Anti-tumor small-molecule drug conjugate including PI3K inhibitor, and synthetic method and application of anti-tumor small-molecule drug conjugate

Abstract

The invention innovatively provides a small molecule drug conjugate for the first time, the small molecule drug conjugate takes a PI3K inhibitor as an active drug, takes extracellular HSP90 as a targeting ligand, and takes an alkane chain, a PEG chain and a nitrogen-containing heterocyclic ring as linker, and the general formula of the small molecule drug conjugate is as shown in formula (I). The invention also discloses the small molecule drug conjugate and a preparation method thereof, and application of the small molecule drug conjugate in preparation of anti-tumor and anti-colorectal cancer drugs. The invention also provides a PI3K inhibitor and a synthesis method thereof, and application of the PI3K inhibitor in synthesis of small molecule drug conjugates and preparation of anti-tumor and anti-colorectal cancer drugs. The PI3K inhibitor has an efficient PI3K inhibition effect, an anti-tumor effect, an anti-colorectal cancer effect and the like, and has a wide potential application prospect. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical synthesis and chemical engineering, and specifically relates to an anti-tumor small molecule drug conjugate including a PI3K inhibitor, and a synthesis method and application thereof. Background Art

[0002] The phosphoinositide 3-kinase (PI3K) / protein kinase B (AKT) / mammalian target of rapamycin (mTOR) signaling pathway is one of the most important signaling pathways in mammalian cells. It inhibits apoptosis and promotes proliferation in cells by affecting the activation state of multiple downstream effector molecules. Abnormalities in the PI3K-AKT-mTOR signaling pathway are closely related to the occurrence of tumors. Drug regulation of a single target in the pathway is not enough to prevent all activated oncogenic signals, which may lead to the activation of other compensatory pathways, offset the efficacy of the drug, or even produce drug resistance. Therefore, it is urgent to develop multi-target drugs for the treatment of cancer.

[0003] Small molecule drug-conjugate (SMDC) is a new drug delivery system, which consists of three parts: targeting ligand, linker and active drug. It has the potential to target tumor cells, allowing the payload to quickly accumulate in tumor cells and penetrate deep into the tumor core, and slowly release the payload over time, thereby continuously delivering the drug to the tumor while quickly clearing it from the plasma, thereby reducing overall toxicity.

[0004] Studies have shown that there is great room for development in applying the basic principles of constructing SMDC to the research of PI3K inhibitors. Summary of the invention

[0005] The purpose of the present invention is to provide a class of anti-tumor small molecule drug conjugates and their synthesis methods and applications. The conjugate includes a PI3K inhibitor as an active drug portion, an Hsp90 inhibitor as a targeting ligand portion, and an alkane chain or PEG chain of different lengths as a connecting chain portion. This type of compound has obvious anti-colorectal cancer activity and excellent selectivity, and can be further developed as an anti-colorectal cancer drug. Specifically, the present invention uses extracellular HSP90 (eHSP90) as a target of tumor tissue, and couples a highly active PI3K inhibitor with an HSP90 ligand through a Linker (cleavable, non-cleavable) to form a small molecule conjugate. The cleavable SMDC masks the active site of the PI3K inhibitor, binds to the extracellular HSP90 to enter the cell, and releases the drug (PI3K inhibitor) at a fixed point in the tumor tissue enzymatic cleavage. The non-cleavable SMDC binds to the extracellular HSP90 and is transported into the cell to directly exert its efficacy.

[0006] The present invention provides a small molecule drug conjugate, specifically, a small molecule drug conjugate with a PI3K inhibitor as an active drug, an extracellular Hsp90 as a targeting ligand, and a linker (including an alkane chain, a PEG chain, a nitrogen-containing heterocycle, etc.), and its structural formula is shown in formula (I):

[0007]

[0008] Wherein, the R1 is

[0009] Wherein, the R2 is an extracellular Hsp90 ligand, and its structure is shown in formula (II) or formula (III):

[0010]

[0011] Wherein, the linker is an alkane chain, a PEG chain and a nitrogen-containing heterocycle, which includes Five-membered nitrogen-containing heterocycle, substituted five-membered nitrogen-containing heterocycle, six-membered nitrogen-containing heterocycle, substituted six-membered nitrogen-containing heterocycle; wherein the heterocyclic group contains 1-3 N or O heteroatoms, and the value of n is 1-3.

[0012] Preferably, the small molecule drug conjugates include those shown in the following formulas (CC-1), (CC-3), (CC-6), (CC-7), (CC-8), (CC-9), (CC-11), and (CC-13):

[0013]

[0014] Preferably, the small molecule drug conjugate comprises the following:

[0015]

[0016] In the present invention, the small molecule drug conjugate is a small molecule drug conjugate with a PI3K inhibitor as an active drug, extracellular Hsp90 as a targeting ligand, and an alkane chain, a PEG chain and a nitrogen-containing heterocycle as a linker. The small molecule drug conjugate includes a PI3K inhibitor-HSP90 ligand.

[0017] In the small molecule drug conjugate of the present invention, the structure on the left side of the linker is a PI3K inhibitor, and the structure on the right side of the linker is an extracellular Hsp90 ligand. The linker includes but is not limited to an alkane chain, a PEG chain, and a nitrogen-containing heterocycle.

[0018] The present invention also provides a PI3K inhibitor, the structure of which is shown in the following formula (IV):

[0019]

[0020] In the formula (IV), R1 is

[0021] Preferably, the PI3K inhibitor includes but is not limited to the structure shown in the following formula (CC-M-1):

[0022]

[0023] Preferably, in a specific embodiment, the PI3K inhibitor can effectively inhibit four subtypes of PI3K kinase, including PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ, and the structural formulas of the inhibitors are as follows:

[0024]

[0025] Further preferably, the PI3K inhibitor includes the following:

[0026]

[0027] The PI3K inhibitor can be used to synthesize the small molecule drug conjugate, and / or can be directly used alone as an active ingredient in a pharmaceutical composition.

[0028] The present invention also provides a pharmaceutical composition, which comprises the small molecule drug conjugate and / or the PI3K inhibitor. Further, the pharmaceutical composition also comprises other pharmaceutically acceptable carriers or excipients. Further, the pharmaceutical composition can be formulated into an injectable fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup, a transdermal patch or an excipient.

[0029] The present invention also provides the application of the small molecule drug conjugate, the PI3K inhibitor and / or the pharmaceutical composition, which can be used in the preparation of PI3K inhibitors, anti-tumor, anti-colorectal cancer drugs or products.

[0030] The present invention also proposes the use of the small molecule drug conjugate, the PI3K inhibitor, and / or the pharmaceutical composition in the preparation of drugs or products for inhibiting the proliferation, growth, infiltration and migration of colorectal cancer cells, or promoting the apoptosis of colon cancer cells.

[0031] In the present invention, the tumors and cancers include but are not limited to colon cancer, rectal cancer, breast cancer, bile duct cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, lymphoma, prostate cancer, etc.

[0032] The present invention also proposes a method for synthesizing the PI3K inhibitor, which includes Suzuki coupling, Click reaction, etc. The synthesis method is to use 3-amino-5-bromo-2-methoxypyridine and 2,4-difluorobenzenesulfonyl chloride as raw materials, perform a substitution affinity reaction in pyridine as a solvent, and react at room temperature for 48 hours to obtain an intermediate compound I-1. Then, compound I-2 is obtained by Miyaura borylation reaction in a 1,4-dioxane solvent system. Then, 3-amino-6-chloropyridazine and chloroacetaldehyde are used as raw materials, and the imidazo[1,2-b]pyridazine mother core I-3 is obtained by ring closure under the action of sodium bicarbonate. Compound I-3 and boric acid ester I-2 are subjected to Suzuki cross-coupling reaction to obtain compound I-4, and then iodinated with NIS to obtain compound I-5. I-5 is subjected to Sonogashira reaction and TMS protection group removal reaction to obtain terminal alkynyl compound I-6. I-6 is subjected to Click reaction with three different azide compounds to obtain compounds CC-P-1, CC-M-1 and CC-N-1-1, and CC-N-1-1 is deprotected in trifluoroacetic acid dichloromethane solution to obtain compound CC-N-1. The reaction process of the method includes the following steps:

[0033]

[0034]

[0035] Wherein, the R1 is

[0036] The raw material 3-amino-5-bromo-2-methoxypyridine (10.00g, 49.30mmol) was placed in a flask, pyridine (40mL) was added to the reaction bottle, and stirred in an ice bath until dissolved, and 2,4-difluorobenzenesulfonyl chloride (15.68g, 73.80mmol) was added thereto after the internal temperature dropped to 0°C, and the ice bath was removed after stirring for 5min under ice bath conditions, and the reaction was placed at room temperature and stirred for 48h. After the reaction was completed, the reaction solution was spin-dried to obtain a brown oil using a rotary evaporator, and dichloromethane (200mL) and water (250mL) were added to extract three times, and 10% citric acid aqueous solution (200mL) was washed three times to remove pyridine in the reaction system, and finally the organic phase was combined and spin-dried to obtain a brown oil. Ethyl acetate (EA) and petroleum ether (PE) were added for pulping, and 17.64g of brown solid I-1 was finally obtained by suction filtration, with a yield of 94.7%.

[0037] Compound I-1 was placed in a 500mL three-necked flask, 1,4-dioxane (250mL) was added thereto and stirred to dissolve, and then diboric acid pinacol ester (11.08g, 43.66mmol), tricyclohexylphosphine (2.23g, 7.94mmol), Pd2(dba)3 (3.63g, 3.96mmol), potassium acetate (11.68g, 119.04mmol) were added to the reaction system, nitrogen was replaced three times, and the reaction was stirred at 110°C under nitrogen protection for 4h. After the reaction was completed, diatomaceous earth was filtered, the reaction solution was spin-dried, dichloromethane (200mL) and water (250mL) were added to extract three times, and ether and petroleum ether (PE) were added to the concentrated organic phase for pulping, and 16.82g of white compound I-2 was obtained by suction filtration, with a yield of 99%.

[0038] Compound 3-amino-6-chloropyridazine (8.58 g, 66.25 mmol), chloroacetaldehyde (10.40 g, 132.50 mmol) and sodium bicarbonate (8.35 g, 99.38 mmol) were placed in a 500 mL pear-shaped flask, 95% ethanol (200 mL) was used as a solvent, and then the reaction was placed in a 90°C oil bath and refluxed for 16 h. After the reaction was completed, the solvent in the reaction system was dried using a rotary evaporator, the sample was dry mixed, and the sample was separated by column chromatography. The ratio of petroleum ether to ethyl acetate in the mobile phase was 2:1, and 7.30 g of yellow solid I-3 was obtained, with a yield of 73%.

