An antitumor small molecule drug conjugate, a synthesis method and application thereof

Abstract

The application first innovatively proposes a kind of small molecule drug conjugate, which is a kind of small molecule drug conjugate with PI3K inhibitor as active drug, extracellular HSP90 as targeting ligand, alkane chain, PEG chain and nitrogen-containing heterocycle as linker, and general formula as shown in formula (I). The application also discloses the small molecule drug conjugate, a preparation method thereof, and application of the small molecule drug conjugate in preparation of antitumor and anti-colorectal cancer drugs. The application further provides a PI3K inhibitor, a synthesis method thereof, and application of the PI3K inhibitor in synthesis of small molecule drug conjugate and preparation of antitumor and anti-colorectal cancer drugs. The application has high PI3K inhibition effect, antitumor, colorectal cancer and other effects, and has wide potential application prospect.

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 a crucial signaling pathway in mammalian cells. It inhibits apoptosis and promotes proliferation by influencing the activation state of multiple downstream effector molecules. Abnormalities in the PI3K-AKT-mTOR signaling pathway are closely associated with tumorigenesis. Drug regulation of a single target in the pathway is insufficient to prevent all activated oncogenic signals and may lead to the activation of other compensatory pathways, counteracting drug efficacy and even causing drug resistance. Therefore, the development of multi-targeted drugs for cancer treatment is urgently needed.

[0003] Small molecule drug-conjugates (SMDCs) are novel drug delivery systems consisting of a targeting ligand, a linker, and an active drug (payload). SMDCs have the potential to target tumor cells, allowing the payload to rapidly accumulate in tumor cells and penetrate deep into the tumor core. They then slowly release the payload over time, resulting in sustained drug delivery to the tumor while rapidly clearing from the plasma, thereby reducing overall toxicity.

[0004] Studies have shown that there is great potential for 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 conjugates include a PI3K inhibitor as the active drug portion, an Hsp90 inhibitor as the targeting ligand portion, and alkane chains or PEG chains of varying lengths as the connecting chain portion. This class of compounds has significant anti-colorectal cancer activity and excellent selectivity, and can be further developed as anti-colorectal cancer drugs. Specifically, the present invention utilizes extracellular HSP90 (eHSP90) as a target for tumor tissue, and couples a highly active PI3K inhibitor to the HSP90 ligand via a linker (cleavable or 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 is cleaved by enzymes in the tumor tissue to release the drug (PI3K inhibitor) at a specific site. The non-cleavable SMDC binds to the extracellular HSP90, is transported into the cell, and directly exerts its drug effect.

[0006] The present invention provides a small molecule drug conjugate, specifically, a small molecule drug conjugate comprising 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.), the structural formula of which 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 represented by 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 type of small molecule drug conjugate with a PI3K inhibitor as the active drug, extracellular Hsp90 as the targeting ligand, and an alkane chain, a PEG chain, and a nitrogen-containing heterocycle as the linker. The small molecule drug conjugate includes a PI3K inhibitor-HSP90 ligand.

[0017] In the small molecule drug conjugates 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 comprising the small molecule drug conjugate and / or the PI3K inhibitor. Furthermore, the pharmaceutical composition also includes other pharmaceutically acceptable carriers or excipients. Furthermore, the pharmaceutical composition can be formulated into an injectable fluid, aerosol, cream, gel, pill, capsule, syrup, transdermal patch, or excipient.

