An NSD2 degrading agent and its application

Abstract

This invention discloses an NSD2 degrading agent and its applications, comprising compounds of formula I, II, or III, or pharmaceutically acceptable salts, stereoisomers, prodrugs, solvates, and deuterated compounds thereof. The compounds of formula I, II, or III, or pharmaceutically acceptable salts, stereoisomers, prodrugs, solvates, and deuterated compounds of this invention, are formed by combining a SET domain ligand with a primary amine degrading ligand, adamantane, or a VHL E3 ligase ligand to form the structure shown. The compounds of this invention can recruit FBXO22 or VHL E3 ligases to degrade NSD2, or achieve degradation via an adamantane hydrophobic tag, and can simultaneously degrade two isoforms of NSD2: long and RE-IIBP, exhibiting excellent antitumor effects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically the field of targeted protein degradation technology, and specifically relates to a class of NSD2 degrading agents and their uses. Background Technology

[0002] Nuclear receptor-binding SET domain protein (NSD) is a class of histone lysine methyltransferases. The SET domain is its catalytic domain. It belongs to the SETD2-Like family of the SET superfamily and has three subtypes: NSD1, 2, and 3. It participates in the dimethylation of lysine 36 of histone H3 (H3K36me2). The NSD2 subtype, also known as MMSET (MM, multiple myeloma) and WHSC1 (Wolf-Hirschhorn syndrome candidate 1), has three expression products—long, short, and RE-IIBP—due to alternative splicing. Its conserved regions mainly consist of the PWWP1 and PWWP2 domains (rich in proline-tryptophan-tryptophan-proline), multiple PHD zinc finger structures, a catalytically functional SET domain, pre-SET (AWS) and post-SET domains, an HMG box structure, and a cysteine-histidine-rich C5HCH domain. RE-IIBP contains the SET domain but not the PWWP1 domain, while the long subtype contains both the SET and PWWP1 domains. NSD2 plays a vital physiological role by regulating the expression of multiple genes through epigenetic modification. The loss or inactivation of NSD2 can lead to developmental abnormalities and disrupt the immune system, and is associated with the pathogenesis of congenital diseases such as Rauch-Steindl syndrome and Wolf-Hirschhorn syndrome. Conversely, overexpression and abnormally enhanced activity of NSD2 are closely related to the pathogenesis of various hematological malignancies and malignant tumors (J Med Chem, 64(2021)14913-14929). In conclusion, NSD2 is an important target for cancer treatment.

[0003] 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 at the histone H3K36 site by binding to the SET and PWWP1 domains of the NSD2 protein. However, the mechanism of action of these inhibitors means they cannot regulate other non-enzymatic functions of NSD2, or abnormal methylation at other histone sites. Furthermore, inhibitors are difficult to effectively intervene in NSD2 E1099K gain-of-function mutants in malignant tumors, while degraders can degrade these mutants, offering advantages in tumor therapeutics compared to inhibitors. Degraders based on PROTAC (proteolysis-targeting chimeras) technology can utilize the ubiquitin-protease system to degrade NSD2, thereby regulating the non-enzymatic functions of NSD2.

[0004] The three isoforms of NSD2—long, short, and RE-IIBP—possess 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 promote tumor proliferation and migration by aberrantly methylating sites such as H3K4, H3K27, and H3K79 (Sci Rep, 5(2015)12485). These non-enzymatic functions of NSD2 proteins cannot be interfered with by NSD2 inhibitors, but can be regulated by NSD2 degrading agents. However, since RE-IIBP does not contain the PWWP1 domain, existing degrading agents that act on the PWWP1 domain cannot degrade RE-IIBP. The degrading agents reported in the literature only degrade the long and short isoforms of NSD2. Currently, there are no degrading agents that can simultaneously degrade the RE-IIBP isoform of NSD2. Therefore, existing techniques cannot effectively intervene in the pro-cancer non-enzymatic functions of the NSD2 isoform RE-IIBP. Summary of the Invention

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

[0006] The purpose of this invention is to provide a degrading agent that can simultaneously and efficiently degrade the long and RE-IIBP isomers of NSD2.

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

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

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

[0010] An NSD2 degrading agent is provided.

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

[0012] To provide 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 as follows:

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

[0015]

[0016] Among them, each R 1 Independently selected from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl or hydroxy halogenated 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 formulas I, II, and III is -(L 1 ) n -(L 2 ) n -(L 3 ) n -(L 4 ) n -(L 5 ) n -; where 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 from 0 to 20.

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

[0020] The compounds of this invention, or their pharmaceutically acceptable salts, stereoisomers, prodrugs, solvates, and deuterated compounds, are formed by conjugating a SET domain ligand with a primary amine degradation ligand, adamantane, or a VHL E3 ligase ligand to form the structures shown. The compounds of this invention can recruit FBXO22 or VHL E3 ligases to degrade NSD2, or achieve degradation via an adamantane hydrophobic tag, and can simultaneously degrade two isoforms of NSD2: long and RE-IIBP. Specifically, the adamantane hydrophobic tag can induce heat shock protein-mediated degradation of NSD2; the VHL ligand can recruit VHL E3 ubiquitin ligases to degrade NSD2, and NSD2 is an important pathogenic protein promoting the proliferation of multiple myeloma; simultaneously, the primary amine compounds of this invention have good NSD2 degrading activity because aliphatic primary amines have the function of recruiting FBXO22 E3 ligases.

[0021] Unlike previous NSD2 inhibitors that inhibit enzyme function by occupying catalytic or H3 binding sites, the compound of this invention, when used as an NSD2 degrader, recruits E3 ligands through E3 ligands, induces ubiquitination and degradation of the target protein, and directly reduces the content of NSD2 and / or its isomers. This achieves dual regulation of its enzymatic and non-enzymatic functions, thus exhibiting potential anti-tumor advantages.

