra: HRMS (ESI) calculated as C 54 H 67 F3N 13 O6S1082.5005[M+H] + , the detection result is 1082.5016.

[0144] Example 9

[0145] A compound, named ND-103, has the following structure:

[0146]

[0147] The above compound (ND-103) is: (2S, 4R)-1-((2S)-2-(4-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)butylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0148] The above compound was prepared by general method A with M7 and MV03 as reactants to give ND-103 as a light yellow solid (TFA salt) with a yield of 46%.

[0149] The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.87(s,1H),8.48(s,1H),8.25–8.18(m,2H),7.62–7.52(m,1H),7.46–7.38(m,4H ),7.37–7.30(m,2H),7.27(s,1H),7.23–7.11(m,1H),6.31–6.07(m,1H),5.65(q,J=16.2Hz,3H),4.99(q,J=7. 1Hz,1H),4.70–4.50(m,5H),4.44–4.34(m,1H),3.93–3.69(m,2H),3.66(s,1H),3.04–2.87(m,4H),2.47(s,3H ),2.34–2.26(m,2H),2.22–2.12(m,2H),1.98–1.85(m,2H),1.84–1.66(m,5H),1.52–1.45(m,4H),1.02(s,9H).

[0150] High resolution mass spectra: HRMS (ESI) calculated as C 53 H 65 F3N 13 O6S1068.4848[M+H] + , the detection result is 1068.4850.

[0151] Example 10

[0152] A compound, named ND-102, has the following structure:

[0153]

[0154] The above compound (ND-102) is: (2S, 4R)-1-((2S)-2-(3-(2-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)propionamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0155] The above compound was prepared by general method A with M7 and MV02 as reactants to give ND-102 as a colorless solid (TFA salt) in a yield of 39%.

[0156] The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.77(s,1H),8.39–8.34(m,1H),8.17–8.08(m,2H),7.45(d,J =8.2Hz,1H),7.38–7.23(m,4H),7.22–7.12(m,2H),7.10–7.04(m,1H),6.24–5.95(m,1H),5 .64–5.49(q,J=16.1,15.5Hz,2H),4.99–4.82(m,1H),4.58–4.29(m,4H),3.61–3.44(m,5H) ,2.92–2.76(m,4H),2.37(s,3H),2.12–1.92(m,4H),1.67–1.37(m,6H),0.99–0.83(m,9H).

[0157] High resolution mass spectra: HRMS (ESI) calculated as C 52 H 63 F3N 13 O6S1054.4692[M+H] + , the detection result is 1054.4698.

[0158] Embodiment 11

[0159] A compound, named ND-101, has the following structure:

[0160]

[0161] The above compound (ND-101) is: (2S, 4R)-1-((2S)-2-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)acetylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0162] The above compound was prepared by general method A with M7 and MV01 as reactants to give ND-101 as a light yellow solid (HCOOH salt) with a yield of 36%.

[0163] The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.87(s,1H),8.60–8.52(m,2H),8.47(s,1H),7.62–7.58(m,1 H),7.47–7.26(m,4H),7.21–7.13(m,1H),6.17–5.91(m,1H),5.78–5.54(m,2H),5.06–4.9 3(m,1H),4.72–4.54(m,4H),4.09–3.96(m,2H),3.88–3.67(m,2H),3.19–2.84(m,4H),2.4 7(s,3H),2.30–1.98(m,2H),1.96–1.67(m,2H),1.49(dd,J=7.1,3.7Hz,2H),1.01(s,9H).

[0164] High resolution mass spectra: HRMS (ESI) calculated as C 51 H 61 F3N 13 O6S1040.4535[M+H] + , the detection result is 1040.4543.

[0165] Example 12

[0166] A compound, named ND-112, has the following structure:

[0167]

[0168] ND-112 is prepared by general method A using intermediates MV12 and M7 as raw materials. It is a colorless solid with a yield of about 53%.

[0169] The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.86(s,1H),8.56–8.43(m,2H),8.33–8.27(m,1H),8.25–8.18(m,2H),7.63–7.55 (m,1H),7.46–7.34(m,4H),7.25–7.13(m,2H),6.43–6.15(t,J=55.7Hz,1H),5.70–5.53(m,2H),4.99(q,J=6.9 Hz,1H),4.69–4.50(m,4H),4.45–4.40(brs,1H),3.93–3.82(m,2H),3.62–3.41(m,10H),3.22–2.90(m,6H),2. 46(s,3H),2.35–2.14(m,4H),2.02–1.78(m,6H),1.55–1.45(m,2H),1.24(dd,J=20.4,6.7Hz,2H),1.01(s,9H).

[0170] Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 60 H 78 F3N 14 O 10 S + 1243.6[M+H] + , the detection result is 1243.7.

[0171] Preparation of intermediate MV12, i.e. (2S,4R)-1-((S)-1-amino-17-(tert-butyl)-11,15-dioxo-3,6,9-trioxa-12,16-diazaoctadecyl-18-oxyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide: 50 mg of MV2 and 35 mg of 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-oic acid were dissolved in 5 mL of acetonitrile, 0.2 mL of DIPEA was added, and then 40 mg of HATU condensation agent was added. After stirring at room temperature for 30 minutes, the solvent and alkali were removed by rotary evaporation, and 2 mL of trifluoroacetic acid was added. The reaction was continued at room temperature for 30 minutes, and then concentrated to obtain a crude product. The crude product was purified by reverse phase column chromatography (C18, acetonitrile: water 0.1% formic acid) to obtain about 45 mg of the product. LC-MS (ESI) calculated C 34 H 53 N6O8S + 705.4[M+H] + , the detection result is 705.4.

[0172] Embodiment 13

[0173] A compound, named ND-113, has the following structure:

[0174]

[0175] ND-113 was prepared by general method A using intermediates MV13 and M7 as raw materials. It is a colorless solid with a yield of about 55%.

[0176] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.86(s,1H),8.55–8.43(m,2H),8.31(s,1H),8.23(s ,1H),7.63–7.57(m,1H),7.47–7.36(m,4H),7.23–7.15(m,1H),6.46–6.16(m,1H), 5.62(s,2H),4.99(q,J=7.0Hz,1H),4.69–4.39(m,5H),3.91–3.47(m,12H),3.27–2 .92(m,4H),2.58–2.17(m,7H),2.01–1.80(m,4H),1.54–1.40(m,2H),1.01(s,9H).