[0029] The present invention also provides the use 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 synthesis method for the PI3K inhibitor, which involves Suzuki coupling, Click reaction, and other methods. The synthesis method involves using 3-amino-5-bromo-2-methoxypyridine and 2,4-difluorobenzenesulfonyl chloride as raw materials, conducting a substitution affinity reaction in pyridine as a solvent, and reacting at room temperature for 48 hours to obtain the intermediate compound I-1. Compound I-2 is then obtained through a Miyaura borylation reaction in a 1,4-dioxane solvent system. Then, using 3-amino-6-chloropyridazine and chloroacetaldehyde as raw materials, a ring closure is performed under the action of sodium bicarbonate to obtain the imidazo[1,2-b]pyridazine core I-3. Compound I-3 and boronate ester I-2 undergo a Suzuki cross-coupling reaction to obtain compound I-4, which is then iodinated with NIS to obtain compound I-5. I-5 undergoes a Sonogashira reaction and a TMS protection group removal reaction to obtain a terminal alkynyl compound I-6. I-6 undergoes a click reaction with three different azide compounds to obtain compounds CC-P-1, CC-M-1, and CC-N-1-1. CC-N-1-1 is deprotected in a trifluoroacetic acid and 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 (10.00 g, 49.30 mmol) was placed in a flask, pyridine (40 mL) was added to the reaction flask, and stirred in an ice bath until dissolved. After the internal temperature dropped to 0°C, 2,4-difluorobenzenesulfonyl chloride (15.68 g, 73.80 mmol) was added thereto. After stirring for 5 minutes in an ice bath, the ice bath was removed and the reaction was stirred at room temperature for 48 hours. After the reaction was completed, the reaction solution was dried using a rotary evaporator to obtain a brown oil. Dichloromethane (200 mL) and water (250 mL) were added and extracted three times. The mixture was washed three times with 10% citric acid aqueous solution (200 mL) to remove pyridine from the reaction system. Finally, the organic phases were combined and dried to obtain a brown oil. Ethyl acetate (EA) and petroleum ether (PE) were added to the slurry and filtered to obtain 17.64 g of a brown solid I-1 with a yield of 94.7%.

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

[0038] The 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 with 95% ethanol (200 mL) as the solvent. The reaction mixture was then refluxed in a 90°C oil bath with stirring for 16 hours. After the reaction, the solvent was dried using a rotary evaporator. The sample was dry-mixed and separated by column chromatography using a mobile phase of petroleum ether to ethyl acetate in a ratio of 2:1 to obtain 7.30 g of I-3 as a yellow solid in a 73% yield.

[0039] Compound I-3 (4.50 g, 47.70 mmol) was placed in a 500 mL three-necked flask, N,N-dimethylformamide (250 mL) and 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), and potassium carbonate (6.10 g, 44.12 mmol) were added to the reaction system. The nitrogen was replaced three times, and the reaction was stirred at 120 ° C under nitrogen protection for 4 hours. After the reaction, the reaction mixture was filtered with diatomaceous earth, the reaction solution was dried, and dichloromethane and water were added and extracted three times. The organic phase was concentrated and dried, and the sample was mixed and separated by column chromatography using a silica gel column. 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). The nitrogen atmosphere was purged three times and the mixture was stirred at room temperature under nitrogen for 12 h. After the reaction, 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 in a 92% yield.

[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. The nitrogen atmosphere was replaced three times and stirred for 0.5 h under nitrogen protection. Triethylamine (2.79 g, 27.60 mmol) and trimethylethynylsilane (2.71 g, 27.60 mmol) were then added by injection. The ice bath was removed and the reaction mixture was slowly heated to 45°C and refluxed with stirring for 12 h. After the reaction was completed, the mixture was filtered off with diatomaceous earth, the reaction system solvent was dried using a rotary evaporator, the sample was dry-mixed, and the sample was separated by column chromatography with a mobile phase ratio of petroleum ether to ethyl acetate of 2:1. 1.94 g of a yellow solid intermediate was obtained with a yield of 42%.

[0042] The resulting intermediate (1.94 g, 3.78 mmol) was placed in a round-bottom flask and dissolved in tetrahydrofuran (100 mL). TBAF (2.39 g, 7.56 mmol) was added to the reaction solution, and the reaction was stirred at 25°C for 1 h. After completion of the reaction, the solvent was dried using a rotary evaporator, the sample was dry-mixed, and separated by column chromatography using a mobile phase of petroleum ether to ethyl acetate in a 1:1 ratio. This afforded 1.50 g of I-6 as a yellow solid, with a yield of 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. The nitrogen was replaced three times and the reaction was stirred at room temperature under nitrogen protection for 3 hours. After the reaction was completed, the reaction solution was dried, the sample was dry-mixed, and separated by neutral alumina column chromatography. The mobile phase was dichloromethane and anhydrous ethanol in a ratio of 50:1. 1.32 g of yellow solid CC-P-1 was obtained with a yield of 62%.