[0022] In particular, the NSD2 alternatively spliced ​​isoform RE-IIBP possesses unique non-enzymatic functions and is closely related to tumorigenesis. Preferred embodiments of the present invention can simultaneously achieve the degradation of both NSD2-RE-IIBP and the long form of NSD2, which has not been reported in existing literature and represents significant innovation. The preferred embodiment of the NSD2 degrading agent of the present invention, under the same experimental conditions, exhibits more potent inhibitory activity against multiple myeloma cell lines compared to known inhibitors, and shows less toxicity to non-tumor, non-tumor normal cell lines, demonstrating significant therapeutic advantages.

[0023] The primary amine compounds of the present invention have good NSD2 degradation agent activity. Preferably, the S-configuration primary amine compounds have better effects. More preferably, the S-configuration ND-311B with an 11-carbon chain length has the best activity. Too short a chain length or too long a chain length is detrimental to degradation activity.

[0024] Furthermore, the R-configuration enantiomer ND-311A (the enantiomer of ND-311B) of this invention exhibits relatively weak degradative activity and SET binding affinity, indicating that NSD2 degradation depends on the binding ability of the S-configuration SET ligand. The short-chain aliphatic compound ND-(303-309) with the S-configuration SET ligand shows relatively weak degradative activity, suggesting that NSD2 degradation requires the synergistic effect of both the SET ligand and a suitable-length aliphatic primary amine ligand.

[0025] Examples ND-311P and ND-311M are tertiary amine and amide structures derived from ND-311B, and have relatively weak degradation activity, indicating 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 an NSD2 degrading agent.

[0028] According to one embodiment of the present invention, the pharmaceutically acceptable salt is: a suitable salt formed by the compound involved in the present invention with an organic acid, organic base, inorganic acid, or 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-methylpropanediol, triisopropanolamine, ethylenediamine, hexamethylethylenediamine, morpholine, piperidine, piperazine, cyclohexylamine, tributylamine, dodecylamine, dimethyldodecylamine; triethylamine benzylamine, 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 formulas I, II, and III using known salt-forming methods.

[0029] According to one embodiment of the present invention, the long-chain aliphatic amine linked to the linking group in Formula I has 7-17 carbons. When the number of carbons is less than 7 or greater than 17, the compound will be inactive or have 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 Alkyl group.

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

[0033] First linking group, Second linking group, Third linking group Fourth linking group, Fifth linking group, The sixth linking group, Seventh linker group Eighth linking group, Ninth linking group, The tenth linking 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 with this structure is named compound ND-311B in this invention. Compound ND-311B is a further preferred compound in the embodiments of this invention. ND-311B exhibits stronger multiple myeloma cell inhibitory activity than known SET inhibitors and can significantly alter the H3K36 methylation level in multiple myeloma and prostate cancer cells. At the same time, it achieves the simultaneous degradation of both RE-IIBP and the long isomer, showing good prospects for disease treatment.

[0043] According to one embodiment of the present invention, a portion of the compounds are synthesized via the following synthetic route, wherein the compounds are named in this invention as compounds ND-(101-117), ND-(202-210), ND-(303-312), ND-311M, or ND-311P:

[0044]

[0045] Specifically, according to a second aspect of the invention, therein lies an NSD2 degrading agent:

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

[0047] A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt, stereoisomer, prodrug, solvate, and deuterated compound thereof, and further comprising pharmaceutically acceptable excipients.

[0048] According to one embodiment of the present invention, the excipients are selected from: lactose, microcrystalline cellulose, methylcellulose, sodium hydroxymethylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, starch, dextrin, cellulose derivatives, polyvinyl alcohol, gelatin, polyethylene glycol, polyvinyl alcohol, brown sugar, distilled water, ethanol, starch paste, antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the subject, as well as aqueous and non-aqueous sterile suspending agents, 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 gastrointestinal or non-gastrointestinal administration.

[0050] Another aspect of the present invention provides a medicament for treating diseases mediated by SET domain proteins. It includes compounds as described in the embodiments of the first aspect above, or pharmaceutically acceptable salts, stereoisomers, prodrugs, solvates, and deuterated compounds thereof. Because this application employs all the technical solutions of the aforementioned compounds or pharmaceutically acceptable salts, stereoisomers, prodrugs, solvates, and deuterated compounds, it possesses at least all the beneficial effects of the technical solutions described in the above embodiments.

[0051] Preferably, a drug for preparing a treatment for 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 diseases are selected from: multiple myeloma, leukemia, prostate cancer, lymphoma, lung cancer, breast cancer, stomach cancer, liver cancer, head and neck malignant tumors, pancreatic cancer, and ovarian cancer.

[0054] According to one embodiment of the present invention, the diseases mediated by the SET domain-containing 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 carcinoma, hepatocellular carcinoma, gastric cancer, intestinal cancer, membranous adenocarcinoma, 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 tumor, head and neck cancer, and hematologic malignancies.

[0056] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

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

[0058] Figure 1 This is a diagram showing the intermediate synthesis steps, separation steps, and absolute configuration identification steps in route 1 of the embodiment.

[0059] Figure 2 The graph shows the degradation activity and inhibitory activity against multiple myeloma cell proliferation of compounds NSD2 in Examples 1-17.

[0060] Figure 3 The graph shows the degradation activity and inhibitory activity against multiple myeloma cell proliferation of compounds NSD2 in Examples 18-23.

[0061] Figure 4 The diagram shows the changes in the content of NSD2 and its long form, RE-IIBP, 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 graphs after treatment with the compounds in Examples 24-34.