[0177] Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 58 H 75 F3N 13 9S + 1186.5[M+H]+, detected as 1186.7.

[0178] Intermediate MV13 ((2S, 4R)-1-((S)-1-amino-14-(tert-butyl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide) and intermediate MV14 ((2S, 4R)-1-((S)- 2-(5-(4-(4-aminobutyryl)piperazin-1-yl)pentylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide) was prepared with reference to known literature (J.Med.Chem.2020,63,7510-7528; CN115260158).

[0179] Embodiment 14

[0180] A compound, named ND-114, has the following structure:

[0181]

[0182] The above compound (ND-114) is: (2S,4R)-1-((2S)-2-(5-(4-(4-(2-(5-(3-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)butyryl)piperazine-1-yl)pentanamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0183] The above compound ND-114 is prepared by general method A using intermediates MV14 and M7 as raw materials, and is a colorless solid with a yield of about 45%.

[0184] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.87–8.82(m,1H),8.57–8.44(m,1H),8.34(s,1H),8.29–8.16(m,2H),7 .71–7.61(m,1H),7.57–7.45(m,1H),7.47–7.28(m,4H),7.15–7.07(m,1H),6.50–6.15(m,1H),5.69– 5.55(m,2H),4.66–4.38(m,3H),3.93–3.52(m,5H),3.20(t,J=6.9Hz,1H),3.07–2.68(m,6H),2.45(s ,3H),2.34–2.06(m,4H),2.01–1.72(m,6H),1.71–1.44(m,5H),1.38–1.21(m,2H),1.15–0.90(m,9H).

[0185] High resolution mass spectra: HRMS (ESI) calculated as C 62 H 81 F3N 15 7S + 1236.6111[M+H] + , the detection result is 1236.6106.

[0186] The synthesis method of the intermediate 2-(5-(4-((6-(bis(tert-butoxycarbonyl)amino)-9H-purin-9-yl)methyl)-5-(3-(tert-butoxycarbonyl)amino)piperidin-1-yl)pyridin-2-yl)-2-fluorophenoxy)acetic acid (M7-H) is the same as the synthesis method of the intermediate M7, except that the starting synthetic raw material of M7 is replaced by 3-Boc aminopiperidine without difluoroethyl from MN5, and the final intermediate M7-H obtained by the synthesis is an analogue of M7 without difluoroethyl, and LC-MS (ESI) calculation is C 39 H 50 FN8O9 + 793.4[M+H] + , the detection result was 793.4.

[0187] Embodiment 15

[0188] A compound, named ND-115, has the following structure:

[0189]

[0190] ND-115 is prepared by general method A using intermediates MV11 and M7-H as raw materials. It is a light yellow solid with a yield of about 55%.

[0191] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.88(s,1H),8.51(s,1H),8.37(s,1H),8.30(s,1H),7.72–7.62(m,1H),7.58–7.51 (m,1H),7.45–7.39(m,4H),7.37–7.33(m,1H),7.09(t,J=8.6Hz,1H),5.73–5.61(m,2H),5.00(q,J=7.0Hz,1H), 4.63–4.42(m,5H),3.91–3.73(m,3H),3.63–3.42(m,2H),3.25(t,J=7.0Hz,2H),3.15–2.95(m,4H),2.47(s,3H) ,2.33–2.09(m,4H),2.03–1.91(m,2H),1.87–1.70(m,2H),1.64–1.46(m,7H),1.38–1.17(m,12H),1.03(s,9H).

[0192] Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 59 H 79 FN 13 O6S + 1116.6[M+H]+ , the detection result is 1116.7.

[0193] Example 16

[0194] A compound, named ND-116, has the following structure:

[0195]

[0196] The above compound is: (2R, 4S)-1-((2R)-2-(5-((1-(5-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)pentanoyl)piperidin-4-yl)amino)pentanoylamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0197] The above compound was prepared by general method A using intermediates MV16 and M7-4 as raw materials, and was obtained as a colorless solid with a yield of about 45%.

[0198] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ9.01–8.79(m,1H),8.54(s,1H),8.42(s,1H),8.31(s,1H),7.61–7.52(m,1H), 7.50–7.32(m,4H),7.27(s,1H),7.11(t,J=9.6Hz,1H),6.46–6.20(m,1H),5.81–5.64(m,2H),5.37–5.30(m, 1H),4.61–4.40(m,4H),4.19–4.01(m,3H),3.90–3.51(m,8H),3.20–2.96(m,5H),2.56–2.33(m,8H),2.19( t,J=7.5Hz,2H),2.05–1.91(m,6H),1.90–1.66(m,6H),1.61–1.46(m,5H),1.32–1.25(m,12H),1.05(s,9H).

[0199] Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 62 H 82 F3N 14 O6S + 1207.6[M+H]+, detected as 1207.7.

[0200] Preparation of intermediate M7-4 (5-(5-(4-((6-(bis(tert-butyloxycarbonyl)amino)-9H-purin-9-yl)methyl)-5-(3-((tert-butyloxycarbonyl)amino)-3-(2,2-difluoroethyl)piperidin-1-yl)pyridin-2-yl)-2-fluorophenoxy)pentanoic acid): The preparation of intermediate M7-4 is similar to that of intermediate M7. 120 mg of M5 and 60 mg of benzyl bromovalerate were dissolved in 5 mL of acetonitrile, and then 100 mg of 300 mesh potassium carbonate was added and the reaction was stirred at room temperature overnight. The reaction solution was filtered and concentrated, and then subjected to reverse phase column chromatography (C18, acetonitrile: water 0.1% formic acid) to obtain about 95 mg of M6-4. M6-4 was dissolved in ethanol again, 100 mg of 10% Pd(OH)2 / C was added, and hydrogenation was carried out at normal pressure overnight. The reaction solution was filtered and concentrated to obtain 80 mg of M7-4, a colorless semi-solid, with a yield (two steps) of about 59%. LC-MS (ESI) calculated as C 44 H 58 F3N8O9 + 899.4[M+H] + , the detection result was 899.5.

