A compound for targeted degradation of BUB1B and BUB1 proteins, its preparation method and application

Abstract

The present invention discloses a compound, its optical isomers and pharmaceutically acceptable salts thereof that target the degradation of BUB1B and BUB1 proteins, which have a significant degradation effect on BUB1B and BUB1 proteins in cancers such as non-small cell lung cancer, colorectal cancer and large B lymphoma, and significantly inhibit the proliferation of related cells, and can be developed as a new anti-tumor drug, having broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of drugs, and particularly relates to a compound for targeted degradation of BUB1B and BUB1 proteins, a preparation method thereof, and an application thereof. Background Art

[0002] The spindle assembly checkpoint (SAC, also known as the mitotic checkpoint) is a major cell cycle control mechanism in mitosis. It is responsible for generating genetically identical daughter cells by ensuring the fidelity of chromosome segregation. The SAC is a signaling cascade that prevents chromosome missegregation by arresting the cell cycle in mitosis until all chromosomes are correctly attached to the mitotic spindle.

[0003] Complete inactivation of the SAC is lethal to malignant tumor cells, resulting in a high degree of chromosomal instability that exceeds the adaptive capacity of cancer cells. Notably, normal cells seem to be less sensitive to death caused by SAC inhibition compared to cancer cells. Since aneuploid cancer cells are more prone to chromosome missegregation, they spend more time in metaphase to align abnormal numbers of chromosomes. In addition, these cells may experience protein imbalance or other dysregulations, leading to synthetic lethality. Therefore, intervening in SAC function is a potential tumor-targeted therapy strategy.

[0004] BubR1 (i.e., BUB1B) and BUB1 are paralogous serine / threonine kinases that are key proteins in the SAC. BUB1B binds to unattached centromeres, helps to stabilize centromere-MT attachment and align chromosomes, and forms part of the MCC. One study showed that BUB1B acts as a pseudokinase in SAC signaling, mainly through a scaffolding function, while other research groups found that the function of BUB1B extends beyond the SAC, including roles in DNA repair, ciliogenesis, and senescence. BUB1 is required for chromosome congression, localization of SAC proteins to centromeres, and establishment and maintenance of efficient bipolar chromosome attachment to the spindle MT. High expression of BUB1 occurs in multiple human tumor types and is usually associated with poor clinical prognosis. In addition, BUB1 has been shown to bind to the transforming growth factor-β (TGFβ) receptor, thereby triggering invasive cell growth. These findings suggest that there may be a crosstalk point between SAC and TGFβ signaling and further support targeting BUB1 as an anticancer therapy.

[0005] Targeted protein degradation technology is expected to specifically intervene in BUB1B and BUB1 by inducing the formation of new protein-protein interaction surfaces. In addition, since the degrader molecules remove the protein itself, all its enzymatic and non-enzymatic functions can be inhibited simultaneously. Therefore, we designed and synthesized a series of degrading agents and found that the active molecules can significantly degrade BUB1B and BUB1 proteins in non-small cell lung cancer, colorectal cancer, and large B lymphoma. Moreover, the molecules with degrading activity can significantly inhibit the proliferation of related cells by causing cell cycle arrest and cohesion defects of sister chromatids, showing great therapeutic potential in non-small cell lung cancer, colorectal cancer, and large B lymphoma. Summary of the Invention

[0006] The object of the present invention is to provide a compound for targeted degradation of BUB1B and BUB1 proteins, its optical isomers, and its pharmaceutically acceptable salts, which have a significant degrading effect on BUB1B and BUB1 proteins in cancers such as non-small cell lung cancer, colorectal cancer, and large B lymphoma and can significantly inhibit the proliferation of related cells, and can be developed as a new anti-tumor drug, having broad application prospects.

[0007] To achieve the above object, the present invention provides a compound represented by formula (I), its optical isomers, and its pharmaceutically acceptable salts:

[0008]

[0009] In the general formula, PL is a known affinity small molecule targeting CDK12 protein; E3 ligase ligand is a DCAF16 ligand; Linker is a linking group.

[0010] PL is a known affinity small molecule targeting CDK12 protein, and its specific structure is the U series T series P series

[0011] The E3 ligase ligand is a protease small molecule ligand encoded by the DCAF16 gene with ubiquitination function, and its specific structure is

[0012] Linker is a linking group, representing -alkylene or -alkoxy, wherein the -alkylene or -alkoxy is an optionally straight-chain or branched-chain alkylene or alkoxy interrupted one or more times by one or more groups selected from the following groups, such as -(CH2) n -, -(CH2) n CO-, -NR1(CH2) n CO-, -NR2(CH2) n -, -(OCH2CH2O)n -,-(CH2CH2O) n -,-(OCH2CH2OCH2) n -,-(CH2CH2OCH2) n -,-(CH2CH2OCH2CH2) n -,alkenylene, alkynylene, cycloalkylene, heteroarylene or any combination thereof, where n represents a natural number from 1 to 20, and R1 and R2 each independently select H or are C 1-10 alkyl group.

[0013] Preferably, the Linker is selected from the following structures:

[0014] n1 is an integer from 1 to 15;

[0015] n2 is an integer from 1 to 6.

[0016] As a more specific preference:

[0017] As an embodiment, the compound has the structure shown by the following formula:

[0018]

[0019] n2 is 2, 3, 4.

[0020] Preferably, the compounds for degrading BUB1B and BUB1 proteins provided by the present invention include but are not limited to the compounds shown in Table 1 below: Table 1

[0021]

[0022]

[0023]

[0024]

[0025]

[0026] Preferably, the pharmaceutically acceptable salt is one or more of the hydrochloride, trifluoroacetate, phosphate, and sulfate corresponding to any of the above-mentioned compounds.

[0027] Preferably, the compound includes:

[0028] UKOP3-1: N-(2-(2-(2-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide hydrochloride

[0029] UKOP3-2: N-(2-(2-(2-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide trifluoroacetate

[0030] UKOP3-3: N-(2-(2-(2-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide phosphate

[0031] UKOP3-4: N-(2-(2-(2-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide sulfate

[0032] The preparation of the compounds for degrading BUB1B and BUB1 proteins in the present invention includes the following steps:

[0033] (1) Synthesis of U series CDK12 ligands

[0034] N-Boc-trans-1,4-cyclohexanediamine and p-bromoiodobenzene react through Buchwald–Hartwig coupling reaction to obtain compound U-1. Then, the secondary phenylamine of compound U-1 reacts with benzyl isocyanate under the action of triethylamine to form urea, obtaining compound U-2. After removing Boc in hydrogen chloride-ethyl acetate solution, it undergoes aromatic nucleophilic substitution reaction with 6-fluoronicotinonitrile to obtain compound U-3, and the latter reacts with 2-hydroxypyridine-5-boronic acid pinacol ester through Suzuki coupling reaction to obtain the U series CDK12 ligand compound U-L.

[0035]

[0036] Synthetic Route of U-Series CDK12 Ligand Compound U-L

[0037] Reaction reagents and conditions: (a) Pd2(dba)3, Xantphos, t BuONa, toluene, 80 °C, 2.5 h; (b) Et3N, THF, 60 °C, 8 h; (c) i. 4N HCl in EtOAc, 25 °C; ii. DIPEA, NMP, 0 - 100 °C, 8 h; (d) Pd(PPh3)4, Cs2CO3, toluene, EtOH, H2O, 16 h.

[0038] (2) Synthesis of T-Series CDK12 Ligands

[0039] Compound T-1 first undergoes an aromatic nucleophilic substitution reaction with (R)-3-amino-1-Boc-piperidine to obtain compound T-2, and then the Boc group is removed in a hydrogen chloride - ethyl acetate solution to obtain the T-series CDK12 ligand compound T-L.

[0040]

[0041] Synthetic Route of T-Series CDK12 Ligand Compound T-L

[0042] Reaction reagents and conditions: (a) DIPEA, NMP, 0 - 110 °C, 6 h; (b) 4N HCl in EtOAc, 25 °C.

[0043] (3) Synthesis of P-Series PROTACs

[0044] Commercially available 4,5-dichloro-2-nitroaniline undergoes a nucleophilic substitution reaction with Linker chain LPn or LCm to obtain intermediates 5Pn or 5Cm. Compounds 5Pn or 5Cm are first reduced to aniline by tin dichloride, then condensed with Boc-glycine, and then dehydrated and cyclized under heating in acetic acid to obtain intermediates 6Pn or 6Cm. 6Pn or 6Cm are deprotected by a hydrochloric acid - ethyl acetate solution and then undergo a nucleophilic substitution reaction with commercially available 2,6-dichloro-9-(1-methylpyrazol-4-yl)-9H-purine to obtain compounds 7Pn or 7Cm. 7Pn or 7Cm undergo a nucleophilic substitution reaction with morpholine at high temperature to obtain 8Pn or 8Cm. Compounds 8Pn or 8Cm are deprotected under hydrogen and palladium on carbon and then condensed with D-L-acyl chloride to obtain the target products PKP1 - 4, PKC9, PKC11, PKC13.

[0045]

[0046]

[0047] Synthetic Route of P-Series PROTAC Molecules

[0048] Reaction Reagents and Conditions: (a) DIPEA, NMP, 90 °C, 12 h; (b) i. SnCl2·2H2O, EtOAc, 70 °C, 3 h; ii. Boc-Gly-OH, EDCI, HOBt, DIPEA, CH2Cl2, 0 - 25 °C, 12 h; iii. AcOH, 70 °C, 3 h; (c) i. 4N HCl in EtOAc, 25 °C, 12 h; ii. 2,6-dichloro-9-(1-methyl-1H-pyrazol-4-yl)-9H-purine, DIPEA, i PrOH, 80 °C, 2 h; (d) NMP, 200 °C, 3 h; (e) i. Pd / C, H2 (1 atm), EtOH, 80 °C, 12 h; ii. D-L-acyl chloride, DIPEA, DMF, 0 - 25 °C, 12 h.

[0049] (4) Synthesis of Linker Chain

[0050] When the degree of polymerization of the target linker chain is 1 or 2, commercially available amino alcohol raw materials can be reacted with Boc anhydride to obtain compounds 1a and 1b respectively. When the degree of polymerization of the target linker chain is greater than 2, commercially available polyethylene glycol (with a degree of polymerization 2 less than that of the target linker chain) raw materials can be reacted with compound 2a through Williamson ether synthesis to obtain compounds 1c and 1d respectively. Subsequently, compounds 1a - d are reacted with p-toluenesulfonyl chloride to obtain polyethylene glycol linker chains 2a - d.

[0051] Commercially available bromohydrins undergo nucleophilic substitution reactions with potassium phthalimide to obtain compounds 3a - c. Subsequently, the phthaloyl protection is removed by hydrazine hydrate to obtain free amino groups, which are then reacted with Boc anhydride to obtain compounds 4a - c. The latter are reacted with p-toluenesulfonyl chloride to obtain linker chains 5a - c.

[0052] Commercially available diamines SPn or SCm undergo nucleophilic substitution reactions with benzyl chloroformate to obtain linker chains LPn or LCm.

[0053]

[0054]

[0055] Synthetic Routes of Linker Chains 2a - d, 5a - c, LPn, and LCm

[0056] Reaction reagents and conditions: (a) Boc2O, Et3N, CH2Cl2, 0 - 25 °C, 3 h; (b) NaH, 1,4 - dioxane, 25 - 60 °C, 16 h; (c) TsCl, Et3N, DMAP, CH2Cl2, 0 - 25 °C, 2 h; (d) K2CO3, DMF, 60 °C, 12 h; (e) i. N2H4·H2O, EtOH, 80 °C, 5 h; ii. Boc2O, Et3N, CH2Cl2, 0 - 25 °C, 3 h; (f) Et3N, CH2Cl2, 0 - 25 °C, 8 h.

[0057] (5) Synthesis of E3 ligase DCAF16 ligand D - L

[0058] 6 - Hydroxy - 1,2,3,4 - tetrahydroquinoline undergoes a nucleophilic substitution reaction with chloroacetyl chloride to obtain compound D - 1. Compound D - 1 undergoes a nucleophilic substitution reaction with tert - butyl bromoacetate, and the resulting product removes the tert - butyl ester protection in trifluoroacetic acid - dichloromethane solution to obtain the DCAF16 ligand compound D - L.

[0059]

[0060] Synthesis route of DCAF16 ligand compound D - L

[0061] Reaction reagents and conditions: (a) NaOH, 1,4 - dioxane, H2O, 0 - 25 °C, 4 h; (b) i. Cs2CO3, DMF, 0 - 25 °C, 3 h; ii. 25% TFA in CH2Cl2, 25 °C.

[0062] (5) Synthesis of target compounds UKNP1 - 4, UKOP1 - 4, UKNC9, UKNC11, UKNC13, UKOC9, UKOC11, UKOC13

[0063] The target - head compound U - L undergoes a nucleophilic substitution reaction with linker chains 2a - d, 5a - c to obtain compounds I - N - 1 - 7, I - O - 1 - 7. The DCAF16 ligand compound D - L reacts with oxalyl chloride to prepare D - L - acyl chloride, which immediately reacts with compounds I - N - 1 - 7, I - O - 1 - 7 after removing the Boc protection to obtain the target PROTAC molecules UKNP1 - 4, UKOP1 - 4, UKNC9, UKNC11, UKNC13, UKOC9, UKOC11, UKOC13.

[0064]

[0065]

[0066] Synthetic Routes of U-Series PROTAC Molecules

[0067] Reaction reagents and conditions: (a) oxalyl chloride, CH2Cl2, 0 - 25 °C, 2 h; (b) K2CO3, DMF, 60 °C, 12 h; (c) i. 4N HCl in EtOAc, 25 °C; ii. DIPEA, DMF, 0 - 25 °C, 3 h.

[0068] (6) Synthesis of Target Compounds TKP1 - 4, TKC9, TKC11, and TKC13

[0069] The nucleophilic substitution reaction of the CDK12 ligand compound T-L with the linker chains 2a - d, 5a - c gives compounds TP1 - 4, TC9, TC11, and TC13. The DCAF16 ligand compound D-L reacts with oxalyl chloride to prepare D-L-acyl chloride, which immediately reacts with the compounds TP1 - 4, TC9, TC11, and TC13 after deprotecting the Boc group to obtain the target PROTAC molecules TKP1 - 4, TKC9, TKC11, and TKC13.

[0070]

[0071] Synthetic Routes of T-Series PROTAC Molecules

[0072] Reaction reagents and conditions: (a) K2CO3, DMF, 60 °C, 12 h; (b) i. 4N HCl in EtOAc, 25 °C; ii. DIPEA, DMF, 0 - 25 °C, 3 h.

[0073] In some embodiments of the present invention, a pharmaceutical composition is provided, wherein the pharmaceutical composition contains a therapeutically effective amount of any one of the said compounds or its pharmaceutically acceptable salts, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0074] In some embodiments of the present invention, the said compounds or their pharmaceutically acceptable salts or pharmaceutical compositions significantly degrade BUB1B and BUB1 proteins in cancers such as non-small cell lung cancer, colorectal cancer, and large B-cell lymphoma, and are expected to become a tumor-targeted treatment strategy.

[0075] In some embodiments of the present invention, the use of any one of the said compounds or their pharmaceutically acceptable salts or pharmaceutical compositions is characterized in that the diseases are selected from cancers, such as non-small cell lung cancer, colorectal cancer, and large B-cell lymphoma.

[0076] Definitions and Explanations

[0077] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indefinite or unclear merely because it is not specifically defined and should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commodity or its active ingredient.

[0078] It should be understood that the substitutions and combinations of substitutions described herein, whether or not explicitly stated, refer to substitutions that conform to the valence of the substituted member. For example, substitution applied to a carbon member refers to the tetravalence of C; when applied to a nitrogen member, it refers to the trivalence of N; when generally indicating a positive charge, it refers to a four-bond nitrogen member. The valence-allowed options are part of the art.

[0079] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0080] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared from compounds having specific substituents found in the present invention with relatively non-toxic acids or bases. When a compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of a base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When a compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of an acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, where the inorganic acids include, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, hydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, where the organic acids include, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid and similar acids; also including salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can thus be converted into either a base or an acid addition salt.

[0081] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing acid radicals or basic groups by conventional chemical methods. Generally, the preparation method of such salts is to react these compounds in the form of free acids or bases with a stoichiometric amount of appropriate bases or acids in water or organic solvents or a mixture of both.

[0082] The term "isomer" means that the compounds of the present invention can exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomer- or diastereoisomer-enriched mixtures, and all these mixtures are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included within the scope of the present invention.

[0083] Unless otherwise specified, "(D)" or "(+)" represents dextrorotation, "(L)" or "(-)" represents levorotation, and "(DL)" or "(±)" represents racemic.

[0084] Unless otherwise specified, a solid wedge bond and a dashed wedge bond represent the absolute configuration of a stereocenter, a solid straight bond and a dashed straight bond represent the relative configuration of a stereocenter, and a wavy line represents a solid wedge bond or a dashed wedge bond or a wavy line represents a solid straight bond and a dashed straight bond

[0085] In addition, when they appear simultaneously, it represents that the relative configuration of the two connected groups is a trans structure, that is, one is upward and the other is downward. For example in, it represents that the configurations of Ra and Rb are trans, one is upward and the other is downward (or one is downward and the other is upward), but it has no restrictive effect on the configuration of the connected six-membered ring.

[0086] The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by substituents, which may include deuterium and variants of hydrogen, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents can be arbitrary based on what is chemically achievable.

[0087] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted with 0 - 2 R's, the group may optionally be substituted with up to two R's, and each R has independent options in each case. In addition, combinations of substituents and / or their variants are permitted only if such combinations result in a stable compound.

[0088] Unless otherwise specified, numerical ranges represent all integers including the numbers at both ends of the range. Unless otherwise specified, integers from 0 - 10 represent 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; integers from 1 - 5 represent 1, 2, 3, 4, 5; integers from 1 - 3 represent 1, 2, 3; C 1-3 Alkyl represents C1, C2, C3 alkyl; C 1-6 Alkyl represents C1, C2, C3, C4, C5, C6 alkyl; C 3-6 Cycloalkyl represents C3, C4, C5, C6 cycloalkyl, and so on.

[0089] Experiments show that:

[0090] (1) The compounds of the present invention have a certain degradation effect on BUB1B and BUB1 in H1299 cells, HCT116 cells, SU - DHL - 4 and SU - DHL - 6 cells.

[0091] (2) Compound UKOP3 has a strong proliferation inhibitory effect on H1299 cells, HCT116 cells, SU - DHL - 4 and SU - DHL - 6 cells, with IC50 values of 117.2 nM, 445.7 nM, 27.86 nM and 26.85 nM respectively. At the same time, it was found that at a given concentration, UKOP3 - N, Warhead and DCAF16 - P3 have no obvious proliferation inhibition on H1299 cells. Therefore, it shows that the proliferation inhibitory effect of UKOP3 does not come from the inhibitory effect of the warhead or the DCAF16 ligand alone, but is the effect of the drug itself.

[0092] (3) The compounds of the present invention can significantly inhibit the proliferation of H1299 cells, HCT116 cells, SU-DHL-4 and SU-DHL-6 cells, while Warhead, DCAF16-P3 and UKOP3-N compounds have less effect on cell proliferation. Therefore, the compounds of the present invention have potential therapeutic effects on related cancers.

[0093] (4) The compounds of the present invention can cause G2 / M phase arrest of the H1299 cell cycle, thereby inhibiting cell proliferation, while their warhead, DCAF16-P3 ligand and negative control compound UKOP3 have less effect on the cell cycle. This further illustrates that the compounds of the present invention have potential therapeutic effects on related cancers.

[0094] (5) The compounds of the present invention can significantly cause sister chromatid cohesion defects in H1299 cells, further indicating that the compounds of the present invention have potential therapeutic effects on related cancers.