[0039] Compound I-3 (4.50 g, 47.70 mmol) was placed in a 500 mL three-necked flask, N, N-dimethylformamide (250 mL), H2O (30 mL) were added thereto and stirred to dissolve, then I-2 (15.67 g, 59.60 mmol), Pd(PPh3)4 (3.40 g, 2.94 mmol), potassium carbonate (6.10 g, 44.12 mmol) were added to the reaction system, nitrogen was replaced three times, and the reaction was stirred at 120 ° C for 4 h under nitrogen protection. After the reaction, diatomaceous earth was filtered, the reaction solution was spin-dried, dichloromethane and water were added for extraction three times, the organic phase was concentrated and dried, and the sample was mixed by dry method, and a silica gel column was used for column chromatography separation, and the mobile phase used a dichloromethane and methanol system (DCM: MeOH = 50: 1). 5.50 g of white solid I-4 was obtained, with a yield of 45%.

[0040] Compound I-4 (5.00 g, 11.99 mmol) and NIS (4.05 g, 17.98 mmol) were added to N,N-dimethylformamide (150 mL), and nitrogen was replaced three times. The mixture was stirred at room temperature for 12 h under nitrogen protection. After the reaction was completed, the reaction solution was slowly added to water to precipitate a yellow solid, which was filtered and vacuum dried to obtain 6.02 g of compound I-5 with a yield of 92%.

[0041] Compound I-5 (5.00 g, 9.20 mmol) was dissolved in tetrahydrofuran (200 mL) and placed in an ice bath until the temperature dropped to 0 ° C. Cuprous iodide (0.18 mg, 0.92 mmol) and Pd (PPh3) 2Cl2 (0.65 g, 0.92 mmol) were added thereto, nitrogen was replaced three times, and stirred for 0.5 h under nitrogen protection, and then triethylamine (2.79 g, 27.60 mmol) and trimethylethynylsilane (2.71 g, 27.60 mmol) were added by injection, the ice bath was removed, and the reaction was slowly heated to 45 ° C and refluxed for 12 h. After the reaction was completed, diatomaceous earth was filtered, the reaction system solvent was dried using a rotary evaporator, the sample was dry mixed, and the sample was separated by column chromatography, and the ratio of petroleum ether to ethyl acetate was 2: 1. 1.94 g of yellow solid intermediate was obtained with a yield of 42%.

[0042] The obtained intermediate (1.94 g, 3.78 mmol) was placed in a round-bottom flask, dissolved in tetrahydrofuran (100 mL), TBAF (2.39 g, 7.56 mmol) was added to the reaction solution, stirred at 25°C for 1 h, and monitored by TLC. After the reaction was completed, the solvent of the reaction system was dried using a rotary evaporator, the sample was dry-mixed, and the sample was separated by column chromatography. The ratio of petroleum ether to ethyl acetate in the mobile phase was 1:1. 1.50 g of yellow solid I-6 was obtained, and the yield was 90%.

[0043] Compound I-6 (1.5 g, 3.4 mmol), 1-(3-azidopropyl)piperidin-4-ol (0.94 g, 5.1 mmol) and thiophene-2-carboxylic acid ketone (Ⅰ) were placed in a 100 mL three-necked flask, and tetrahydrofuran (30 mL) was added thereto. The nitrogen was replaced three times, and the reaction was stirred at room temperature for 3 h under nitrogen protection. After the reaction was completed, the reaction solution was dried, the sample was dry-mixed, and the neutral alumina column was used for separation. The ratio of dichloromethane and anhydrous ethanol as the mobile phase was 50:1. 1.32 g of yellow solid CC-P-1 was obtained, and the yield was 62%.

[0044] The present invention also proposes a method for synthesizing the small molecule drug conjugate, which comprises preparing the small molecule drug conjugate through Claisen ester condensation reaction, amide condensation, etc. The synthesis method is that CC-P-1 and Linker are first subjected to an ester condensation reaction to obtain a key intermediate, and the designed alkane chain can be directly purchased as a Linker from the market. Under the condition of EDCI as a condensation agent, CC-P-1 reacts with N-Boc-glycine through hydroxyl and carboxyl groups to obtain compound CC-P-2, and then the Boc protecting group is removed in a dichloromethane solution of trifluoroacetic acid to obtain compound CC-P-4. CC-P-4 and compound 17 are subjected to a final amide condensation reaction using a combination of N,N,N',N'-tetramethylchloroformamide hexafluorophosphate (TCFH) and N-methylimidazole (NMI) to obtain compound CC-1.

[0045] The method comprises the following steps:

[0046]

[0047] Reagents and reaction conditions were: (a) DMAP, DIPEA, EDCI, DCM, rt, 12 h; (b) TFA, DCM, rt, 2 h; (c) TCFH, NMI, DMF, rt, 12 h.

[0048] The method comprises the following steps: dissolving N-Boc-glycine (126.13 mg, 0.72 mmol) in anhydrous dichloromethane, adding DMAP (117.28 mg, 0.96 mmol) and DIPEA (216.72 mg, 1.68 mmol) thereto, and stirring for 20 min under ice bath conditions. After the internal part is cooled to 0°C, EDCI (184.03 mg, 0.96 mmol) is added thereto, stirring at 0°C for 5 min and then stirring at room temperature for 20 min, and CC-P-1 (300.00 mg, 0.48 mmol) is added thereto and stirred at room temperature for overnight reaction. After the reaction is completed, the reaction system solvent is dried by a rotary evaporator, wet sample is loaded, and the sample is separated by column chromatography, and the ratio of mobile phase dichloromethane to anhydrous methanol is 50:1. The target product CC-P-2256.75 mg is obtained as a yellow solid, with a yield of 68%. CC-P-2 (126.13 mg, 0.72 mmol) was dissolved in anhydrous dichloromethane, and a dichloromethane solution of TFA (0.5 mL) was added thereto, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was dried to obtain a brown-yellow oil CC-P-4, which was directly used in the next step without treatment. Compound CC-P-4 (69.97 mg, 0.10 mmol), 17 (50.00 mg, 0.114 mmol), TCFH (47.90 mg, 0.17 mmol), and NMI (32.70 mg, 0.40 mmol) were added to anhydrous DMF (5 mL) and stirred at room temperature overnight. After the reaction was completed, the reaction solution was dried, washed three times with DCM, washed three times with a saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, concentrated the organic phase, loaded by dry method, and purified by column chromatography. The ratio of mobile phase dichloromethane to anhydrous methanol was 20:1, and one thousandth of triethylamine was added. 1.21 mg of yellow solid CC-15 was obtained with a yield of 41%.

[0049] The beneficial effects of the present invention include: the present invention innovatively proposes a class of small molecule drug conjugates for the first time, which are small molecule drug conjugates with PI3K inhibitors as active drugs, extracellular HSP90 as targeting ligands, and alkane chains, PEG chains and nitrogen-containing heterocycles as linkers. Compared with the treatment of tumors and colorectal cancer with a single PI3K inhibitor, it has the effects of strong targeting, obvious inhibitory effect, low toxicity, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0051] Figure 1 HPLC monitoring results of compound CC-11 plasma stability test, ***p<0.001, nsp>0.05. The left figure is the HPLC test peak area of ​​compound CC-11, and the right figure is the residual percentage of compound CC-11 cleavage.

[0052] Figure 2 Schematic diagram of the hydrolysis products of compound CC-11.

[0053] Figure 3 This is the H-NMR chart of the compound CC-M-1 (PI3K inhibitor) of the present invention.

[0054] Figure 4 This is the C-NMR chart of the compound CC-M-1 (PI3K inhibitor) of the present invention.

[0055] Figure 5 This is the H-NMR chart of the compound CC-P-1 (PI3K inhibitor) of the present invention.

[0056] Figure 6 This is the C-NMR chart of the compound CC-P-1 (PI3K inhibitor) of the present invention.

[0057] Figure 7 This is the H-NMR chart of the compound CC-N-1-1 (PI3K inhibitor) of the present invention.

[0058] Figure 8 This is the C-NMR chart of the compound CC-N-1-1 (PI3K inhibitor) of the present invention.

[0059] Fig. 9 This is the H-NMR chart of the compound CC-1 (small molecule conjugate) of the present invention.

[0060] Fig.10 This is the C-NMR chart of the compound CC-1 (small molecule conjugate) of the present invention.

[0061] Fig.11 This is the H-NMR chart of the compound CC-3 (small molecule conjugate) of the present invention.

[0062] Fig.12 This is the C-NMR chart of the compound CC-3 (small molecule conjugate) of the present invention.

[0063] Fig.13 This is the H-NMR chart of the compound CC-6 (small molecule conjugate) of the present invention.

[0064] Fig.14 This is the C-NMR chart of the compound CC-6 (small molecule conjugate) of the present invention.

[0065] Fig.15 This is the H-NMR chart of the compound CC-7 (small molecule conjugate) of the present invention.

[0066] Fig.16 This is the C-NMR chart of the compound CC-7 (small molecule conjugate) of the present invention.

[0067] Fig.17 This is the H-NMR chart of the compound CC-8 (small molecule conjugate) of the present invention.

[0068] Fig.18 This is the C-NMR chart of the compound CC-8 (small molecule conjugate) of the present invention.

[0069] Fig.19 This is the H-NMR chart of the compound CC-9 (small molecule conjugate) of the present invention.

[0070] Fig. 20 This is the C-NMR chart of the compound CC-9 (small molecule conjugate) of the present invention.

[0071] Fig.21 This is the H-NMR chart of the compound CC-10 (small molecule conjugate) of the present invention.

[0072] Fig. 22 This is the C-NMR chart of the compound CC-10 (small molecule conjugate) of the present invention.

[0073] Fig.23 This is the H-NMR chart of the compound CC-11 (small molecule conjugate) of the present invention.

[0074] Fig.24 This is the C-NMR chart of the compound CC-11 (small molecule conjugate) of the present invention.

[0075] Fig.25 This is the H-NMR chart of the compound CC-12 (small molecule conjugate) of the present invention.

[0076] Fig.26 This is the C-NMR chart of the compound CC-12 (small molecule conjugate) of the present invention.

[0077] Fig. 27 This is the H-NMR chart of the compound CC-13 (small molecule conjugate) of the present invention.

[0078] Fig.28 This is the C-NMR chart of the compound CC-13 (small molecule conjugate) of the present invention.

[0079] Fig.29 This is the H-NMR chart of the compound CC-14 (small molecule conjugate) of the present invention.

[0080] Fig.30 This is the C-NMR chart of the compound CC-14 (small molecule conjugate) of the present invention.

[0081] Fig.31 The graph shows the antitumor activity of compounds CC-M-1 and CC-11 in the HCT116 xenograft tumor model. DETAILED DESCRIPTION

[0082] The present invention is further described in detail in conjunction with the following specific examples and drawings, and the protection content of the present invention includes but is not limited to the following examples. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention has no special restrictions.