[0044] The present invention also provides a method for synthesizing the small molecule drug conjugate, which comprises preparing the small molecule drug conjugate through a Claisen ester condensation reaction, an amide condensation reaction, and the like. The synthesis method comprises first reacting CC-P-1 with a linker through an ester condensation reaction to obtain a key intermediate. The designed alkane chain used as the linker can be directly purchased commercially. Using EDCI as a condensing agent, CC-P-1 reacts with N-Boc-glycine via the hydroxyl and carboxyl groups to obtain compound CC-P-2. The Boc protecting group is then removed in a dichloromethane solution of trifluoroacetic acid to obtain compound CC-P-4. CC-P-4 and compound 17 are then 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), and stirring in an ice bath for 20 minutes. After the internal temperature is cooled to 0°C, EDCI (184.03 mg, 0.96 mmol) is added, stirring at 0°C for 5 minutes, then at room temperature for 20 minutes. CC-P-1 (300.00 mg, 0.48 mmol) is added, and the mixture is stirred at room temperature overnight. After the reaction is completed, the solvent in the reaction system is dried using a rotary evaporator, the sample is wet-loaded, and the sample is separated by column chromatography with a mobile phase of dichloromethane and anhydrous methanol in a ratio of 50:1. The target product CC-P-2256.75 mg is obtained as a yellow solid in a yield of 68%. CC-P-2 (126.13 mg, 0.72 mmol) was dissolved in anhydrous dichloromethane, and a solution of TFA (0.5 mL) in dichloromethane was added. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was dried to obtain a brown oil, CC-P-4, which was used directly in the next reaction 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 aqueous NaCl solution, dried over anhydrous sodium sulfate, and the organic phase was concentrated and dry-loaded. The sample was separated and purified by column chromatography using a mobile phase of dichloromethane and anhydrous methanol in a ratio of 20:1, and 1 / 1000 triethylamine was added. 1.21 mg of yellow solid CC-15 was obtained with a yield of 41%.

[0049] The present invention offers the following beneficial effects: It innovatively proposes a class of small-molecule drug conjugates, comprising a PI3K inhibitor as the active agent, an extracellular HSP90 as the targeting ligand, and an alkane chain, a PEG chain, and a nitrogen-containing heterocycle as the linker. Compared to single PI3K inhibitors for the treatment of tumors and colorectal cancer, these conjugates exhibit enhanced targeting, significant inhibitory effects, and reduced toxicity. 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 the plasma stability test of compound CC-11, ***p<0.001, nsp>0.05. The left figure shows the peak area of ​​compound CC-11 HPLC test, and the right figure shows 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 spectrum of the compound CC-M-1 (PI3K inhibitor) of the present invention.

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

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

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

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

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

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

[0060] Figure 10 C-NMR spectrum of compound CC-1 (small molecule conjugate) of the present invention.

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

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

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

[0064] Figure 14 C-NMR spectrum of compound CC-6 (small molecule conjugate) of the present invention.

[0065] Figure 15 H-NMR chart of the compound CC-7 (small molecule conjugate) of the present application.

[0066] Figure 16 C-NMR chart of the compound CC-7 (small molecule conjugate) of the present application.

[0067] Figure 17 H-NMR chart of the compound CC-8 (small molecule conjugate) of the present application.

[0068] Figure 18 C-NMR chart of the compound CC-8 (small molecule conjugate) of the present application.

[0069] Figure 19 H-NMR chart of the compound CC-9 (small molecule conjugate) of the present application.

[0070] Figure 20 C-NMR chart of the compound CC-9 (small molecule conjugate) of the present application.

[0071] Figure 21 H-NMR chart of the compound CC-10 (small molecule conjugate) of the present application.

[0072] Figure 22 C-NMR chart of the compound CC-10 (small molecule conjugate) of the present application.

[0073] Figure 23 H-NMR chart of the compound CC-11 (small molecule conjugate) of the present application.

[0074] Figure 24 C-NMR chart of the compound CC-11 (small molecule conjugate) of the present application.

[0075] Figure 25 H-NMR chart of the compound CC-12 (small molecule conjugate) of the present application.

[0076] Figure 26 C-NMR chart of the compound CC-12 (small molecule conjugate) of the present application.

[0077] Figure 27 H-NMR chart of the compound CC-13 (small molecule conjugate) of the present application.

[0078] Figure 28 C-NMR chart of the compound CC-13 (small molecule conjugate) of the present application.