[0201] Preparation of intermediate MV16 ((2R, 4S)-1-((R)-3,3-dimethyl-2-(5-(piperidin-4-ylamino)pentaamino)butyryl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide): Intermediate MV16 was prepared by reductive amination of intermediate MV4 and N-Boc-4-piperidone. 60 mg of MV4 and 50 mg of N-Boc-4-piperidone were dissolved in a HFIP-DCE mixed solution (5 ml 1:3), and 30 mg of sodium triacetoxyborohydride was added in three batches, and stirred overnight at room temperature. After adding a small amount of water to quench the reaction, the crude product was concentrated by rotary evaporation. 2 mL of trifluoroacetic acid was added to the crude product and stirred for half an hour, then concentrated again. After reverse phase column chromatography (C18, acetonitrile: water 0.1% trifluoroacetic acid), about 41 mg of the product was obtained as a light yellow oil with a yield of about 60%. LC-MS (ESI) calculated as C 33 H 51 N6O4S + 627.4[M+H] + , the detection result is 627.5.

[0202] Embodiment 17

[0203] A compound, named ND-117, has the following structure:

[0204]

[0205] The above compound is: (2R, 4S)-1-((2R)-2-(6-((1-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)pentanoyl)piperidin-4-yl)amino)hexanamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0206] The above compound is prepared by general method A using intermediates MV17 and M7-4 as raw materials, and is a colorless solid with a yield of about 45%.

[0207] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.96(s,1H),8.56(s,1H),8.47(s,1H),8.35(s,1H),7.50–7.55( m,1H),7.48–7.37(m,4H),7.34(s,1H),7.32–7.22(m,1H),7.17–7.05(m,1H),6.46–6.21(m,1H ),5.89–5.71(m,2H),4.70–4.40(m,5H),4.17–4.06(m,3H),3.91–3.50(m,7H),3.27–2.96(m, 7H), 2.53 (t, J = 7.3Hz, 2H), 2.48 (s, 3H), 2.38–1.91 (m, 12H), 1.91–1.23 (m, 24H), 1.04 (s, 9H).

[0208] Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 63 H 84 F3N 14 O6S + 1221.6[M+H] + , the detection result is 1221.7.

[0209] Preparation of intermediate MV17 ((2R, 4S)-1-((R)-3,3-dimethyl-2-(6-(piperidin-4-ylamino)hexanaphthoyl)butanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide): Intermediate MV17 was prepared by reductive amination of intermediate MV5 and N-Boc-4-piperidone. 60 mg of MV5 and 50 mg of N-Boc-4-piperidone were dissolved in a HFIP-DCE mixed solution (5 ml 1:3), and 35 mg of sodium triacetoxyborohydride was added in three batches, and stirred overnight at room temperature. After adding a small amount of water to quench the reaction, the crude product was concentrated by rotary evaporation. 2 mL of trifluoroacetic acid was added and stirred for half an hour, then concentrated again. After reverse phase column chromatography (C18, acetonitrile: water 0.1% trifluoroacetic acid), about 39 mg of the product was obtained as a light yellow oil with a yield of about 55%. LC-MS (ESI) calculated as C 34 H 53 N6O4S + 641.4[M+H] + , the detection result is 641.4.

[0210] Embodiment 18

[0211] A compound, named ND-202, has the following structure:

[0212]

[0213] The above compound is: 2-((3R, 5R, 7R)-adamantan-1-yl)-N-(2-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)ethyl)acetamide)acetamide)acetamide A02 formate.

[0214] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.52(s,1H),8.45(s,1H),8.31(s,1H),8.22(s,1H ),7.67–7.60(m,1H),7.46(s,1H),7.43–7.37(m,1H),7.23–7.16(m,1H),6.45– 6.16(m,1H),5.63(s,2H),4.62(s,2H),3.42–3.34(m,3H),3.24(t,J=10.3Hz,2 H),3.05–2.91(m,2H),2.41–2.25(m,2H),1.98–1.81(m,6H),1.67–1.50(m,9H).

[0215] Liquid chromatography-mass spectrometry: LC-MS (ESI) calculated as C 40 H 50 F3N 10 O3 + 775.4[M+H] + , the detection result is 775.5.

[0216] Embodiment 19

[0217] A compound, named ND-204, has the following structure:

[0218]

[0219] The above compound is: 2-((3R, 5R, 7R)-adamantan-1-yl)-N-(4-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)acetamido)butyl)acetamide.

[0220] The H NMR spectrum is: 1 H NMR(500MHz, Methanol-d4)δ8.55(s,1H),8.40(s,1H),8.29(s,1H),7.63–7.54(m,1H),7.50–7.44(m,1H),7.43–7.31(m,2H),7.22–7.14(m,1H),6 .48–6.20(m,1H),5.76–5.63(m,2H),4.62(s,2H),3.76–3.56(m,3H),3.2 5–2.91(m,6H),2.55–2.40(m,2H),2.13–1.81(m,6H),1.67–1.50(m,13H).

[0221] High resolution mass spectra: HRMS (ESI) calculated as C 42 H 54 F3N 10 O3 + 803.4327[M+H] + , the detection result is 803.4319.

[0222] Embodiment 20

[0223] A compound, named ND-206, has the following structure:

[0224]

[0225] The above compound is: 2-((3R, 5R, 7R)-adamantan-1-yl)-N-(6-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)hexyl)acetamide.

[0226] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.51(s,1H),8.27(s,1H),8.25(s,1H),7.40–7.33(m,2H),7.23–7.16(m,1H),6.40–6.07( m,1H),5.72–5.55(m,2H),4.64(s,2H),3.30–2.87(m,9H),2.30–2.13(m,2H),2.01–1.69(m,12H),1.68–1.34(m,13H).

[0227] Embodiment 21

[0228] A compound, named ND-208, has the following structure:

[0229]

[0230] The above compound is: 2-((3R, 5R, 7R)-adamantan-1-yl)-N-(8-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)octyl)acetamide formate.

[0231] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.53(s,1H),8.36(s,1H),8.24–8.20(m,2H),7.80(s ,1H),7.68–7.58(m,1H),7.52(s,1H),7.49–7.42(m,1H),7.25–7.17(m,1H),6.49 –6.21(m,1H),5.69–5.56(m,2H),4.64(s,2H),3.28–2.91(m,6H),2.51–2.39(m,2 H),2.17–1.89(m,8H),1.75–1.58(m,11H),1.48–1.31(m,4H),1.20–1.07(m,7H).

[0232] High resolution mass spectra: HRMS (ESI) calculated as C46 H 62 F3N 10 O3 + 859.4953[M+H] + , the detection result is 859.4959.