[0083] Preparation method of PI3K inhibitor:

[0084]

[0085] The raw material 3-amino-5-bromo-2-methoxypyridine (10.00g, 49.30mmol) was placed in a 250mL pear-shaped flask, pyridine (40mL) was added to the reaction bottle, and stirred in an ice bath until dissolved, and 2,4-difluorobenzenesulfonyl chloride (15.68g, 73.80mmol) was added thereto after the internal temperature dropped to 0°C, and the ice bath was removed after stirring for 5min under ice bath conditions, and the reaction was placed at room temperature and stirred for 48h. After the reaction was completed, the reaction solution was spin-dried using a rotary evaporator to obtain a brown oil, and dichloromethane (200mL) and water (250mL) were added to extract three times, and 10% citric acid aqueous solution (200mL) was washed three times to remove pyridine in the reaction system, and finally the organic phase was combined and spin-dried to obtain a brown oil. Ethyl acetate (EA) and petroleum ether (PE) were added for pulping, and 17.64g of brown solid I-1 was finally obtained by suction filtration, with a yield of 94.7%.

[0086] Compound I-1 was placed in a 500mL three-necked flask, 1,4-dioxane (250mL) was added thereto and stirred to dissolve, and then diboric acid pinacol ester (11.08g, 43.66mmol), tricyclohexylphosphine (2.23g, 7.94mmol), Pd2(dba)3 (3.63g, 3.96mmol), potassium acetate (11.68g, 119.04mmol) were added to the reaction system, nitrogen was replaced three times, and the reaction was stirred at 110°C under nitrogen protection for 4h. After the reaction was completed, diatomaceous earth was filtered, the reaction solution was spin-dried, dichloromethane (200mL) and water (250mL) were added to extract three times, and ether and petroleum ether (PE) were added to the concentrated organic phase for pulping, and 16.82g of white compound I-2 was obtained by suction filtration, with a yield of 99%.

[0087]

[0088] Compound 3-amino-6-chloropyridazine (8.58 g, 66.25 mmol), chloroacetaldehyde (10.40 g, 132.50 mmol) and sodium bicarbonate (8.35 g, 99.38 mmol) were placed in a 500 mL pear-shaped flask, 95% ethanol (200 mL) was used as a solvent, and then the reaction was placed in a 90°C oil bath and refluxed for 16 h. After the reaction was completed, the solvent in the reaction system was dried using a rotary evaporator, the sample was dry mixed, and the sample was separated by column chromatography. The ratio of petroleum ether to ethyl acetate in the mobile phase was 2:1, and 7.30 g of yellow solid I-3 was obtained, with a yield of 73%.

[0089] Compound I-3 (4.50 g, 47.70 mmol) was placed in a 500 mL three-necked flask, N, N-dimethylformamide (250 mL), H2O (30 mL) were added thereto and stirred to dissolve, then I-2 (15.67 g, 59.60 mmol), Pd(PPh3)4 (3.40 g, 2.94 mmol), potassium carbonate (6.10 g, 44.12 mmol) were added to the reaction system, nitrogen was replaced three times, and the reaction was stirred at 120 ° C for 4 h under nitrogen protection. After the reaction, diatomaceous earth was filtered, the reaction solution was spin-dried, dichloromethane and water were added for extraction three times, the organic phase was concentrated and dried, and the sample was mixed by dry method, and a silica gel column was used for column chromatography separation, and the mobile phase used a dichloromethane and methanol system (DCM: MeOH = 50: 1). 5.50 g of white solid I-4 was obtained, with a yield of 45%.

[0090] Compound I-4 (5.00 g, 11.99 mmol) and NIS (4.05 g, 17.98 mmol) were added to N,N-dimethylformamide (150 mL), and nitrogen was replaced three times. The mixture was stirred at room temperature for 12 h under nitrogen protection. After the reaction was completed, the reaction solution was slowly added to water to precipitate a yellow solid, which was filtered and vacuum dried to obtain 6.02 g of compound I-5 with a yield of 92%.

[0091] Compound I-5 (5.00 g, 9.20 mmol) was dissolved in tetrahydrofuran (200 mL) and placed in an ice bath until the temperature dropped to 0 ° C. Cuprous iodide (0.18 mg, 0.92 mmol) and Pd (PPh3) 2Cl2 (0.65 g, 0.92 mmol) were added thereto, nitrogen was replaced three times, and stirred for 0.5 h under nitrogen protection, and then triethylamine (2.79 g, 27.60 mmol) and trimethylethynylsilane (2.71 g, 27.60 mmol) were added by injection, the ice bath was removed, and the reaction was slowly heated to 45 ° C and refluxed for 12 h. After the reaction was completed, diatomaceous earth was filtered, the reaction system solvent was dried using a rotary evaporator, the sample was dry mixed, and the sample was separated by column chromatography, and the ratio of petroleum ether to ethyl acetate was 2: 1. 1.94 g of yellow solid intermediate was obtained with a yield of 42%.

[0092] The obtained intermediate (1.94 g, 3.78 mmol) was placed in a round-bottom flask, dissolved in tetrahydrofuran (100 mL), TBAF (2.39 g, 7.56 mmol) was added to the reaction solution, stirred at 25°C for 1 h, and monitored by TLC. After the reaction was completed, the solvent of the reaction system was dried using a rotary evaporator, the sample was dry-mixed, and the sample was separated by column chromatography. The ratio of petroleum ether to ethyl acetate in the mobile phase was 1:1. 1.50 g of yellow solid I-6 was obtained, and the yield was 90%.

[0093] Compound I-6 (1.5 g, 3.4 mmol), 1-(3-azidopropyl)piperidin-3-ol (0.94 g, 5.1 mmol) and thiophene-2-carboxylic acid ketone (I) were placed in a 100 mL three-necked flask, and tetrahydrofuran (30 mL) was added thereto. The nitrogen was replaced three times, and the reaction was stirred at room temperature for 3 h under nitrogen protection. After the reaction was completed, the reaction solution was spin-dried, the sample was dry-mixed, and the neutral alumina column was used for separation. The ratio of dichloromethane and anhydrous ethanol as the mobile phase was 50:1. 1.32 g of yellow solid CC-M-1 was obtained, and the yield was 62%.

[0094]

[0095] 1H NMR (400MHz, DMSO) δ8.86 (s, 1H), 8.62 (s, 1H), 8.54 (s, 1H), 8.31 (t, J = 4.0Hz, 2H) ,7.89–7.77(m,2H),7.44(t,J=10.1Hz,1H),7.13(t,J=10.1Hz,1H),4.81(s,1H), 4.60(t,J=6.9Hz,2H),3.80(s,3H),3.58(s,1H),2.94–2.91(m,1H),2.76(s,1H), 2.58(s,2H),2.19–2.09(m,4H),1.67(t,J=16.8Hz,2H),1.43(s,1H),1.18(s,1H).

[0096] 13 C NMR (101MHz, DMSO) δ164.8 (dd, J=252.5, 11.1Hz), 159.8 (dd, J=256.5, 12.1Hz), 159.0,149.9,139.9,139.0,136.4,132.0(d,J=4.0Hz),131.9,129.2,127.4,126 .7,125.5(dd,J=14.1,4.0Hz),125.0,122.1,121.5,115.7,112.0(dd,J=21.2,4. 0Hz), 106.0 (t, J = 27.3Hz), 65.3, 60.3, 54.7, 54.0, 53.0, 48.2, 32.4, 26.8, 22.1.

[0097] HRMS (ESI) m / z calcd.for C 28 H 30 F2N9O4S(M+H) + 626.2110, found 626.2096.

[0098] Purity (HPLC): 95.64%

[0099] 1-(3-azidopropyl)piperidin-4-ol was used to replace 1-(3-azidopropyl)piperidin-3-ol in the synthesis of CC-M-1. The synthesis steps were the same as those of CC-M-1 to obtain compound CC-P-1.

[0100]

[0101] 1H NMR (400MHz, DMSO) δ8.87 (s, 1H), 8.57 (dd, J = 21.8, 2.3Hz, 2H), 8.31 (t, J = 4.0Hz, 2H), 7 .88(d,J=9.7Hz,1H),7.83–7.77(m,1H),7.44(td,J=9.5,2.6Hz,1H),7.13(td,J=8.5,2 .7Hz,1H),4.62(t,J=6.9Hz,2H),3.80(s,3H),3.57(s,1H),3.01–2.98(m,2H),2.75(t, J=7.5Hz,2H),2.57(s,2H),2.25(p,J=7.3Hz,2H),1.79–1.74(m,2H),1.52–1.44(m,2H).

[0102] 13 C NMR (101MHz, DMSO) δ164.8 (dd, J=253.5, 11.1Hz), 159.7 (dd, J=256.5, 13.1Hz) ),159.0,149.9,139.8,139.0,136.5,132.0(d,J=7.0Hz),131.9,129.2,127. 4,126.7,125.6(dd,J=12.1,5.1Hz),125.0,122.0,121.5,115.7,112.0(dd,J =22.2, 3.0Hz), 106.1 (t, J = 26.3Hz), 64.3, 54.1, 54.0, 50.3, 48.1, 32.8, 26.5.

[0103] HRMS (ESI) m / z calcd.for C 28 H 30 F2N9O4S(M+H) + 626.2110, found 626.2096.

[0104] Purity(HPLC):98.90%

[0105] 4-(3-azidopropyl)piperazine-1-carboxylic acid tert-butyl ester is used to replace 1-(3-azidopropyl)piperidin-3-ol in the synthesis of CC-M-1. The synthesis steps are the same as CC-M-1 to obtain compound CC-N-1-1.

[0106]

[0107] 1H NMR(400MHz, CDCl3)δ8.68(d,J=2.2Hz,1H),8.63(s,1H),8.45(s,1H),8.38(d, J=2.2Hz,1H),8.06(d,J=9.4Hz,1H),7.77–7.71(m,1H),7.45(d,J=9.4Hz,1H), 6.90–6.82(m,2H),4.58(t,J=7.0Hz,2H),3.95(s,3H),3.31(t,J=5.0Hz,4H),2 .37(t,J=6.9Hz,2H),2.29(t,J=5.0Hz,4H),2.16(p,J=7.0Hz,2H),1.37(s,9H).

[0108] 13 C NMR (101MHz, CDCl3) δ166.3 (dd, J=260.6, 12.1Hz), 159.9 (dd, J=260.6, 12.1H z),155.6,154.7,148.5,140.4,137.0,133.1,132.1(d,J=10.1Hz),126.4,125 .7,125.5,123.3(dd,J=13.1,3.0Hz),121.3,121.1,113.6,112.2(dd,J=22.2 ,3.0Hz),106.0(t,J=26.4Hz),79.6,55.0,54.6,52.9,48.7,28.4,27.5,26.9.