[0095] (6) In addition, the compounds of the present invention can significantly decrease the mitotic index of H1299 cells, indicating that they severely inhibit the activity of the SAC checkpoint, while Warhead, DCAF16-P3 and UKOP3 compounds have less effect on the mitotic index of cells, indicating no effect on SAC activity. Therefore, it is again proved that the compounds of the present invention have potential therapeutic effects on related cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 To verify the degradation effect of compound UKOP3 on BUB1B and BUB1 proteins in H1299 cells.

[0097] Figure 2 To verify the degradation effect of compound UKOP3 on BUB1B and BUB1 proteins in HCT116 cells.

[0098] Figure 3 To verify the degradation effect of compound UKOP3 on BUB1B and BUB1 proteins in SU-DHL-4 cells.

[0099] Figure 4 To verify the degradation effect of compound UKOP3 on BUB1B and BUB1 proteins in SU-DHL-6 cells.

[0100] Figure 5 Effects of multiple compounds on the proliferation of H1299 cells.

[0101] Figure 6 Effects of compound UKOP3 and multiple comparative compounds on the proliferation of H1299 cells.

[0102] Figure 7Effect of compound UKOP3 and multiple comparative compounds on the proliferation of HCT116 cells.

[0103] Figure 8 Effect of compound UKOP3 and multiple comparative compounds on the proliferation of SU-DHL-4 cells.

[0104] Figure 9 Effect of compound UKOP3 and multiple comparative compounds on the proliferation of SU-DHL-6 cells.

[0105] Figure 10 To investigate the effect of UKOP3, Warhead and DCAF16 P3 compounds on the cell cycle arrest of H1299 cells.

[0106] Figure 11 To investigate the effect of UKOP3 compound on the sister chromatid cohesion of H1299 cells.

[0107] Figure 12 To investigate the effect of UKOP3 compound on the mitotic index of H1299 cells. Detailed implementation methods

[0108] The present invention will be described in detail below through examples, but this does not mean any adverse limitation to the present invention. The present invention has been described in detail herein, and its specific implementation methods have also been disclosed. For those skilled in the art, various changes and improvements to the specific implementation methods of the present invention will be obvious without departing from the spirit and scope of the present invention.

[0109] In the examples, the experimental methods without specific conditions are conventional methods and conventional conditions well-known in the art, or are operated according to the conditions recommended by the instrument manufacturer.

[0110] Example 1. Synthesis of the warhead U-L

[0111]

[0112] Reaction reagents and conditions: (a) Pd2(dba)3, Xantphos, t BuONa, toluene, 80 °C, 2.5 h; (b) Et3N, THF, 60 °C, 8 h; (c) i. 4N HCl in EtOAc, 25 °C; ii. DIPEA, NMP, 0 - 100 °C, 8 h; (d) Pd(PPh3)4, Cs2CO3, toluene, EtOH, H2O, 16 h.

[0113] Step a: ((1,4-trans)-4-((4-bromophenyl)amino)cyclohexyl)carbamic acid tert-butyl ester U-1

[0114]

[0115] Under nitrogen protection, N-Boc-trans-1,4-cyclohexanediamine (3.0 g, 14.0 mmol) and p-bromoiodobenzene (4.76 g, 16.8 mmol) were dissolved in anhydrous toluene. Sodium tert-butoxide (2.02 g, 21.0 mmol) and Xantphos (730 mg, 1.26 mmol) were added successively. After displacing with nitrogen three times, Pd2(dba)3 (515 mg, 0.56 mmol) was quickly added. After displacing with nitrogen three more times, the temperature was raised to 70 °C and the reaction was carried out for 2.5 h. After monitoring by TLC that the raw material N-Boc-trans-1,4-cyclohexanediamine was completely reacted, the reaction solution was cooled to room temperature, filtered through diatomaceous earth, the filter cake was washed with ethyl acetate, and the filtrate was concentrated under reduced pressure. The product U-1 (2.71 g, 52%) was obtained as a light yellow solid by separation and purification through flash column chromatography. The molecular formula was C 17 H 25 BrN2O2, LC-MS (ESI, m / z): 369.2 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 7.15 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 7.9 Hz, 1H), 6.49 (d, J = 8.9 Hz, 2H), 5.62 (d, J = 8.0 Hz, 1H), 3.25–3.15 (m, 1H), 3.13–2.99 (m, 1H), 1.93 (d, J = 11.5 Hz, 2H), 1.78 (d, J = 11.0 Hz, 2H), 1.37 (s, 9H), 1.30–1.19 (m, 2H), 1.18–1.07 (m, 2H).

[0116] Step b: ((1,4-trans)-4-(3-benzyl-1-(4-bromophenyl)ureido)cyclohexyl) tert-butyl carbamate U-2

[0117]

[0118] Under nitrogen protection, compound U-1 (2.71 g, 7.34 mmol) was dissolved in anhydrous THF, and anhydrous triethylamine (2.23 g, 22.0 mmol) was added. After stirring at room temperature for 5 min, benzyl isocyanate (3.91 g, 29.4 mmol) was added dropwise to the reaction solution, and the temperature was raised to 60 °C and reacted for 8 h. After TLC monitoring showed that the raw material U-1 was completely reacted, the reaction solution was cooled to room temperature, and ethyl acetate was added to dilute and precipitate a solid. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure and separated and purified by flash column chromatography to obtain product U-2 (3.3 g, 90%) as a light yellow solid, with the molecular formula C 25 H 32 BrN3O3, LC-MS (ESI, m / z): 501.2 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 7.61 (d, J = 8.5 Hz, 2H), 7.25 (t, J = 7.4 Hz, 2H), 7.15 (t, J = 8.9 Hz, 3H), 7.09 (d, J = 8.5 Hz, 2H), 6.67 (d, J = 7.9 Hz, 1H), 5.99 (t, J = 5.9 Hz, 1H), 4.19–4.09 (m, 3H), 2.99–2.87 (m, 1H), 1.71 (d, J = 9.6 Hz, 4H), 1.32 (s, 9H), 1.25–1.17 (m, 2H), 1.03–0.94 (m, 2H).

[0119] Step c: 3-benzyl-1-(4-bromophenyl)-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)urea U-3

[0120]

[0121] Compound U-2 (3.3 g, 6.6 mmol) was treated with a 4N hydrogen chloride-ethyl acetate solution. After TLC monitoring showed that the raw material was completely reacted, the solvent in the reaction solution was distilled off under reduced pressure. The obtained solid and 6-fluoronicotinonitrile (1.76 g, 14.4 mmol) were dissolved in anhydrous NMP. After the reaction system was cooled to 0 °C, DIPEA (5.2 g, 40 mmol) was added dropwise. After the addition was complete, the temperature was raised to 100 °C and reacted for 8 h. After TLC monitoring showed that the reaction was complete, the reaction solution was poured into 100 mL of water and extracted with dichloromethane (50 mL x 3). The organic phases were combined and concentrated under reduced pressure. The obtained residue was suspended in ethyl acetate and stirred at room temperature for 15 min. The suspension was filtered through a suction funnel, and the filter cake was washed with a 50% ethyl acetate / petroleum ether mixture. After the filter cake solid was dried under vacuum, product U-3 (2.14 g, 62%) was obtained as an off-white powdery solid, with the molecular formula C26 H 26 BrN5O, LC-MS (ESI, m / z): 504.1 [M+H] + ; 1 H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 7.56 (d, J = 8.5 Hz, 2H), 7.40 (dd, J = 8.8, 2.1 Hz, 1H), 7.29 (t, J = 7.2 Hz, 2H), 7.23 (d, J = 7.1 Hz, 1H), 7.16 (d, J = 7.1 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H), 6.26 (d, J = 8.8 Hz, 1H), 5.19 (d, J = 8.0 Hz, 1H), 4.64–4.49 (m, 1H), 4.36 (d, J = 5.8 Hz, 2H), 4.27 (t, J = 5.8 Hz, 1H), 3.53 (s, 1H), 2.10 (d, J = 11.8 Hz, 2H), 1.95 (d, J = 11.5 Hz, 2H), 1.41 (q, J = 13.4 Hz, 2H), 1.25 (q, J = 15.1, 13.8 Hz, 2H).

[0122] Step d: 3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(6-oxo-1,6-dihydropyridin-3-yl)phenyl)urea U-L

[0123]

[0124] Under nitrogen protection, compound U-3 (2.0 g, 4.0 mmol), 2-hydroxypyridine-5-boronic acid pinacol ester (884 mg, 4.8 mmol) and cesium carbonate (3.9 g, 12 mmol) were suspended in a mixture of toluene, ethanol and water (toluene:ethanol:water = 40:40:1). After purging with nitrogen three times, tetrakis(triphenylphosphine)palladium (462 mg, 0.4 mmol) was quickly added. After purging with nitrogen three more times, the temperature was raised to 100 °C and the mixture was stirred vigorously for 16 h. After monitoring the reaction to completion by LC-MS, the reaction was quenched with water and extracted with ethyl acetate (50 mL x 3). The combined organic phases were concentrated under reduced pressure and purified by flash column chromatography to give the product U-L (1.12 g, 54%) as an off-white solid, with the molecular formula C 31 H 30 N6O2, LC-MS (ESI, m / z): 519.2 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H), 8.29 (d, J = 2.4 Hz, 1H), 7.88 (dd, J = 9.6, 2.8 Hz, 1H), 7.77 (d, J = 2.9 Hz, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.60 (dd, J = 8.9, 2.4 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.30–7.25 (m, 2H), 7.22–7.16 (m, 5H), 6.46 (dd, J = 9.3, 6.8 Hz, 2H), 5.83 (t, J = 6.0 Hz, 1H), 4.38–4.23 (m, 1H), 4.16 (d, J = 6.0 Hz, 2H), 3.49 (s, 1H), 1.92 (d, J = 8.8 Hz, 2H), 1.82 (d, J = 11.1 Hz, 2H), 1.32 (q, J = 11.3 Hz, 2H), 1.16 (q, J = 13.8 Hz, 2H).

[0125] Example 2. Synthesis of Linker

[0126]

[0127] Reaction reagents and conditions: (a) Boc2O, Et3N, CH2Cl2, 0 - 25 °C, 3 h; (b) NaH, 1,4 - dioxane, 25 - 60 °C, 16 h; (c) TsCl, Et3N, DMAP, CH2Cl2, 0 - 25 °C, 2 h; (d) K2CO3, DMF, 60 °C, 12 h; (e) i. N2H4·H2O, EtOH, 80 °C, 5 h; ii. Boc2O, Et3N, CH2Cl2, 0 - 25 °C, 3 h; (f) Et3N, CH2Cl2, 0 - 25 °C, 8 h.

[0128] Step a:

[0129] (1) tert-Butyl (2-(2-hydroxyethoxy)ethyl)carbamate 1a

[0130]

[0131] Dissolve the raw material 2-(2-aminoethoxy)ethan-1-ol (4.26 g, 40 mmol) and triethylamine (6.06 g, 60 mmol) in dichloromethane. After cooling the reaction solution to 0 °C, dropwise add di-tert-butyl dicarbonate (8.8 g, 40.5 mmol). After the addition, slowly raise the temperature to room temperature and react for 3 h. After monitoring by TLC that the raw materials have completely reacted, the obtained reaction solution is directly subjected to the next step of reaction.

[0132] (2)tert-Butyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate 1b

[0133]

[0134] The preparation method is the same as that of compound 1a, using 2-(2-(2-aminoethoxy)ethoxy)ethanol (40 mmol) instead of 2-(2-aminoethoxy)ethanol as the raw material.

[0135] Step b:

[0136] (1)tert-Butyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate 1c

[0137]

[0138] Under nitrogen protection, dissolve diethylene glycol (3.543 g, 33.42 mmol) in anhydrous 1,4-dioxane. While maintaining the reaction temperature at 25 °C, add sodium hydride (900 mg, 22.28 mmol) slowly in three batches at intervals of 20 min. After stirring the reaction solution at room temperature for 2 h, slowly dropwise add the 1,4-dioxane solution of compound 2a (4.000 g, 11.14 mmol) prepared. After the addition is complete, raise the temperature to 60 °C and react for 18 h. After monitoring by TLC that the raw material 2a has completely reacted, add water (100 mL) to quench the reaction, extract with ethyl acetate (60 mL × 3), combine the organic phases and concentrate under reduced pressure, and separate and purify by flash column chromatography to obtain the product 1c (820 mg, 25%) as a pale yellow oily liquid with the molecular formula C 13 H 27 NO6, LC-MS (ESI, m / z): 294.3 [M + H] + .

[0139] (2)tert-Butyl (14-hydroxy-3,6,9,12-tetraoxaoctadecyl)carbamate 1d

[0140]

[0141] The preparation method is the same as that of compound 1c, using triethylene glycol (12 mmol) instead of diethylene glycol as the raw material, to obtain the product 1d (1.05 g, 26%) as a pale yellow oily liquid with the molecular formula C 15 H 31 NO7, LC-MS (ESI, m / z): 338.2 [M + H] + .

[0142] Step c:

[0143] (1) 2-(2-((tert-Butoxycarbonyl)amino)ethoxy)ethyl 4-methylbenzenesulfonate 2a

[0144]

[0145] At 0 °C, triethylamine (6.06 g, 60 mmol) and 4-dimethylaminopyridine (244 mg, 2.0 mmol) were added to the reaction solution of unpurified compound 1a. Finally, a dichloromethane solution of prepared p-toluenesulfonyl chloride (7.8 g, 41 mmol) was slowly added dropwise. After the addition was complete, the reaction mixture was warmed to room temperature and stirred for 2 h. After monitoring by TLC to confirm the complete reaction of starting material 1a, the reaction solution was poured into 200 mL of water and extracted with dichloromethane (150 mL × 3). The combined organic phases were concentrated under reduced pressure and purified by flash column chromatography to obtain product 2a (19.4 g, 90%) as a white waxy solid with the molecular formula C 16 H 25 NO6S, LC-MS (ESI, m / z): 360.2 [M+H] + .

[0146] (2) 2,2-Dimethyl-4-oxo-3,8,11-trioxa-5-azatridecan-13-yl 4-methylbenzenesulfonate 2b

[0147]

[0148] The preparation method was the same as that of compound 2a. The reaction solution of unpurified compound 1b was used instead of the reaction solution of unpurified compound 1a as the starting material to obtain product 2b (22.2 g, 92%) as a pale yellow oily liquid with the molecular formula C 18 H 29 NO7S, LC-MS (ESI, m / z): 404.2 [M+H] + .

[0149] (3) 2,2-Dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-yl 4-methylbenzenesulfonate 2c

[0150]

[0151] The preparation method was the same as that of compound 2a. A dichloromethane solution of compound 1c was used instead of the reaction solution of unpurified compound 1a as the starting material to obtain product 2c (1.10 g, 88%) as a pale yellow oily liquid with the molecular formula C 20 H 33 NO8S, LC-MS (ESI, m / z): 448.2 [M+H] + .

[0152] (4) 4-Methylbenzenesulfonic acid 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadec-19-yl ester 2d

[0153]

[0154] Prepared in the same way as compound 2a, using the dichloromethane solution of compound 1d to replace the reaction solution of unpurified compound 1a as the raw material, the product 2d (1.2 g, 84%) was obtained as a pale yellow oily liquid, with the molecular formula C 22 H 37 NO9S, LC-MS (ESI, m / z): 492.3 [M+H] + .

[0155] (5) 4-Methylbenzenesulfonic acid 9-((tert-butoxycarbonyl)amino)nonyl ester 5a

[0156]

[0157] Prepared in the same way as compound 2a, using the dichloromethane solution of compound 4a to replace the reaction solution of unpurified compound 1a as the raw material, the product 5a (1.6 g, 89%) was obtained as a white solid, with the molecular formula C 21 H 35 NO5S, LC-MS (ESI, m / z): 414.2 [M+H] + .

[0158] (6) 4-Methylbenzenesulfonic acid 11-((tert-butoxycarbonyl)amino)undecyl ester 5b

[0159]

[0160] Prepared in the same way as compound 2a, using the dichloromethane solution of compound 4b to replace the reaction solution of unpurified compound 1a as the raw material, the product 5b (2.18 g, 86%) was obtained as a white solid, with the molecular formula C 23 H 39 NO5S, LC-MS (ESI, m / z): 442.3 [M+H] + .

[0161] (7) 4-Methylbenzenesulfonic acid 13-((tert-butoxycarbonyl)amino)tridecyl ester 5c

[0162]

[0163] Prepared in the same way as compound 2a, using the dichloromethane solution of compound 4c to replace the reaction solution of unpurified compound 1a as the raw material, the product 5c (1.45 g, 81%) was obtained as a white solid, with the molecular formula C 25H 43 NO5S, LC-MS (ESI, m / z): 470.3 [M+H] + .

[0164] Step d:

[0165] (1) 2-(11-Hydroxyundecyl)isoindoline-1,3-dione 3b

[0166]

[0167] Dissolve 11-bromo-1-undecanol (2.51 g, 10.0 mmol) and potassium phthalimide (1.62 g, 11.0 mmol) in anhydrous DMF, heat to 60 °C and react for 12 h. After monitoring the reaction of the raw material 11-bromo-1-undecanol to completion by TLC, pour the reaction solution into 200 mL of water, extract with ethyl acetate (150 mL x 3), combine the organic phases, concentrate under reduced pressure, and separate and purify by flash column chromatography to obtain the product 3b (2.32 g, 73%) as a white solid, with the molecular formula C 19 H 27 NO3, LC-MS (ESI, m / z): 318.2 [M+H] + .

[0168] (2) 2-(9-Hydroxynonyl)isoindoline-1,3-dione 3a

[0169]

[0170] The preparation method is the same as that of compound 3b, using 9-bromo-1-alcohol to replace 11-bromo-1-undecanol as the raw material, to obtain the product 3a (2.1 g, 67%) as a white solid, with the molecular formula C 17 H 23 NO3, LC-MS (ESI, m / z): 290.2 [M+H] + .

[0171] (3) 2-(13-Hydroxytridecyl)isoindoline-1,3-dione 3c

[0172]

[0173] The preparation method is the same as that of compound 3b, using 13-bromotridecane-1-ol to replace 11-bromo-1-undecanol as the raw material, to obtain the product 3c (2.8 g, 72%) as a white solid, with the molecular formula C 21 H 31 NO3, LC-MS (ESI, m / z): 346.2 [M+H] + .

[0174] Step e:

[0175] (1) tert-Butyl (11-hydroxyundecyl)carbamate 4b

[0176]

[0177] Dissolve compound 3b in ethanol, and dropwise add 80% hydrazine hydrate (2.2 g, 44 mmol) at room temperature. Heat the reaction mixture to 80 °C and reflux for 5 h. After monitoring by TLC that the raw material 3b has completely reacted, cool the reaction mixture to 0 °C to precipitate a solid, filter it through diatomaceous earth, wash the filter cake with pre-cooled ethanol, concentrate the filtrate under reduced pressure, dissolve the obtained white solid in dichloromethane, add triethylamine (740 mg, 7.3 mmol), cool the reaction mixture to 0 °C, and then slowly add di-tert-butyl dicarbonate (2.4 g, 11.0 mmol) dropwise. After the addition, raise the temperature to room temperature and react for 3 h. After monitoring by TLC that the reaction is complete, pour the reaction mixture into 150 mL of water, extract with dichloromethane (100 mL × 3), combine the organic phases, concentrate under reduced pressure, and separate and purify by flash column chromatography to obtain product 4b (1.65 g, 79%) as a white solid with the molecular formula C 16 H 33 NO3, LC-MS (ESI, m / z): 288.3 [M+H] + .

[0178] (2) tert-Butyl (9-hydroxynonyl)carbamate 4a

[0179]

[0180] The preparation method is the same as that of compound 4b, using compound 3a instead of compound 3b as the raw material to obtain product 4a (1.8 g, 70%) as a white solid with the molecular formula C 14 H 29 NO3, LC-MS (ESI, m / z): 260.2 [M+H] + .