[0062] Figure 5The graphs show the inhibitory activity of the compound and control compound from Example 25 against KMS11 multiple myeloma cells and their cytotoxicity against human embryonic kidney cells 293T. Detailed Implementation

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

[0064] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0065] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of the present invention.

[0066] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0067] In the examples, compounds ND-(101-117), ND-(202-210), ND-(303-312), ND-311M, and ND-311P were synthesized via 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: react at -78°C first, 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: methyl 2-bromo-5-fluoroisonicotinic acid, Pd(dppf)Cl₂, Cs₂CO₃, 1,4-dioxane-water; Conditions: reflux under 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 (k) Reagents: H2, Pd(OH)2 / C, AcOH, ethanol, room temperature; (l) Reagents: benzyl bromoacetate, K2CO3, acetonitrile, room temperature; (m) Reagents: H2, Pd(OH)2, ethanol, room temperature; (n) Reagents: HATU, DIPEA, DMF, room temperature; (o) Reagents: CF3COOH, DCM, room temperature.

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

[0072] In Route 1, the synthesis of intermediate methyl 2-(3-(benzyloxy)-4-fluorophenyl)-5-fluoroisonicotinic acid (M1) was performed as follows: 28.01 g of methyl 2-bromo-5-fluoroisonicotinic acid, 30.05 g of SM2, 1.50 g of Pd(dppf)Cl2, and 78.30 g of Cs2CO3 were dissolved in 250 mL of 1,4-dioxane / water (4:1). The reaction mixture was refluxed under an Ar atmosphere for 2 h and then filtered through diatomaceous earth. The filtrate was then concentrated under reduced pressure and the resulting mixture was further purified by rapid chromatography (EA:PE = 2-30%) to give 25.93 g of M1, a yellow solid, in a yield of ~61%. The 1H NMR spectrum was as follows: 1¹H NMR (500MHz, Chloroform-d) δ 8.67–8.58 (m, 1H), 8.11 (d, J = 5.4Hz, 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). LC-MS (ESI) was calculated as C1. 20 H 16 F2NO3 356.1 [M+H] + The result was 356.1.

[0073] In Route 1, the synthesis of intermediate methyl 2-(3-(benzyloxy)-4-fluorophenyl)-5-(3-(tert-butoxycarbonyl)amino)-3-(2,2-difluoroethyl)piperidin-1-yl)isonicotinic acid (M2) was performed as follows: 13.51 g of M1 and 10.07 g of MN5 were dissolved in 25 mL of DMSO, and then 8 mL of DIPEA was added. The reaction mixture was stirred at 120 °C for 12 hours. After cooling to room temperature, the reaction mixture was diluted with 100 mL of water and then extracted with EA (80 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. Further purification was performed by rapid chromatography (EA:PE = 10-50%) to obtain 12.85 g of M2, a yellow oil, with a yield of ~56%. The 1H NMR spectrum was as follows: 1 ¹H NMR (600MHz, 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). LC-MS (ESI) calculated as C1. 32 H 37 F3N3O5 600.3[M+H] + The test result was 600.5.

[0074] In Route 1, the synthesis 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) was performed 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. Further purification was performed by rapid chromatography (EA:PE = 20-50%) to give 7.01 g of M3, a pale yellow solid, in a yield of ~61%. The proton NMR spectrum is as follows: 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 result was 572.4.

[0075] In Route 1, the synthesis 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-purine-6-yl)(tert-butoxycarbonyl)carbamate (M4) proceeded as follows: 6.29 g of M3 was dissolved in 60 mL of dry DCM, cooled to 0 °C, and then 2.5 eq. 3.27 g of SOCl2 was added dropwise. After stirring at 0 °C for 30 min, TLC showed that the reaction was complete. The reaction was quenched with 100 mL of Na2CO3 at 0 °C. The resulting mixture was extracted with DCM (100 mL × 3). The combined DCM solutions were washed with saturated NaCl aqueous solution, dried over Mg2SO4, and concentrated under reduced pressure to obtain the crude chlorinated product, which was used directly in the next step. 3.85 g of the chlorinated product N was then... 6 -diBoc-adenine, 3.05 g K₂CO₃ dissolved in 50 mL acetonitrile. The reaction mixture was stirred at rt for 18 hours, then filtered through a celite pad. The filtrate was then concentrated under reduced pressure and further purified by rapid chromatography (C18 column, acetonitrile:water 0.1%, FA = 10-100%) to give 5.18 g M₄, a white powder, yield ~53% (2 steps). The 1H NMR spectrum is as follows: 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 0.77–5.40(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 test result was 889.7.

[0076] In Route 1, the synthesis 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-purine-6-yl)carbamate (M5) proceeded as follows: 5.05 g of M4 was dissolved in 50 mL of ethanol, followed by the addition of 0.50 g of Pd(OH)2 / C and 1 mL of CH3COOH. The reaction was stirred for 36 hours under a 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 rapid chromatography (C18 column, acetonitrile:water 0.1%, FA = 10-100%) to give 3.63 g of M5, a white powder, with a yield of ~81%. The 1H NMR spectrum was as follows: 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 C1. 39 H 50 F3N8O7 799.4 [M+H] + The test result was 799.5.

[0077] In Route 1, 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) was synthesized as follows: 3.50 g of M5, 1.05 g of benzyl bromoacetate, and 1.20 g of K2CO3 were 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 rapid chromatography (C18 column, acetonitrile:water 0.1%, FA = 10-100%) to give 2.07 g of M6, a pale yellow powder, with a yield of ~50%. The 1H NMR spectrum was as follows: 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 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 test result was 947.7.