[0109] HRMS (ESI) m / z calcd.for C 32 H 40 F2N 10 O5S(M+H) + 711.2637, found 711.2606.

[0110] Purity (HPLC): 96.77%

[0111]

[0112] Compound CC-N-1-1 (116 mg, 0.15 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (0.5 mL) was slowly added dropwise, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was dried to obtain a crude product CC-N-1, which was used directly in the next step without purification.

[0113] Example 1

[0114]

[0115] 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamide)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-3-yl(5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxylate)glycine CC-6

[0116] Specific synthesis steps of CC-6:

[0117] Dissolve N-Boc-glycine (126.13 mg, 0.72 mmol) in anhydrous dichloromethane, add DMAP (117.28 mg, 0.96 mmol) and DIPEA (216.72 mg, 1.68 mmol) and stir for 20 min in an ice bath. After the internal temperature is cooled to 0°C, add EDCI (184.03 mg, 0.96 mmol) and stir at 0°C for 5 min and then at room temperature for 20 min. Add CC-M-1 (300.00 mg, 0.48 mmol) and stir at room temperature overnight. After the reaction is completed, use a rotary evaporator to dry the reaction system solvent, wet sample loading, column chromatography to separate the sample, and the ratio of mobile phase dichloromethane to anhydrous methanol is 50:1.

[0118] The target product CC-M-2 was obtained as a yellow solid, 256.75 mg, with a yield of 68%.

[0119]

[0120] 1 H NMR (400MHz, CDCl3) δ8.74–8.68(m,2H),8.51–8.45(m,2H),8.12(d,J=9.5Hz,1H),7.86–7. 79(m,1H),7.51(d,J=9.5Hz,1H),6.94–6.89(m,2H),5.14(s,1H),4.89–4.87(m,1H),4.68– 4.56(m,2H),4.01(s,3H),3.90(d,J=1.0Hz,2H),2.77–2.74(m,1H),2.52(s,1H),2.44(t,J =7.0Hz,2H),2.28–2.20(m,4H),1.82(s,1H),1.71(s,1H),1.55–1.43(m,2H),1.43(s,9H).

[0121] 13C NMR(101MHz, CDCl3) δ169.8,166.2(dd,J=259.4,11.6Hz),159.9(dd,J=259.2,13.1Hz),1 55.9,155.8,148.5,140.5,138.8,136.9,133.1,132.2(d,J=11.0Hz),126.3,126.1,125. 6,123.4(dd,J=13.7,3.8Hz),121.9,121.1,121.1,113.6,112.2(dd,J=22.1,3.5Hz),105 .9(t,J=25.5Hz),79.9,70.7,56.8,54.8,54.5,53.2,42.6,29.4,28.3,27.7,26.9,22.6.

[0122] CC-M-2 (126.13 mg, 0.72 mmol) was dissolved in anhydrous dichloromethane, and a dichloromethane solution of TFA (0.5 mL) was added thereto, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was dried to obtain a brown-yellow oil CC-M-4, which was used directly in the next step without treatment.

[0123] Compound CC-M-4 (69.97 mg, 0.10 mmol), HSP90 inhibitor 17 (50.00 mg, 0.114 mmol), TCFH (47.90 mg, 0.17 mmol), NMI (32.70 mg, 0.40 mmol) were added to anhydrous DMF (5 mL) and stirred at room temperature overnight. After the reaction was completed, the reaction solution was spin-dried, washed three times with DCM, washed three times with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, concentrated the organic phase, dry-loaded, and purified by column chromatography. The ratio of dichloromethane to anhydrous methanol was 20:1, and 1 / 1000 triethylamine was added. Yellow solid CC-651.21 mg was obtained with a yield of 41%.

[0124]

[0125] 1H NMR(400MHz,DMSO)δ9.80(s,1H),9.70(s,1H),9.19(t,J=4.7Hz,1H),8.81(d,J=1.7Hz,1H),8.78(s,1H),8.54(s,1H),8.34–8.31(m,2H),7.88(d,J=9.7Hz,1H),7.84–7.78(m,1H),7.53(t,J=9.8Hz,1H),7.21–7.17(m,5H),6.73(s,1H),6.47(s,1H),4.74(s,1H),4.57(t,J=7.0Hz,2H),3.98(s,2H),3.78(s,3H),3.54(s,4H),3.42(s,2H),3.01–2.94(m,1H),2.82–2.79(m,1H),2.57(s,1H),2.41(t,J=6.4Hz,2H),2.33(s,4H),2.18–2.10(m,4H),1.76(s,1H),1.62(s,1H),1.47–1.42(m,1H),1.31–1.26(m,1H),0.90(d,J=5.4Hz,6H).

[0126] 13 C NMR(101MHz,DMSO)δ169.0,167.4,165.4(dd,J=254.5,12.1Hz),159.8(dd,J=257.5,13.1Hz),160.8,158.9,158.0,157.1,155.3,149.5,142.6,139.0,137.0,136.2,132.3(d,J=5.0Hz),132.0,129.6,129.1,128.8,128.3,126.8,126.1,125.7(dd,J=13.1,4.0Hz),125.3,122.1,121.9,121.6,115.7,115.6,112.4(dd,J=25.2,3.0Hz),106.3(t,J=26.3Hz),104.9,103.2,70.3,66.5,62.5,56.7,54.8,54.2,53.5,52.8,48.4,29.3,27.4,26.8,25.8,22.8,22.5.

[0127] HRMS(ESI)m / z calcd.for C 54 H 57 F2N 12 O 10 S(M+H) +1103.4009, found 1103.4041.

[0128] Purity (HPLC): 98.16%

[0129] Example 2

[0130] Synthesis of 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonylamino)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-3-yl 3-(5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamido)propanoate CC-7.

[0131]

[0132] First, Boc-β-alanine was used to replace N-Boc-glycine in Example 1, and the synthesis steps were the same as CC-M-2 to obtain compound CC-M-3.

[0133]

[0134] The structural confirmation data are as follows: 1 H NMR(400MHz, CDCl3)δ8.72(d,J=13.8Hz,2H),8.50–8.45(m,2H),8.11(d,J=9 .4Hz,1H),7.86–7.81(m,1H),7.51(d,J=9.5Hz,1H),6.97–6.91F(m,2H),5.29 (s,1H),4.90(s,1H),4.64(t,J=7.3Hz,2H),4.00(s,3H),3.37(q,J=7.3Hz,1H ),2.58–2.28(m,10H),1.81(d,J=9.0Hz,2H),1.70–1.54(m,2H),1.41(s,9H).

[0135] 13C NMR(101MHz,CDCl3)δ171.6,165.7(dd,J=259.6,10.1Hz),159.9(dd,J=259.6,13.1Hz),15 5.9,155.9,148.5,140.6,138.8,137.0,133.0,132.2(d,J=10.1Hz),126.5,126.3,125.4, 123.5(dd,J=13.1,4.0Hz),121.7,121.3,121.1,113.7,112.2(dd,J=23.2,2.0Hz),105.9( t, J=25.3Hz),79.3,68.7,56.1,55.0,54.5,53.0,48.4,36.2,34.9,31.6,28.4,26.9,22.7.

[0136] CC-M-3 was used to replace CC-M-2 in Example 1. The synthesis steps of CC-7 were the same as CC-6.

[0137] The structural confirmation data are as follows: 1 H NMR (400MHz, DMSO) δ9.85 (s, 1H), 9.72 (s, 1H), 8.92 (t, J = 6.7Hz, 1H), 8.85–8.80 (m, 2H), 8.57–8.53 (m, 1H), 8.37–8.31 (m,2H),7.91(q,J=7.4Hz,1H),7.82(q,J=7.4Hz,1H),7.58–7.52(m,1H),7.29–7.18(m,5H),6.74–6.71(m,1H),6.52–6 .48(m,1H),4.78(s,1H),4.60(t,J=6.9Hz,2H),3.77(s,3H),3.60–3.43(m,10H),3.01–2.94(m,3H),2.71(s,1H),2.58 (s,1H),2.42(s,6H),2.21–2.18(m,2H),1.72(d,J=31.2Hz,2H),1.50(s,1H),1.41–1.38(m,1H),0.90(d,J=5.4Hz,6H).

[0138] 13C NMR (101MHz, DMSO) δ170.7,167.1,165.5(dd,J=256.5,12.1Hz),159.8(dd,J=258.5,12.1Hz),160.5,158.9,158 .0,157.8,155.2,149.5,143.0,139.0,136.3,132.6,132.3(d,J=6.0Hz),129.5,129.2,128.2,126.9,126.1,125 .5(dd,J=15.1,3.0Hz),125.3,122.1,121.6,121.3,115.8,115.2,112.4(dd,J=24.2,4.0Hz),106.3(t,J=26.3H z),104.8,103.2,68.8,66.2,62.1,55.4,54.7,54.3,53.3,52.5,48.3,45.9,35.6,34.1,26.8,25.8,22.8,21.8.

[0139] HRMS (ESI) m / z calcd.for C 55 H 59 F2N 12 O 10 S(M+H) + 1117.4166, found 1117.4136.

[0140] Purity (HPLC): 95.29%

[0141] Example 3

[0142] Synthesis of 1-(3-(4-(6-(5-(((2,4-difluorophenyl)sulfonyl)methyl)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-3-yl 2-(2-(5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide)ethoxy)acetate CC-8.

[0143]

[0144] First, 2-(2-((tert-butyloxycarbonyl)amino)ethoxy)acetic acid was used to replace N-Boc-glycine in Example 1, and the synthesis steps were the same as CC-M-2 to obtain compound CC-M-6.

[0145]

[0146] The structural confirmation data are as follows:1 H NMR (400MHz, CDCl3) δ8.74–8.67(m,2H),8.50–8.43(m,2H),8.11(t,J=8.3Hz,1H),7.86–7 .80(m,1H),7.53–7.48(m,1H),6.98–6.89(m,2H),5.24(s,1H),4.93(s,1H),4.64–4.59(m, 2H),4.07(s,2H),4.01(s,3H),3.61–3.57(m,2H),3.33–3.31(m,2H),2.78(s,1H),2.57(s ,1H),2.49(s,2H),2.35–2.22(m,4H),1.83(s,1H),1.73(s,1H),1.57(s,2H),1.43(s,9H).

[0147] 13 C NMR(101MHz, CDCl3) δ169.9,166.2(dd,J=258.6,11.7Hz),159.9(dd,J=259.3,12.8Hz),156.0 ,155.8,148.5,140.5,138.8,136.9,136.9,132.9,132.2(d,J=10.6Hz),126.3,126.1,125.5, 123.5(dd,J=13.1,4.0Hz),121.8,121.2,121.1,113.7,112.2(dd,J=24.2,3.0Hz),105.9(t,J =25.3Hz),79.2,70.8,70.1,68.3,60.4,56.7,54.9,54.5,48.6,40.4,29.2,28.4,27.5,22.4.