[0181] (3) tert-Butyl (13-hydroxytridecyl)carbamate 4c

[0182]

[0183] The preparation method is the same as that of compound 4b, using compound 3c instead of compound 3b as the raw material to obtain product 4c (1.62 g, 66%) as a white solid with the molecular formula C 18 H 37 NO3, LC-MS (ESI, m / z): 316.3 [M+H] + .

[0184] Step f:

[0185] (1) Benzyl (2-(2-aminoethoxy)ethyl)carbamate

[0186]

[0187] Dissolve the raw materials 2,2'-oxybis(ethyl-1-amine) (5 g, 48 mmol) and triethylamine (14.54 g, 144 mmol) in dichloromethane. After cooling the reaction solution to 0 °C, add benzyl chloroformate (24.6 g, 144 mmol) dropwise. After the addition is complete, slowly raise the temperature to room temperature and react for 8 h. After monitoring the reaction to completion by TLC, pour the reaction solution into 150 mL of water and extract with dichloromethane (100 mL × 3). Combine the organic phases and concentrate under reduced pressure. Purify by flash column chromatography to obtain the product LP1 (3.08 g, 27%) as a white solid, with the molecular formula C 12 H 18 N2O3, LC-MS (ESI, m / z): 239.1 [M+H] + .

[0188] (2) Benzyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate LP2

[0189]

[0190] The preparation method is the same as that of compound LP1. Use 2,2'-ethane-1,2-diylbisoxy-bis(ethyl-1-amine) to replace 2,2'-oxybis(ethyl-1-amine) as the raw material to obtain the product LP2 (3.62 g, 32%) as a white solid, with the molecular formula C 14 H 22 N2O4, LC-MS (ESI, m / z): 283.2 [M+H] + .

[0191] (3) Benzyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate LP3

[0192]

[0193] The preparation method is the same as that of compound LP1. Use 2,2'-(oxybis(ethane-2,1-diyl))bis(oxy))bis(ethyl-1-amine) to replace 2,2'-oxybis(ethyl-1-amine) as the raw material to obtain the product LP3 (2.8 g, 36%) as a white solid, with the molecular formula C 16 H 26 N2O5, LC-MS (ESI, m / z): 327.2 [M+H] + .

[0194] (4)Benzyl (14-amino-3,6,9,12-tetraoxatetradecyl)carbamate LP4

[0195]

[0196] The preparation method is the same as that of compound LP1. Using 3,6,9,12-tetraoxatetradecane-1,14-diamine to replace 2,2'-oxybis(ethyl-1-amine) as the raw material, the product LP4 (2.56 g, 26%) was obtained as a white solid, and the molecular formula is C 18 H 30 N2O6, LC-MS (ESI, m / z): 371.2 [M+H] + .

[0197] (5)Benzyl (9-aminononyl)carbamate LC3

[0198]

[0199] The preparation method is the same as that of compound LP1. Using nonane-1,9-diamine to replace 2,2'-oxybis(ethyl-1-amine) as the raw material, the product LC3 (2.41 g, 22%) was obtained as a white solid, and the molecular formula is C 17 H 28 N2O2, LC-MS (ESI, m / z): 293.2 [M+H] + .

[0200] (6)Benzyl (11-aminoundecyl)carbamate LC4

[0201]

[0202] The preparation method is the same as that of compound LP1. Using undecane-1,11-diamine to replace 2,2'-oxybis(ethyl-1-amine) as the raw material, the product LC4 (1.9 g, 28%) was obtained as a white solid, and the molecular formula is C 19 H 32 N2O2, LC-MS (ESI, m / z): 321.3 [M+H] + .

[0203] (7)Benzyl (13-aminotridecyl)carbamate LC5

[0204]

[0205] The preparation method is the same as that of compound LP1. Using tridecane-1,13-diamine as the raw material, the product LC5 (1.95 g, 26%) was obtained as a white solid, and the molecular formula is C 21 H 36N2O2, LC-MS (ESI, m / z): 349.3 [M+H] + .

[0206] Example 3. Synthesis of DCAF16 Ligand

[0207]

[0208] Reaction reagents and conditions: (a) NaOH, 1,4-dioxane, H2O, 0 - 25 °C, 4 h; (b) i. Cs2CO3, DMF, 0 - 25 °C, 3 h; ii. 25% TFA in CH2Cl2, 25 °C.

[0209] Step a: 2-Chloro-1-(6-hydroxy-3,4-dihydroquinolin-1(2H)-yl)ethan-1-one D-1

[0210]

[0211] Dissolve 6-hydroxy-1,2,3,4-tetrahydroquinoline (5.0 g, 41.5 mmol) in 1,4-dioxane. Under ice bath conditions, add an aqueous solution of sodium hydroxide (2.0 g, 50.0 mmol) with a concentration of 0.8 M. Under nitrogen protection, slowly dropwise add chloroacetyl chloride (5.1 g, 45.6 mmol). After the addition, raise the temperature to room temperature and react for 4 h. After monitoring by TLC that the raw material 6-hydroxy-1,2,3,4-tetrahydroquinoline has completely reacted, add 100 mL of water for dilution, slowly dropwise add dilute hydrochloric acid with a concentration of 1 N to adjust the pH of the reaction solution to 3, extract with ethyl acetate (80 mL x 3), combine the organic phases and concentrate under reduced pressure, and separate and purify by flash column chromatography to obtain the product D-1 (7.4 g, 79%) as a white solid, with the molecular formula C 11 H 12 ClNO2, LC-MS (ESI, m / z): 226.1 [M+H] + .

[0212] Step b: 2-((1-(2-Chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetic acid D-L

[0213]

[0214] Dissolve compound D-1 (6.75 g, 30 mmol) in anhydrous DMF, and add cesium carbonate (14.7 g, 45 mmol). Cool the reaction system to 0 °C, and slowly add tert-butyl bromoacetate (7.3 g, 37.5 mmol). After the addition is complete, raise the temperature to room temperature and react for 3 h. After monitoring the complete reaction of compound D-1 by TLC, pour the reaction solution into 100 mL of water, extract with ethyl acetate (50 mL × 3), combine the organic phases and concentrate under reduced pressure. The pale yellow solid obtained by separation and purification by flash column chromatography is treated with a 25% (v / v) trifluoroacetic acid-dichloromethane solution. After monitoring the complete reaction by TLC, distill off the solvent and excess trifluoroacetic acid under reduced pressure. The remaining residue is dissolved in dichloromethane, and then an excess of pre-cooled ethyl acetate is added to precipitate a solid. Filter through a Buchner funnel, and wash the filter cake with pre-cooled ethyl acetate. After vacuum drying the filter cake solid, product D-L (5.1 g, 60%) is obtained as a white powdery solid with the molecular formula C 13 H 14 ClNO4, LC-MS (ESI, m / z): 284.1 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.46 (d, J = 110.6 Hz, 1H), 6.74 (dd, J = 14.2, 5.5 Hz, 2H), 4.65 (s, 2H), 4.47 (s, 2H), 3.66 (t, J = 6.3 Hz, 2H), 2.67 (t, J = 7.1 Hz, 2H), 1.94–1.77 (m, 2H).

[0215] Example 4. Synthesis of intermediate D-L acyl chloride

[0216]

[0217] Under nitrogen protection, dissolve compound D-L in anhydrous dichloromethane. Cool the reaction system to 0 °C, and slowly add oxalyl chloride (1.1 equiv.). After the addition is complete, raise the temperature to room temperature and react for 2 h. Take a small amount of the reaction solution and dilute it in methanol. After monitoring the complete reaction of compound D-L by TLC (the acyl chloride reacts with methyl ester to form a methyl ester derivative), distill off the solvent in the reaction solution under reduced pressure, and dry the residue under vacuum (avoid contact with moisture during the process) to obtain the crude product D-L acyl chloride (2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetyl chloride) with the molecular formula C 13 H 13 Cl2NO3, LC-MS (ESI, m / z): 298.1 [M+H] + (Compound D-L acyl chloride is unstable in water, and the data here are for the methyl ester derivative product).

[0218] Example 5. Synthesis of Intermediates I-N-1 and I-O-1

[0219] tert-Butyl (2-(2-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)ethoxy)ethyl)carbamate I-N-1;

[0220] tert-Butyl (2-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl))pyridin-2-yl)oxy)ethoxy)ethyl)carbamate I-O-1.

[0221]

[0222] Under nitrogen protection, compound U-L (155 mg, 0.3 mmol) and compound 2a (161 mg, 0.36 mmol) were dissolved in anhydrous DMF, potassium carbonate (124 mg, 0.9 mmol) was added, and the temperature was raised to 60 °C for reaction for 12 h. After monitoring the complete reaction of compound U-L by TLC, the reaction solution was poured into 20 mL of water and extracted with ethyl acetate (15 mL × 3). The organic phases were combined and concentrated under reduced pressure. The products were separated and purified by flash column chromatography to obtain product I-N-1 (86 mg, 41%) as a white solid with the molecular formula C 40 H 47 N7O5, LC-MS (ESI, m / z): 706.1 [M+H] + . Product I-O-1 (44 mg, 21%) was a white solid with the molecular formula C 40 H 47 N7O5, LC-MS (ESI, m / z): 706.1 [M+H] + .

[0223] Example 6. Synthesis of Intermediates I-N-2 and I-O-2

[0224] tert-Butyl (2-(2-(2-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)ethoxy)ethoxy)ethyl)carbamate I-N-2;

[0225]

[0226] tert-Butyl (2-(2-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethyl)carbamate I-O-2.

[0227]

[0228] The preparation method is the same as that of Compound I-N-1 and I-O-1. Using Compound 2b as the raw material instead of 2a, the product I-N-2 (96 mg, 43%) was obtained as a white solid, with the molecular formula C 42 H 51 N7O6, LC-MS (ESI, m / z): 750.4 [M+H] + . The product I-O-2 (50 mg, 22%) was obtained as a white solid, with the molecular formula C 42 H 51 N7O6, LC-MS (ESI, m / z): 750.4 [M+H] + .

[0229] Example 7. Synthesis of Intermediates I-N-3 and I-O-3

[0230] tert-Butyl (2-(2-(2-(2-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl))ureido)phenyl)-2-oxopyridin-1(2H)-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamate I-N-3;

[0231]

[0232] tert-Butyl (2-(2-(2-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)))cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate I-O-3.

[0233]

[0234] The preparation method is the same as that of Compound I-N-1 and I-O-1. Using Compound 2c as the raw material instead of 2a, the product I-N-3 (107 mg, 45%) was obtained as a white solid, with the molecular formula C 44 H 55 N7O7, LC-MS (ESI, m / z): 794.4 [M+H] + . The product I-O-3 (50 mg, 21%) was obtained as a white solid, with the molecular formula C44 H 55 N7O7, LC-MS (ESI, m / z): 794.4 [M+H] + .

[0235] Example 8. Synthesis of Intermediate I-N-4 and I-O-4

[0236] tert-Butyl (14-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)-3,6,9,12-tetraoxatetradecyl)carbamate I-N-4;

[0237]

[0238] tert-Butyl (14-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)-3,6,9,12-tetraoxatetradecyl)carbamate I-O-4.

[0239]

[0240] The preparation method is the same as that of Compounds I-N-1 and I-O-1. Using Compound 2d instead of 2a as the raw material, the product I-N-4 (115 mg, 46%) was obtained as a white solid, with the molecular formula C 46 H 59 N7O8, LC-MS (ESI, m / z): 838.5 [M+H] + . The product I-O-4 (56 mg, 22%) was obtained as a white solid, with the molecular formula C 46 H 59 N7O8, LC-MS (ESI, m / z): 838.4 [M+H] + .

[0241] Example 9. Synthesis of Intermediate I-N-5 and I-O-5

[0242] (9-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopiperidin-1(2H)-yl)nonyl)carbamic acid tert-butyl ester I-N-5;

[0243]

[0244] (9 - ((5 - (4 - (3 - benzyl - 1 - ((1,4 - trans) - 4 - ((5 - cyanopyridin - 2 - yl)amino)cyclohexyl)ureido)phenyl)pyridin - 2 - yl)oxy)nonyl)carbamic acid tert - butyl ester I - O - 5。

[0245]

[0246] The preparation method is the same as that of compound I - N - 1 and I - O - 1. Using compound 5a instead of 2a as the raw material, the product I - N - 5 (105 mg, 49%) is obtained as a white solid, with the molecular formula C 45 H 57 N7O4, LC - MS (ESI, m / z): 760.5[M + H] + . The product I - O - 5 (76 mg, 32%) is obtained as a white solid, with the molecular formula C 45 H 57 N7O4, LC - MS (ESI, m / z): 760.5[M + H] + .

[0247] Example 10. Synthesis of intermediates I - N - 6 and I - O - 6

[0248] (11 - (5 - (4 - (3 - benzyl - 1 - ((1,4 - trans) - 4 - ((5 - cyanopyridin - 2 - yl)amino)cyclohexyl)ureido)phenyl) - 2 - oxopyridin - 1(2H) - yl)undecyl)carbamic acid tert - butyl ester I - N - 6;

[0249]

[0250] (11 - ((5 - (4 - (3 - benzyl - 1 - ((1,4 - trans) - 4 - ((5 - cyanopyridin - 2 - yl)amino)cyclohexyl)ureido)phenyl)pyridin - 2 - yl)oxy)undecyl)carbamic acid tert - butyl ester I - O - 6。

[0251]

[0252] The preparation method is the same as that of compound I - N - 1 and I - O - 1. Using compound 5b instead of 2a as the raw material, the product I - N - 6 (117 mg, 47%) is obtained as a white solid, with the molecular formula C 47 H 61 N7O4, LC - MS (ESI, m / z): 788.5[M + H] + . The product I - O - 6 (63 mg, 26%) is obtained as a white solid, with the molecular formula C 47 H 61 N7O4, LC - MS (ESI, m / z): 788.5[M + H]+ .

[0253] Example 11. Synthesis of Intermediate I-N-7 and I-O-7

[0254] (13-(5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)tridecyl)tert-butyl carbamate I-N-7;

[0255]

[0256] (13-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)tridecyl)tert-butyl carbamate I-O-7.

[0257]

[0258] The preparation method is the same as that of Compounds I-N-1 and I-O-1. Using Compound 5c instead of 2a as the raw material, the product I-N-7 (109 mg, 45%) was obtained as a white solid, with the molecular formula C 49 H 65 N7O4, LC-MS (ESI, m / z): 816.5 [M+H] + . The product I-O-7 (72 mg, 33%) was obtained as a white solid, with the molecular formula C 49 H 65 N7O4, LC-MS (ESI, m / z): 816.5 [M+H] + .

[0259] Example 12. Synthesis of Target Compounds UKNP1 and UKOP1

[0260] N-(2-(2-(5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-ylamino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNP1

[0261]

[0262] Compound I-N-1 (91 mg, 0.12 mmol) was treated with a 4 N hydrogen chloride - ethyl acetate solution. After monitoring the complete reaction of the starting material by TLC, the solvent was removed by distillation under reduced pressure. The resulting residue was dried in vacuo and dissolved in anhydrous DMF. The reaction system was cooled to 0 °C, and DIPEA (155 mg, 1.2 mmol) was slowly added dropwise. Under nitrogen protection, the crude product of freshly prepared D-L acyl chloride (0.132 mmol) was dissolved in anhydrous DMF and transferred via a newly opened syringe. It was slowly added dropwise to the above reaction system at 0 °C. After the addition, the reaction mixture was warmed to room temperature and reacted for 3 h. After monitoring the complete reaction by LC-MS, the reaction solution was poured into 25 mL of water and extracted with ethyl acetate (15 mL x 3). The organic phases were combined and concentrated under reduced pressure. The product UKNP1 (21 mg, 20%) was obtained as an off-white solid by separation and purification through flash column chromatography. The molecular formula was C 48 H 51 ClN8O6, LC-MS (ESI, m / z): 871.4 [M + H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.7 Hz, 1H), 8.08 (d, J = 2.9 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.86 (dd, J = 9.5, 2.7 Hz, 1H), 7.67–7.62 (m, 2H), 7.59 (dd, J = 8.9, 2.4 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.41–7.16 (m, 8H), 6.81–6.72 (m, 2H), 6.49 (d, J = 9.5 Hz, 1H), 6.46 (d, J = 8.9 Hz, 1H), 5.87 (t, J = 6.1 Hz, 1H), 4.41 (s, 2H), 4.33–4.24 (m, 1H), 4.19–4.11 (m, 4H), 3.70 (t, J = 5.5 Hz, 2H), 3.64 (t, J = 6.3 Hz, 2H), 3.55–3.52 (m, 1H), 3.40 (t, J = 5.9 Hz, 2H), 3.22 (q, J = 5.9 Hz, 2H), 2.65 (s, 2H), 1.9–1.77 (m, 6H), 1.30–1.08 (m, 6H).

[0263] N-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOP1

[0264]

[0265] The preparation method is the same as that of compound UKNP1. Using compound I-O-1 instead of I-N-1 as the raw material, the product UKOP1 (21 mg, 38%) is obtained as an off-white solid with the molecular formula C 48 H 51 ClN8O6, LC-MS (ESI, m / z): 871.4 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 2.8 Hz, 1H), 8.29 (d, J = 2.5 Hz, 1H), 8.14–8.01 (m, 2H), 7.79–7.72 (m, 2H), 7.60 (dd, J = 8.9, 2.4 Hz, 1H), 7.49 (d, J = 7.6 Hz, 1H), 7.45–7.00 (m, 8H), 6.93 (d, J = 8.7 Hz, 1H), 6.85–6.74 (m, 2H), 6.47 (d, J = 8.8 Hz, 1H), 5.91 (t, J = 6.2 Hz, 1H), 4.44 (s, 2H), 4.43–4.38 (m, 2H), 4.36–4.26 (m, 1H), 4.16 (d, J = 6.1 Hz, 2H), 3.79–3.73 (m, 2H), 3.65 (t, J = 6.3 Hz, 2H), 3.60–3.57 (m, 1H), 3.46 (t, J = 5.9 Hz, 2H), 3.30 (q, J = 5.8 Hz, 2H), 2.65 (s, 2H), 1.95–1.81 (m, 6H), 1.38–1.26 (m, 6H).

[0266] Example 13. Synthesis of target compounds UKNP2 and UKOP2

[0267] N-(2-(2-(2-(5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNP2

[0268]

[0269] The preparation method is the same as that of compound UKNP1. Using compound I-N-2 instead of I-N-1 as the raw material, the product UKNP2 (21 mg, 20%) is obtained as an off-white solid with the molecular formula C 50 H 55ClN8O7, LC-MS (ESI, m / z): 915.4 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.7 Hz, 1H), 8.08 (d, J = 2.7 Hz, 1H), 8.01 (t, J = 5.9 Hz, 1H), 7.85 (dd, J = 9.5, 2.7 Hz, 1H), 7.66–7.62 (m, 2H), 7.59 (dd, J = 8.9, 2.4 Hz, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.35–7.12 (m, 8H), 6.80–6.72 (m, 2H), 6.49 (d, J = 9.5 Hz, 1H), 6.46 (d, J = 8.9 Hz, 1H), 5.87 (t, J = 6.2 Hz, 1H), 4.39 (s, 2H), 4.33–4.24 (m, 1H), 4.19–4.11 (m, 4H), 3.70 (t, J = 5.5 Hz, 2H), 3.64 (t, J = 6.3 Hz, 2H), 3.55–3.52 (m, 2H), 3.51–3.45 (m, 3H), 3.40 (t, J = 5.9 Hz, 2H), 3.22 (q, J = 5.9 Hz, 2H), 2.65 (s, 2H), 1.93–1.78 (m, 6H), 1.29–1.08 (m, 6H); 13 C NMR (100 MHz, DMSO-D6) δ 167.6, 165.2, 160.7, 160.5, 159.2, 156.5, 153.1, 141.3, 138.9, 138.4, 137.6, 136.8, 135.6, 131.7, 131.6, 128.0, 126.8, 126.3, 126.1, 124.9, 119.5, 119.1, 116.8, 114.3, 112.3, 109.1, 104.7, 94.0, 69.7, 69.5, 68.9, 67.9, 67.0, 53.2, 48.5, 48.2, 43.5, 42.9, 40.8, 38.2, 31.3, 30.2, 26.3, 23.2.