[0079] Figure 29 H-NMR chart of the compound CC-14 (small molecule conjugate) of the present application.

[0080] Figure 30 C-NMR spectrum of compound CC-14 (small molecule conjugate) of the present invention.

[0081] Figure 31 Figure 2 shows the antitumor activity of compounds CC-M-1 and CC-11 in the HCT116 xenograft tumor model. DETAILED DESCRIPTION

[0082] The present invention will be further described in detail with reference to the following specific examples and accompanying drawings. The protection content of the present invention includes but is not limited to the following examples. The processes, conditions, reagents, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the field and are not particularly limited by the present invention.

[0083] Preparation method of PI3K inhibitor:

[0084]

[0085] The raw material 3-amino-5-bromo-2-methoxypyridine (10.00 g, 49.30 mmol) was placed in a 250 mL pear-shaped flask. Pyridine (40 mL) was added to the reaction flask and stirred in an ice bath until dissolved. After the internal temperature dropped to 0°C, 2,4-difluorobenzenesulfonyl chloride (15.68 g, 73.80 mmol) was added. After stirring in an ice bath for 5 minutes, the ice bath was removed and the reaction was stirred at room temperature for 48 hours. After the reaction was completed, the reaction solution was dried using a rotary evaporator to obtain a brown oil. Dichloromethane (200 mL) and water (250 mL) were added and extracted three times. The reaction system was washed three times with 10% citric acid aqueous solution (200 mL) to remove pyridine. Finally, the organic phases were combined and dried to obtain a brown oil. Ethyl acetate (EA) and petroleum ether (PE) were added to the slurry and filtered to obtain 17.64 g of a brown solid I-1 with a yield of 94.7%.

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

[0087]

[0088] The 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 with 95% ethanol (200 mL) as the solvent. The reaction mixture was then refluxed in a 90°C oil bath with stirring for 16 hours. After the reaction, the solvent was dried using a rotary evaporator. The sample was dry-mixed and separated by column chromatography using a mobile phase of petroleum ether to ethyl acetate in a ratio of 2:1 to obtain 7.30 g of I-3 as a yellow solid in a 73% yield.

[0089] Compound I-3 (4.50 g, 47.70 mmol) was placed in a 500 mL three-necked flask, N,N-dimethylformamide (250 mL) and 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), and potassium carbonate (6.10 g, 44.12 mmol) were added to the reaction system. The nitrogen was replaced three times, and the reaction was stirred at 120 ° C under nitrogen protection for 4 hours. After the reaction, the reaction mixture was filtered with diatomaceous earth, the reaction solution was dried, and dichloromethane and water were added and extracted three times. The organic phase was concentrated and dried, and the sample was mixed and separated by column chromatography using a silica gel column. 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). The nitrogen atmosphere was purged three times and the mixture was stirred at room temperature under nitrogen for 12 h. After the reaction, 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 in a 92% yield.

[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. The nitrogen atmosphere was replaced three times and stirred for 0.5 h under nitrogen protection. Triethylamine (2.79 g, 27.60 mmol) and trimethylethynylsilane (2.71 g, 27.60 mmol) were then added by injection. The ice bath was removed and the reaction mixture was slowly heated to 45°C and refluxed with stirring for 12 h. After the reaction was completed, the mixture was filtered off with diatomaceous earth, the reaction system solvent was dried using a rotary evaporator, the sample was dry-mixed, and the sample was separated by column chromatography with a mobile phase ratio of petroleum ether to ethyl acetate of 2:1. 1.94 g of a yellow solid intermediate was obtained with a yield of 42%.