[0078] In Route 1, the synthesis of 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) was performed as follows: 2.05 g of M6 was dissolved in 50 mL of ethanol, followed by the addition of 0.50 g of Pd(OH)2 / C and 1 mL of CH3COOH. The reaction was stirred for 36 hours under a 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 rapid chromatography (C18 column, acetonitrile:water 0.1%, FA = 10-100%) to give 1.61 g of M7, a pale yellow powder, in ~87% yield. The 1H NMR spectrum was as follows: 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 0.94 (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 C1. 41 H 52 F3N8O9 857.4[M+H] + The result was 857.6.

[0079] In Route 1, a benzoyl group that absorbs ultraviolet light is 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 intermediate M4 was performed using a C3 IH chiral column and methanol-carbon dioxide SFC supercritical fluid chromatography to obtain two enantiomers, (-)-M4A with a specific rotation [α]. D 20 -28.3 (c 0.7, EtOH) and (+)-M4B specific curl [α] D 20 +26.0 (c 0.5, EtOH).

[0081] In Route 1, the synthesis of the intermediate (-)-M4A-Bz used for configuration identification was as follows: 30 mg of (-)-M4A was dissolved in 2 mL of DCM before adding 1 mL of CF3COOH. After stirring at room temperature for 30 minutes, the reaction mixture was concentrated to dryness to obtain the crude amine product. The amine intermediate was then dissolved in 4 mL of DMF, followed by the addition of 0.3 mL of DIPEA and 5 mg of DMAP. 12 mg of benzoyl chloride was added to the stirred solution at 40 °C. After stirring at 40 °C for 20 minutes, LC-MS monitoring showed that the reaction was complete. The mixture was concentrated under reduced pressure, and the residue was further purified by rapid chromatography (C18 column, acetonitrile:water 0.1%, TFA = 10-100%) to give 5 mg of (-)-M4A-Bz as a yellow semi-solid, with a yield of ~20%. The 1H NMR spectrum was as follows: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 as C 45 H 40 F3N8O3 + 797.3170[M+H] + The result was 797.3168.

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

[0083] In the embodiments, the final target compound was synthesized using general method A and general method B (method B for primary amine compounds).

[0084] The preparation of ND-(V01-V11), ND-(CO2-C11), and ND-L11P in this embodiment of the invention employs general method A, including the amide condensation process and subsequent deprotection process. Typical procedure: Before adding HATU (1.1 equivalents), 30 mg (0.035 mmol) of M7, 5 equivalents of DIPEA, and 1.0 equivalents of the corresponding amine intermediates (MV01-MV11, MCO2-MC12, PL11) are mixed in 3-5 mL of CH3CN. After LC-MS monitoring shows the reaction is complete, the reaction is quenched with 0.5 mL of water and concentrated to dryness. The residue is then dissolved in 2 mL of CF3COOH to remove the Boc group. After the reaction was confirmed by LC-MS monitoring, the concentrated mixture was purified by rapid chromatography (C18 column, acetonitrile:water:0.1% TFA = 10-100%, and in some cases, 0.1% formic acid aqueous solution) to obtain ND-(V01-V11), ND-(CO2-C11), and ND-L11P as TFA or FA salts, with a two-step yield of approximately 35% to 60%.

[0085] The preparation of ND-(303-312) and ND-311M in this embodiment of the invention employs a general method B, involving the amide condensation and subsequent deprotection. The typical steps differ slightly from Method A, with the addition of excess alkyl primary amine and solvent to avoid side reactions. 30 mg (0.035 mmol) of M7, 10 equivalents of DIPEA, and 8.0 equivalents of the corresponding alkyl primary amine intermediates (M303-M312, M311M) are mixed in 10 mL of acetonitrile. Then, 1.1 equivalents of HATU are added. After LC-MS monitoring indicates the reaction is complete, the reaction is quenched with 0.5 mL of water, and the solvent is concentrated to remove it. The residue is then dissolved in 3 mL of CF3COOH to remove the Boc group. After the reaction was completed as monitored by LC-MS, the TFA salts of ND-(303-312) and ND-311M were concentrated and purified by rapid chromatography (C18 column, acetonitrile:water 0.1%, TFA = 5-100%), with an overall yield of approximately 30-45%. 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamidodecanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide·TFA.

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

[0091] The proton NMR spectrum is as follows: 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 as follows: 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 spectrometry is: HRMS (ESI) calculated as C 61 H 81 F3N 13 O6S1180.6100[M+H] + The result was 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)undecanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0099] The above-mentioned compound was used as reactants to prepare ND-110 by conventional method A, which was a pale yellow solid (TFA salt) with a yield of 53%.

[0100] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 60 H 79 F3N 13 O6S1166.5944[M+H] + The result was 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)decanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0106] The above compound was prepared by conventional method A using M7 and MV09 as reactants to obtain ND-109, which was a pale yellow solid (TFA salt) with a yield of 46%.

[0107] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 59 H 77 F3N 13 O6S1152.5787[M+H] + The result was 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)nonamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0113] The above compound was prepared by conventional method A using M7 and MV08 as reactants to obtain ND-108, which was a pale yellow solid (TFA salt) with a yield of 49%.

[0114] The proton NMR spectrum is as follows: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 spectrometry is: HRMS (ESI) calculated as C 58 H 75 F3N 13 O6S1138.5631[M+H] + The result was 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)octamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0120] The above compound was prepared by conventional method A using M7 and MV07 as reactants to obtain ND-107, which was a pale yellow solid (TFA salt) with a yield of 38%.

[0121] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 57 H 73 F3N 13 O6S1124.5474[M+H] + The result was 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)acetamido)-3,3-dimethylbutamido)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0127] The above compound was prepared by conventional method A using M7 and MV06 as reactants to obtain ND-106, which was a pale yellow solid (TFA salt) with a yield of 47%.