[0270] N-(2-(2-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOP2

[0271]

[0272] The preparation method is the same as that of compound UKNP1. Using compound I-O-2 to replace I-N-1 as the raw material, the product UKOP2 (21 mg, 38%) is obtained as an off-white solid with the molecular formula C 50 H 55 ClN8O7, LC-MS (ESI, m / z): 915.4 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 2.6 Hz, 1H), 8.29 (d, J = 2.4 Hz, 1H), 8.12–8.02 (m, 2H), 7.79–7.71 (m, 2H), 7.60 (dd, J = 8.9, 2.4 Hz, 1H), 7.49 (d, J = 7.3 Hz, 1H), 7.45–7.00 (m, 8H), 6.93 (d, J = 8.6 Hz, 1H), 6.87–6.74 (m, 2H), 6.47 (d, J = 8.8 Hz, 1H), 5.91 (t, J = 6.2 Hz, 1H), 4.46 (s, 2H), 4.43–4.40 (m, 2H), 4.36–4.26 (m, 1H), 4.16 (d, J = 5.9 Hz, 2H), 3.78–3.73 (m, 2H), 3.65 (t, J = 6.4 Hz, 2H), 3.60–3.57 (m, 2H), 3.56–3.48 (m, 3H), 3.46 (t, J = 5.9 Hz, 2H), 3.30 (q, J = 5.8 Hz, 2H), 2.66 (s, 2H), 1.97–1.79 (m, 6H), 1.28–1.16 (m, 6H); 13 C NMR (100 MHz, DMSO-D6) δ 167.7, 165.2, 162.7, 159.3, 156.5, 155.0, 153.1, 144.7, 141.3, 138.5, 137.7, 137.4, 136.4, 131.8, 131.6, 128.6, 128.1, 127.2, 126.8, 126.3, 125.1, 119.2, 114.3, 112.3, 112.3, 110.9, 108.9, 94.0, 69.8, 69.6, 68.9, 68.8, 67.1, 65.1, 53.3, 48.6, 43.5, 42.9, 40.8, 38.3, 31.3, 30.3, 26.3, 23.3.

[0273] Example 14. Synthesis of target compounds UKNP3 and UKOP3

[0274] N-(2-(2-(2-(2-(5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNP3

[0275]

[0276] The preparation method is the same as that of compound UKNP1. Using compound I-N-3 to replace I-N-1 as the raw material, the product UKNP3 (43 mg, 35%) is obtained as a pale yellow solid, with the molecular formula C 52 H 59 ClN8O8, LC-MS (ESI, m / z): 959.4 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.5 Hz, 1H), 8.08 (d, J = 2.9 Hz, 1H), 8.04 (t, J = 5.8 Hz, 1H), 7.86 (dd, J = 9.5, 2.7 Hz, 1H), 7.67–7.62 (m, 2H), 7.60 (dd, J = 8.9, 2.4 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.43–7.06 (m, 8H), 6.82–6.73 (m, 2H), 6.50 (d, J = 9.5 Hz, 1H), 6.46 (d, J = 9.0 Hz, 1H), 5.87 (t, J = 6.1 Hz, 1H), 4.43 (s, 2H), 4.35–4.25 (m, 1H), 4.15 (dd, J = 14.4, 5.7 Hz, 4H), 3.69 (t, J = 5.6 Hz, 2H), 3.65 (t, J = 6.4 Hz, 2H), 3.53 (dd, J = 6.3, 3.4 Hz, 2H), 3.51–3.42 (m, 7H), 3.40 (t, J = 6.1 Hz, 2H), 3.26 (q, J = 6.7 Hz, 2H), 2.66 (s, 2H), 1.92–1.79 (m, 6H), 1.35–1.10 (m, 6H); 1313C NMR (100 MHz, DMSO-D6) δ 167.7, 165.2, 161.5, 160.7, 159.2, 156.5, 153.1, 141.3, 138.9, 138.4, 137.6, 136.8, 135.6, 131.7, 131.6, 128.0, 126.8, 126.3, 126.1, 125.0, 119.5, 119.1, 116.8, 114.3, 112.3, 109.0, 104.7, 94.0, 69.8, 69.7, 69.7, 69.6, 68.8, 67.8, 67.1, 53.2, 48.5, 48.2, 43.5, 42.9, 40.8, 38.2, 31.3, 30.3, 26.3, 23.2.

[0277] N-(2-(2-(2-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOP3

[0278]

[0279] The preparation method is the same as that of compound UKNP1. Using compound I-O-3 instead of I-N-1 as the raw material, the product UKOP3 (25 mg, 41%) was obtained as an off-white solid, with the molecular formula C 52 H 59 ClN8O8, LC-MS (ESI, m / z): 959.4 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 2.9 Hz, 1H), 8.29 (d, J = 2.5 Hz, 1H), 8.06 (dd, J = 8.7, 2.6 Hz, 2H), 7.79–7.72 (m, 2H), 7.60 (dd, J = 8.9, 2.4 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.43–7.01 (m, 8H), 6.93 (d, J = 8.7 Hz, 1H), 6.85–6.75 (m, 2H), 6.47 (d, J = 8.9 Hz, 1H), 5.91 (t, J = 6.1 Hz, 1H), 4.45 (s, 2H), 4.44–4.39 (m, 2H), 4.38–4.27 (m, 1H), 4.17 (d, J = 6.1 Hz, 2H), 3.79–3.72 (m, 2H), 3.65 (t, J = 6.3 Hz, 2H), 3.58 (dd, J = 5.7, 2.9 Hz, 2H), 3.56–3.52 (m, 2H), 3.55–3.49 (m, 5H), 3.45 (t, J = 5.9 Hz, 2H), 3.29 (q, J = 5.9 Hz, 2H), 2.67 (s, 2H), 1.90–1.73 (m, 6H), 1.36–1.10 (m, 6H); 13 13C NMR (100 MHz, DMSO-D6) δ 167.7, 165.2, 162.7, 159.3, 156.5, 155.0, 153.1, 144.7, 141.3, 138.5, 137.7, 137.4, 136.4, 131.8, 131.6, 128.6, 128.0, 127.2, 126.8, 126.3, 125.0, 119.1, 114.3, 112.4, 112.3, 110.9, 109.0, 94.0, 69.9, 69.8, 69.8, 69.6, 68.8, 68.8, 67.1, 65.0, 53.3, 48.6, 43.5, 43.0, 40.8, 38.3, 31.3, 30.3, 26.4, 23.2.

[0280] Example 15. Synthesis of Target Compounds UKNP4 and UKOP4

[0281] N-(14-(5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)-3,6,9,12-tetraoxatetradecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNP4

[0282]

[0283] The preparation method is the same as that of compound UKNP1. Using compound I-N-4 to replace I-N-1 as the raw material, the product UKNP4 (14 mg, 36%) is obtained as an off-white solid with the molecular formula C 54 H 63 ClN8O9, LC-MS (ESI, m / z): 1003.5 [M+H] + ; 1 1H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 3.0 Hz, 1H), 8.08 (d, J = 2.9 Hz, 1H), 8.05 (t, J = 5.8 Hz, 1H), 7.87 (dd, J = 9.5, 2.7 Hz, 1H), 7.67–7.63 (m, 2H), 7.60 (dd, J = 9.1, 2.2 Hz, 1H), 7.49 (d, J = 7.3 Hz, 1H), 7.33–7.13 (m, 8H), 6.84–6.72 (m, 2H), 6.50 (d, J = 9.5 Hz, 1H), 6.46 (d, J = 9.3 Hz, 1H), 5.86 (t, J = 5.8 Hz, 1H), 4.44 (s, 2H), 4.34–4.25 (m, 1H), 4.14 (dd, J = 10.6, 5.5 Hz, 4H), 3.69 (t, J = 5.4 Hz, 2H), 3.65 (t, J = 6.4 Hz, 2H), 3.53 (dd, J = 5.8, 3.0 Hz, 2H), 3.51–3.42 (m, 11H), 3.40 (t, J = 5.8 Hz, 2H), 3.26 (q, J = 5.9 Hz, 2H), 2.66 (s, 2H), 1.96–1.77 (m, 6H), 1.30–1.18 (m, 6H).

[0284] N-(14-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOP4

[0285]

[0286] The preparation method is the same as that of compound UKNP1. Using compound I-O-4 to replace I-N-1 as the raw material, the product UKOP4 (16 mg, 32%) is obtained as an off-white solid, with the molecular formula C 54 H 63ClN8O9, LC-MS (ESI, m / z): 1003.5 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 2.5 Hz, 1H), 8.28 (d, J = 2.3 Hz, 1H), 8.06 (dd, J = 8.7, 2.6 Hz, 2H), 7.78–7.73 (m, 2H), 7.60 (dd, J = 9.0, 2.5 Hz, 1H), 7.49 (d, J = 6.9 Hz, 1H), 7.34–7.13 (m, 8H), 6.94 (d, J = 8.6 Hz, 1H), 6.85–6.75 (m, 2H), 6.46 (d, J = 9.1 Hz, 1H), 5.91 (t, J = 6.1 Hz, 1H), 4.45 (s, 2H), 4.43–4.39 (m, 2H), 4.31 (m, 1H), 4.16 (d, J = 5.8 Hz, 2H), 3.78–3.73 (m, 2H), 3.65 (t, J = 6.3 Hz, 2H), 3.57 (dd, J = 5.8, 3.0 Hz, 2H), 3.54–3.48 (m, 11H), 3.44 (t, J = 6.0 Hz, 2H), 3.28 (q, J = 5.9 Hz, 2H), 2.66 (s, 2H), 1.95–1.80 (m, 6H), 1.24–1.14 (m, 6H); 13 C NMR (100 MHz, DMSO-D6) δ 167.7, 165.2, 162.8, 159.3, 156.5, 155.0, 153.2, 144.7, 141.3, 138.5, 137.7, 137.4, 136.4, 131.8, 131.6, 128.6, 128.1, 127.2, 126.8, 126.3, 125.1, 119.2, 114.4, 112.4, 112.3, 110.9, 109.0, 94.0, 69.9, 69.9, 69.8, 69.8, 69.8, 69.6, 68.9, 68.8, 67.1, 65.1, 53.3, 48.6, 43.6, 42.9, 40.7, 38.3, 31.3, 30.3, 26.3, 23.3.

[0287] Example 16. Synthesis of Target Compounds UKNC9 and UKOC9

[0288] N-(9-(5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)nonyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNC9

[0289]

[0290] The preparation method is the same as that of compound UKNP1. Using compound I-N-5 to replace I-N-1 as the raw material, the product UKNC9 (16 mg, 22%) is obtained as an off-white solid, and the molecular formula is C 53 H 61 ClN8O5, LC-MS (ESI, m / z): 925.4 [M+H] + ; 1 1H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.5 Hz, 1H), 8.12 (d, J = 2.9 Hz, 1H), 8.07 (t, J = 5.8 Hz, 1H), 7.88 (dd, J = 9.5, 2.7 Hz, 1H), 7.68–7.60 (m, 2H), 7.58 (dd, J = 8.9, 2.5 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.45–7.06 (m, 8H), 6.81–6.76 (m, 2H), 6.52 (d, J = 9.5 Hz, 1H), 6.44 (d, J = 9.0 Hz, 1H), 5.87 (t, J = 6.2 Hz, 1H), 4.43 (s, 2H), 4.41–4.35 (m, 2H), 4.29–4.2 (m, 1H), 4.14 (d, J = 5.8 Hz, 2H), 3.86–3.76 (m, 2H), 3.64 (t, J = 6.6 Hz, 2H), 3.45 (td, J = 5.6, 4.7 Hz, 1H), 3.24 (q, J = 5.8 Hz, 2H), 1.97–1.56 (m, 10H), 1.37–1.12 (m, 16H).

[0291] N-(9-(5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)nonyl)-2-(1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOC9

[0292]

[0293] The preparation method is the same as that of compound UKNP1. Using compound I-O-5 instead of I-N-1 as the raw material, the product UKNC11 (18 mg, 25%) is obtained as an off-white solid with the molecular formula C 53 H 61 ClN8O5, LC-MS (ESI, m / z): 925.4 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.51 (d, J = 2.6 Hz, 1H), 8.27 (d, J = 2.4 Hz, 1H), 8.12 (d, J = 2.9 Hz, 1H), 8.07 (t, J = 5.6 Hz, 1H), 7.71–7.67 (m, 2H), 7.60 (dd, J = 8.7, 2.4 Hz, 1H), 7.48 (d, J = 7.9 Hz, 1H), 7.47–7.12 (m, 8H), 6.88–6.76 (m, 2H), 6.48 (d, J = 9.4 Hz, 1H), 6.46 (d, J = 8.8 Hz, 1H), 5.94 (t, J = 6.2 Hz, 1H), 4.45 (s, 2H), 4.42–4.33 (m, 2H), 4.31–4.24 (m, 1H), 4.18 (d, J = 5.8 Hz, 2H), 3.81–3.76 (m, 2H), 3.56 (t, J = 6.4 Hz, 2H), 3.47 (td, J = 5.6, 4.8 Hz, 1H), 3.15 (q, J = 5.8 Hz, 2H), 1.96–1.56 (m, 10H), 1.34–1.13 (m, 16H).

[0294] Example 17. Synthesis of target compounds UKNC11 and UKOC11

[0295] N-(11-(5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)undecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNC11

[0296]

[0297] The preparation method is the same as that of compound UKNP1. Using compound I-N-6 instead of I-N-1 as the raw material, the product UKNC11 (15 mg, 29%) is obtained as an off-white solid with the molecular formula C 55 H 65 ClN8O5, LC-MS (ESI, m / z): 953.5 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.4 Hz, 1H), 8.11 (d, J = 2.9 Hz, 1H), 8.08 (t, J = 5.7 Hz, 1H), 7.89 (dd, J = 9.4, 2.6 Hz, 1H), 7.68–7.61 (m, 2H), 7.60 (dd, J = 8.9, 2.5 Hz, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.44–7.06 (m, 8H), 6.82–6.76 (m, 2H), 6.51 (d, J = 9.5 Hz, 1H), 6.47 (d, J = 9.0 Hz, 1H), 5.84 (t, J = 6.2 Hz, 1H), 4.43 (s, 2H), 4.41–4.33 (m, 2H), 4.29–4.21 (m, 1H), 4.15 (d, J = 5.9 Hz, 2H), 3.84–3.73 (m, 2H), 3.69 (t, J = 6.4 Hz, 2H), 3.47 (td, J = 5.7, 4.7 Hz, 1H), 3.24 (q, J = 5.8 Hz, 2H), 1.99–1.55 (m, 10H), 1.38–1.15 (m, 20H).

[0298] N-(11-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)undecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOC11

[0299]

[0300] The preparation method is the same as that of compound UKNP1. Using compound I-O-6 instead of I-N-1 as the raw material, the product UKOC11 (16 mg, 32%) was obtained as an off-white solid with the molecular formula C 55 H 65 ClN8O5, LC-MS (ESI, m / z): 953.5 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J = 2.5 Hz, 1H), 8.28 (d, J = 2.4 Hz, 1H), 8.12 (d, J = 2.7 Hz, 1H), 8.04 (t, J = 5.8 Hz, 1H), 7.77–7.69 (m, 2H), 7.60 (dd, J = 8.9, 2.4 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.44–7.07 (m, 8H), 6.86–6.74 (m, 2H), 6.50 (d, J = 9.4 Hz, 1H), 6.47 (d, J = 8.9 Hz, 1H), 5.91 (t, J = 6.2 Hz, 1H), 4.43 (s, 2H), 4.40–4.33 (m, 2H), 4.31–4.24 (m, 1H), 4.16 (d, J = 5.8 Hz, 2H), 3.81–3.74 (m, 2H), 3.69 (t, J = 6.5 Hz, 2H), 3.51 (td, J = 5.6, 4.8 Hz, 1H), 3.15 (q, J = 5.8 Hz, 2H), 1.99–1.61 (m, 10H), 1.35–1.16 (m, 20H).

[0301] Example 18. Synthesis of Target Compounds UKNC13 and UKOC13

[0302] N-(13-(5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)tridecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNC13

[0303]

[0304] The preparation method is the same as that of compound UKNP1. Using compound I-N-7 instead of I-N-1 as the raw material, the product UKNC13 (24 mg, 28%) was obtained as an off-white solid, with the molecular formula C 57 H 69 ClN8O5, LC-MS (ESI, m / z): 981.5 [M + H] + ; 11H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.4 Hz, 1H), 8.12 (d, J = 2.9 Hz, 1H), 8.06 (t, J = 5.7 Hz, 1H), 7.88 (dd, J = 9.4, 2.7 Hz, 1H), 7.69–7.61 (m, 2H), 7.60 (dd, J = 8.8, 2.5 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.44–7.06 (m, 8H), 6.82–6.76 (m, 2H), 6.53 (d, J = 9.4 Hz, 1H), 6.46 (d, J = 9.0 Hz, 1H), 5.86 (t, J = 6.2 Hz, 1H), 4.44 (s, 2H), 4.41–4.35 (m, 2H), 4.28–4.2 (m, 1H), 4.15 (d, J = 5.8 Hz, 2H), 3.84–3.76 (m, 2H), 3.63 (t, J = 6.5 Hz, 2H), 3.47 (td, J = 5.6, 4.6 Hz, 1H), 3.21 (q, J = 5.8 Hz, 2H), 1.96–1.56 (m, 10H), 1.35–1.15 (m, 24H).

[0305] N-(13-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)tridecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOC13

[0306]

[0307] The preparation method is the same as that of compound UKNP1, using compound I-O-7 to replace I-N-1 as the raw material, and the product UKOC13 (24 mg, 25%) is obtained as an off-white solid, with the molecular formula C 57 H 69 ClN8O5, LC-MS (ESI, m / z): 981.5 [M + H] + ; 11H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 2.5 Hz, 1H), 8.28 (d, J = 2.4 Hz, 1H), 8.11 (d, J = 2.9 Hz, 1H), 8.1 (t, J = 5.7 Hz, 1H), 7.73–7.67 (m, 2H), 7.62 (dd, J = 8.6, 2.4 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.49–7.12 (m, 8H), 6.87–6.79 (m, 2H), 6.48 (d, J = 9.5 Hz, 1H), 6.47 (d, J = 8.6 Hz, 1H), 5.91 (t, J = 6.2 Hz, 1H), 4.45 (s, 2H), 4.44–4.35 (m, 2H), 4.28–4.21 (m, 1H), 4.17 (d, J = 5.9 Hz, 2H), 3.84–3.76 (m, 2H), 3.63 (t, J = 6.4 Hz, 2H), 3.49 (td, J = 5.7, 4.6 Hz, 1H), 3.19 (q, J = 5.8 Hz, 2H), 1.98–1.56 (m, 10H), 1.36–1.15 (m, 24H).

[0308] Example 19. Synthesis of the target head T-L

[0309]

[0310] Reaction reagents and conditions: (a) DIPEA, NMP, 0 - 110 °C, 6 h; (b) 4N HCl in EtOAc, 25 °C.