[0233] Embodiment 22

[0234] A compound, named ND-209, has the following structure:

[0235]

[0236] The above compound is: 2-((3R, 5R, 7R)-adamantan-1-yl)-N-(9-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)nonyl)acetamide.

[0237] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.51(s,1H),8.31(s,1H),8.22(s,1H),7.81(s, 1H),7.65–7.52(m,1H),7.49–7.26(m,2H),7.25–7.07(m,1H),6.45–6.13(m, 1H),5.70–5.56(m,2H),4.63(s,2H),3.76–3.51(m,3H),3.27–2.91(m,6H),2 .52–2.22(m,2H),1.91–1.60(m,12H),1.51–1.27(m,12H),1.24–1.03(m,8H).

[0238] High resolution mass spectra: HRMS (ESI) calculated as C 47 H 64 F3N 10 O3 + 873.5109[M+H] + , the detection result is 873.5101.

[0239] Embodiment 23

[0240] A compound, named ND-210, has the following structure:

[0241]

[0242] The above compound is: 2-((3r,5r,7r)-adamantan-1-yl)-N-(10-(2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)decyl)acetamide.

[0243] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.53(s,1H),8.40–8.29(m,2H),8.22(s,1H),7.81 (s,1H),7.65–7.57(m,1H),7.50–7.37(m,2H),7.20(dd,J=10.9,8.5Hz,1H),6.4 6–6.17(m,1H),5.63(s,2H),4.63(s,2H),3.28–2.90(m,8H),2.53–2.43(m,2H) ,1.99–1.90(m,6H),1.75–1.56(m,12H),1.50–1.30(m,6H),1.30–1.06(m,10H).

[0244] High resolution mass spectra: HRMS (ESI) calculated as C 48 H 66 F3N 10 O3 + 887.5266[M+H] + , detected 887.5278

[0245] Embodiment 24

[0246] A compound, named ND-311P, has the following structure:

[0247]

[0248] The above compound is: (S)-N-(11-(2-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetylamino)undecyl)benzamide.

[0249] The above compound was prepared by general method A with (+)-M7 and N-(1-aminoundecyl)benzamide as reactants to give ND-311P as a light yellow solid (TFA salt) in a yield of 50%.

[0250] The H NMR spectrum is: 1H NMR(600MHz, Methanol-d4)δ8.54(s,1H),8.45(s,1H),8.32(s,1H),7.81–7.77(m,2H),7.71–7. 67(m,1H),7.57–7.54(m,1H),7.53–7.49(m,2H),7.46–7.41(m,3H),7.36(s,1H),7.08(t,J=8.6H z,1H),6.44–6.21(m,1H),5.79–5.68(m,2H),4.60(s,2H),3.38–3.35(m,5H),3.26–3.14(m,5H), 2.56–2.40(m,2H),2.14–1.88(m,2H),1.65–1.55(m,5H),1.53–1.46(m,2H),1.43–1.29(m,18H).

[0251] 19 F NMR (471MHz, Methanol-d4) delta -77.06, -114.50, -134.74.

[0252] High resolution mass spectra: HRMS (ESI) calculated as C 44 H 56 F3N 10 O3 829.4483[M+H] + , the detection result is 829.4488.

[0253] Embodiment 25

[0254] A compound, named ND-311B, has the following structure:

[0255]

[0256] The above compound is: (S)-2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminoundecyl)acetamide.

[0257] The above ND-311B(S) was prepared by general method B with (+)-M7 and M311 as reactants, colorless solid (TFA salt), yield 35%.

[0258] The H NMR spectrum is: 1H NMR(600MHz,Methanol-d4)δ8.54(s,1H),8.42(s,1H),8.30(s,1H),7.72–7.67(m,1H),7.62–7 .54(m,1H),7.37(s,1H),7.11(t,J=8.5Hz,1H),6.46–6.23(m,1H),5.76–5.65(m,2H),4.60(s, 2H),3.41–3.33(m,2H),3.26(t,J=7.2Hz,2H),3.19–2.96(m,3H),2.90(t,J=7.8Hz,2H),2.55– 2.40(m,2H),2.11–1.84(m,4H),1.64(p,J=7.6Hz,2H),1.55–1.49(m,2H),1.43–1.28(m,18H).

[0259] The carbon NMR spectrum is: 13 C NMR(150MHz,Methanol-d4)δ170.36,163.21,162.92,162.69,155.05(d,J=158.0Hz),153 .93,150.47,149.14,147.39,146.26,144.72,144.40,142.94,136.32,122.15,120.55,11 7.78,116.45,115.58,114.87,69.73,59.73,55.43,54.87,46.42,43.50,40.79,40.14,32.45,31.05,30.72,30.56,30.51,30.45,30.34,30.21,28.61,27.92,27.85,27.46,22.09.

[0260] 19 F NMR(471MHz,Methanol-d4)δ-76.95,-114.50,-134.63.[α] D 20 +29.0 (c 0.5, EtOH).

[0261] High resolution mass spectra: HRMS (ESI) calculated as C 37 H 52 F3N 10 O2 725.4221[M+H] + , the detection result is 725.4216.

[0262] Embodiment 26

[0263] A compound, named ND-311, has the following structure:

[0264]

[0265] Synthesis of racemate 2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminoundecyl)acetamide.

[0266] Racemic ND-311 was prepared by general method B using (+)-M7 and M311 as reactants as a white solid (TFA salt) in 39% yield.

[0267] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.55(s,1H),8.43(s,1H),8.33(s,1H),7.63–7.56(m,1H),7.48– 7.38(m,1H),7.36(s,1H),7.22–7.15(m,1H),6.47–6.23(m,1H),5.78–5.68(m,2H),4.61(s,2 H),3.40–3.35(brs,2H),3.24(t,J=7.0Hz,2H),3.21–3.00(m,3H),2.90(t,J=7.8Hz,2H),2.5 4–2.39(m,2H),2.13–1.91(m,4H),1.67–1.58(m,2H),1.50–1.43(m,2H),1.39–1.20(m,18H).

[0268] High resolution mass spectra: HRMS (ESI) calculated as C 37 H 52 F3N 10 O2 725.4221[M+H] + , the detection result is 725.4215.

[0269] Embodiment 27

[0270] A compound, named ND-311A, has the following structure:

[0271]

[0272] The above compound is: (R)-2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminoundecyl)acetamide.