[0148] CC-M-6 was used to replace CC-M-2 in Example 1. The synthesis steps of CC-8 were the same as those of CC-6.

[0149] The structural confirmation data are as follows: 1H NMR(400MHz,DMSO)δ9.78(s,1H),9.67(s,1H),8.81(s,3H),8.55(s,1H),8.35(s,1H),8.32(s,1H),7.91(dd,J=9.6,3.0Hz,1H),7.80(q,J=8.0Hz,1H),7.54(t,J=8.8Hz,1H),7.24–7.18(m,5H),6.72(s,1H),6.46(s,1H),4.77(s,1H),4.57(s,1H),4.08(s,2H),3.77(s,2H),3.55(s,6H),3.45–3.36(m,6H),3.00–2.93(m,1H),2.82(s,1H),2.58(s,1H),2.44–2.36(m,6H),2.14–2.11(m,2H),1.76(s,1H),1.62(s,1H),1.46(s,1H),1.34(s,1H),0.89(d,J=6.8Hz,6H).

[0150] 13 C NMR(101MHz,DMSO)δ170.0,166.9,165.4(dd,J=254.5,11.1Hz),159.8(dd,J=257.5,13.1Hz),160.6,158.8,157.9,157.9,155.2,149.5,142.4,139.0,137.0,136.2,132.3(d,J=8.1Hz),131.9,129.4,129.2,128.9,128.2,126.8,126.1,125.8(dd,J=15.1,4.0Hz),125.3,122.1,121.7,121.6,115.7,115.2,112.4(dd,J=23.2,4.0Hz),106.3(t,J=25.3Hz),104.9,103.1,69.9,69.3,67.9,66.5,62.5,56.7,54.8,54.2,53.5,52.8,48.4,46.1,29.4,27.3,25.8,22.8,22.6.

[0151] HRMS(ESI)m / z calcd.for C 56 H 61 F2N 12 O 11 S(M+H) + 1147.4272,found 1147.4294.

[0152] Purity (HPLC): 98.01%

[0153] Example 4

[0154] Synthesis of 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonylamino)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-3-yl 2-(2-(2-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide)ethoxy)acetate CC-9.

[0155]

[0156] First, 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatridecane-13-oic acid was used to replace N-Boc-glycine in Example 1, and the synthesis steps were the same as CC-M-2 to obtain compound CC-M-7.

[0157]

[0158] The structural confirmation data are as follows: 1 H NMR (400MHz, CDCl3) δ8.68–8.62(m,2H),8.45–8.38(m,2H),8.05(dd,J=9.5,4.4Hz,1H),7.79–7.75(m,1H),7.44(dd, J=9.5,4.4Hz,1H),6.91–6.83(m,2H),5.03(s,1H),4.86–4.83(m,1H),4.55(t,J=2,0Hz,2H),4.04(d,J=4.4Hz,2H),3 .95(d,J=4.4Hz,3H),3.63–3.61(m,2H),3.58–3.56(m,2H),3.47(q,J=4.8Hz,2H),3.26–3.23(m,2H),2.70(d,J=11.7 Hz,1H),2.47(s,1H),2.40–2.36(m,2H),2.17–2.11(m,4H),1.77(s,1H),1.64(s,1H),1.52–1.43(m,2H),1.36(s,9H).

[0159] 13C NMR(101MHz, CDCl3)δ169.8,166.3(dd,J=257.6,10.1Hz),159.9(dd,J=257.6,14.1Hz),156.0, 155.7,148.5,140.5,138.8,136.9,133.0,132.2(d,J=10.1Hz),126.3,126.0,125.5,123.4(dd ,J=14.1,3.0Hz),121.9,121.2,121.1,113.7,112.2(dd,J=25.2,3.0Hz),105.9(t,J=26.3Hz), 79.2,70.8,70.3,70.2,68.6,56.9,54.9,54.5,53.2,48.7,40.4,29.7,29.4,28.4,27.7,22.6.

[0160] CC-M-7 was used to replace CC-M-2 in Example 1. The synthesis steps of CC-9 were the same as those of CC-6.

[0161] The structural confirmation data are as follows: 1 H NMR (400MHz, DMSO) δ9.78(s,1H),9.68(s,1H),8.85–8.78(m,3H),8.55(s,1H),8.34(s,1H),8.32(s,1H),7.89(d,J=9.7H z,1H),7.81(q,J=8.0Hz,1H),7.54(t,J=9.8Hz,1H),7.25–7.17(m,5H),6.73(s,1H),6.47(s,1H),4.74(s,1H),4.57(t,J =7.5Hz,2H),4.08(s,2H),3.78(s,3H),3.56–3.35(m,12H),3.01–2.94(m,1H),2.80(d,J=10.3Hz,2H),2.58(s,2H),2.43 –2.35(m,6H),2.19–2.10(m,4H),1.75(s,1H),1.60(s,1H),1.47–1.42(m,1H),1.32–1.26(m,1H),0.90(d,J=7.1Hz,6H).

[0162] 13C NMR (101MHz, DMSO) δ170.0,166.9,165.4(dd,J=255.5,12.1Hz),159.8(dd,J=259.5,12.1Hz),160.6,158.9,158.0,157.9,1 55.2,149.5,142.6,139.0,137.0,136.2,132.3(d,J=6.1Hz),132.1,129.4,129.2,129.0,128.2,126.9,126.1,125.7(dd,J =13.1,3.0Hz),125.3,122.1,121.7,121.6,115.7,115.2,112.4(dd,J=22.2,2.0Hz),106.3(t,J=26.3Hz),104.9,103.2,70 .4,69.9,68.9,68.2,66.5,62.5,56.7,54.8,54.2,53.5,52.8,48.4,46.1,29.3,27.3,26.8,25.8,22.8,22.5.HRMS(ESI)m / z calcd.for C 58 H 65 F2N 12 O 12 S(M+H) + 1191.4534, found 1191.4526.

[0163] Purity (HPLC): 97.46%

[0164] Example 5

[0165] Synthesis of 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonylamino)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-3-yl 1-(5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)isoxazol-3-yl)-1-oxo-5,8,11-trioxo-2-azetidine-13-carboxylate CC-10.

[0166]

[0167] First, 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxo-5-azahexadecane-16-oic acid was used to replace N-Boc-glycine in Example 1, and the synthesis steps were the same as CC-M-2 to obtain compound CC-M-8.

[0168]

[0169] The structural confirmation data are as follows: 1 H NMR(400MHz, CDCl3)δ8.73(s,1H),8.71(s,1H),8.47–8.46(m,2H),8.12(d,J=9.5Hz,1H),7 .88–7.82(m,1H),7.53(d,J=9.5Hz,1H),6.99–6.92(m,2H),5.16(s,1H),4.97(s,1H),4.64( t,J=7.0Hz,2H),4.15(s,2H),4.00(s,3H),3.73–3.60(m,8H),3.55–3.52(m,2H),3.31(q,J =6.0Hz,2H),2.66(s,2H),2.60(s,2H),2.31(s,4H),1.85(s,2H),1.61(s,2H),1.43(s,9H).

[0170] 13 C NMR (101MHz, CDCl3) δ 169.8, 166.1 (dd, J = 259.6, 11.1Hz), 159.8 (dd, J = 260.6, 13.1Hz), 156.0, 148. 6,140.6,138.8,136.9,132.9,132.8,132.2(d,J=10.1Hz),126.5,126.3,125.4,123.5(dd,J=14.1, 3.0Hz),121.8,121.2,121.1,121.1,113.8,112.2(dd,J=22.2,4.0Hz),105.9(t,J=26.3Hz),79.1,7 0.8,70.5,70.4,70.2,70.2,68.6,54.9,54.4,53.0,48.5,45.9,40.3,29.0,28.4,26.9,22.6,22.6.

[0171] CC-M-8 was used to replace CC-M-2 in Example 1. The synthesis steps of CC-10 were the same as those of CC-6.

[0172] The structural confirmation data are as follows: 1H NMR(400MHz,DMSO)δ9.82(s,1H),9.70(s,1H),8.82(s,3H),8.55(s,1H),8.35(s,1H),8.33(s,1H),7.91(d,J=9.4Hz,1H),7.81(q,J=8.0Hz,1H),7.56(t,J=9.8Hz,1H),7.28–7.18(m,5H),6.73(s,1H),6.48(s,1H),4.81(s,1H),4.59(t,J=7.1Hz,2H),4.09(s,2H),3.77(s,3H),3.59–3.35(m,18H),3.01–2.89(m,3H),2.68(s,2H),2.43(s,6H),2.19–2.16(m,2H),1.76(s,1H),1.66(s,1H),1.47(s,1H),1.39(s,1H),0.90(d,J=6.8Hz,6H).

[0173] 13 C NMR(101MHz,DMSO)δ169.9,166.9,165.5(dd,J=255.5,11.1Hz),159.8(dd,J=258.5,13.1Hz),160.6,158.9,158.0,155.2,149.5,143.2,139.0,136.3,132.7,132.4(d,J=8.1Hz),129.5,129.4,129.3,128.2,126.9,126.1,125.5(dd,J=14.1,4.0Hz),125.3,122.1,121.6,121.1,115.8,115.1,112.4(dd,J=22.2,4.0Hz),106.3(t,J=26.3Hz),104.9,103.2,70.4,70.1,70.0,69.1,68.9,68.1,66.2,62.1,56.1,54.6,54.3,53.2,52.5,48.3,46.0,28.8,26.8,25.9,22.8,22.5,21.9.HRMS(ESI)m / z calcd.forC 60 H 69 F2N 12 O 13 S(M+H) + 1235.4796,found 1235.4768.

[0174] Purity(HPLC):98.29%

[0175] Example 6

[0176]

[0177] Synthesis of 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-3-yl(4-((1-(2,4-dihydroxy-5-isopropylbenzoyl)indolin-5-yl)amino)-4-oxobutyryl)glycine CC-11.

[0178] HSP90 inhibitor 7 was used to replace HSP90 inhibitor 17 in Example 1. The synthesis steps of CC-11 were the same as those of CC-6.