[0311] Step a: tert-Butyl (R)-3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidine-1-carboxylate T-2

[0312]

[0313] Under nitrogen protection, commercially available starting material T-1 (3.45 g, 10 mmol) and (R)-3-amino-1-Boc-piperidine (2.2 g, 11 mmol) were dissolved in anhydrous NMP. After cooling the reaction system to 0 °C, DIPEA (3.9 g, 30 mmol) was added dropwise. After the addition was complete, the temperature was raised to 110 °C and the reaction was carried out for 6 h. After monitoring the complete reaction of starting material T-1 by TLC, the reaction solution was poured into 100 mL of water and extracted with ethyl acetate (50 mL x 3). The combined organic phases were concentrated under reduced pressure and purified by flash column chromatography to obtain the product T-2 (4.8 g, 95%) as a light brown solid, with the molecular formula C 23 H 32ClN5O4S, LC-MS(ESI, m / z): 510.2 [M+H] + ; 1 H NMR(400 MHz, DMSO-d6) δ 9.49(s, 1H), 8.74(d, J = 60.9 Hz, 1H), 8.14(s, 1H), 7.82(d, J = 8.0 Hz, 1H), 7.72(s, 1H), 7.42 - 7.07(m, 2H), 3.68(s, 2H), 3.49 - 3.37(m, 1H), 3.24 - 2.55(m, 2H), 2.49 - 2.27(m, 1H), 1.97 - 1.57(m, 4H), 1.55 - 1.20(m, 9H), 1.17(dd, J = 7.2, 2.1 Hz, 6H).

[0314] Step b: (R)-5-chloro-N 4 -(2-(isopropylsulfonyl)phenyl)-N 2 -(piperidin-3-yl)pyrimidine-2,4-diamine hydrochloride T-L

[0315]

[0316] Compound T-2 was treated with a 4N hydrogen chloride-ethyl acetate solution. After monitoring the complete reaction of the starting material T-2 by TLC, the solvent in the reaction solution was distilled off under reduced pressure to obtain the product T-L as a pale yellow solid, with the molecular formula C 18 H 24 ClN5O2S, LC-MS(ESI, m / z): 410.2 [M+H] + .

[0317] Example 20. Synthesis of intermediate TP1

[0318] (R)-(2-(2-(3-((5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)ethoxy)ethyl) carbamate tert-butyl ester TP1

[0319]

[0320] Prepared in the same manner as compounds I-N-1 and I-O-1, using compound T-L instead of U-L as the starting material, to obtain the product TP1 (118 mg, 53%) as a white solid, with the molecular formula C 27 H 41 ClN6O5S, LC-MS(ESI, m / z): 597.3 [M+H] + .

[0321] Example 21. Synthesis of intermediate TP2

[0322] (R)-(2-(2-(2-(3-((5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)ethoxy)ethoxy)ethyl)carbamic acid tert-butyl ester TP2

[0323]

[0324] The preparation method is the same as that of Compound I-N-1 and I-O-1. Using Compound T-L to replace U-L and Compound 2b to replace 2a as raw materials, the product TP2 (124 mg, 51%) is obtained as a white solid with the molecular formula C 29 H 45 ClN6O6S, LC-MS (ESI, m / z): 641.3 [M+H] + .

[0325] Example 22. Synthesis of Intermediate TP3

[0326] (R)-(2-(2-(2-(2-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamic acid tert-butyl ester TP3

[0327]

[0328] The preparation method is the same as that of Compound I-N-1 and I-O-1. Using Compound T-L to replace U-L and Compound 2c to replace 2a as raw materials, the product TP3 (104 mg, 47%) is obtained as a white solid with the molecular formula C 31 H 49 ClN6O7S, LC-MS (ESI, m / z): 685.3 [M+H] + .

[0329] Example 23. Synthesis of Intermediate TP4

[0330] (R)-(14-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)-3,6,9,12-tetraoxatetradecyl)carbamic acid tert-butyl ester TP4

[0331]

[0332] The preparation method is the same as that of Compound I-N-1 and I-O-1. Using Compound T-L to replace U-L and Compound 2d to replace 2a as raw materials, the product TP4 (131 mg, 52%) is obtained as a white solid with the molecular formula C 33 H 53ClN6O8S, LC-MS(ESI, m / z): 729.3 [M+H] + .

[0333] Example 24. Synthesis of Intermediate TC9

[0334] (R)-(9-(3-((5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)nonyl) carbamate tert-butyl ester TC9

[0335]

[0336] The preparation method is the same as that of Compound I-N-1 and I-O-1. Using Compound T-L to replace U-L and Compound 5a to replace 2a as raw materials, the product TC9 (142 mg, 54%) is obtained as a white solid, and the molecular formula is C 32 H 51 ClN6O4S, LC-MS(ESI, m / z): 651.3 [M+H] + .

[0337] Example 25. Synthesis of Intermediate TC11

[0338] (R)-(11-(3-((5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)undecyl) carbamate tert-butyl ester TC11

[0339]

[0340] The preparation method is the same as that of Compound I-N-1 and I-O-1. Using Compound T-L to replace U-L and Compound 5b to replace 2a as raw materials, the product TC11 (101 mg, 49%) is obtained as a white solid, and the molecular formula is C 34 H 55 ClN6O4S, LC-MS(ESI, m / z): 679.4 [M+H] + .

[0341] Example 26. Synthesis of Intermediate TC13

[0342] (R)-(13-(3-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)tridecyl) carbamate tert-butyl ester TC13

[0343]

[0344] The preparation method is the same as that of Compound I-N-1 and I-O-1. Using Compound T-L to replace U-L and Compound 5c to replace 2a as raw materials, the product TC13 (113 mg, 57%) is obtained as a white solid, with the molecular formula C 36 H 59 ClN6O4S, LC-MS (ESI, m / z): 707.4 [M+H] + .

[0345] Example 27. Synthesis of the target compound TKP1

[0346] (R)-N-(2-(2-(3-((5-chloro-4-((2-isopropylsulfonylphenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide TKP1

[0347]

[0348] The preparation method is the same as that of Compound UKNP1. Using Compound TP1 to replace I-N-1 as the raw material, the product TKP1 (27 mg, 38%) is obtained as an off-white solid, with the molecular formula C 35 H 45 Cl2N7O6S, LC-MS (ESI, m / z): 762.3 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.74 (d, J = 61.8 Hz, 1H), 8.17 (t, J = 4.9 Hz, 1H), 8.18 (s, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.73 (s, 1H), 7.45 (d, J = 119.6 Hz, 1H), 7.39 - 7.21 (m, 2H), 6.72 (dd, J = 14.5, 5.8 Hz, 2H), 4.64 (s, 2H), 4.46 (s, 2H), 3.72 (d, J = 61.3 Hz, 2H), 3.68 (t, J = 6.6 Hz, 2H), 3.54 - 3.36 (m, 5H), 3.16 (td, J = 5.9, 4.7 Hz, 2H), 3.11 - 2.53 (m, 4H), 2.49 - 2.27 (m, 3H), 1.99 - 1.51 (m, 6H), 1.15 (dd, J = 7.1, 2.8 Hz, 6H).

[0349] Example 28. Synthesis of the target compound TKP2

[0350] (R)-N-(2-(2-(2-(3-((5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide TKP2

[0351]

[0352] The preparation method is the same as that of compound UKNP1. Using compound TP2 to replace I-N-1 as the raw material, the product TKP2 (31 mg, 36%) was obtained as an off-white solid, and the molecular formula is C 37 H 49 Cl2N7O7S, LC-MS (ESI, m / z): 806.3 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.73 (d, J = 62.7 Hz, 1H), 8.18 (t, J = 4.8 Hz, 1H), 8.19 (s, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.72 (s, 1H), 7.43 (d, J = 118.9 Hz, 1H), 7.38 - 7.11 (m, 2H), 6.79 (dd, J = 14.1, 5.6 Hz, 2H), 4.69 (s, 2H), 4.45 (s, 2H), 3.69 (d, J = 63.2 Hz, 2H), 3.66 (t, J = 6.4 Hz, 2H), 3.51 - 3.37 (m, 9H), 3.15 (td, J = 5.8, 4.9 Hz, 2H), 3.12 - 2.55 (m, 4H), 2.48 - 2.29 (m, 3H), 1.97 - 1.52 (m, 6H), 1.17 (dd, J = 7.5, 2.6 Hz, 6H).

[0353] Example 29. Synthesis of the target compound TKP3

[0354] (R)-N-(2-(2-(2-(2-(3-((5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide TKP3

[0355]

[0356] The preparation method is the same as that of compound UKNP1. Using compound TP3 to replace I-N-1 as the raw material, the product TKP3 (25 mg, 31%) was obtained as an off-white solid, and the molecular formula is C39 H 53 Cl₂N₇O₈S, LC-MS (ESI, m / z): 850.3 [M+H] + ; 1 H NMR (400 MHz, DMSO-d₆) δ 9.48 (s, 1H), 8.71 (d, J = 62.7 Hz, 1H), 8.18 (t, J = 4.9 Hz, 1H), 8.16 (s, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.73 (s, 1H), 7.44 (d, J = 115.8 Hz, 1H), 7.42 - 7.11 (m, 2H), 6.71 (dd, J = 14.2, 5.4 Hz, 2H), 4.68 (s, 2H), 4.46 (s, 2H), 3.67 (d, J = 62.4 Hz, 2H), 3.67 (t, J = 6.3 Hz, 2H), 3.52 - 3.37 (m, 13H), 3.16 (td, J = 5.6, 4.8 Hz, 2H), 3.12 - 2.54 (m, 4H), 2.48 - 2.27 (m, 3H), 1.97 - 1.54 (m, 6H), 1.17 (dd, J = 7.5, 2.2 Hz, 6H).

[0357] Example 30. Synthesis of the target compound TKP4

[0358] (R)-N-(14-(3-((5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)-3,6,9,12-tetraoxatetradecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide TKP4

[0359]

[0360] The preparation method is the same as that of compound UKNP1. Using compound TP4 instead of I-N-1 as the raw material, the product TKP4 (22 mg, 35%) was obtained as an off-white solid, and the molecular formula is C 41 H 57 Cl₂N₇O₉S, LC-MS (ESI, m / z): 894.3 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.72 (d, J = 61.9 Hz, 1H), 8.18 (t, J = 4.7 Hz, 1H), 8.18 (s, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.71 (s, 1H), 7.42 (d, J = 116.9 Hz, 1H), 7.41 - 7.11 (m, 2H), 6.73 (dd, J = 14.4, 5.5 Hz, 2H), 4.68 (s, 2H), 4.46 (s, 2H), 3.67 (d, J = 62.4 Hz, 2H), 3.67 (t, J = 6.3 Hz, 2H), 3.52 - 3.36 (m, 17H), 3.15 (td, J = 5.7, 4.9 Hz, 2H), 3.13 - 2.54 (m, 4H), 2.47 - 2.29 (m, 3H), 1.96 - 1.53 (m, 6H), 1.16 (dd, J = 7.4, 2.4 Hz, 6H).

[0361] Example 31. Synthesis of the target compound TKC9

[0362] (R)-N-(9-(3-(5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)nonyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide TKC9

[0363]

[0364] The preparation method was the same as that of compound UKNP1. Using compound TC9 instead of I-N-1 as the raw material, the product TKC9 (18 mg, 34%) was obtained as an off-white solid with the molecular formula C 40 H 55 Cl2N7O5S, LC-MS (ESI, m / z): 816.3 [M + H] + ; 11H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.77 (d, J = 60.6 Hz, 1H), 8.17 (t, J = 4.8 Hz, 1H), 8.15 (s, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.74 (s, 1H), 7.43 (d, J = 117.8 Hz, 1H), 7.41 - 7.11 (m, 2H), 6.72 (dd, J = 13.8, 5.6 Hz, 2H), 4.66 (s, 2H), 4.47 (s, 2H), 3.68 (d, J = 62.8 Hz, 2H), 3.64 (t, J = 6.1 Hz, 2H), 3.49 - 3.37 (m, 1H), 3.17 (td, J = 5.7, 4.8 Hz, 2H), 3.14 - 2.57 (m, 4H), 2.49 - 2.26 (m, 3H), 1.98 - 1.54 (m, 10H), 1.36 - 1.13 (m, 16H).

[0365] Example 32. Synthesis of the target compound TKC11

[0366] (R)-N-(11-(3-(5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)undecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide TKC11

[0367]

[0368] The preparation method was the same as that of compound UKNP1. Using compound TC11 instead of I-N-1 as the raw material, the product TKC11 (28 mg, 31%) was obtained as an off-white solid, and the molecular formula was C 42 H 59 Cl2N7O5S, LC-MS (ESI, m / z): 844.4 [M + H] + ; 11H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 1H), 8.75 (d, J = 61.9 Hz, 1H), 8.19 (t, J = 4.9 Hz, 1H), 8.15 (s, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.72 (s, 1H), 7.44 (d, J = 112.6 Hz, 1H), 7.41 - 7.09 (m, 2H), 6.73 (dd, J = 14.1, 5.5 Hz, 2H), 4.67 (s, 2H), 4.46 (s, 2H), 3.69 (d, J = 61.4 Hz, 2H), 3.66 (t, J = 6.2 Hz, 2H), 3.48 - 3.37 (m, 1H), 3.15 (td, J = 5.6, 4.7 Hz, 2H), 3.13 - 2.55 (m, 4H), 2.47 - 2.27 (m, 3H), 1.96 - 1.56 (m, 10H), 1.33 - 1.11 (m, 20H).

[0369] Example 33. Synthesis of the target compound TKC13

[0370] (R)-N-(13-(3-(5-chloro-4-((2-isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)piperidin-1-yl)tridecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide TKC13

[0371]

[0372] The preparation method was the same as that of compound UKNP1, using compound TC13 instead of I-N-1 as the raw material, and the product TKC13 (22 mg, 28%) was obtained as an off-white solid with the molecular formula C 44 H 63 Cl2N7O5S, LC-MS (ESI, m / z): 872.4 [M + H] + ; 11H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 1H), 8.72 (d, J = 63.1 Hz, 1H), 8.16 (t, J = 4.8 Hz, 1H), 8.15 (s, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.73 (s, 1H), 7.46 (d, J = 111.6 Hz, 1H), 7.43 - 7.07 (m, 2H), 6.76 (dd, J = 14.3, 5.6 Hz, 2H), 4.66 (s, 2H), 4.47 (s, 2H), 3.68 (d, J = 63.9 Hz, 2H), 3.67 (t, J = 6.3 Hz, 2H), 3.47 - 3.37 (m, 1H), 3.15 (td, J = 5.7, 4.8 Hz, 2H), 3.14 - 2.56 (m, 4H), 2.47 - 2.25 (m, 3H), 1.99 - 1.56 (m, 10H), 1.35 - 1.15 (m, 24H).

[0373] Example 34. Synthesis of Intermediate 5P1

[0374] (((2-(5-Amino-2-chloro-4-nitrophenyl)amino)ethoxy)ethyl)carbamic acid benzyl ester 5P1

[0375]

[0376] 4,5-Dichloro-2-nitroaniline (964 mg, 4.68 mmol) and LP1 (1.1 g, 4.68 mmol) were added to a round-bottom flask. After the flask was evacuated and purged with N2 twice, anhydrous NMP (7 mL) and DIPEA (2.4 mL, 14.04 mmol) were added via syringe. The resulting mixture was stirred at 90 °C for 12 h. The mixture was cooled to room temperature. H2O (70 mL) was added and the mixture was extracted with EtOAc (50 mL x 2). The combined organic layers were dried over anhydrous Na2SO4. After concentration in vacuo, the residue was subsequently purified by column chromatography to give the product 5P1 as a yellow solid (1.37 g, 72%), with the molecular formula C 18 H 21 ClN4O5, LC-MS (ESI, m / z): 409.1 [M+H] + .

[0377] Example 35. Synthesis of Intermediate 5P2

[0378] (2-(2-(2-((5-Amino-2-chloro-4-nitrophenyl)amino)ethoxy)ethoxy))ethyl)carbamic acid benzyl ester 5P2

[0379]

[0380] The preparation method is the same as that of compound 5P1. Using compound LP2 to replace LP1 as the raw material, the product 5P2 (1.1 g, 78%) is obtained as a yellow solid with the molecular formula C 20 H 25 ClN4O6, LC-MS (ESI, m / z): 453.2 [M+H] + .

[0381] Example 36. Synthesis of intermediate 5P3

[0382] (2-(2-(2-(2-((5-Amino-2-chloro-4-nitrophenyl)amino)ethoxy)ethoxy)ethoxy)ethyl)carbamic acid benzyl ester 5P3

[0383]

[0384] The preparation method is the same as that of compound 5P1. Using compound LP3 to replace LP1 as the raw material, the product 5P3 (1.42 g, 66%) is obtained as a yellow solid with the molecular formula C 22 H 29 ClN4O7, LC-MS (ESI, m / z): 497.2 [M+H] + .

[0385] Example 37. Synthesis of intermediate 5P4

[0386] (14-((5-Amino-2-chloro-4-nitrophenyl)amino)-3,6,9,12-tetraoxatetradecyl)carbamic acid benzyl ester 5P4

[0387]

[0388] The preparation method is the same as that of compound 5P1. Using compound LP4 to replace LP1 as the raw material, the product 5P4 (1.09 g, 65%) is obtained as a yellow solid with the molecular formula C 24 H 33 ClN4O8, LC-MS (ESI, m / z): 541.2 [M+H] + .

[0389] Example 38. Synthesis of intermediate 5C3

[0390] (9-((5-Amino-2-chloro-4-nitrophenyl)amino)nonyl)carbamic acid benzyl ester 5C3

[0391]

[0392] The preparation method is the same as that of compound 5P1. Using compound LC3 to replace LP1 as the raw material, the product 5C3 (1.3 g, 79%) is obtained as a yellow solid with the molecular formula C23 H 31 ClN4O4, LC-MS(ESI, m / z): 463.2 [M+H] + .

[0393] Example 39. Synthesis of Intermediate 5C4

[0394] (11-(5-Amino-2-chloro-4-nitrophenyl)amino)undecyl)carbamic acid benzyl ester 5C4

[0395]

[0396] Prepared in the same manner as compound 5P1, using compound LC4 instead of LP1 as the raw material, to obtain the product 5C4 (1.21 g, 72%) as a yellow solid, with the molecular formula C 25 H 35 ClN4O4, LC-MS(ESI, m / z): 491.2 [M+H] + .

[0397] Example 40. Synthesis of Intermediate 5C5

[0398] (13-((5-Amino-2-chloro-4-nitrophenyl)amino)tridecyl)carbamic acid benzyl ester 5C5

[0399]

[0400] Prepared in the same manner as compound 5P1, using compound LC5 instead of LP1 as the raw material, to obtain the product 5C5 (1.14 g, 75%) as a yellow solid, with the molecular formula C 27 H 39 ClN4O4, LC-MS(ESI, m / z): 519.3 [M+H] + .

[0401] Example 41. Synthesis of Intermediate 6P1

[0402] (((6-((2-(2-(((Benzyloxy)carbonyl)amino)ethoxy)ethyl)amino)-5-chloro-1H-benzo[d]imidazol-2-yl)methyl)carbamic acid tert-butyl ester 6P1

[0403]

[0404] 5P1 (1.37 g, 2.64 mmol) was mixed with tin(II) chloride hydrate (3.57 g, 15.84 mmol) in a round-bottom flask equipped with a stir bar. After purging with nitrogen three times, EtOAc (30 mL) was added via syringe. The mixture was stirred at 70 °C for 3 h until 5P1 was completely consumed (monitored by TLC). The reaction was quenched by adding 2.0 M NaOH solution, and the aqueous phase was extracted twice with EtOAc. The combined organic phases were dried over anhydrous Na2SO4 and concentrated in vacuo to give the crude product which was used directly in the next step without further purification. The crude 1,2-phenylenediamine (967 mg, 2.56 mmol) and N-Boc-glycine (447 mg, 2.56 mmol) were mixed in a round-bottom flask equipped with a stir bar. Subsequently, anhydrous CH2Cl2 (20 mL), EDCI (476 mg, 3.07 mmol), HOBt (548 mg, 3.58 mmol) and DIPEA (4.45 mL, 25.6 mmol) were added, and the mixture was stirred at room temperature for 12 h. The reaction was quenched by adding H2O (80 mL), the organic phase was separated from the aqueous phase and extracted twice with CH2Cl2. The combined organic layers were dried over anhydrous Na2SO4. After concentration in vacuo, the residue was then treated with glacial acetic acid and heated at 70 °C for 3 h. After cooling to room temperature, EtOAc (20 mL) and saturated NaHCO3 solution were gently added to quench the reaction. The organic phase was separated from the aqueous phase, extracted three times with EtOAc and dried over Na2SO4. The combined organic phases were dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography to give the product 6P1 (526 mg, 38%) as a brown solid, with the molecular formula C 25 H 32 ClN5O5, LC-MS (ESI, m / z): 518.2 [M+H] + .