[0273] ND-311A(R) was prepared by General Method B with (-)-M7 and M311 as reactants as a colorless solid (TFA salt) in 32% yield.

[0274] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.53(s,1H),8.40(s,1H),8.28(s,1H),7.73–7.65(m,1H),7.62– 7.51(m,1H),7.39(s,1H),7.11(t,J=8.6Hz,1H),6.51–6.19(m,1H),5.76–5.60(m,2H),4.60( s,2H),3.45–3.34(m,2H),3.26(t,J=7.2Hz,2H),3.17–2.96(m,3H),2.90(t,J=7.7Hz,2H),2. 57–2.38(m,2H),2.11–1.86(m,4H),1.67–1.58(m,2H),1.56–1.47(m,2H),1.42–1.26(m,18H).

[0275] 19 F NMR(471MHz,Methanol-d4)δ-77.06,-114.49,-134.74.[α] D 20 -30.0 (c 0.5, EtOH).

[0276] High resolution mass spectra: HRMS (ESI) calculated as C 37 H 52 F3N 10 O2 725.4221[M+H] + , the detection result is 725.4217.

[0277] Embodiment 28

[0278] A compound, named ND-312, has the following structure:

[0279]

[0280] The above compound is: (S)-2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminododecyl)acetamide.

[0281] ND-312 was prepared by General Procedure B with (+)-M7 and M312 as reactants as a light yellow solid (TFA salt) in 30% yield.

[0282] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.53(s,1H),8.41(s,1H),8.29(s,1H),7.72–7.66(m,1H), 7.61–7.51(m,1H),7.38(s,1H),7.11(t,J=8.6Hz,1H),6.49–6.21(m,1H),5.75–5.62(m, 2H),4.60(s,2H),3.37–3.33(m,2H),3.26(t,J=7.2Hz,2H),3.08–2.87(m,3H),2.54–2. 41(m,2H),2.13–1.89(m,4H),1.68–1.59(m,2H),1.55–1.48(m,2H),1.43–1.23(m,20H).

[0283] High resolution mass spectra: HRMS (ESI) calculated as C 38 H 54 F3N 10 O2 + 739.4378[M+H] + , the detection result is 739.4379.

[0284] Embodiment 29

[0285] A compound, named ND-309, has the following structure:

[0286]

[0287] The above compound is: (S)-2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminononyl)acetamide.

[0288] ND-309 was prepared from (+)-M7 and M309 by general method B as a white solid (TFA salt) in 39% yield.

[0289] The H NMR spectrum is: 1H NMR(500MHz,Methanol-d4)δ8.54(s,1H),8.42(s,1H),8.31(s,1H),7.72–7.65(m,1H),7.59–7 .53(m,1H),7.48(t,J=7.8Hz,1H),7.40–7.32(m,2H),7.11(t,J=8.6Hz,1H),6.47–6.19(m,1H), 5.78–5.64(m,2H),4.60(s,2H),3.60–3.36(m,2H),3.26(t,J=7.2Hz,2H),3.13–2.85(m,6H),2 .54–2.40(m,2H),2.11–1.91(m,4H),1.68–1.58(m,2H),1.56–1.50(m,2H),1.40–1.31(m,14H).

[0290] The carbon NMR spectrum is: 13 C NMR(125MHz,Methanol-d4)δ170.28,154.57(d,J C-F =115.1Hz),153.07,150.45,148.60,148.13,146.15,144.93,144.32,143.02,138.73,134.05,131.11,128.66,126.31,124.11,120.23,1 16.90,115.92,115.76,69.53,59.69,55.45,54.87,54.35,43.57,40 .79,40.15,32.47,31.39,30.34,30.16,28.58,27.84,27.39,22.11.

[0291] 19 F NMR(471MHz, Methanol-d4)δ-77.21,-114.59,-134.87.

[0292] High resolution mass spectra: HRMS (ESI) calculated as C 35 H 48 F3N 10 O2 + 697.3908[M+H] + , the detection result is 697.3924.

[0293] Embodiment 30

[0294] A compound, named ND-307, has the following structure:

[0295]

[0296] The above compound is: (S)-2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminoheptyl)acetamide.

[0297] ND-307 was prepared by General Method B with (+)-M7 and M307 as reactants as a light yellow solid (TFA salt) in 45% yield.

[0298] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.55(s,1H),8.47(s,1H),8.35(s,1H),7.71–7.64(m,1H),7.59– 7.51(m,1H),7.33(s,1H),7.11(t,J=8.6Hz,1H),6.46–6.20(m,1H),5.84–5.66(m,2H),4.60( s,2H),3.38–3.32(m,2H),3.27(t,J=7.2Hz,2H),3.18–2.96(m,2H),2.90(t,J=7.7Hz,3H),2. 59–2.39(m,2H),2.17–1.92(m,3H),1.70–1.60(m,2H),1.60–1.52(m,2H),1.45–1.28(m,8H).

[0299] High resolution mass spectra: HRMS (ESI) calculated as C 33 H 44 F3N 10 O2 669.3595[M+H] + , the detection result is 669.3593.

[0300] Embodiment 31

[0301] A compound, named ND-306, has the following structure:

[0302]

[0303] The above compound is: (S)-2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminohexyl)acetamide.

[0304] ND-306 was prepared by General Method B with (+)-M7 and M306 as reactants as a pale yellow solid (TFA salt) in a yield of 41%.

[0305] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.54(s,1H),8.44(s,1H),8.32(s,1H),7.72–7.64(m,1H),7 .62–7.51(m,1H),7.35(s,1H),7.11(t,J=8.6Hz,1H),6.50–6.21(m,1H),5.83–5.63(m,2 H),4.60(s,2H),3.40–3.34(m,2H),3.29–3.26(m,2H),3.24–2.87(m,5H),2.55–2.41(m, 2H), 2.12–1.91 (m, 4H), 1.64 (p, J = 7.2Hz, 2H), 1.57 (p, J = 7.2Hz, 2H), 1.46–1.31 (m, 6H).

[0306] The carbon NMR spectrum is: 13 C NMR(125MHz,Methanol-d4)δ170.39,155.03,154.65,153.62(d,J C-F =136.3Hz),150.44,149.55,148.09,146.15,144.59,144.43,142.98,134.13,124.08,120.27,116.92,116.46,11 5.91,115.75,69.55,61.59,59.74,55.43,54.89,43.50,40.66,39.91,32.47,30.21,28.50,27.28,26.98,22.11.