[0179] The structural confirmation data are as follows: 1 H NMR(400MHz,DMSO)δ9.91(s,1H),9.77(s,1H),9.65(s,1H),8.86(s,1H),8.59(s,1H),8.54(s,1H),8.33–8.2 7(m,3H),7.87–7.80(m,2H),7.57(s,1H),7.42(t,J=9.9Hz,1H),7.26(s,1H),7.13(t,J=8.4Hz,1H),6.99(s,1 H),6.44(s,1H),4.67(s,1H),4.58(s,2H),3.97(s,2H),3.80(s,3H),3.12–3.03(m,7H),2.74(d,J=11.5Hz,1H ),2.37–2.34(m,4H),2.11–2.01(m,5H),1.73(s,1H),1.59(s,1H),1.46–1.37(m,2H),1.15(d,J=25.7Hz,6H).

[0180] 13C NMR (101MHz, DMSO) δ172.3,170.4,169.8,167.7,165.5(dd,J=255.5,11.1Hz),159.8(dd,J=258.6,14.1Hz),158 .9,157.1,153.5,149.5,143.0,139.0,138.7,136.3,135.6,133.4,132.3(d,J=4.0Hz),126.9,126.0,125.5(dd ,J=14.13.0Hz),125.3,122.1,121.6,121.3,117.7,116.4,115.8,115.7,112.4(dd,J=22.2,3.0Hz),106.3(t,J =26.3Hz),102.9,69.8,56.5,54.7,54.3,52.7,49.3,48.4,41.3,31.9,30.5,28.0,27.2,26.8,26.3,23.1,22.5.

[0181] HRMS (ESI) m / z calcd.for C 52 H 55 F2N 12 O 10 S(M+H) + 1077.3853, found 1077.3875.

[0182] Purity (HPLC): 98.64%

[0183] Example 7

[0184] Synthesis of 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamide)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-3-yl 3-(4-((1-(2,4-dihydroxy-5-isopropylbenzoyl)indolin-5-yl)amino)-4-oxobutanamide)propanoate CC-12.

[0185]

[0186] HSP90 inhibitor 7 was used to replace HSP90 inhibitor 17 in Example 1. The synthesis steps of CC-12 were the same as CC-7.

[0187] The structural confirmation data are as follows: 1H NMR(400MHz,DMSO)δ9.87(s,1H),9.76(s,1H),9.63(s,1H),8.84(s,1H),8.63(s,1H),8.54(s,1H),8.33–8.29(m,2H),7.95(s,1H),7.87–7.79(m,2H),7.55(s,1H),7.44(t,J=9.7Hz,1H),7.26(s,1H),7.14(t,J=8.4Hz,1H),6.98(s,1H),6.41(s,1H),4.66(s,1H),4.57(t,J=7.0Hz,2H),3.95(t,J=8.3Hz,2H),3.79(s,3H),3.26–3.00(m,7H),2.77–2.75(m,1H),2.41–2.37(m,6H),2.11–2.00(m,5H),1.75(s,1H),1.59(s,1H),1.40–1.38(m,2H),1.15(d,J=18.0Hz,6H).

[0188] 13 C NMR(101MHz,DMSO)δ171.8,171.1,170.5,167.7,165.4(dd,J=254.5,12.1Hz),159.8(dd,J=258.6,14.1Hz),158.9,157.1,153.5,149.5,142.8,139.0,138.6,136.3,135.5,133.4,132.3(d,J=5.1Hz),126.9,126.0,125.6(dd,J=14.14.0Hz),125.3,122.1,121.6,117.7,116.3,115.8,115.7,112.4(dd,J=25.2,4.0Hz),106.3(t,J=26.3Hz),102.9,69.3,56.7,54.8,54.3,52.8,49.3,48.4,35.2,34.5,32.0,30.7,29.2,28.0,27.2,26.8,26.3,23.1,22.5.

[0189] HRMS(ESI)m / z calcd.for C 53 H 57 F2N 12 O 10 S(M+H) + 1091.4009,found 1091.4019.

[0190] Purity (HPLC): 96.43%

[0191] Example 8

[0192] Synthesis of 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonylamino)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-3-yl 2-(2-(4-((1-(2,4-dihydroxy-5-isopropylbenzoyl)indolin-5-yl)amino)-4-oxobutanamido)ethoxy)acetate CC-13.

[0193]

[0194] HSP90 inhibitor 7 was used to replace HSP90 inhibitor 17 in Example 1. The synthesis steps of CC-13 were the same as those of CC-8.

[0195] The structural confirmation data are as follows: 1 H NMR(400MHz,DMSO)δ9.88(s,1H),9.75(s,1H),9.62(s,1H),8.81(s,2H), 8.55(s,1H),8.35(s,1H),8.33(s,1H),7.96–7.89(m,2H),7.80(q,J=8.7H z,1H),7.55(t,J=8.4Hz,2H),7.26–7.18(m,2H),6.98(s,1H),6.41(s,1H ),4.76(s,1H),4.58(t,J=7.0Hz,2H),4.07(s,2H),3.95(t,J=8.4Hz,2H), 3.77(s,3H),3.45(t,J=5.9Hz,2H),3.40–3.34(m,4H),3.21(q,J=6.0Hz, 2H),3.13–3.07(m,1H),3.02(t,J=8.4Hz,2H),2.84(d,J=12.1Hz,1H),2.6 0(s,1H),2.46–2.38(m,2H),2.13(t,J=7.1Hz,2H),1.76(s,1H),1.63(s, 1H), 1.46 (d, J = 10.1Hz, 1H), 1.33 (d, J = 8.6Hz, 1H), 1.12 (d, J = 6.8Hz, 6H).

[0196] 13C NMR(101MHz,DMSO)δ171.8,170.5,170.0,167.8,165.5(dd,J=256.5,11.1Hz),159.8(dd,J=259.6,11.1Hz),158.9, 157.2,153.5,149.5,142.9,139.0,138.7,136.3,135.6,133.4,132.3(d,J=10.1Hz),126.9,126.0,125.6(dd,J=14 .1,4.0Hz),125.3,122.1,121.6,121.4,117.7,116.4,115.7,115.6,112.4(dd,J=25.2,4.0Hz),106.3(t,J=25.3Hz ),102.9,70.0,69.6,68.0,56.5,54.8,54.3,52.7,49.3,48.4,38.9,32.1,30.8,29.2,28.0,27.1,26.3,23.1,22.3.

[0197] HRMS (ESI) m / z calcd.for C 54 H 59 F2N 12 O 11 S(M+H) + 1121.4115, found 1121.4133.

[0198] Purity (HPLC): 95.38%

[0199] Example 9

[0200] Synthesis of N1-(2-(4-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazine-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperazine-1-yl)-2-oxoethyl)-N4-(1-(2,4-dihydroxy-5-isopropylbenzoyl)indoline-5-yl)succinamide CC-14.

[0201]

[0202] Reagents and conditions: (a) TCFH, NMI, DMF, rt, 12h; (b) TFA, DCM, rt, 2h; (c) TCFH, NMI, DMF, rt, 12h.

[0203] Compound CC-N-1 (100.00 mg, 0.16 mmol), N-Boc-glycine (57.38 mg, 0.33 mmol), TCFH (68.90 mg, 0.25 mmol), NMI (47.10 mg, 0.57 mmol) were added to anhydrous DMF (5 mL) and stirred at room temperature overnight. After the reaction was completed, the reaction solution was spin-dried, washed three times with DCM water, washed three times with a saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, the organic phase was concentrated, dry-loaded, and separated by column chromatography using a silica gel column. The mobile phase used a dichloromethane and anhydrous methanol system (DCM: MeOH: = 25: 1) to obtain a yellow solid CC-N-299.81 mg, with a yield of 79%.

[0204] Tert-butyl (2-(4-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperazin-1-yl)-2-oxoethyl)carbamate (CC-N-2)

[0205]

[0206] 1 HNMR (400MHz, CDCl3) δ8.76 (d, J = 2.2 Hz, 1H), 8.71 (s, 1H), 8.51 (s, 1H), 8.46 (d, J = 2. 2Hz,1H),8.13(d,J=9.4Hz,1H),7.84–7.79(m,1H),7.53(d,J=9.4Hz,1H),6.97–6.89 (m,2H),4.66(t,J=7.0Hz,2H),4.01(s,3H),3.92(d,J=4.4Hz,2H),3.60(t,J=10.1Hz ,2H),3.36(t,J=10.1Hz,2H),2.49–2.41(m,6H),2.25(p,J=6.9Hz,2H),1.45(s,9H).

[0207] 13C NMR(101MHz, CDCl3)δ166.3(dd,J=259.6,12.1Hz),166.7,159.9(dd,J=259.6,12.1Hz),1 55.8,155.8,148.5,140.5,138.8,136.9,133.0,132.1(d,J=11.1Hz),126.4,125.9,125. 5,123.3(dd,J=13.1,3.0Hz),121.9,121.3,121.2,121.1,113.7,112.2(dd,J=22.2,4.0H z), 106.0 (t, J = 26.3Hz), 79.7, 54.6, 54.5, 52.7, 52.6, 48.5, 44.2, 42.2, 41.9, 28.4, 27.4.

[0208] CC-N-2 (81.90 mg, 0.10 mmol) was dissolved in anhydrous dichloromethane, and a dichloromethane solution of TFA (0.5 mL) was added thereto, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was dried to obtain a brown-yellow oil CC-N-3, which was used directly in the next step without treatment.

[0209] Compound CC-N-3 (71.23 mg, 0.11 mmol), 17 (40.00 mg, 0.097 mmol), TCFH (40.74 mg, 0.14 mmol), NMI (27.84 mg, 0.34 mmol) were added to anhydrous DMF (5 mL) and stirred at room temperature overnight. After the reaction was completed, the reaction solution was spin-dried, washed three times with DCM water, washed three times with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, the organic phase was concentrated, dry-loaded, and separated by column chromatography using a silica gel column. The mobile phase used a dichloromethane and anhydrous methanol system (DCM: MeOH: = 20: 1), and 1 / 1000 triethylamine was added. A yellow solid CC-1438.12 mg was obtained with a yield of 37%.

[0210] N1-(2-(4-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperazin-1-yl)-2-oxoethyl)-N4-(1-(2,4-dihydroxy-5-isopropylbenzoyl)indolin-5-yl)succinamide (CC-14)

[0211]

[0212] 11H NMR (400 MHz, DMSO) δ 9.92 (s, 1H), 9.78 (s, 1H), 9.66 (s, 1H), 8.82 (s, 2H), 8.57 (s, 1H), 8.35 (s, 1H), 8.33 (s, 1H), 8.01 (t, J = 5.6 Hz, 1H), 7.91 (d, J = 9.5 Hz, 1H), 7.82 (q, J = 8.6 Hz, 1H), 7.56 (d, J = 11.6 Hz, 2H), 7.27–7.19 (m, 2H), 6.98 (s, 1H), 6.44 (s, 1H), 4.61 (t, J = 7.1 Hz, 2H), 3.99–3.92 (m, 4H), 3.78 (s, 3H), 3.46–3.35 (m, 8H), 3.13–3.02 (m, 5H), 2.20–2.16 (m, 6H), 1.13 (d, J = 7.0 Hz, 6H).