[0405] Example 42. Synthesis of Intermediate 6P2

[0406] (2-(2-(2-((2-(((tert-Butoxycarbonyl)amino)methyl)-5-chloro-1H-benzo[d]imidazol-6-yl)amino)ethoxy)ethoxy)ethyl)carbamic acid benzyl ester 6P2

[0407]

[0408] The preparation method was the same as that of compound 6P1, using compound 5P2 instead of 5P1 as the starting material, to give the product 6P2 (642 mg, 42%) as a brown solid, with the molecular formula C 27 H 36 ClN5O6, LC-MS (ESI, m / z): 562.2 [M+H] + .

[0409] Example 43. Synthesis of Intermediate 6P3

[0410] ((5-Chloro-6-((3-oxo-1-phenyl-2,7,10,13-tetraoxa-4-azapentadec-15-yl)amino)-1H-benzo[d]imidazol-2-yl)methyl)carbamic acid tert-butyl ester 6P3

[0411]

[0412] The preparation method was the same as that of compound 6P1, using compound 5P3 instead of 5P1 as the raw material, and the product 6P3 (499 mg, 39%) was obtained as a brown solid, with the molecular formula C 29 H 40 ClN5O7, LC-MS (ESI, m / z): 606.3 [M+H] + .

[0413] Example 44. Synthesis of Intermediate 6P4

[0414] ((5-Chloro-6-((3-oxo-1-phenyl-2,7,10,13,16-pentaoxa-4-azaoctadec-18-yl)amino)-1H-benzo[d]imidazol-2-yl)methyl)carbamic acid tert-butyl ester 6P4

[0415]

[0416] The preparation method was the same as that of compound 6P1, using compound 5P4 instead of 5P1 as the raw material, and the product 6P4 (549 mg, 44%) was obtained as a brown solid, with the molecular formula C 31 H 44 ClN5O8, LC-MS (ESI, m / z): 650.3 [M+H] +

[0417] Example 45. Synthesis of Intermediate 6C3

[0418] ((6-((9-((Benzyloxycarbonyl)amino)nonyl)amino)-5-chloro-1H-benzo[d]imidazol-2-yl)methyl)carbamic acid tert-butyl ester 6C3

[0419]

[0420] The preparation method was the same as that of compound 6P1, using compound 5C3 instead of 5P1 as the raw material, and the product 6C3 (521 mg, 41%) was obtained as a brown solid, with the molecular formula C 30 H 42 ClN5O4, LC-MS (ESI, m / z): 572.3 [M+H] +.

[0421] Example 46. Synthesis of Intermediate 6C4

[0422] ((6 - ((11 - (((Benzyloxy)carbonyl)amino)undecyl)amino)-5 - chloro - 1H - benzimidazol - 2 - yl)methyl)carbamic acid tert - butyl ester 6C4

[0423]

[0424] The preparation method is the same as that of compound 6P1, using compound 5C4 instead of 5P1 as the raw material, and the product 6C4 (528 mg, 40%) is obtained as a brown solid with the molecular formula C 32 H 46 ClN5O4, LC - MS (ESI, m / z): 600.3[M + H] + .

[0425] Example 47. Synthesis of Intermediate 6C5

[0426] tert - Butyl ((6 - ((13 - (((benzyloxy)carbonyl)amino)tridecyl)amino)-5 - chloro - 1H - benzimidazol - 2 - yl)methyl)carbamic acid tert - butyl ester 6C5

[0427]

[0428] The preparation method is the same as that of compound 6P1, using compound 5C5 instead of 5P1 as the raw material, and the product 6C5 (512 mg, 39%) is obtained as a brown solid with the molecular formula C 34 H 50 ClN5O4, LC - MS (ESI, m / z): 628.4[M + H] + .

[0429] Example 48. Synthesis of Intermediate 7P1

[0430] (2 - (2 - ((5 - chloro - 2 - (((2 - chloro - 9 - (1 - methyl - 1H - pyrazol - 4 - yl)-9H - purin - 6 - yl)amino)methyl)-1H - benzimidazol - 6 - yl)amino)ethoxy)ethyl)carbamic acid benzyl ester 7P1

[0431]

[0432] 6P1 (526 mg, 1.02 mmol) was treated with 4 N HCl at room temperature for 12 h. The mixture was concentrated in vacuo and mixed with 2,6-dichloro-9-(1-methylpyrazol-4-yl)-9H-purine (286 mg, 1.07 mmol) and DIPEA (1.77 mL, 10.2 mmol) in a round-bottom flask. After the flask was evacuated and purged with nitrogen twice, isopropanol (6 mL) was added via syringe. The resulting mixture was stirred at 80 °C for 2 h and then cooled to room temperature. H2O was added and the mixture was extracted three times with EtOAc (15 mL). The combined organic layers were dried over anhydrous Na2SO4, concentrated in vacuo, and the residue was then purified by column chromatography to afford 7P1 (432 mg, 65%) as a brown solid, with the molecular formula C 29 H 29 Cl2N 11 O3, LC-MS (ESI, m / z): 650.2 [M+H] + .

[0433] Example 49. Synthesis of Intermediate 7P2

[0434] (2-(2-(2-((5-chloro-2-(((2-chloro-9-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)ethoxy)ethoxy)ethyl)carbamic acid benzyl ester 7P2

[0435]

[0436] Prepared in the same manner as compound 7P1, using compound 6P2 instead of 6P1 as the starting material, to afford the product 7P2 (457 mg, 69%) as a brown solid, with the molecular formula C 31 H 33 Cl2N 11 O4, LC-MS (ESI, m / z): 694.2 [M+H] + .

[0437] Example 50. Synthesis of Intermediate 7P3

[0438] (2-(2-(2-(2-((5-chloro-2-(((2-chloro-9-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)carbamic acid benzyl ester 7P3

[0439]

[0440] The preparation method was the same as that of compound 7P1, using compound 6P3 to replace 6P1 as the raw material, and the product 7P3 (464 mg, 64%) was obtained as a brown solid with the molecular formula C 33 H 37 Cl2N 11 O5, LC-MS (ESI, m / z): 738.2 [M+H] + .

[0441] Example 51. Synthesis of intermediate 7P4

[0442] (14 - ((5-chloro-2 - (((2-chloro-9-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)-3,6,9,12-tetraoxatetradecyl)carbamic acid benzyl ester 7P4

[0443]

[0444] The preparation method was the same as that of compound 7P1, using compound 6P4 to replace 6P1 as the raw material, and the product 7P4 (421 mg, 62%) was obtained as a brown solid with the molecular formula C 35 H 41 Cl2N 11 O6, LC-MS (ESI, m / z): 782.3 [M+H] + .

[0445] Example 52. Synthesis of intermediate 7C3

[0446] (9 - ((5-chloro-2 - (((2-chloro-9-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)nonyl)carbamic acid benzyl ester 7C3

[0447]

[0448] The preparation method was the same as that of compound 7P1, using compound 6C3 to replace 6P1 as the raw material, and the product 7C3 (424 mg, 61%) was obtained as a brown solid with the molecular formula C 34 H 39 Cl2N 11 O2, LC-MS (ESI, m / z): 704.3 [M+H] + .

[0449] Example 53. Synthesis of intermediate 7C4

[0450] (11-((5-chloro-2-(((2-chloro-9-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)undecyl)carbamic acid benzyl ester 7C4

[0451]

[0452] The preparation method is the same as that of compound 7P1. Using compound 6C4 to replace 6P1 as the raw material, the product 7C4 (441 mg, 68%) is obtained as a brown solid, and the molecular formula is C 36 H 43 Cl2N 11 O2, LC-MS (ESI, m / z): 732.3 [M+H] + .

[0453] Example 54. Synthesis of intermediate 7C5

[0454] (13-((5-chloro-2-(((2-chloro-9-(1-methyl-1H-pyrazol-4-yl)-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)tridecyl)carbamic acid benzyl ester 7C5

[0455]

[0456] The preparation method is the same as that of compound 7P1. Using compound 6C5 to replace 6P1 as the raw material, the product 7C5 (424 mg, 61%) is obtained as a brown solid, and the molecular formula is C 38 H 47 Cl2N 11 O2, LC-MS (ESI, m / z): 760.3 [M+H] + .

[0457] Example 55. Synthesis of intermediate 8P1

[0458] (2-(2-((5-chloro-2-(((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)ethoxy)ethyl)carbamic acid benzyl ester 8P1

[0459]

[0460] To a round-bottom flask equipped with a stir bar, 7P1 (432 mg, 0.66 mmol), morpholine (579 mg, 6.6 mmol) and DIPEA (1.15 mL, 6.6 mmol) were added. After purging with nitrogen twice, anhydrous NMP (3 mL) was added via syringe. The resulting mixture was stirred at 200 °C for 3 h and then cooled to room temperature. H2O (10 mL) was added and the mixture was extracted 3 times with EtOAc. The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was then purified by column chromatography to afford the product 8P1 (360 mg, 77%) as a brown solid, having the molecular formula C 33 H 37 ClN 12 O4, LC-MS (ESI, m / z): 701.3 [M+H] + .

[0461] Example 56. Synthesis of Intermediate 8P2

[0462] (2-(2-(2-(5-chloro-2-(((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholin-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)ethoxy)ethoxy)ethyl) carbamate 8P2

[0463]

[0464] Prepared in the same manner as compound 8P1, using compound 7P2 in place of 7P1 as the starting material, to afford the product 8P2 (324 mg, 68%) as a brown solid, having the molecular formula C 35 H 41 ClN 12 O5, LC-MS (ESI, m / z): 745.3 [M+H] + .

[0465] Example 57. Synthesis of Intermediate 8P3

[0466] (2-(2-(2-(2-((5-chloro-2-(((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholin-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)-amino)ethoxy)ethoxy)ethoxy)ethyl) carbamate 8P3

[0467]

[0468] Prepared in the same manner as compound 8P1, using compound 7P3 in place of 7P1 as the starting material, to afford the product 8P3 (381 mg, 78%) as a brown solid, having the molecular formula C 37 H 45ClN 12 O6, LC-MS (ESI, m / z): 789.3 [M+H] + .

[0469] Example 58. Synthesis of Intermediate 8P4

[0470] (14 - ((5-chloro-2 - (((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)-3,6,9,12-tetraoxatetradecyl)carbamic acid benzyl ester 8P4

[0471]

[0472] The preparation method is the same as that of compound 8P1, using compound 7P4 instead of 7P1 as the raw material, and the product 8P4 (311 mg, 65%) is obtained as a brown solid, with the molecular formula C 39 H 49 ClN 12 O7, LC-MS (ESI, m / z): 833.3 [M+H] + .

[0473] Example 59. Synthesis of Intermediate 8C3

[0474] (9-(5-chloro-2 - (((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl-1H-benzo[d]imidazol-6-yl)amino)nonyl)carbamic acid benzyl ester 8C3

[0475]

[0476] The preparation method is the same as that of compound 8P1, using compound 7C3 instead of 7P1 as the raw material, and the product 8C3 (331 mg, 74%) is obtained as a brown solid, with the molecular formula C 38 H 47 ClN 12 O3, LC-MS (ESI, m / z): 755.4 [M+H] + .

[0477] Example 60. Synthesis of Intermediate 8C4

[0478] (11 - ((5-chloro-2 - (((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)undecyl)carbamic acid benzyl ester 8C4

[0479]

[0480] The preparation method is the same as that of compound 8P1. Using compound 7C4 to replace 7P1 as the raw material, the product 8C4 (303 mg, 71%) is obtained as a brown solid, with the molecular formula C 40 H 51 ClN 12 O3, LC-MS (ESI, m / z): 783.4 [M+H] + .

[0481] Example 61. Synthesis of intermediate 8C5

[0482] (13 - ((5-chloro-2 - (((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)tridecyl)carbamic acid benzyl ester 8C5

[0483]

[0484] The preparation method is the same as that of compound 8P1. Using compound 7C5 to replace 7P1 as the raw material, the product 8C5 (352 mg, 74%) is obtained as a brown solid, with the molecular formula C 42 H 55 ClN 12 O3, LC-MS (ESI, m / z): 811.4 [M+H] + .

[0485] Example 62. Synthesis of the target compound PKP1

[0486] N-(2-(2 - ((5-chloro-2 - (((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)ethoxy)ethyl)-2 - ((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide PKP1

[0487]

[0488] The preparation method is the same as that of compound UKNP1. Using compound 8P1 to replace I-N-1 as the raw material, the product PKP1 (39 mg, 44%) is obtained as an off-white solid, with the molecular formula C 38 H 43 Cl2N 13 O5, LC-MS (ESI, m / z): 832.3 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H), 8.56 (s, 1H), 8.31 (t, J = 5.9 Hz, 1H), 7.84 (t, J = 5.0 Hz, 1H), 7.70 (s, 1H), 7.64 (s, 1H), 7.38 (s, 1H), 7.01–6.94 (m, 2H), 6.90 (d, J = 9.0 Hz, 1H), 6.82 (dd, J = 9.0, 2.2 Hz, 1H), 6.57 (dd, J = 2.1, 1.0 Hz, 1H), 4.78 (d, J = 5.9 Hz, 2H), 4.53 (s, 2H), 4.18–4.08 (m, 1H), 4.01–3.90 (m, 1H), 3.82 (d, J = 7.4 Hz, 5H), 3.71–3.63 (m, 8H), 3.58 (q, J = 3.9 Hz, 4H), 3.50 (dt, J = 5.3, 4.2 Hz, 2H), 3.35 (dt, J = 5.0, 4.3 Hz, 2H), 2.81 (td, J = 7.2, 0.9 Hz, 2H), 2.09–1.90 (m, 2H).

[0489] Example 63. Synthesis of the target compound PKP2

[0490] N-(2-(2-(2-((5-chloro-2-(((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide PKP2

[0491]

[0492] The preparation method was the same as that of compound UKNP1, using compound 8P2 instead of I-N-1 as the raw material, and the product PKP2 (34 mg, 41%) was obtained as an off-white solid with the molecular formula C 40 H 47 Cl2N 13 O6, LC-MS (ESI, m / z): 876.3 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.56 (s, 1H), 8.33 (t, J = 5.9 Hz, 1H), 7.82 (t, J = 5.1 Hz, 1H), 7.70 (s, 1H), 7.65 (s, 1H), 7.37 (s, 1H), 7.01–6.96 (m, 2H), 6.91 (d, J = 9.3 Hz, 1H), 6.84 (dd, J = 9.1, 2.4 Hz, 1H), 6.55 (dd, J = 2.3, 1.1 Hz, 1H), 4.77 (d, J = 5.9 Hz, 2H), 4.51 (s, 2H), 4.17–4.08 (m, 1H), 4.03–3.91 (m, 1H), 3.81 (d, J = 7.4 Hz, 5H), 3.74–3.63 (m, 12H), 3.57 (q, J = 3.8 Hz, 4H), 3.52 (dt, J = 5.3, 4.1 Hz, 2H), 3.35 (dt, J = 5.3, 4.1 Hz, 2H), 2.85 (td, J = 7.5, 0.7 Hz, 2H), 2.19–1.92 (m, 2H).

[0493] Example 64. Synthesis of the target compound PKP3

[0494] N-(2-(2-(2-((5-Chloro-2-(((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide PKP3

[0495]

[0496] The preparation method is the same as that of compound UKNP1, using compound 8P3 instead of I-N-1 as the raw material, and the product PKP3 (46 mg, 42%) is obtained as an off-white solid with the molecular formula C 42 H 51 Cl2N 13 O7, LC-MS (ESI, m / z): 920.3 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H), 8.55 (s, 1H), 8.32 (t, J = 5.9 Hz, 1H), 7.82 (t, J = 5.3 Hz, 1H), 7.74 (s, 1H), 7.63 (s, 1H), 7.38 (s, 1H), 7.06–6.93 (m, 2H), 6.91 (d, J = 9.1 Hz, 1H), 6.85 (dd, J = 9.3, 2.4 Hz, 1H), 6.54 (dd, J = 2.2, 1.3 Hz, 1H), 4.79 (d, J = 5.9 Hz, 2H), 4.54 (s, 2H), 4.15–4.09 (m, 1H), 4.01–3.89 (m, 1H), 3.80 (d, J = 7.4 Hz, 5H), 3.76–3.63 (m, 16H), 3.55 (q, J = 3.9 Hz, 4H), 3.51 (dt, J = 5.2, 4.3 Hz, 2H), 3.33 (dt, J = 5.2, 4.1 Hz, 2H), 2.86 (td, J = 7.4, 0.9 Hz, 2H), 2.11–1.91 (m, 2H).

[0497] Example 65. Synthesis of the target compound PKP4

[0498] N-(14-((5-chloro-2-(((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)-3,6,9,12-tetraoxatetradecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide PKP4

[0499]

[0500] The preparation method was the same as that of compound UKNP1, using compound 8P4 instead of I-N-1 as the raw material, and the product PKP4 (31 mg, 39%) was obtained as an off-white solid with the molecular formula C 44 H 55 Cl2N 13 O8, LC-MS (ESI, m / z): 964.4 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H), 8.54 (s, 1H), 8.31 (t, J = 5.7 Hz, 1H), 7.83 (t, J = 5.2 Hz, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 7.39 (s, 1H), 7.07–6.94 (m, 2H), 6.91 (d, J = 9.2 Hz, 1H), 6.84 (dd, J = 9.2, 2.6 Hz, 1H), 6.55 (dd, J = 2.1, 1.3 Hz, 1H), 4.78 (d, J = 5.9 Hz, 2H), 4.55 (s, 2H), 4.12–4.08 (m, 1H), 4.01–3.86 (m, 1H), 3.80 (d, J = 7.5 Hz, 5H), 3.75–3.63 (m, 20H), 3.54 (q, J = 3.8 Hz, 4H), 3.51 (dt, J = 5.1, 4.4 Hz, 2H), 3.32 (dt, J = 5.1, 4.1 Hz, 2H), 2.87 (td, J = 7.5, 0.9 Hz, 2H), 2.13–1.94 (m, 2H).

[0501] Example 66. Synthesis of the target compound PKC9

[0502] N-(9-((5-Chloro-2-(((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)nonyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide PKC9

[0503]

[0504] The preparation method was the same as that of compound UKNP1. Using compound 8C3 instead of I-N-1 as the raw material, the product PKC9 (37 mg, 46%) was obtained as an off-white solid, with the molecular formula C 43 H 53 Cl2N 13 O4, LC-MS (ESI, m / z): 886.4 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H), 8.55 (s, 1H), 8.31 (t, J = 5.9 Hz, 1H), 7.84 (t, J = 5.2 Hz, 1H), 7.77 (s, 1H), 7.64 (s, 1H), 7.38 (s, 1H), 7.08–6.95 (m, 2H), 6.93 (d, J = 9.1 Hz, 1H), 6.85 (dd, J = 9.1, 2.5 Hz, 1H), 6.54 (dd, J = 2.1, 1.2 Hz, 1H), 4.78 (d, J = 5.9 Hz, 2H), 4.54 (s, 2H), 4.18–4.08 (m, 1H), 4.01–3.91 (m, 1H), 3.82 (d, J = 7.4 Hz, 5H), 3.72–3.61 (m, 8H), 3.30 (q, J = 5.3 Hz, 2H), 3.13 (td, J = 5.6, 4.8 Hz, 2H), 2.81 (td, J = 7.2, 0.9 Hz, 2H), 2.09–1.89 (m, 2H), 1.64 (tt, J = 7.3, 5.3 Hz, 2H), 1.57–1.46 (m, 2H), 1.35 (dq, J = 8.0, 6.6 Hz, 2H), 1.31–1.18 (m, 8H).