[0128] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 56 H 71 F3N 13 O6S1110.5318[M+H] + The result obtained was 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)hexanediamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0134] The above compound was prepared by conventional method A using M7 and MV05 as reactants to obtain ND-105, which was a pale yellow solid (TFA salt) in 50% yield.

[0135] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 55 H 69 F3N 13 O6S1096.5161[M+H] + The value was 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)pentamine)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0141] The above-mentioned compound was used as reactants to prepare ND-104 by conventional method A. The result was a pale yellow solid (HCOOH salt) with a yield of 35%.

[0142] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 54 H 67 F3N 13 O6S1082.5005[M+H] + The result obtained was 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamido)butamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

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

[0149] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 53 H 65 F3N 13 O6S1068.4848[M+H] + The result was 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)acetamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

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

[0156] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 52 H 63 F3N 13 O6S1054.4692[M+H] + The result was 1054.4698.

[0158] Example 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)acetamido)acetamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

[0162] The above compound was prepared by conventional method A using M7 and MV01 as reactants to obtain ND-101, which was a pale yellow solid (HCOOH salt) with a yield of 36%.

[0163] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 51 H 61 F3N 13 O6S1040.4535[M+H] + The result was 1040.4543.

[0165] Example 12

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

[0167]

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

[0169] The proton NMR spectrum is as follows: 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 result was 1243.7.

[0171] Preparation of intermediate MV12, namely (2S,4R)-1-((S)-1-amino-17-(tert-butyl)-11,15-dioxo-3,6,9-trioxa-12,16-diazaoctadecyl-18-oxy)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide: 50 mg MV2 and 35 mg 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-oic acid were dissolved in 5 mL acetonitrile. 0.2 mL DIPEA was added, followed by 40 mg HATU condensing agent. The mixture was stirred at room temperature for 30 minutes, and the solvent and alkali were removed by rotary evaporation. 2 mL trifluoroacetic acid was then added, and the mixture was reacted at room temperature for 30 minutes. The mixture was then concentrated to obtain the crude product. The crude product was subjected to reversed-phase column chromatography (C18, acetonitrile:water 0.1% formic acid) to yield approximately 45 mg of product. LC-MS (ESI) yielded a C1 value. 34 H 53 N6O8S + 705.4[M+H] + The result was 705.4.

[0172] Example 13

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

[0174]

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

[0176] The proton NMR spectrum is as follows: 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 O9S + 1186.5[M+H]+, the result of the test is 1186.7.

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

[0179] Example 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)acetamyl)butyryl)piperazin-1-yl)pentamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

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

[0184] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 62 H 81 F3N 15 O7S + 1236.6111[M+H] + The result obtained was 1236.6106.

[0186] The synthesis method of 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 that of intermediate M7, except that the starting material for M7 is replaced by 3-Boc aminopiperidine without difluoroethyl, instead of MN5. The resulting intermediate M7-H is an analog of M7 without difluoroethyl, and its LC-MS (ESI) value is calculated to be C0. 39 H 50 FN8O9 + 793.4[M+H] + The result was 793.4.

[0187] Example 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 pale yellow solid with a yield of approximately 55%.

[0191] The proton NMR spectrum is as follows: 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 result was 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)pentanoyl)piperidin-4-yl)amino)pentanoyl)-3,3-dimethylbutyryl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazolyl-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, as a colorless solid with a yield of about 45%.

[0198] The proton NMR spectrum is as follows: 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]+, the result of the detection is 1207.7.

[0200] Preparation of intermediate M7-4 (5-(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)valerate): 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. The mixture was stirred overnight at room temperature. After filtration and concentration, the reaction solution was subjected to reversed-phase column chromatography (C18, acetonitrile: water 0.1% formic acid) to obtain approximately 95 mg of M6-4. M6-4 was redissolved in ethanol, and 100 mg of 10% Pd(OH)2 / C was added. The mixture was hydrogenated overnight at atmospheric pressure. After filtration and concentration, 80 mg of M7-4, a colorless semi-solid, was obtained, with a yield (two steps) of approximately 59%. LC-MS (ESI) calculated the concentration as C. 44 H 58 F3N8O9 + 899.4 [M+H] + The test result was 899.5.

[0201] Preparation of intermediate MV16 ((2R,4S)-1-((R)-3,3-dimethyl-2-(5-(piperidin-4-ylamino)pentamine)butyryl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide): Intermediate MV16 was prepared by the reductive amination reaction of intermediate MV4 and N-Boc-4-piperidinone. 60 mg of MV4 and 50 mg of N-Boc-4-piperidinone were dissolved in an HFIP-DCE mixture (5 mL 1:3), and 30 mg of sodium triacetoxyborohydride was added in three portions, with stirring overnight at room temperature. After quenching the reaction with a small amount of water, the product was concentrated by rotary evaporation. The crude product was then added to 2 mL of trifluoroacetic acid and stirred for half an hour, followed by further concentration. Reversed-phase column chromatography (C18, acetonitrile:water, 0.1% trifluoroacetic acid) yielded approximately 41 mg of a pale yellow oily product, with a yield of approximately 60%. LC-MS (ESI) calculated the product to be C1. 33 H 51 N6O4S + 627.4 [M+H] + The test result was 627.5.

[0202] Example 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-(5-(3-amino-3-(2,2-difluoroethyl)piperidin-1-yl)-4-((6-amino-9H-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)valeryl)piperidin-4-yl)amino)hexamamide)-3,3-dimethylbutyryl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide.

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

[0207] The proton NMR spectrum is as follows: 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 result was 1221.7.