[0213] 13 13C NMR (101 MHz, DMSO) δ 171.9, 170.5, 167.8, 167.3, 165.5 (dd, J = 257.6, 12.1 Hz), 159.8 (dd, J = 258.6, 13.1 Hz), 158.9, 157.2, 153.5, 149.4, 143.2, 139.0, 138.7, 136.2, 135.6, 133.4, 132.6, 132.4 (d, J = 9.1 Hz), 126.9, 126.0, 125.3, 122.0, 121.6, 121.1, 117.7, 116.4, 115.7, 115.7, 112.5 (dd, J = 22.2, 3.0 Hz), 106.4 (t, J = 26.3 Hz), 102.9, 55.4, 54.7, 54.3, 52.9, 52.5, 49.3, 48.4, 45.9, 32.1, 30.7, 27.1, 26.3, 23.1.

[0214] HRMS (ESI) m / z calcd. for C 51 H 54 F2N 13 O9S (M + H) + 1062.3856, found 1062.3965.

[0215] Purity (HPLC): 98.47%

[0216] Example 10

[0217] Synthesis of 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl(5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-(morpholinomethyl)phenyl)isoxazole-3-carbonyl)glycine ester CC-1

[0218]

[0219] Reagents and conditions: (a) DMAP, DIPEA, EDCI, DCM, rt, 12 h; (b) TFA, DCM, rt, 2 h; (c) TCFH, NMI, DMF, rt, 12 h.

[0220] Dissolve N-Boc-glycine (126.13 mg, 0.72 mmol) in anhydrous dichloromethane, add DMAP (117.28 mg, 0.96 mmol) and DIPEA (216.72 mg, 1.68 mmol) and stir for 20 min in an ice bath. After the internal temperature is cooled to 0°C, add EDCI (184.03 mg, 0.96 mmol) and stir at 0°C for 5 min and then at room temperature for 20 min. Add CC-P-1 (300.00 mg, 0.48 mmol) and stir at room temperature overnight. After the reaction is completed, use a rotary evaporator to dry the reaction system solvent, wet sample loading, column chromatography to separate the sample, and the ratio of mobile phase dichloromethane to anhydrous methanol is 50:1.

[0221] The target product CC-P-2256.75 mg was obtained as a yellow solid with a yield of 68%.

[0222]

[0223] The structural characterization data are as follows: 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl(tert-butoxycarbonyl)glycine ester (CC-P-2):

[0224] 1HNMR(400MHz, CDCl3)δ8.64(d,J=18.3Hz,2H),8.43(s,1H),8.38(s,1H),8.05(d,J=9.4Hz,1H) ,7.78–7.72(m,1H),7.44(d,J=9.4Hz,1H),6.86(q,J=8.2Hz,2H),5.14(s,1H),4.93–4.76(m,1H ),4.59(d,J=5.1Hz,2H),3.94(s,3H),3.83(t,J=5.4Hz,2H),2.69(d,J=12.5Hz,1H),2.46(s,1H ),2.42(t,J=7.5Hz,2H),2.27–2.15(m,4H),1.74–1.67(m,2H),1.52–1.47(m,2H),1.36(s,9H).

[0225] 13 C NMR(101MHz, CDCl3)δ168.8,165.2(dd,J=260.6,12.1Hz),158.9(dd,J=259.6,13.1Hz), 154.8,154.8,147.5,139.5,137.8,135.9,132.0,131.2(d,J=11.1Hz),125.3,125.1,12 4.5,122.4(dd,J=14.1,4.0Hz),120.8,120.2,120.1,113.1,112.7,111.2(dd,J=22.2,4 .0Hz), 104.9 (t, J = 26.3Hz), 78.9, 69.4, 55.6, 53.9, 53.5, 52.2, 47.6, 28.7, 27.3, 26.4.

[0226] CC-P-2 (126.13 mg, 0.72 mmol) was dissolved in anhydrous dichloromethane, and a dichloromethane solution of TFA (0.5 mL) was added thereto, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was dried to obtain a brown-yellow oil CC-P-4, which was used directly in the next step without treatment.

[0227] Compound CC-P-4 (69.97 mg, 0.10 mmol), 17 (50.00 mg, 0.114 mmol), TCFH (47.90 mg, 0.17 mmol), NMI (32.70 mg, 0.40 mmol) were added to anhydrous DMF (5 mL) and stirred at room temperature overnight. After the reaction was completed, the reaction solution was spin-dried, washed three times with DCM, washed three times with saturated NaCl aqueous solution, dried over anhydrous sodium sulfate, concentrated the organic phase, dry-loaded, and purified by column chromatography. The ratio of mobile phase dichloromethane to anhydrous methanol was 20:1, and 1 / 1000 triethylamine was added. Yellow solid CC-151.21 mg was obtained with a yield of 41%.

[0228] 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl(5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-(morpholinomethyl)phenyl)isoxazole-3-carbonyl)glycine ester (CC-1)

[0229]

[0230] 1 H NMR(400MHz,DMSO)δ9.83(s,1H),9.73(s,1H),9.27–9.21(m,1H),8.86–8.81(m,2H),8.56–8.54(m,1H),8.36–8 .33(m,2H),7.93–7.89(m,1H),7.84–7.79(m,1H),7.55(t,J=8.7Hz,1H),7.26–7.17(m,5H),6.73(s,1H),6.48(s ,1H),4.84–4.77(m,1H),4.67–4.56(m,2H),3.99(s,2H),3.77(s,3H),3.56(s,4H),3.48(d,J=7.2Hz,2H),2.97 (p,J=6.5Hz,3H),2.77(s,2H),2.38–2.27(m,6H),2.15(s,2H),1.93(s,2H),1.75(s,2H),0.90(d,J=6.8Hz,6H).

[0231] 13C NMR (101MHz, DMSO) δ169.0,167.4,165.5(dd,J=255.5,13.1Hz),159.8(dd,J=258.5,13.1Hz),160.9,159.0,15 8.0,157.2,155.3,149.5,142.9,139.0,136.4,136.2,132.3(d,J=7.1Hz),129.6,129.3,128.9,128.3,126.9,1 26.1,125.7(dd,J=15.1,3.0Hz),125.3,125.3,122.1,121.6,121.5,115.8,115.5,112.4(dd,J=23.2,3.0Hz),1 06.3(t,J=25.3Hz),104.8,103.2,66.3,62.3,54.7,54.3,54.0,53.4,49.7,48.0,41.6,29.0,26.8,25.8,22.8.

[0232] HRMS (ESI) m / z calcd.for C 54 H 57 F2N 12 O 10 S(M+H) + 1103.4009, found 1103.4041.

[0233] Purity(HPLC):97.70%

[0234]

[0235] The synthesis method of compounds CC-P-7 to 9 is the same as CC-P-2

[0236] 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetate (CC-P-7):

[0237]

[0238] 1HNMR(400MHz, CDCl3)δ8.77–8.71(m,2H),8.51–8.45(m,2H),8.14–8.10(m,1H),7.87–7.82 (m,1H),7.55–7.50(m,1H),6.98–6.91(m,2H),5.16(s,1H),4.89(s,1H),4.66(t,J=5.7Hz,2 H),4.08–4.07(m,2H),4.03–4.01(m,3H),3.63–3.59(m,2H),3.35–3.34(m,2H),2.74(s,2H) ,2.54(s,2H),2.39(s,2H),2.31–2.27(m,2H),1.95(s,2H),1.76–1.73(m,2H),1.44(s,9H).

[0239] 13 C NMR (101MHz, CDCl3) δ169.9,166.2(dd,J=260.6,11.1Hz),159.9(dd,J=259.6,13.1Hz),156.0,155.8,148.5,140.5,138.8,136.9,133.0, 132.1(d,J=10.1Hz),126.3,126.0,125.5,123.4(dd,J=14.1,5.1Hz),121.8,121.2,121.2,113.7,112.2(dd,J=22.2,4.0Hz),105.9(t,J=

[0240] 26.3Hz),79.3,70.8,68.3,54.8,54.5,50.4,48.6,45.8,40.4,30.3,28.4,27.6.

[0241] Embodiment 11

[0242] Synthesis of 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl 2-(2-(5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamido)ethoxy)acetate (CC-3):

[0243]

[0244] The synthesis method of compound CC-P-7 is the same as CC-P-2 in Example 10;

[0245] 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl 2-(2-((tert-butoxycarbonyl)amino)ethoxy)acetate (CC-P-7):

[0246]

[0247] 1 H NMR (400MHz, CDCl3) δ8.77–8.71(m,2H),8.51–8.45(m,2H),8.14–8.10(m,1H),7.87–7.82( m,1H),7.55–7.50(m,1H),6.98–6.91(m,2H),5.16(s,1H),4.89(s,1H),4.66(t,J=5.7Hz,2H ),4.08–4.07(m,2H),4.03–4.01(m,3H),3.63–3.59(m,2H),3.35–3.34(m,2H),2.74(s,2H) ,2.54(s,2H),2.39(s,2H),2.31–2.27(m,2H),1.95(s,2H),1.76–1.73(m,2H),1.44(s,9H).

[0248] 13 C NMR(101MHz,CDCl3)δ169.9,166.2(dd,J=260.6,11.1Hz),159.9(dd,J=259.6,13.1Hz),1 56.0,155.8,148.5,140.5,138.8,136.9,133.0,132.1(d,J=10.1Hz),126.3,126.0,125. 5,123.4(dd,J=14.1,5.1Hz),121.8,121.2,121.2,113.7,112.2(dd,J=22.2,4.0Hz),105 .9(t,J=26.3Hz),79.3,70.8,68.3,54.8,54.5,50.4,48.6,45.8,40.4,30.3,28.4,27.6.

[0249] The synthesis method of compound CC-P-10 is the same as CC-P-4 in Example 10.

[0250] The synthesis method of compound CC-3 is the same as CC-14 in Example 10.

[0251] 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-b]pyridazin-3-yl)-1H-1,2,3-triazol-1-yl)propyl)piperidin-4-yl 2-(2-(5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamido)ethoxy)acetate (CC-3):

[0252]

[0253] 1 H NMR(400MHz,DMSO)δ9.82(s,1H),9.71(s,1H),8.85(s,2H),8.66(s,1H),8.56(s,1H),8.32(s,1H),8.31(s,1H) ,7.88(d,J=9.5Hz,1H),7.81(q,J=7.9Hz,1H),7.46(t,J=10.0Hz,1H),7.24–7.13(m,5H),6.72(s,1H),6.49(s,1 H),4.75(s,1H),4.60(t,J=2.0Hz,1H),4.10(s,2H),3.80(s,3H),3.58–3.53(m,6H),3.42–3.37(m,4H),2.99–2 .94(m,3H),2.72(s,2H),2.33(s,6H),2.17–2.14(m,2H),1.83(s,2H),1.61–1.59(m,2H),0.90(d,J=7.0Hz,6H).