[0505] Example 67. Synthesis of the target compound PKC11

[0506] N-(11-(5-Chloro-2-(((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-yl)amino)methyl)-1H-benzo[d]imidazol-6-yl)amino)undecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide PKC11

[0507]

[0508] The preparation method was the same as that of compound UKNP1, using compound 8C4 instead of I-N-1 as the raw material to obtain the product PKC11 (43 mg, 41%) as an off-white solid, with the molecular formula C 45 H 57 Cl2N 13 O4, LC-MS (ESI, m / z): 914.4 [M + H] + ; 11H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.54 (s, 1H), 8.31 (t, J = 5.8 Hz, 1H), 7.85 (t, J = 5.2 Hz, 1H), 7.76 (s, 1H), 7.63 (s, 1H), 7.37 (s, 1H), 7.07–6.96 (m, 2H), 6.93 (d, J = 9.4 Hz, 1H), 6.84 (dd, J = 9.3, 2.4 Hz, 1H), 6.56 (dd, J = 2.1, 1.4 Hz, 1H), 4.78 (d, J = 5.9 Hz, 2H), 4.53 (s, 2H), 4.17–4.09 (m, 1H), 4.01–3.90 (m, 1H), 3.83 (d, J = 7.5 Hz, 5H), 3.71–3.61 (m, 8H), 3.33 (q, J = 5.2 Hz, 2H), 3.14 (td, J = 5.7, 4.7 Hz, 2H), 2.85 (td, J = 7.1, 0.9 Hz, 2H), 2.08–1.87 (m, 2H), 1.65 (tt, J = 7.2, 5.1 Hz, 2H), 1.55–1.46 (m, 2H), 1.34 (dq, J = 8.3, 6.5 Hz, 2H), 1.32–1.14 (m, 12H).

[0509] Example 68. Synthesis of the target compound PKC13

[0510] N-(13-(5-chloro-2-((9-(1-methyl-1H-pyrazol-4-yl)-2-morpholinyl-9H-purin-6-ylamino)methyl)-1H-benzimidazol-6-ylamino)tridecyl)-2-(1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide PKC13

[0511]

[0512] The preparation method was the same as that of compound UKNP1, using compound 8C5 instead of I-N-1 as the raw material, and the product PKC13 (37 mg, 46%) was obtained as an off-white solid with the molecular formula C 47 H 61 Cl2N 13 O4, LC-MS (ESI, m / z): 942.4 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H), 8.53 (s, 1H), 8.34 (t, J = 5.9 Hz, 1H), 7.86 (t, J = 5.2 Hz, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 7.38 (s, 1H), 7.08–6.97 (m, 2H), 6.93 (d, J = 9.5 Hz, 1H), 6.85 (dd, J = 9.1, 2.5 Hz, 1H), 6.55 (dd, J = 2.3, 1.5 Hz, 1H), 4.79 (d, J = 5.9 Hz, 2H), 4.52 (s, 2H), 4.16–4.09 (m, 1H), 4.02–3.91 (m, 1H), 3.85 (d, J = 7.4 Hz, 5H), 3.74–3.62 (m, 8H), 3.33 (q, J = 5.2 Hz, 2H), 3.16 (td, J = 5.7, 4.7 Hz, 2H), 2.82 (td, J = 7.4, 0.9 Hz, 2H), 2.09–1.85 (m, 2H), 1.64 (tt, J = 7.1, 5.1 Hz, 2H), 1.55–1.44 (m, 2H), 1.36 (dq, J = 8.3, 6.5 Hz, 2H), 1.31–1.12 (m, 16H).

[0513] Example 69. Synthesis of Negative Compound Warhead

[0514] 3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)-1-(4-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)phenyl)urea Warhead

[0515]

[0516] The preparation method was the same as that of compound U-L, using 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one instead of 2-hydroxypyridine-5-boronic acid pinacol ester as the raw material to obtain the product Warhead (126 mg, 49%) as an off-white solid, with the molecular formula C 32 H 32 N6O2, LC-MS (ESI, m / z): 533.3 [M+H] + ; 11H NMR (400 MHz, DMSO-d6) δ 8.29 (d, J = 2.7 Hz, 1H), 7.86 (dd, J = 9.5, 2.7 Hz, 1H), 7.74 (d, J = 2.8 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.58 (dd, J = 8.1, 2.7 Hz, 1H), 7.44 (d, J = 7.4 Hz, 1H), 7.36–7.29 (m, 2H), 7.21–7.12 (m, 5H), 6.48 (dd, J = 9.1, 6.5 Hz, 2H), 5.81 (t, J = 6.1 Hz, 1H), 4.39–4.26 (m, 1H), 4.12 (d, J = 6.3 Hz, 2H), 3.48 (s, 1H), 3.36 (s, 3H), 1.94 (d, J = 8.3 Hz, 2H), 1.81 (d, J = 12.4 Hz, 2H), 1.34 (q, J = 12.3 Hz, 2H), 1.14 (q, J = 12.8 Hz 2H).

[0517] Example 70. Synthesis of the negative compound DCAF16-P3

[0518] 2 - ((1-(2-Chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)-N-(2,5,8,11-tetraoxatridecan-13-yl)acetamide DCAF16-P3

[0519]

[0520] Dissolve commercially available 3,6,9,12-tetraoxatridecanamine (207 mg, 1.2 mmol) in anhydrous DMF and cool to 0 °C. Slowly add DIPEA (465 mg, 3.6 mmol). Under nitrogen protection, dissolve the freshly prepared crude D-L-acyl chloride (1.2 mmol) in anhydrous DMF and transfer it through a newly opened syringe. Slowly add it dropwise to the above reaction system at 0 °C. After the addition, raise the temperature to room temperature and react for 3 h. After monitoring the reaction to completion by LC-MS, pour the reaction solution into 25 mL of water and extract with ethyl acetate (15 mL × 3). Combine the organic phases and concentrate under reduced pressure. Purify by flash column chromatography to obtain the product DCAF16-P3 (226 mg, 40%) as a white solid with the molecular formula C 22 H 33 ClN2O7, LC-MS (ESI, m / z): 473.2 [M + H] + ; 11H NMR (400 MHz, DMSO-d6) δ 7.84 (t, J = 5.0 Hz, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.82 (dd, J = 9.0, 2.2 Hz, 1H), 6.57 (dd, J = 2.0, 1.0 Hz, 1H), 4.53 (s, 2H), 4.16–4.09 (m, 1H), 4.03–3.91 (m, 1H), 3.83 (s, 2H), 3.66 (t, J = 0.9 Hz, 8H), 3.62–3.54 (m, 6H), 3.40 (s, 3H), 3.35 (dt, J = 5.0, 4.2 Hz, 2H), 2.81 (td, J = 7.2, 0.9 Hz, 2H), 2.10–1.89 (m, 2H).

[0521] Example 71. Synthesis of Negative Compound UKOP3-N

[0522]

[0523] Reaction reagents and conditions: (a) NaOH, 1,4-dioxane, H2O, 0 - 25 °C, 4 h; (b) i. Cs2CO3, DMF, 0 - 25 °C, 3 h; ii. 25% TFA in CH2Cl2, 25 °C, 3 h; (c) CH2Cl2, 0 - 25 °C, 3 h; (d) i. 4N HCl in EtOAc, 25 °C, 12 h; ii. D-LN-acyl chloride, DIPEA, DMF, 0 - 25 °C, 3 h.

[0524] Step a: 1-(6-Hydroxy-3,4-dihydroquinolin-1(2H)-yl)propan-1-one D-1N

[0525]

[0526] Prepared in the same way as compound D-1, using propionyl chloride instead of chloroacetyl chloride as the raw material, the product D-1N (1.1 g, 71%) was obtained as a white solid, with the molecular formula C 12 H 15 NO2, LC-MS (ESI, m / z): 206.1 [M + H] + .

[0527] Step b: 2-((1-Propionyl-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetic acid D-LN

[0528]

[0529] The preparation method is the same as that of compound D-L, using D-1N to replace D-1 as the raw material, and the product D-LN (760 mg, 61%) is obtained as a white solid with the molecular formula C 14 H 17 NO4, LC-MS (ESI, m / z): 264.1 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 13.1 (s, 1H), 7.45 (d, J = 112.5 Hz, 1H), 6.73 (dd, J = 14.1, 5.1 Hz, 2H), 4.47 (s, 2H), 3.66 (t, J = 6.3 Hz, 2H), 2.67 (t, J = 7.1 Hz, 2H), 2.24 (q, J = 7.7 Hz, 2H), 1.94–1.77 (m, 2H), 1.06 (t, J = 7.7 Hz, 3H).

[0530] Step c: 2-((1-Propionyl-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetyl chloride D-LN-acylchloride

[0531]

[0532] The preparation method is the same as that of compound D-L-acyl chloride, using D-LN to replace D-L as the raw material, and the crude product D-LN-acyl chloride is obtained with the molecular formula C 14 H 16 ClNO3, LC-MS (ESI, m / z): 278.1 [M+H] + (Compound D-LN-acyl chloride is unstable in water, and the data here are for the methyl ester derivative).

[0533] Step d: N-(2-(2-(2-(2-((5-(4-(3-Benzyl-1-(1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-propionyl-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOP3-N

[0534]

[0535] The preparation method is the same as that of compound UKNP1, using compound I-O-3 to replace I-N-1 and D-LN-acyl chloride to replace D-L-acyl chloride as the raw materials, and the product UKOP3-N (33 mg, 35%) is obtained as an off-white solid with the molecular formula C 53H 62 N8O8, LC-MS (ESI, m / z): 939.5 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 2.4 Hz, 1H), 8.1 (d, J = 2.8 Hz, 1H), 8.02 (t, J = 5.9 Hz, 1H), 7.85 (dd, J = 9.4, 2.8 Hz, 1H), 7.69–7.61 (m, 2H), 7.57 (dd, J = 8.8, 2.3 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.42–7.06 (m, 8H), 6.86–6.72 (m, 2H), 6.54 (d, J = 9.4 Hz, 1H), 6.45 (d, J = 9.1 Hz, 1H), 5.88 (t, J = 6.1 Hz, 1H), 4.43 (s, 2H), 4.34–4.26 (m, 1H), 4.14 (dd, J = 14.6, 5.9 Hz, 2H), 3.68 (t, J = 5.6 Hz, 2H), 3.61 (t, J = 6.6 Hz, 2H), 3.51 (dd, J = 6.4, 3.1 Hz, 2H), 3.51–3.41 (m, 7H), 3.38 (t, J = 6.2 Hz, 2H), 3.25 (q, J = 6.6 Hz, 2H), 2.65 (s, 2H), 2.27 (q, J = 7.5 Hz, 2H), 1.91–1.72 (m, 6H), 1.35–1.16 (m, 6H), 1.05 (t, J = 7.6 Hz, 3H).

[0536] Example 72. Synthesis of UKOP3-1, UKOP3-2, UKOP3-3, and UKOP3-4

[0537] N-(2-(2-(2-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide hydrochloride UKOP3-1

[0538]

[0539] Dissolve UKOP3 (20 mg, 20.9 μmol) in ethyl acetate, add 21 μL of 1N hydrochloric acid. Monitor the reaction by TLC until the raw materials are completely reacted. After concentration under reduced pressure, UKOP3-1 is obtained as a beige solid.

[0540] N-(2-(2-(2-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide trifluoroacetate UKOP3-2

[0541]

[0542] Dissolve UKOP3 (15 mg, 15.6 μmol) in ethyl acetate, add 16 μL of 1N hydrochloric acid. Detect by TLC that the raw material reaction is complete. After concentration under reduced pressure, UKOP3-2 is obtained as a beige solid.

[0543] N-(2-(2-(2-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide phosphate UKOP3-3

[0544]

[0545] Dissolve UKOP3 (21 mg, 21.9 μmol) in ethyl acetate, add 22 μL of 1N hydrochloric acid. Detect by TLC that the raw material reaction is complete. After concentration under reduced pressure, UKOP3-3 is obtained as a beige solid.

[0546] N-(2-(2-(2-(2-((5-(4-(3-Benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide sulfate UKOP3-4

[0547]

[0548] Dissolve UKOP3 (16 mg, 16.7 μmol) in ethyl acetate, add 17 μL of 1N hydrochloric acid. Detect by TLC that the raw material reaction is complete. After concentration under reduced pressure, UKOP3-4 is obtained as a beige solid.

[0549] Example 72. Biological activity evaluation

[0550] I. Detection of the degradation effect of compounds on BUB1B and BUB1 by Western blot

[0551] Using Warhead and UKOP3-N compounds as negative controls, the degradation of BUB1B and BUB1 proteins in H1299 cell line, HCT116 cell line, SU-DHL-4 and SU-DHL-6 cell lines by compounds was determined by Western blot.

[0552] Experimental materials:

[0553] Cell lines: H1299 cells, HCT116 cells, SU-DHL-4 and SU-DHL-6 cells

[0554] Media: RPMI-1640 Medium, McCoy’s 5A Medium

[0555] For H1299, SU-DHL-4 and SU-DHL-6: RPMI-1640 Medium + 10% Hyclone serum

[0556] For HCT116: McCoy’s 5A Medium + 10% Hyclone serum

[0557] Drug preparation method: Dissolve the drug in DMSO to make a 50 mM stock solution and dilute it to the corresponding concentration according to a certain ratio.

[0558] 1. In vitro culture and drug administration of H1299 cells

[0559] (1) In vitro culture of H1299 cells:

[0560] The selected H1299 cells were cultured in a 37 °C constant temperature incubator containing 5% CO2, and the culture conditions were RPMI-1640 Medium + 10% Hyclone serum. When the cell density reached 70-90%, passage was carried out for subsequent experiments.

[0561] (2) Seeding cells on plates: Digest the cells and add the cell suspension into 1.5 mL Eppendorf tubes, centrifuge at 1,000 rpm for 5 min, resuspend the cells with 2 mL of culture medium and count. Seed the cell suspension in 6-well plates, with 100,000 cells per well, and place for 24 h until all the cells adhere to the wall.

[0562] (3) Drug administration to cells: Dissolve the compound in DMSO, add 1 μL of the compound to each well, and the final concentrations are 200 nM respectively. Incubate in a 37 °C, 5% CO2 cell culture incubator for 24 h, with the DMSO group as the blank control.

[0563] 2. In vitro culture and administration of HCT116 cells

[0564] (1) In vitro culture of HCT116 cells:

[0565] The selected HCT116 cells were cultured in a 37°C constant temperature incubator with 5% CO2. The culture conditions were McCoy’s 5A Medium + 10% Hyclone serum. When the cell density reached 70 - 90%, subculture was performed for subsequent experiments.

[0566] (2) Seeding cells on plates: Digest the cells and add the cell suspension into a 1.5 mL Eppendorf tube. Centrifuge at 1,000 rpm for 5 min, resuspend the cells with 2 mL of culture medium and count them. Seed the cell suspension into a 6-well plate, with 100,000 cells per well, and place it for 24 h until all the cells adhere to the plate.

[0567] (3) Administration of compounds to cells: Dissolve the compound in DMSO, add 1 μL of the compound to each well, and the final concentrations were 400 nM respectively. Incubate in a 37°C cell culture incubator with 5% CO2 for 24 h, using the DMSO group as the blank control.

[0568] 3. In vitro culture and administration of SU-DHL-4 cells

[0569] (1) In vitro culture of SU-DHL-4 cells:

[0570] The selected SU-DHL-4 cells were cultured in a 37°C constant temperature incubator with 5% CO2. The culture conditions were RPMI-1640 Medium + 10% Hyclone serum. When the cell density reached 70 - 90%, subculture was performed for subsequent experiments.

[0571] (2) Seeding cells on plates: Digest the cells and add the cell suspension into a 1.5 mL Eppendorf tube. Centrifuge at 1,000 rpm for 5 min, resuspend the cells with 2 mL of culture medium and count them. Seed the cell suspension into a 6-well plate, with 100,000 cells per well, and place it for 24 h until all the cells adhere to the plate.

[0572] (3) Administration of compounds to cells: Dissolve the compound in DMSO, add 1 μL of the compound to each well, and the final concentrations were 400 nM respectively. Incubate in a 37°C cell culture incubator with 5% CO2 for 24 h, using the DMSO group as the blank control.

[0573] 4. In vitro culture and administration of SU-DHL-6 cells

[0574] (1) In vitro culture of SU-DHL-6 cells:

[0575] The selected SU-DHL-6 was cultured in an incubator at 37°C with 5% CO2. The culture conditions were RPMI-1640 Medium + 10% Hyclone serum. Subculture was performed when the cell density reached 70-90% for future experiments.

[0576] (2) Seeding cells: Digest the cells and add the cell suspension into a 1.5 mL Eppendorf tube. Centrifuge at 1,000 rpm for 5 min, resuspend the cells with 2 mL of culture medium and count them. Seed the cell suspension into a 6-well plate, with 100,000 cells per well, and incubate for 24 h until all cells adhere to the plate.

[0577] (3) Treating cells with compounds: Dissolve the compounds in DMSO. Add 1 μL of the compound to each well, with the final concentrations being 400 nM. Incubate in a cell culture incubator at 37°C with 5% CO2 for 24 h, using the DMSO group as the blank control.

[0578] 5. Cell lysis

[0579] At the end of the action time, collect the cells and wash them once with PBS; Add the corresponding volume of 4% SDS to lyse the cells according to the cell amount, and sonicate until the cells are no longer viscous; Centrifuge at 12,000 rpm at room temperature for 30 min; Transfer the supernatant to a new EP tube for protein quantification.

[0580] 6. Protein quantification

[0581] Dilute the 2 mg / mL BSA standard product in half successively to obtain the concentrations used for the standard curve, which are 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.0625 mg / mL; Calculate according to 200 μL of solution A and 4 μL of solution B in the BCA quantification kit for each sample. Take the corresponding volumes of solution A and solution B (volume ratio 50:1) and mix them evenly; Take 10 μL of BSA with different concentrations and samples and add them into a 96-well plate respectively, then add 200 μL of the evenly mixed solution A and solution B, gently tap to mix evenly and place in the dark at 37°C for reaction for 30 min; After the reaction, measure the absorbance value at 562 nm, and calculate the protein concentration of the sample using the standard curve.

[0582] 7. Western Blot

[0583] (1) Preparation of protein samples

[0584] Take 20 μg of protein and add a certain amount of 6× Loading Buffer according to the volume to make the final concentration 1× Loading Buffer; Heat and denature at 95°C for 10 min. After cooling, centrifuge and mix evenly for Western Blot experiment, and store the remaining samples at -80°C.

[0585] (2) Gel preparation

[0586] a. Separating gel: Select the concentration of the separating gel to be prepared according to the molecular weight of the target protein. First, assemble the gel cassette: Clamp the thick and thin glass plates tightly and keep the bottom surface flat. Add the separating gel reagents to the centrifuge tube in sequence and vortex to mix well. After mixing, add the separating gel between the two glass plates, and then add an appropriate amount of isopropanol on the upper layer of the gel surface and wait for it to solidify.

[0587] b. Stacking gel: Discard the isopropanol on the upper layer of the separating gel and wash the remaining residual liquid with triple-distilled water. Prepare the stacking gel and vortex to mix well. After mixing, add the stacking gel between the two glass plates, insert the gel comb, and wait for it to solidify.