[0307] 19 F NMR (471MHz, Methanol-d4) delta -77.28, -114.57, -134.83.

[0308] High resolution mass spectra: HRMS (ESI) calculated as C 32 H 42 F3N 10 O2 + 655.3439[M+H] + , the detection result is 655.3437.

[0309] Embodiment 32

[0310] A compound, named ND-305, has the following structure:

[0311]

[0312] The above compound is: (S)-2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminopentyl)acetamide.

[0313] ND-305 was prepared by general procedure B using (+)-M7 and ML05 as reactants as a light yellow solid (TFA salt) in 33% yield.

[0314] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.54(s,1H),8.44(s,1H),8.32(s,1H),7.71–7.65(m,1H),7 .56(d,J=8.7Hz,1H),7.35(s,1H),7.12(t,J=8.7Hz,1H),6.49–6.14(m,1H),5.81–5.65(m ,2H),4.61(s,2H),3.38–3.34(m,2H),3.30–3.28(m,2H),3.16–2.86(m,4H),2.58–2.40(m ,2H),2.13–1.91(m,4H),1.67(p,J=7.5Hz,2H),1.59(p,J=7.5Hz,2H),1.47–1.33(m,4H).

[0315] High resolution mass spectra: HRMS (ESI) calculated as C 31 H 40 F3N 10 O2 641.3282[M+H] + , the detection result is 641.3279.

[0316] Embodiment 33

[0317] A compound, named ND-303, has the following structure:

[0318]

[0319] The above compound is: (S)-2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminopropyl)acetamide.

[0320] (ND-303 was prepared by General Method B using (+)-M7 and M303 as reactants to obtain ND-303 as a colorless solid (TFA salt) in 31% yield.

[0321] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.57(s,1H),8.49(s,1H),8.36(s,1H),7.70–7.63(m,1 H),7.60–7.49(m,1H),7.36–7.32(m,1H),7.14(t,J=8.6Hz,2H),6.46–6.16(m,2H),5 .88–5.68(m,2H),4.65(s,2H),3.44–3.35(m,4H),3.18–2.99(m,2H),2.95(t,J=7.4 Hz,2H),2.56–2.42(m,2H),2.13–1.97(m,2H),1.92–1.82(m,2H),1.36–1.29(m,2H).

[0322] The carbon NMR spectrum is: 13 C NMR(125MHz,Methanol-d4)δ171.37,159.18,158.84,154.60(d,J C-F =109.7Hz),153.08,150.45,150.11,147.98,146.17,144.49,142.99,134.33,124.10,120.29,118.36,117.23,116.97, 116.46,115.96,115.80,114.97,114.57,112.71,69.49,59.77,55.41,54.89,43.47,38.26,36.82,32.46,28.77,22.09.

[0323] 19 F NMR (471MHz, Methanol-d4)δ 19 F NMR (471MHz, Methanol-d4) delta -76.86, -77.39, -114.53, -134.54.

[0324] High resolution mass spectra: HRMS (ESI) calculated as C 29 H 36 F3N 10 O2 + 613.2969[M+H] +, the detection result is 613.2964.

[0325] Embodiment 34

[0326] A compound, named ND-311M, has the following structure:

[0327]

[0328] The above compound is: (S)-2-(5-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purin-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-(methylamino)undecyl)acetamide.

[0329] ND-311M was prepared by general method B using (+)-M7 and N-methylundecane-1,1-diamine as reactants. It is a light yellow solid (TFA salt) with a yield of 35%.

[0330] The H NMR spectrum is: 1 H NMR(500MHz,Methanol-d4)δ8.56(s,1H),8.44(s,1H),8.34(s,1H),7.60–7.54(m,1H),7.45–7.3 8(m,1H),7.34(s,1H),7.23–7.15(m,1H),6.47–6.18(m,1H),5.82–5.64(m,2H),4.60(s,2H),3.42 –3.32(m,2H),3.24(t,J=7.0Hz,2H),3.22–3.06(m,3H),3.03–2.93(m,4H),2.87(s,3H),2.70–2. 41(m,2H),2.12–1.91(m,5H),1.75–1.55(m,3H),1.52–1.43(t,J=7.0Hz,2H),1.39–1.20(m,18H).

[0331] 19 F NMR (471MHz, Methanol-d4) delta -73.70, -75.20, -77.18, -114.60, -134.56.

[0332] High resolution mass spectra: HRMS (ESI) calculated as C 38 H 54 F3N 10 O2 + 739.4378[M+H] + , the detection result is 739.4382.

[0333] In addition to the above-mentioned example compounds, the known positive control compound used in the following anti-tumor activity and biological activity tests, namely, NSD2 SET inhibitor compound CAS: 2604512-02-7, was prepared by the synthesis method reported in WO2021028854.

[0334] Biological activity and anti-tumor efficacy testing:

[0335] Compounds were tested for NSD degradation activity and H3K36 methylation levels:

[0336] The test conditions are as follows:

[0337] The cell culture conditions of the three cells involved in the present invention are as follows: KMS11 and 22RV1 cells were cultured in RPMI 1640 medium (BasalMedia, #L210KJ). All cell culture media additionally contained 10% fetal bovine serum, and 293T cells used DMEM plus 10% fetal bovine serum, as well as 1% antibiotic and anti-mycoplasma penicillin-streptomycin-Saveit (PSS) solution (Hanbio, HB-PSS-100). All cells were grown in a humidified incubator at 37.0°C, CO 2 The concentration is 5.0%. Mycoplasma testing is performed twice a month to ensure that the cell culture medium is free of mycoplasma.

[0338] Immunoblotting and antibodies used: Cell samples were lysed with SDS sample buffer. The lysed samples were then heat denatured at 99°C for 15 minutes. Subsequently, proteins were separated using 8% and 10% acrylamide SDS-PAGE. Nitrocellulose membranes (GE Healthcare) were used to transfer the separated proteins. The membranes were then blocked in 5 mL of 5% skim milk (diluted with TBST) at room temperature for 60 minutes. After blocking, the membranes were incubated with primary antibodies for 14-18 hours with gentle agitation. The membranes were washed three times with 5 mL of TBST, and after washing for 15 minutes, the membranes were incubated with the corresponding secondary antibodies at a dilution of 1:10 000 for 60 minutes. The membranes were washed three times with 5 ml of TBST, and after washing for 15 minutes, the membranes were incubated with SuperSignal WestDura (Thermo Scientific, #34076) for 1 minute to remove excess solution. The chemiluminescent signals were captured using the GE ImageQuant LAS 4000 system.