[0209] Preparation of intermediate MV17 ((2R,4S)-1-((R)-3,3-dimethyl-2-(6-(piperidin-4-ylamino)hexanoyl)butyryl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide): Intermediate MV17 was prepared by the reductive amination reaction of intermediate MV5 and N-Boc-4-piperidinone. 60 mg of MV5 and 50 mg of N-Boc-4-piperidinone were dissolved in an HFIP-DCE mixture (5 mL 1:3), and 35 mg of sodium triacetoxyborohydride was added in three portions, with stirring overnight at room temperature. After quenching the reaction with a small amount of water, the product was concentrated by rotary evaporation. The crude product was then added to 2 mL of trifluoroacetic acid and stirred for half an hour, followed by further concentration. Reversed-phase column chromatography (C18, acetonitrile:water, 0.1% trifluoroacetic acid) yielded approximately 39 mg of a pale yellow oily product, with a yield of approximately 55%. LC-MS (ESI) calculated the product to be C1. 34 H 53 N6O4S + 641.4[M+H] + The result was 641.4.

[0210] Example 18

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

[0212]

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

[0214] The proton NMR spectrum is as follows: 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 test result was 775.5.

[0216] Example 19

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

[0218]

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

[0220] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 42 H 54 F3N 10 O3 + 803.4327[M+H] + The result was 803.4319.

[0222] Example 20

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

[0224]

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

[0226] The proton NMR spectrum is as follows: 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] Example 21

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

[0229]

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

[0231] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C46 H 62 F3N 10 O3 + 859.4953[M+H] + The result obtained was 859.4959.

[0233] Example 22

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

[0235]

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

[0237] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 47 H 64 F3N 10 O3 + 873.5109[M+H] + The result was 873.5101.

[0239] Example 23

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

[0241]

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

[0243] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 48 H 66 F3N 10 O3 + 887.5266[M+H] + The measured value was 887.5278.

[0245] Example 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)acetamido)undecyl)benzamide.

[0249] The above compound was prepared by conventional method A using (+)-M7 and N-(1-aminoundecyl)benzamide as reactants to obtain ND-311P, which was a pale yellow solid (TFA salt) in 50% yield.

[0250] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 44 H 56 F3N 10 O3 829.4483[M+H] + The result was 829.4488.

[0253] Example 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-mentioned ND-311B(S) was prepared by general method B, using (+)-M7 and M311 as reactants, as a colorless solid (TFA salt) with a yield of 35%.

[0258] The proton NMR spectrum is as follows: 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 as follows: 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 spectrometry is: HRMS (ESI) calculated as C 37 H 52 F3N 10 O2 725.4221[M+H] + The result was 725.4216.

[0262] Example 26

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

[0264]

[0265] Synthesis of the racemic form: 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 conventional method B, using (+)-M7 and M311 as reactants, as a white solid (TFA salt) in 39% yield.

[0267] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 37 H 52 F3N 10 O2 725.4221[M+H] + The value was 725.4215.

[0269] Example 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 using (-)-M7 and M311 as reactants, as a colorless solid (TFA salt) in 32% yield.

[0274] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 37 H 52 F3N 10 O2 725.4221[M+H] + The result was 725.4217.

[0277] Example 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 method B using (+)-M7 and M312 as reactants, as a pale yellow solid (TFA salt) in 30% yield.

[0282] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 38 H 54 F3N 10 O2 + 739.4378[M+H] + The result was 739.4379.

[0284] Example 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminononyl)acetamide.

[0288] ND-309 was prepared by conventional method B using (+)-M7 and M309 as reactants. It was a white solid (TFA salt) with a yield of 39%.

[0289] The proton NMR spectrum is as follows: 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 as follows: 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) delta -77.21, -114.59, -134.87.

[0292] High-resolution mass spectrometry is: HRMS (ESI) calculated as C 35 H 48 F3N 10 O2 + 697.3908[M+H] + The result was 697.3924.

[0293] Example 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminoheptyl)acetamide.

[0297] ND-307 was prepared by conventional method B using (+)-M7 and M307 as reactants, and was a pale yellow solid (TFA salt) with a yield of 45%.

[0298] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 33 H 44 F3N 10 O2 669.3595 [M+H] + The result was 669.3593.

[0300] Example 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminohexyl)acetamide.

[0304] ND-306 was prepared by conventional method B using (+)-M7 and M306 as reactants, as a pale yellow solid (TFA salt) in 41% yield.

[0305] The proton NMR spectrum is as follows: 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 as follows: 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 spectrometry is: HRMS (ESI) calculated as C 32 H 42 F3N 10 O2 + 655.3439[M+H] + The result was 655.3437.

[0309] Example 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-purine-9-yl)methyl)pyridin-2-yl)-2-fluorophenoxy)-N-(11-aminopentyl)acetamide.

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

[0314] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 31 H 40 F3N 10 O2 641.3282 [M+H] + The result was 641.3279.

[0316] Example 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. The reactants were colorless solids (TFA salts) with a yield of 31%.

[0321] The proton NMR spectrum is as follows: 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 as follows: 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 (471 MHz, Methanol-d4)δ 19 F NMR (471MHz, Methanol-d4) delta -76.86, -77.39, -114.53, -134.54.

[0324] High-resolution mass spectrometry is: HRMS (ESI) calculated as C 29 H 36 F3N 10 O2 + 613.2969[M+H] +The result was 613.2964.

[0325] Example 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 was a pale yellow solid (TFA salt) with a yield of 35%.

[0330] The proton NMR spectrum is as follows: 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 spectrometry is: HRMS (ESI) calculated as C 38 H 54 F3N 10 O2 + 739.4378[M+H] + The result was 739.4382.

[0333] In addition to the compounds in the examples above, the known positive control compound used in the antitumor activity and bioactivity tests below, namely the NSD2 SET inhibitor compound CAS:2604512-02-7, was prepared by the synthetic method reported in WO2021028854.