[0588] (3) Electrophoresis

[0589] a. Preparation: Install the prepared gel cassette into the electrophoresis tank and place it in the electrophoresis instrument. Add the diluted 1×Tricine buffer to the middle electrophoresis tank and add 1×Running buffer to the outer tank, and let it stand for several minutes.

[0590] b. Loading: Vortex the sample to mix well, pipette a certain amount of the sample (5 - 10 μL) for loading. After loading, turn on the electrophoresis instrument. First, set it to the constant voltage mode, electrophorese at 70 V for 10 min, and confirm that the current is normal. After the sample enters the separating gel, adjust the voltage to 130 V. When the sample runs to the appropriate position, stop the electrophoresis.

[0591] (4) Transfer and blocking

[0592] a. Transfer: Add a sponge, thick filter paper, and thin filter paper to the transfer cassette in sequence. Take the gel: Use a gel spatula to remove the stacking gel block at the edge and carefully remove the separating gel and place it in the center of the filter paper. Apply the membrane: Stick the PVDF membrane pre-activated with methanol to the gel from one side and expel the air bubbles. Then add thin filter paper, thick filter paper, and sponge in sequence and clamp the transfer cassette well to assemble the transfer cassette in sequence. Add an appropriate amount of transfer buffer to the transfer tank, add an appropriate amount of water to the outer layer of the transfer tank and place an ice pack to keep the transfer tank in ice water to prevent a large amount of heat from being released during the transfer process and affecting the transfer effect.

[0593] b. Blocking: After the transfer is completed, open the transfer cassette, remove the gel and PVDF membrane (if the transfer is successful, it can be seen that the protein Marker has been completely transferred to the PVDF membrane). Then cut the PVDF membrane according to the molecular weight of the target protein and place the membrane in 5% skim milk and block it at room temperature for 60 min.

[0594] (5) Incubate with antibodies

[0595] a. Incubate with primary antibody: After blocking, discard the milk and wash the PVDF membrane 3 times with TBST (15 min, 5 min, 5 min). Then add an appropriate amount of primary antibody (usually diluted at a ratio of primary antibody: antibody diluent = 1:1000) and incubate overnight on a shaker at 4°C.

[0596] b. Incubate with secondary antibody: Recover the primary antibody and wash 3 times with TBST (15 min, 5 min, 5 min). Then add the secondary antibody (usually prepared at a ratio of secondary antibody: 5% skim milk = 1:5000) and incubate on a horizontal shaker at room temperature for 60 min.

[0597] (6) Exposure

[0598] a. Wash the membrane: Discard the secondary antibody and wash 3 times with TBST (15 min, 5 min, 5 min).

[0599] b. Exposure: Prepare the ECL luminescent solution: Solution A: Solution B = 1:1. First, blot the residual TBST on the strip with filter paper, then use forceps to pick up the strip and place it on the exposure board. Drop the ECL developing solution on the strip to cover it and let it stand for about 1 min. Put the exposure board into the AI800 exposure machine and select an appropriate time for exposure.

[0600] (7) Gray-scale analysis: Open the Image software and open the resulting white-background black-strip image obtained by exposure through this software. After removing the gray background of the gel image, use the rectangular marquee tool to select the strip to be analyzed to obtain a peak-shaped graph. Then use the straight line tool to enclose each small peak graph. Finally, select the magic wand tool and click on each closed small peak in turn to obtain the gray-scale value of the corresponding strip.

[0601] (8) Calculation of degradation rate:

[0602] Table 2 Results of the degradation of BUB1B and BUB1 by the compound in H1299 cells

[0603]

[0604]

[0605] Table 3 Results of the degradation of BUB1B and BUB1 by the compound in HCT116 cells

[0606]

[0607] Table 4 Results of the degradation of BUB1B and BUB1 by the compound in SU-DHL-4 cells

[0608]

[0609]

[0610] Table 5 Degradation results of BUB1B and BUB1 by the compound on SU-DHL-6 cells

[0611]

[0612] Experimental results: As shown in Tables 2, 3, 4 and 5 and Appendix Figure 1 、 2 、3 and 4, the compounds of the present invention have a certain degradation effect on BUB1B and BUB1 on H1299 cells, HCT116 cells, SU-DHL-4 and SU-DHL-6 cells.

[0613] II. Detection of the inhibitory effect of the compound on the proliferation of H1299 cells, HCT116 cells and SU-DHL-4 cells by sulforhodamine B (SRB) staining

[0614] Using Warhead, DCAF16-P3, and UKOP3-N compounds as negative controls, the proliferation of H1299 cell line, HCT116 cell line, SU-DHL-4 and SU-DHL-6 cell lines was determined by sulforhodamine B (SRB) staining and CCK8.

[0615] First, seed the cells in a 96-well plate at a density of 3000 per well and then administer the drug for 72 h, followed by detection. For H1299 cells and HCT116 cells, at the end of the action time, discard the culture medium in the 96-well plate, add 70 μL of PBS to wash once, then add 70 μL of 10% trichloroacetic acid to fix the cells at 4 °C for 1 h; rinse the trichloroacetic acid with tap water and then dry in an oven; then add 70 μL of SRB staining solution and stain at room temperature for 20 min; discard the SRB staining solution, wash it clean with 1% glacial acetic acid, and dry it in an oven; add 150 μL of 10 mM Tris-base dissolution solution, dissolve it on a shaker for 15 min, and then measure the absorbance value at 540 nm.

[0616] For SU-DHL-4 and SU-DHL-6 cells, at the end of the action time, add 10 μL of CCK8 reagent to each well, incubate at 37 °C in the dark for 4 h, mix well, and then measure the absorbance value A at 450 nm. And calculate the proliferation inhibition rate of each well according to the formula Inhibition%=1 - A 给药组 / A DMSO组 Calculate the proliferation inhibition rate of each well, and take the average value of the inhibition rates obtained from three replicate wells.

[0617] Table 6 Proliferation inhibition of the compound on H1299 cells

[0618]

[0619]

[0620] Table 7 Proliferation inhibition of compounds on HCT116 cells

[0621]

[0622] Table 8 Proliferation inhibition of compounds on SU-DHL-4 cells

[0623]

[0624] Table 9 Proliferation inhibition of compounds on SU-DHL-6 cells

[0625]

[0626] According to Tables 6 to 9 and Figures 5 - 9 the experimental results: Compound UKOP3 has strong proliferation inhibition effects on H1299 cells, HCT116 cells, SU-DHL-4 and SU-DHL-6 cells, and the IC50 values are 117.2 nM, 445.7 nM, 27.86 nM and 26.85 nM respectively. To determine the effect of compound UKOP3, we made a horizontal comparison of UKOP3 with the negative control compound UKOP3-N, the warhead, and the ligand derivative DCAF16-P3 of the DCAF16 E3 ligase, and found that at the given concentration, UKOP3-N, Warhead and DCAF16-P3 have no obvious proliferation inhibition on H1299 cells. Therefore, it shows that the proliferation inhibition effect of UKOP3 does not come from the inhibitory effects of the warhead or the DCAF16 ligand alone, but is the effect of the drug itself.

[0627] The compounds of the present invention can significantly inhibit the proliferation of H1299 cells, HCT116 cells, SU-DHL-4 and SU-DHL-6 cells, while the compounds of Warhead, DCAF16-P3 and UKOP3-N have little effect on cell proliferation. Therefore, the compounds of the present invention have potential therapeutic effects on related cancers.

[0628] III. Propidium Iodide (PI) staining and flow cytometry to detect the cell cycle arrest of compounds on H1299 cells

[0629] The cell cycle consists of the following four stages, including the G0 / G1 phase in which cell size increases, the S phase in which DNA is replicated, the G2 phase in which the cell prepares for division, and the mitotic M phase. Among them, the chromosome multiplicity in the G0 / G1 phase is 2N, in the S phase is 2N - 4N, and in the G2 / M phase is 4N. Since propidium iodide can produce fluorescence after binding to double-stranded DNA, and the fluorescence intensity is proportional to the content of double-stranded DNA. Therefore, after the DNA in the cell is stained with propidium iodide, the DNA content of the cell can be measured by flow cytometry, and the cell cycle can be further analyzed.

[0630] Seed H1299 cells in a six-well plate at a cell density of 200,000 per well, administer UKOP3, Warhead, and DCAF16-P3200 nM for 48 hours, and give an equal amount of DMSO as the control group. Detect the cell cycle of H1299 cells by PI staining combined with flow cytometry.

[0631] (1) Permeabilization: Collect the cells, wash them once with PBS, centrifuge at 300 g for 5 min, discard the supernatant, and add

[0632] 75% ethanol solution and punch holes at -20°C overnight.

[0633] (2) RNase digestion: Collect the cells, wash them twice with PBS, centrifuge at 300 g for 5 min, discard the supernatant, and add 3 μL of RNase and incubate at 37°C for 1 h.

[0634] (3) Staining: Add 5 μL of PI dye and incubate at room temperature in the dark for 20 min.

[0635] (4) Find the main cell population and set the gate by adjusting the voltages of FSC and SSC, adjust the flow rate to 200

[0636] cells per second, the excitation wavelength is 488 nm, record the data of the PE channel at a wavelength of 575 nm, collect about 1×10 4 cells, and use FlowJo 7.6 software to uniformly set the gate and analyze the proportion of cells in each cell cycle, that is, the number of cells in each cell cycle accounts for the total number of cells measured in this sample.

[0637] Table 10 Effects of compounds on the cell cycle of H1299 cells

[0638] Compound Number G0 / G1 (%) S(％) G2 / M (%) DMSO 49 16.4 29.8 UKOP3 38.3 19.7 38.2 Warhead 47.8 19.4 30.2 DCAF16P3 49.9 20.6 28.6 UKOP3-N 48.5 21.3 25.8

[0639] From Table 10 and Figure 10Experimental results: The compound of the present invention can cause G2 / M phase arrest of the H1299 cell cycle, thereby inhibiting cell proliferation, while its warhead, DCAF16-P3 ligand, and the negative control compound UKOP3-N have less impact on the cell cycle. Therefore, the compound of the present invention has potential therapeutic effects on related cancers.

[0640] IV. Synchronize the cell cycle with colchicine and detect the sister chromatid cohesion of the compound on H1299 cells by Giemsa staining

[0641] Cell division requires the replication of all chromosomes and then their separation into two identical sister chromatids. Sister chromatid cohesion holds the sister chromatids together until they begin to separate correctly during the metaphase-to-anaphase transition, with a normal morphology as shown in Normal in the appendix. Figure 6 The common morphology of sister chromatid cohesion defects is Railroad, which is further divided into Severe and Mild according to the degree of the defect, as shown in Severe and Mild in the appendix. Figure 11 Sister chromatid cohesion defects may further lead to mitotic arrest. We used the Gimesa staining method combined with a high-power microscope to observe the sister chromatid cohesion of the compound on H1299 cells. The specific operation steps are as follows.

[0642] Seed H1299 cells in a T25 cell culture dish at a cell density of 1 million per dish. Administer 200 nM of UKOP3 for 24 hours, and use an equal amount of DMSO as the control group. Synchronize the cell cycle with colchicine and detect the sister chromatid cohesion of H1299 cells by Giemsa staining.

[0643] Prepare metaphase spreads from the 3rd to 5th generation cell lines. Treat exponentially growing cells with 10 μg / ml of colchicine for 6 - 10 hours. Collect cells by trypsin digestion and then centrifugation. Incubate the collected cells in a 0.075 mM NaCl hypotonic solution at 37°C for 20 minutes, and then suspend them in a 3:1 methanol:acetic acid fixation solution 3 times, 30 minutes each time. Prepare slides by adding the fixed metaphase cells to a clean slide and spreading them. Dry the slides at 37°C, stain them with Giemsa solution for 5 - 10 minutes, and then take pictures and observe them under a high-power microscope. Collect the sister chromatid cohesion morphologies of 30 cells in each group, and respectively count the proportions of cells with sister chromatid cohesion morphologies of Normal, Severe Railroad, and Mild Railroad in the total number of cells in each group.

[0644] Table 11 Effects of the compound on sister chromatid cohesion of H1299 cells

[0645] Compound Number Normal (%) Severe Railroad (%) Mild Railroad (%) DMSO 68 32 0 UKOP3 45 50 5 UKOP3-N 73 24 3

[0646] Experimental results: As shown in the appendix Figure 11 and Table 11, the proportion of cells with normal sister chromatid cohesion morphology in the blank control group was 68%, the proportion of cells with Severe Railroad morphology was 32%, and the proportion of cells with Mild Railroad morphology was 0%. In the negative control compound UKOP3-N group, the proportion of cells with normal sister chromatid cohesion morphology was 73%, the proportion of cells with Severe Railroad morphology was 24%, and the proportion of cells with Mild Railroad morphology was 3%. In the UKOP3 group, the proportion of cells with normal sister chromatid cohesion morphology was 45%, the proportion of cells with Severe Railroad morphology was 50%, and the proportion of cells with Mild Railroad morphology was 5%, with an increase in the proportion of Severe Railroad morphology defects. Therefore, the compound of the present invention can significantly cause sister chromatid cohesion defects in H1299 cells and has potential therapeutic effects on related cancers.

[0647] V. Immunofluorescence and Nocodazole Detection of the Effect of Compounds on the Mitotic Index of H1299 Cells

[0648] The cell cycle stage can be divided into the mitotic cell stage (such as the cell being in prophase, prometaphase, metaphase, anaphase, telophase or cytokinesis) or the non-mitotic cell stage (such as the cell being in the resting phase or interphase). In the relevant aspects and embodiments of the present invention, the determination of cells in mitosis is based on one or more characteristics, including but not limited to cell size, cell morphology, and nuclear morphology, etc. MI is the ratio of the number of mitotic cells to non-mitotic cells in the sample.

[0649] Nocodazole can bind to β-tubulin in microtubules, thereby inhibiting the formation of related disulfide bonds and the dynamic changes of microtubules, and further inhibiting the function of the spindle during mitosis. Due to the presence of the SAC checkpoint, the cell cycle cannot proceed normally. Therefore, the function of the cell SAC checkpoint can be detected by MI. In the relevant aspects and embodiments of the present invention, we used compound immunofluorescence and Nocodazole to detect the effect of compounds on the mitotic index (Mitotic Index) of H1299 cells.

[0650] H1299 cells were seeded in six-well plates with a cell density of 200,000 per well. After 24 hours of treatment with 200 nM of UKOP3, Warhead, and DCAF16-P3, an equal amount of DMSO was used as the control group. After 24 hours of blocking with 100 ng / mL Nocodazole, the mitotic index of H1299 cells was detected by immunofluorescence staining.

[0651] Immunofluorescence experiment steps:

[0652] 1. Fixation and Permeabilization

[0653] 1) Wash the coverslips on which H1299 cells have been grown in the culture plate three times with 1×PBS, each time for 3 minutes.

[0654] 2) Fix the slides with 4% paraformaldehyde for 15 min, and wash the slides three times with 1×PBS, each time for 3 min.

[0655] 3) 0.5% Triton X-100 (prepared in 1×PBS) was used to permeabilize the cells for 15 min at room temperature, and the slides were washed three times with 1×PBS, each time for 3 min.

[0656] 2. Closure

[0657] Blot 1×PBS with absorbent paper, add 5% normal serum (similar to or consistent with the species of the secondary antibody) on the slide, and block at room temperature for 1 h.

[0658] 3. Antibody Incubation

[0659] 1) Use absorbent paper to absorb the blocking solution. Without washing, add a sufficient amount of diluted DAPI primary antibody to each slide and place it in a wet box and incubate overnight at 4°C.

[0660] 2) Add fluorescent secondary antibody: Wash the slides three times with PBST, each time for 3 min. After absorbing the excess liquid on the slides with absorbent paper, add the diluted fluorescent secondary antibody and incubate in a wet box at 37°C for 1 h. Wash the slides three times with PBST, each time for 3 min.

[0661] 4. Fixed photo taking

[0662] Use absorbent paper to absorb the liquid on the slide, seal the slide with sealing solution containing anti-fluorescence quenching agent, and observe and collect images under a fluorescence microscope.

[0663] Table 12 Effects of compounds on the mitotic index of H1299 cells

[0664] Compound Number Mitotic Index (%) DMSO 52.63 UKOP3 2.04 Warhead 58.3 DCAF16-P3 65.21 UKOP3-N 57.83

[0665] From Table 12 and Figure 12From the experimental results, it can be seen that the compound of the present invention can significantly decrease the mitotic index of H1299 cells, indicating that it severely inhibits the activity of the SAC checkpoint, while the Warhead, DCAF16-P3, and UKOP3 compounds have little effect on the mitotic index of cells, indicating no effect on SAC activity. Therefore, the compound of the present invention has potential therapeutic effects on related cancers.

Claims

1. A PROTAC compound for degrading BUB1B and BUB1 proteins, characterized in that, The general structural formula of the PROTAC compounds that degrade BUB1B and BUB1 proteins is shown in Formula (I), or is an optical isomer of the structure shown in Formula (I) or a pharmaceutically acceptable salt thereof; PL—Linker-DCAF16 ligand (Ⅰ) In Formula (I), PL is: Among them indicates that the relative configuration of the two keys is trans, that is, the relative orientations are opposite; The DCAF16 ligand is Linker is a linking group, and Linker is selected from the following structures: n1 is an integer from 1 to 15; n2 is an integer from 1 to 6.

2. The PROTAC compound for degrading BUB1B and BUB1 proteins according to claim 1, characterized in that: Has the structure shown in the following formula: n2 is 2, 3, or 4.

3. The PROTAC compound for degrading BUB1B and BUB1 proteins according to claim 1, wherein The structure is: UKOP0: N-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOP1: N-(2-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethyl-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOP2: N-(2-(2-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOP3: N-(2-(2-(2-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOP4: N-(14-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNP0: N-(2-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1-(2H)-yl)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNP1: N-(2-(2-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNP2: N-(2-(2-(2-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNP3: N-(2-(2-(2-(2-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNP4: N-(14-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)-3,6,9,12-tetraoxatetradecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOC9: N-(9-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)nonyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOC11: N-(11-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)undecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKOC13: N-(13-((5-(4-(3-benzyl-1-(1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)tridecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNC9: N-(9-(5-(4-(3-benzyl-1-(1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)nonyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNC11: N-(11-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)undecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide UKNC13: N-(13-(5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)-2-oxopyridin-1(2H)-yl)tridecyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide.

4. The PROTAC compound for degrading BUB1B and BUB1 proteins according to any one of claims 1 to 3, characterized in that, The pharmaceutically acceptable salt is one or more of the hydrochloride, trifluoroacetate, phosphate, and sulfate corresponding to the compound.

5. The PROTAC compound for degrading BUB1B and BUB1 proteins according to claim 1, characterized in that, The structure is as follows: UKOP3-1: N-(2-(2-(2-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide hydrochloride UKOP3-2: N-(2-(2-(2-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide trifluoroacetate UKOP3-3: N-(2-(2-(2-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide phosphate UKOP3-4: N-(2-(2-(2-(2-((5-(4-(3-benzyl-1-((1,4-trans)-4-((5-cyanopyridin-2-yl)amino)cyclohexyl)ureido)phenyl)pyridin-2-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-2-((1-(2-chloroacetyl)-1,2,3,4-tetrahydroquinolin-6-yl)oxy)acetamide sulfate.

6. A pharmaceutical composition, wherein, The pharmaceutical composition as described above contains a therapeutically effective amount of the compound as claimed in any one of claims 1-5 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

7. Use of the compound as claimed in any one of claims 1-5 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition as claimed in claim 6 in the manufacture of a medicament for treating a disease that can benefit from the degradation of BUB1B and BUB1 proteins.

8. The application according to claim 7, wherein The disease is selected from one or more of non-small cell lung cancer, colorectal cancer and large B lymphoma cancer.

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