[0339] The following primary antibodies were used: NSD1 (SANTA CRUZ BIOTECHNOLOGY, INC., Catalog #sc-130470), NSD2 (Cell Signaling Technology, Catalog #65127S), NSD3 (Cell Signaling Technology, Catalog #92056S), Anti-Histone H3 (mono methyl K36) (Abcam, Catalog #ab176920), Anti-Histone H3 (di methyl K36) (Abcam, Catalog #ab176921), Anti-Histone H3 (tri methyl K36) (Abcam, Catalog #ab282572).

[0340] Compound binding activity test for NSD (SPR)

[0341] The truncated human NSD2 gene was cloned into the pet-21b vector, and a 6×His-HRV 3C site tag was added to the N-terminus. The corresponding plasmid was transformed into Escherichia coli BL21 (DE3) cells and cultured in 1 liter of Luria-Bertani (LB) medium at 37°C for 5 hours, and then 0.4mM IPTG (isopropyl-β-D-1-thiogalactopyranoside) was used to induce NSD2 protein expression at 16°C for 16 hours. The cells were collected by centrifugation (8,000rpm, 4°C, 20 minutes), resuspended in lysis buffer (250mMNaCl, 50mM Tris pH 7.4, 20mM imidazole pH 7.4, 10mMβ-mercaptoethanol) at 4°C, and then sonicated for about 15 minutes. The cell lysate was centrifuged at 18,000 rpm for 1 hour, and the soluble part of the cells was collected. The His-tagged target protein was then separated using a nickel-equipped HiTrap Chelating FF column produced by GE Healthcare. The His-tag was cleaved by incubation with His-tagged PreScission protease overnight, and the solution was re-loaded onto the nickel column for removal. The target protein was further purified using a Superdrex 75 column (GE Healthcare) equilibrated with running buffer (250 mM NaCl, 50 mM Tris pH 7.4, 1 mM DTT). The purified protein was concentrated to about 10 mg / ml and stored in a refrigerator at -80°C. SPR analysis was performed on a Biacore T200 instrument (GE Healthcare) at 25°C to measure the binding affinity of NSD2 to the compound. The NSD2 protein was coupled to a CM5 chip (GE Healthcare) and the optical density reached 6000 RU at a flow rate of 30 μL / min. The path without coupling of NSD2 protein was used as a control reference. The compounds were dissolved in SPR buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.5% v / v DMSO) and diluted to different concentrations. The final 0 μM (no drug) concentration was used as the compound control. The compounds were then injected at a flow rate of 30 μL / min, bound to the immobilized protein for 120 seconds, and dissociated for 300 seconds. Finally, the K values ​​of the compounds for NSD2 protein were obtained using Biacore T200 software version 1.0. D value to assess its binding affinity.

[0342] The compound's proliferation inhibitory activity on KMS11 multiple myeloma cells and cytotoxicity test on human embryonic kidney 293T cells:

[0343] KMS11 and 293T cells were diluted to a density of 10,000 cells / mL with complete growth medium. 100 μL of the diluted cells were then plated on a 96-well plate. About 3 h after seeding, 100 μL of cell culture medium containing compounds was added to each well at serial dilutions. After 8-12 days, cell viability was assessed using the CellTiter-Glo kit (Promega, #G7570).

[0344] Among them, the NSD2 degradation activity and multiple myeloma cell proliferation inhibition activity of compounds ND-101 to ND-117 were determined, such as Figure 2 shown.

[0345] Among them, the NSD2 degradation activity and multiple myeloma cell proliferation inhibition activity of compounds ND-201 to ND-210 were determined, such as Figure 3 shown.

[0346] Among them, the changes in the content of NSD2 and its long form, RE-IIBP, the changes in the methylation levels of NSD1 / 3 and H3K36 after treatment with compounds ND-302 to ND-312, and the SPR affinity test graph are shown in Figure 2. Figure 4 shown.

[0347] Among them, the compound ND-311B was tested for its proliferation inhibition activity on KMS11 multiple myeloma cells and its cytotoxicity on non-tumor normal human embryonic kidney cells 293T. Figure 5 shown.

[0348] According to the instruction manual Figure 2 The experimental results show that the ND-111 compound in the VHL E3 ligand-linked compound exhibits NSD2 degradation activity and KMS multiple myeloma cell proliferation inhibition activity. VHL ligands can recruit VHL E3 ubiquitin ligases to degrade NSD2, which is an important pathogenic protein that promotes the proliferation of multiple myeloma.

[0349] According to the instruction manual Figure 3 The experimental results in the figure show that ND-210, among the adamantane-linked compounds, exhibits NSD2 degradation activity and KMS multiple myeloma cell proliferation inhibition activity. The adamantane hydrophobic tag can induce heat shock protein-mediated degradation of NSD2, which is an important pathogenic protein that promotes the proliferation of multiple myeloma.

[0350] According to the instruction manual Figure 4 The experimental results in the literature show that ND-311B, a primary amine compound, has good NSD2 degradation activity and can simultaneously degrade two tumor-related isomers, NSD2-Long (long form) and RE-IIBP (DC 501.48 and 0.8 μM respectively), and can also degrade NSD1 and NSD2 in the SET family. RE-IIBP has a special non-enzyme function that cannot be regulated by previous inhibitors and is closely related to tumor onset (PLoS One, 9 (2014) e99493; Sci Rep, 5 (2015) 12485). The preferred embodiment of the present invention can achieve the degradation of RE-IIBP, which is helpful for the treatment of malignant tumors associated with RE-IIBP. In addition, the targeted degradation of NSD2 RE-IIBP has not been reported in previous literature. Existing technical means cannot effectively intervene in the cancer-promoting non-enzyme function of NSD2 isomer RE-IIBP to enhance anti-tumor activity. It can be seen that the present invention has significant innovation. At the same time, the attached Figure 4 The Western blot structure in the results showed that the fatty chain length of the S-configured primary amine compound ND-(303-312) and the configuration of the SET ligand were related to the activity. The S-configured ND-311B with an 11-carbon chain length had the best activity, and too short or too long chain lengths were not conducive to degradation activity. Figure 4 The WB and SPR results in the results show that the R-enantiomer ND-311A (enantiomer of ND-311B) has weaker degradation activity and SET binding affinity, which indicates that NSD2 degradation depends on the S-enantiomer SET ligand binding ability. Figure 4 The results shown in the figure show that the short fatty chain compound ND-(303-309) with S-configuration SET ligand has weak degradation activity, which indicates that the degradation of NSD2 requires the SET ligand and the fatty primary amine ligand of appropriate length to work together. It can be seen that the previously reported NSD2 ligand inhibitors cannot achieve degradation effect. Figure 4 The examples ND-311P and ND-311M are tertiary amine and amide structures derived from ND-311B, and have weak degradation activity, which indicates that the NSD2 degradation induced by the preferred examples is primary amine-dependent. As described in the technical background section, the primary amine-dependent characteristics indicate that ND-311B recruits FBXO22 to achieve degradation, which is different from the mechanism of action of previous inhibitors that do not contain long-chain primary amines and cannot degrade proteins.