[0092] The resulting intermediate (1.94 g, 3.78 mmol) was placed in a round-bottom flask and dissolved in tetrahydrofuran (100 mL). TBAF (2.39 g, 7.56 mmol) was added to the reaction solution, and the reaction was stirred at 25°C for 1 h. After completion of the reaction, the solvent was dried using a rotary evaporator, the sample was dry-mixed, and separated by column chromatography using a mobile phase of petroleum ether to ethyl acetate in a 1:1 ratio. This afforded 1.50 g of I-6 as a yellow solid, with a yield of 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. The nitrogen was replaced three times and the reaction was stirred at room temperature under nitrogen protection for 3 hours. After the reaction was completed, the reaction solution was dried, the sample was dry-mixed, and separated by neutral alumina column chromatography with a mobile phase of dichloromethane and anhydrous ethanol in a ratio of 50:1. 11.32 g of yellow solid CC-M-1 was obtained with a yield of 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 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 was used to replace 1-(3-azidopropyl)piperidin-3-ol in the synthesis of CC-M-1. The synthesis steps were 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), and trifluoroacetic acid (0.5 mL) was slowly added dropwise thereto. 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-carbonyl)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 in an ice bath for 20 min. After cooling the mixture to 0°C, add EDCI (184.03 mg, 0.96 mmol), stir at 0°C for 5 min, then at room temperature for 20 min. Add CC-M-1 (300.00 mg, 0.48 mmol), and stir overnight at room temperature. After completion of the reaction, dry the reaction solvent using a rotary evaporator, wet-load the sample, and separate it by column chromatography using a mobile phase consisting of dichloromethane and anhydrous methanol in a ratio of 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] Dissolve CC-M-2 (126.13 mg, 0.72 mmol) in anhydrous dichloromethane, add TFA (0.5 mL) in dichloromethane, and stir at room temperature for 2 h. After the reaction, spin dry the reaction mixture to obtain a brown oil, CC-M-4, which was used directly in the next reaction without further 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), and NMI (32.70 mg, 0.40 mmol) were added to anhydrous DMF (5 mL) and stirred at room temperature overnight. After completion of the reaction, the reaction solution was spin-dried and washed three times with DCM and saturated NaCl solution, then dried over anhydrous sodium sulfate. The organic phase was concentrated and dry-loaded. The sample was purified by column chromatography using a mobile phase of dichloromethane and anhydrous methanol in a ratio of 20:1, and 1 / 1000 triethylamine was added. CC-65 was obtained as a yellow solid (1.21 mg, 41% yield).

[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 (101 MHz, CDC13) δ 171.6, 165.7 (dd, J = 259.6, 10.1 Hz), 159.9 (dd, J = 259.6, 13.1 Hz), 155.9, 155.9, 148.5, 140.6, 138.8, 137.0, 133.0, 132.2 (d, J = 10.1 Hz), 126.5, 126.3, 125.4, 123.5 (dd, J = 13.1, 4.0 Hz), 121.7, 121.3, 121.1, 113.7, 112.2 (dd, J = 23.2, 2.0 Hz), 105.9 (t, J = 25.3 Hz), 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] Replace CC-M-2 in Example 1 with CC-M-3, and the synthesis of CC-7 is the same as CC-6.

[0137] The structure confirmation data are as follows: 1 H NMR (400 MHz, DMSO) δ 9.85 (s, 1H), 9.72 (s, 1H), 8.92 (t, J = 6.7 Hz, 1H), 8.85 - 8.80 (m, 2H), 8.57 - 8.53 (m, 1H), 8.37 - 8.31 (m, 2H), 7.91 (q, J = 7.4 Hz, 1H), 7.82 (q, J = 7.4 Hz, 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 = 6.9 Hz, 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 = 31.2 Hz, 2H), 1.50 (s, 1H), 1.41 - 1.38 (m, 1H), 0.90 (d, J = 5.4 Hz, 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. 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. 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. 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 (400 MHz, DMSO) δ 9.82 (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 = 9.4 Hz, 1H), 7.81 (q, J = 8.0 Hz, 1H), 7.56 (t, J = 9.8 Hz, 1H), 7.28 - 7.18 (m, 5H), 6.73 (s, 1H), 6.48 (s, 1H), 4.81 (s, 1H), 4.59 (t, J = 7.1 Hz, 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 = 6.8 Hz, 6H).

[0173] 13 C NMR (101 MHz, DMSO) δ 169.9, 166.9, 165.5 (dd, J = 255.5, 11.1 Hz), 159.8 (dd, J = 258.5, 13.1 Hz), 160.6, 158.9, 158.0, 155.2, 149.5, 143.2, 139.0, 136.3, 132.7, 132.4 (d, J = 8.1 Hz), 129.5, 129.4, 129.3, 128.2, 126.9, 126.1, 125.5 (dd, J = 14.1, 4.0 Hz), 125.3, 122.1, 121.6, 121.1, 115.8, 115.1, 112.4 (dd, J = 22.2, 4.0 Hz), 106.3 (t, J = 26.3 Hz), 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 for C 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-oxobutanoyl)glycine CC-11.