[0334] Bioactivity and antitumor efficacy testing:

[0335] The NSD degradation activity and H3K36 methylation level of the compound were tested:

[0336] The test conditions are as follows:

[0337] The cell culture conditions for the three cell types involved in this invention are as follows: KMS11 and 22RV1 cells were cultured in RPMI 1640 medium (BasalMedia, #L210KJ). All cell cultures additionally contained 10% fetal bovine serum (FBS), while 293T cells were cultured in DMEM with an additional 10% FBS, along with 1% antibiotics and an anti-mycoplasma penicillin-streptomycin-Saveit (PSS) solution (Hanbio, HB-PSS-100). All cells were grown in a humidified incubator at 37.0°C. 2 The concentration is 5.0%. Mycoplasma is tested 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 denatured at 99°C for 15 minutes. Proteins were subsequently 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 antibody for 14–18 hours with gentle agitation. The membranes were washed three times with 5 mL of TBST for 15 minutes each time, followed by incubation with the appropriate secondary antibody diluted 1:10,000 for 60 minutes. The membranes were washed three times with 5 mL of TBST for 15 minutes each time, followed by incubation with SuperSignal WestDura (Thermo Scientific, #34076) for 1 minute to remove excess solution. Chemiluminescence signals were captured using a 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] SPR (Special Purpose Reactivity Test) of Compounds for NSD

[0341] The truncated human NSD2 gene was cloned into the pet-21b vector, and a 6×His-HRV 3C tag was added to the N-terminus. The corresponding plasmid was transformed into *E. coli* BL21(DE3) cells and cultured in 1 L Luria-Bertani (LB) medium at 37°C for 5 hours. NSD2 protein expression was then induced for 16 hours at 16°C with 0.4 mM IPTG (isopropyl-β-D-1-thiogalactopyranoside). Cells were collected by centrifugation (8,000 rpm, 4°C, 20 min), resuspended in lysis buffer (250 mM NaCl, 50 mM Tris pH 7.4, 20 mM imidazole pH 7.4, 10 mM β-mercaptoethanol) at 4°C, and then sonicated for approximately 15 minutes. Cell lysates were centrifuged at 18,000 rpm for 1 hour, and the soluble fraction was collected. The His-tagged target protein was then separated using a nickel-coated HiTrap Chelating FF column (GE Healthcare). The His-tag was lysed by overnight incubation with His-tagged PreScission protease, and the solution was removed by repacking the nickel column. 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 approximately 10 mg / ml and stored 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) at a flow rate of 30 μL / min to achieve an optical density of 6000 RU. A pathway without NSD2 protein coupling was used as a control. The compound was 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 added) concentration served as the compound control. The compound was then injected at a flow rate of 30 μL / min, binding to the immobilized protein for 120 seconds and dissociating for 300 seconds. Finally, the Kelvin (K) titer of the compound against the NSD2 protein was determined using Biacore T200 software version 1.0. D The value was used to assess its binding affinity.

[0342] The compound's inhibitory activity against the proliferation of KMS11 multiple myeloma cells and its cytotoxicity against human embryonic kidney cells 293T were tested.

[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 into 96-well plates. Approximately 3 hours after seeding, 100 μL of cell culture medium containing the compound was added to each well, serially diluted. Cell viability was assessed using the CellTiter-Glo kit (Promega, #G7570) after 8–12 days.

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

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

[0346] Among them, the changes in the content of NSD2 and its elongated form, RE-IIBP, the changes in the methylation level of NSD1 / 3 and H3K36 after treatment with compounds ND-302 to ND-312, and the SPR affinity test diagram are shown, as follows: Figure 4 As shown.

[0347] Among them, the inhibitory activity of compound ND-311B against the proliferation of KMS11 multiple myeloma cells and the cytotoxicity of non-tumor normal human embryonic kidney cells 293T were tested, such as... Figure 5 As shown.

[0348] According to the instruction manual Figure 2 The experimental results show that the ND-111 compound, linked by the VHL E3 ligand, exhibits NSD2 degrading activity and KMS multiple myeloma cell proliferation inhibitory activity. The VHL ligand can recruit the VHL E3 ubiquitin ligase to degrade NSD2, which is a key pathogenic protein promoting multiple myeloma proliferation.

[0349] According to the instruction manual Figure 3 The experimental results show that ND-210, linked by adamantane, exhibits NSD2 degrading activity and KMS multiple myeloma cell proliferation inhibitory activity. The adamantane hydrophobic tag can induce heat shock protein-mediated degradation of NSD2, which is a key pathogenic protein promoting multiple myeloma proliferation.

[0350] According to the instruction manual Figure 4 The experimental results showed that ND-311B, a primary amine compound, exhibited good NSD2 degrading activity and could simultaneously degrade two tumor-associated isomers, NSD2-Long and RE-IIBP (DC). 50The concentrations are 1.48 μM and 0.8 μM respectively. It can also degrade NSD1 and NSD2 in the SET family. RE-IIBP has unique non-enzymatic functions that cannot be regulated by previous inhibitors and are closely related to tumorigenesis (PLoS One, 9(2014)e99493; Sci Rep, 5(2015)12485). The preferred embodiments of this invention can achieve the degradation of RE-IIBP, which is helpful in treating malignant tumors associated with RE-IIBP. Furthermore, the targeted degradation of NSD2 RE-IIBP has not been reported in previous literature. Existing technologies cannot effectively intervene in the pro-cancer non-enzymatic functions of the NSD2 isoform RE-IIBP to enhance anti-tumor activity. Therefore, this invention has significant innovation. (The appendix...) Figure 4 Western blot analysis revealed that the aliphatic chain length and SET ligand configuration of the S-configuration primary amine compound ND-(303-312) were related to its activity. The S-configuration ND-311B with an 11-carbon chain length exhibited the best activity, while excessively short or long chain lengths were detrimental to degradation activity. (Appendix) Figure 4 The WB and SPR results showed that the R-configuration enantiomer ND-311A (the enantiomer of ND-311B) exhibited weaker degradative activity and SET binding affinity, indicating that NSD2 degradation depends on the binding ability of the S-configuration SET ligand. Furthermore, according to the appendix... Figure 4 The results show that the short-chain aliphatic compound ND-(303-309) with the S-configuration SET ligand exhibits weak degradation activity. This indicates that the degradation of NSD2 requires the synergistic effect of both the SET ligand and a suitable-length aliphatic primary amine ligand. Therefore, it is evident that previously reported NSD2 ligand inhibitors are ineffective in achieving degradation. Figure 4 The examples ND-311P and ND-311M are tertiary amine and amide structures derived from ND-311B, and have weak degradation activity. This indicates that the NSD2 degradation induced by the preferred examples is primary amine dependent. As described in the background section, the primary amine dependent characteristic indicates that ND-311B recruits FBXO22 to achieve degradation, which is different from the mechanism of action of previous inhibitors that cannot degrade proteins without long-chain primary amines.