[0351] As attached Figure 4As shown in the WB results of H3K36 methylation, the preferred embodiment ND-311B of the present invention can reduce the methylation level of H3K36 in the KMS11 multiple myeloma cell line and the 22RV1 prostate cancer cell line, and the reduction of H3K36 methylation in these malignant tumors can have a therapeutic effect of inhibiting tumor proliferation or be used in combination with other drugs to achieve a therapeutic effect (Mol Cell, 44 (2011) 609-620; Nat Genet (2024). DOI: 10.1038 / s41588-024-01893-6).

[0352] According to the instruction manual Figure 4 The experimental results in the literature show that ND-311B, a primary amine compound, has good NSD2 degradation activity and can simultaneously degrade two tumor-related isomers, NSD2-Long (long form) and RE-IIBP (DC 50 1.48 and 0.8 μM respectively), and can also degrade NSD1 and NSD3 in the SET family. RE-IIBP has a special non-enzyme function that cannot be regulated by previous inhibitors and is closely related to tumor onset. The preferred embodiment of the present invention can achieve the degradation of RE-IIBP, which has not been reported in previous literature and has significant innovation. NSD2 and its isomers are important pathogenic proteins that promote the proliferation of multiple myeloma. The preferred embodiment of the present invention, ND-311B, can reduce the methylation level of H3K36 in the KMS11 multiple myeloma cell line and the 22RV1 prostate cancer cell line, and the reduction of H3K36 methylation in these malignant tumors can have a therapeutic effect of inhibiting tumor proliferation or can also be used in combination with other drugs (Nat Genet (2024). DOI: 10.1038 / s41588-024-01893-6).

[0353] As the instruction manual Figure 5 As shown, the preferred embodiment compound ND-311B of the present invention has an inhibitory activity (IC 50 =0.9 μM) was significantly superior to the antitumor activity (IC =0.9 μM) of the known inhibitors containing SET domain ligand structure but without long fatty chain primary amine (NSD2 SET inhibitor compound CAS: 2604512-02-7; WO2021028854) under the same experimental conditions. 50=4.7μM), the anti-tumor activity of the degradation agent increased by 5.2 times. It can be seen that under the same experimental conditions, compared with the known technical inventions, the preferred embodiments of the present invention have significant advantages in anti-tumor efficacy. The anti-tumor mode of action of ND-311B, that is, combining with the SET domain to recruit E3 ligase through long fatty chain primary amines to achieve synchronous degradation of NSD2 long form and RE-IIBP, has a different mechanism from the previously reported SET inhibitors, and the structure of ND-311B connected to long-chain fatty primary amines has not been reported before, and cannot be known from the existing technical documents, which has significant innovation. In addition, as shown in the attached manual Figure 5 As shown, the preferred embodiment compound ND-311B of the present invention has no significant effect on the cell activity of non-tumor normal cells human embryonic kidney cells 293T, while under the same experimental conditions, the above-mentioned known inhibitor (CAS: 2604512-02-7) containing a SET domain ligand structure but without a long fatty chain primary amine has significant cytotoxicity to the 293T non-tumor normal cell line. It can be seen that the preferred embodiment of the present invention can achieve selective killing of malignant tumors, while having little effect on the cell activity of human non-tumor normal cell lines, and has a significant safety advantage.

[0354] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A compound of formula I, formula II or formula III, or a pharmaceutically acceptable salt, stereoisomer, prodrug, solvate or deuterated compound thereof, characterized in that: The structure of the compound of formula I, formula II or formula III is as follows: Among them, each R 1 independently selected from hydrogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl or hydroxy halide C 1-6 alkyl; Each R 2 independently selected from hydrogen, halogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl or C 1-6 Alkoxy; The linking group in formula I, II, and III is -(L 1 ) n -(L 2 ) n -(L 3 ) n -(L 4 ) n -(L 5 ) n -; among them, L 1 , L 2 , L 3 , L 4 , L 5 Each is independently selected from: -CH2-, -O-, -NH-, -CH2CH2O-, piperazine or 4-amino-piperidine; and each n is independently selected from an integer of 0-20.

2. The compound according to claim 1, characterized in that: The carbon number of the long-chain fatty amine connected to the linking group in Formula I is 7-17 carbons.

3. The compound according to claim 1, characterized in that: The R 1 Selected from hydrogen or 4. The compound according to claim 1, characterized in that: The R 2 Selected from hydrogen, fluorine or C 1-6 Alkoxy.

5. The compound according to claim 1, characterized in that: The linking group is selected from one of the following structures: The first linking group, The second linking group, The third linking group, The fourth linking group, The fifth connecting group, The sixth connecting group, The seventh connecting group, The eighth connecting group, The ninth connecting group, A tenth connecting group.

6. The compound according to claim 1, characterized in that: The compound of formula I, formula II or formula III includes:

7. The compound according to claim 1, characterized in that: The compound is:

8. A NSD2 degradation agent, characterized in that: The NSD2 degrading agent includes the compound according to any one of claims 1-7.

9. A drug for treating a disease mediated by a SET domain protein, characterized in that: The medicament comprises the compound according to claims 1-7.

10. The drug according to claim 9, characterized in that The disease is cancer.

11. The drug according to claim 10, characterized in that The disease is selected from: multiple myeloma, leukemia, prostate cancer, lymphoma, lung cancer, breast cancer, stomach cancer, liver cancer, head and neck malignancies, pancreatic cancer, and ovarian cancer.

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