[0254] 13C NMR (101MHz, DMSO) δ170.0, 166.9, 164.9 (dd, J=253.5, 11.1Hz), 159.8 (dd, J=256.5, 13.1Hz), 160.7, 158.9, 158. 0,157.9,155.2,149.8,140.5,139.0,137.2,136.3,132.1(d,J=10.1Hz),129.4,129.2,128.9,128.2,126.8,126 .1,125.6(dd,J=12.1,2.0Hz),125.1,122.1,121.8,121.6,115.6,115.2,112.1(dd,J=22.2,4.0Hz),106.1(t,J= 27.3Hz),104.9,103.2,69.3,67.9,66.6,62.6,54.6,54.1,53.6,50.3,48.4,45.9,30.2,27.4,26.8,25.8,22.8.

[0255] HRMS (ESI) m / z calcd.for C 56 H 61 F2N 12 O 11 S(M+H) + 1147.4272, found 1147.4170.

[0256] Purity(HPLC):99.57%

[0257] Activity Test Example 1 HSP90 Binding Activity

[0258] The present invention firstly tests the HSP90 enzyme binding activity by fluorescence polarization method, which is the key verification of whether such compounds can specifically bind to extracellular HSP90, so as to achieve the design purpose of molecular conjugates to transport drugs to tumor sites by specifically binding to extracellular HSP90. 50 The unit is nM, NVP-AUY922 and AT-13387 are positive controls.

[0259] Table 1 Binding activity of compounds to HSP90

[0260] Compound <![CDATA[IC 50 (nM)]]> Compound <![CDATA[IC 50 (nM)]]> NVP-AUY922 14 CC-9 17 AT-13387 9 CC-10 51 CC-3 6 CC-11 15 CC-6 11 CC-12 12 CC-7 13 CC-13 115 CC-8 9 CC-14 82

[0261] It can be seen from Table 1 that the binding activity of alkane chain as linker to HSP90 is slightly better than that of PEG chain as linker, and the binding activity decreases with the increase of chain length. Most small molecule drug conjugates maintain a certain binding activity compared with positive compounds, ensuring the binding of the final compound to HSP90.

[0262] Activity Test Example 2 Kinase Inhibition Activity Test

[0263] The inhibitory activity of PI3Kα was determined using the KinaseGlo method. The present invention tested the inhibition rate of the compound on PI3Kα at a concentration of 1 nM.

[0264] Table 2 Inhibitory effect of compounds on PI3Kα kinase

[0265]

[0266] It can be seen from Table 2 that at a concentration of 1 nM, the inhibition rate of PI3K inhibitor CC-M-1 on PI3Kα kinase was 86.4%, ensuring the inhibitory effect on PI3Kα kinase. With CC-M-1 as a PI3K inhibitor, the inhibition rate of the cleavable small molecule drug conjugate on PI3Kα kinase was lower than that of the single PI3K inhibitor, achieving the purpose of masking the active site of the single PI3K inhibitor.

[0267] Activity Test Example 3 In vitro anti-colorectal cancer cell proliferation experiment

[0268] The present invention uses Sulforhodamine B (SRB) analysis to evaluate the anti-proliferation ability of compounds CC-6 to CC-13 in colorectal cancer cell lines HT-29 and HCT-116.

[0269] Table 3 Inhibitory effect of compounds on HCT-116 / HT-29 cell proliferation

[0270] Compound <![CDATA[IC 50 (HCT-116,μM)]]> <![CDATA[IC 50 (HT-29,μM)]]> CC-M-1 0.38 1.70 CC-P-1 33.73 ＞50 CC-N-1 3.62 13.93 CC-1 0.37 0.73 CC-3 0.62 2.81 CC-6 0.34 1.01 CC-7 0.22 1.09 CC-8 0.29 1.22 CC-9 0.64 2.08 CC-10 0.78 2.61 CC-11 0.20 0.89 CC-12 0.20 4.88 CC-13 0.48 1.52 CC-14 4.37 71.24

[0271] From Table 2, it can be seen that this class of compounds has a certain inhibitory effect on human colon cancer HCT-116 and HT-29 cells. Among them, the small molecule drug conjugate CC-11 has excellent cell anti-proliferation ability, and the inhibitory effect IC on HCT-116 and HT-29 is 50 0.20 μM and 0.89 μM, respectively, which are superior to the single PI3K inhibitor CC-M-1.

[0272] Activity Test Example 4 Antiproliferative Activity Test of Human Normal Liver Cell Line HL-7702

[0273] The present invention uses Sulforhodamine B (SRB) analysis to evaluate the anti-proliferation ability of compounds CC-3, CC-6 and CC-11 in the human normal liver cell line HL-7702.

[0274] Table 4 Inhibitory effect of compounds on HL-7702 cell proliferation

[0275] Compound <![CDATA[IC 50 (HCT-116,μM)]]> <![CDATA[IC 50 (HT-29,μM)]]> <![CDATA[IC 50 (HL-7702,μM)]]> CC-3 0.62 2.81 1.26 CC-6 0.34 1.01 3.18 CC-11 0.20 0.89 8.60

[0276] In the HL-7702 cell line, the inhibitory effect of compound CC-11 of the present invention on colorectal cancer cells HCT-116 / HT-29 was IC 50 The inhibitory effects on HL-7702 were 0.20 μM and 0.89 μM, while the IC 50 The IC value of the inhibitory effect on HCT-116 and HT-29 is 8.60 μM. 50 The results showed that CC-11 has excellent selectivity, which is 50 and 10 times higher than the original value. Therefore, CC-11 deserves further optimization and evaluation.

[0277] Activity Test Example 5 Plasma Stability Test

[0278] The HSP90 targeting ligand of the small molecule drug conjugate binds to the extracellular HSP90 protein highly expressed in the tumor site and enters the tumor cell through endocytosis. The cleavable small molecule drug conjugate is hydrolyzed in the presence of esterase to release the active drug to exert its effect. Therefore, it is very important to explore whether the cleavable small molecule conjugate is successfully cleaved under the conditions of esterase and the length of its half-life. The present invention monitors the hydrolysis process of compound CC-11 by HPLC and LC-MS.

[0279] like Figure 1 The HPLC monitoring results of the plasma stability test of compound CC-11 are shown, note: ***p<0.001, nsp>0.05. The HPLC monitoring results showed that the half-life of CC-11 was less than 0.5h, and it was almost completely hydrolyzed in 1h. At the same time, LC-MS verified that CC-11 was successfully hydrolyzed under esterase conditions to release the single PI3K inhibitor CC-M-1.

[0280] The hydrolysis products of compound CC-11 are as follows Figure 2 As shown. The results of kinase and cell experiments prove that the compounds of the present invention can specifically bind to HSP90 and have good in vitro anti-tumor and selectivity, and therefore have potential research value. Activity Test Example 6 In vivo Anti-tumor Activity Experiment of Compounds CC-M-1 and CC-11

[0281] The present invention evaluated the antitumor activity of compounds CC-M-1 and CC-11 in the HCT116 xenograft Balb / c nude mouse model. First, HCT-116 cells were subcutaneously inoculated under the right axilla of 5-6 week-old Balb / c nude mice at 5 million / 100ul / mouse. The tumors were grown to 100 mm. 3 At about 24 hours, the nude mice were randomly divided into groups of 6, and CC-M-1 (15 mg / kg) and CC-11 (25.875 mg / kg) were administered by oral gavage. The tumor volumes of the mice were recorded every two days (e.g. Fig.31 A) and body weight (such as Fig.31 B), Fig.31 C shows the schematic diagram of tumor size of 6 mice in different groups.

[0282] like Fig.31 D The results showed that the TGI of CC-M-1 (15 mg / kg) group and CC-11 (25.875 mg / kg) group were 32.95% and 63.12%, respectively, indicating that CC-11 can effectively inhibit tumor growth in mice, and compounds CC-M-1 and CC-11 have almost no effect on the weight of mice.

[0283] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A small molecule drug conjugate, characterized in that: Its structural formula is shown in formula (I): Wherein, the R1 is Wherein, the R2 is an extracellular Hsp90 ligand, and its structure is shown in formula (II) or formula (III): Wherein, the linker is an alkane chain, a PEG chain and a nitrogen-containing heterocycle, which includes Five-membered nitrogen-containing heterocycle, substituted five-membered nitrogen-containing heterocycle, six-membered nitrogen-containing heterocycle, substituted six-membered nitrogen-containing heterocycle; wherein the heterocyclic group contains 1-3 N or O heteroatoms, and the value of n is 1-3.

2. The small molecule drug conjugate according to claim 1, characterized in that: Its structure includes the following formula (CC-1), formula (CC-3), formula (CC-6), formula (CC-7), formula (CC-8), formula (CC-9), formula (CC-11), and formula (CC-13):

3. A PI3K inhibitor, characterized in that Its structure is shown in the following formula (IV): In the formula (IV), R1 is 4. The PI3K inhibitor according to claim 3, characterized in that The PI3K inhibitor can effectively inhibit four subtypes of PI3K kinase, including PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ. The inhibitor structures are shown in formula (CC-M-1), formula (CC-P-1) and formula (CC-N-1), respectively:

5. A pharmaceutical composition, characterized in that It comprises the small molecule drug conjugate according to any one of claims 1-2 and / or the PI3K inhibitor according to any one of claims 3-4; the pharmaceutical composition is formulated into an injectable fluid, aerosol, cream, gel, pill, capsule, syrup, transdermal patch or excipient.

6. Use of the small molecule drug conjugate according to any one of claims 1 to 2, the PI3K inhibitor according to any one of claims 3 to 4, and the pharmaceutical composition according to claim 5 in the preparation of PI3K inhibitors, anti-tumor and anti-colorectal cancer drugs.

7. The use according to claim 6, characterized in that The application includes application in preparing drugs for inhibiting the proliferation, growth, infiltration and migration of colorectal cancer cells, or promoting the apoptosis of colon cancer cells.

8. The use according to claim 6, characterized in that The tumors and / or cancers include colon cancer, rectal cancer, breast cancer, bile duct cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, lymphoma, and prostate cancer.

9. The method for synthesizing a PI3K inhibitor according to claim 3, characterized in that: The method obtains the PI3K inhibitor through Suzuki coupling and click reaction, and the method comprises the following steps: Among them, R1 is 10. The method for synthesizing a small molecule drug conjugate according to claim 1, characterized in that: The synthesis method prepares the small molecule drug conjugate through Claisen esterification reaction and amide condensation; the method comprises the following steps:

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