[0351] As attached Figure 4The WB results of H3K36 methylation show that 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. The reduction of H3K36 methylation in these malignant tumors can have a therapeutic effect of inhibiting tumor proliferation or can 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 showed that ND-311B, a primary amine compound, exhibited good NSD2 degrading activity and could simultaneously degrade two tumor-associated isomers, NSD2-Long and RE-IIBP (DC). 50 The concentrations are 1.48 and 0.8 μM, respectively. It can also degrade NSD1 and NSD3 in the SET family. RE-IIBP has unique non-enzymatic functions that cannot be regulated by previous inhibitors and are closely related to tumorigenesis. The preferred embodiment of this invention can achieve the degradation of RE-IIBP, which has not been reported in previous literature and is highly innovative. NSD2 and its isoforms are important pathogenic proteins that promote the proliferation of multiple myeloma. The preferred embodiment of this invention, ND-311B, can reduce the methylation level of H3K36 in the KMS11 multiple myeloma cell line and the 22RV1 prostate cancer cell line. The reduction of H3K36 methylation in these malignant tumors can have a therapeutic effect of inhibiting tumor proliferation or can be used in combination with other drugs (Nat Genet (2024). DOI:10.1038 / s41588-024-01893-6).

[0353] As per the instruction manual Figure 5 As shown, the preferred embodiment of the present invention, compound ND-311B, exhibits inhibitory activity (IC50) against the proliferation of KMS11 multiple myeloma cells. 50 =0.9 μM) significantly outperformed known inhibitors containing SET domain ligand structures but lacking long aliphatic primary amines (NSD2 SET inhibitor compound CAS: 2604512-02-7; WO2021028854) under the same experimental conditions in terms of antitumor activity (IC50, 0.9 μM). 50=4.7 μM), the antitumor activity of the degrading agent was increased by 5.2 times. Therefore, under the same experimental conditions, compared with known inventions, the preferred embodiment of this invention has a significant advantage in antitumor efficacy. The antitumor mechanism of ND-311B, namely, binding to the SET domain and recruiting E3 ligases through long-chain aliphatic primary amines to achieve simultaneous degradation of NSD2 long and RE-IIBP, differs from previously reported SET inhibitors. Furthermore, the structure of ND-311B linked to a long-chain aliphatic primary amine is not previously reported and cannot be known from existing technical documents, demonstrating significant innovation. In addition, as per the appendix to the specification... Figure 5 As shown, the preferred embodiment of the present invention, compound ND-311B, has no significant effect on the cell viability of non-tumor normal human embryonic kidney cells 293T. However, under the same experimental conditions, the aforementioned known inhibitor (CAS: 2604512-02-7), which contains a SET domain ligand structure but does not contain a long adipose chain primary amine, has significant cytotoxicity to the 293T non-tumor normal cell line. It is evident that the preferred embodiment of the present invention can achieve selective killing of malignant tumors with minimal impact on the cell viability of human non-tumor normal cell lines, demonstrating a significant safety advantage.

[0354] The above are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A compound of formula I, II, or III, or a pharmaceutically acceptable salt thereof and a deuterated compound thereof, characterized in that: The structures of the compounds of formula I, II, or III are as follows: ； Among them, each R 1 Independently selected from hydrogen and C 1-6 Alkyl or halogenated C 1-6 alkyl; R 2 It is a halogen; The linking group in formulas I, II, and III is -(L 1 ) n -(L 2 ) n -(L 3 ) n -(L 4 ) n -(L 5 ) n -; where 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 from 0 to 20.

2. The compound according to claim 1, characterized in that: The number of carbons in the long-chain aliphatic amines linked to the linking group in Formula I is 7-17.

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 It is fluorine.

5. The compound according to claim 1, characterized in that: The linking group is selected from one of the following structures: First linking group, Second linking group, Third linking group Fourth linking group, Fifth linking group, The sixth linking group, Seventh linker group Eighth linking group, Ninth linking group, The tenth linking group.

6. The compound according to claim 1, characterized in that: The compounds of formula I, II or III include: 。 7. The compound according to claim 1, characterized in that: The compound is: 。 8. An NSD2 degrading agent, characterized in that: The NSD2 degrading agent includes the compound according to any one of claims 1-7.

9. A medicament for treating diseases mediated by SET domain proteins, characterized in that: The drug comprises the compounds according to claims 1-7.

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

11. The medicament according to claim 10, characterized in that, The diseases mentioned are selected from: multiple myeloma, leukemia, prostate cancer, lymphoma, lung cancer, breast cancer, stomach cancer, liver cancer, head and neck malignant tumors, pancreatic cancer, and ovarian cancer.

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