[0178] HSP90 inhibitor 17 in Example 1 was replaced by HSP90 inhibitor 7. 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)sulfonamido)-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 those of 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]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.

[0201]

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

[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), and 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 and water, washed three times with a saturated aqueous NaCl solution, and dried over anhydrous sodium sulfate. The organic phase was concentrated, dry-loaded, and separated by column chromatography using a silica gel column with a mobile phase of dichloromethane and anhydrous methanol (DCM:MeOH:=25:1) to obtain 99.81 mg of CC-N-2 as a yellow solid in 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] Dissolve CC-N-2 (81.90 mg, 0.10 mmol) in anhydrous dichloromethane, add TFA (0.5 mL) in dichloromethane, and stir at room temperature for 2 h. After the reaction is complete, spin dry the reaction solution to obtain a brown oil, CC-N-3, which is used directly in the next reaction without further 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), and NMI (27.84 mg, 0.34 mmol) were added to anhydrous DMF (5 mL) and stirred at room temperature overnight. After completion of the reaction, the reaction solution was spin-dried, washed three times with DCM and water, then three times with saturated NaCl solution, and dried over anhydrous sodium sulfate. The organic phase was concentrated, dry-loaded, and separated by column chromatography using a silica gel column with a mobile phase of dichloromethane and anhydrous methanol (DCM:MeOH = 20:1) and 1 / 1000 triethylamine. CC-1438.12 mg of a yellow solid was obtained in a 37% yield.

[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] 1H NMR(400MHz,DMSO)δ9.92(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=5.6Hz,1H),7.91(d,J=9.5Hz,1H),7.82(q,J=8.6Hz,1H),7.56(d,J=11.6Hz,2H),7.27–7.19(m,2H),6.98(s,1H),6.44(s,1H),4.61(t,J=7.1Hz,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=7.0Hz,6H).

[0213] 13 C NMR(101MHz,DMSO)δ171.9,170.5,167.8,167.3,165.5(dd,J=257.6,12.1Hz),159.8(dd,J=258.6,13.1Hz),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=9.1Hz),126.9,126.0,125.3,122.0,121.6,121.1,117.7,116.4,115.7,115.7,112.5(dd,J=22.2,3.0Hz),106.4(t,J=26.3Hz),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] 实施例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 cooling the mixture to 0°C, add EDCI (184.03 mg, 0.96 mmol), stir at 0°C for 5 min, then at room temperature for 20 min. Add CC-P-1 (300.00 mg, 0.48 mmol), and stir overnight at room temperature. After completion of the reaction, dry the reaction solvent using a rotary evaporator, wet-load the sample, and separate it by column chromatography using a mobile phase consisting of dichloromethane and anhydrous methanol in a ratio of 50:1.

[0221] The target product CC-P-22 was obtained as a yellow solid, 56.75 mg, 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 (400 MHz, CDC13) δ 8.64 (d, J = 18.3 Hz, 2H), 8.43 (s, 1H), 8.38 (s, 1H), 8.05 (d, J = 9.4 Hz, 1H), 7.78 - 7.72 (m, 1H), 7.44 (d, J = 9.4 Hz, 1H), 6.86 (q, J = 8.2 Hz, 2H), 5.14 (s, 1H), 4.93 - 4.76 (m, 1H), 4.59 (d, J = 5.1 Hz, 2H), 3.94 (s, 3H), 3.83 (t, J = 5.4 Hz, 2H), 2.69 (d, J = 12.5 Hz, 1H), 2.46 (s, 1H), 2.42 (t, J = 7.5 Hz, 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 (101 MHz, CDC13) δ 168.8, 165.2 (dd, J = 260.6, 12.1 Hz), 158.9 (dd, J = 259.6, 13.1 Hz), 154.8, 154.8, 147.5, 139.5, 137.8, 135.9, 132.0, 131.2 (d, J = 11.1 Hz), 125.3, 125.1, 124.5, 122.4 (dd, J = 14.1, 4.0 Hz), 120.8, 120.2, 120.1, 113.1, 112.7, 111.2 (dd, J = 22.2, 4.0 Hz), 104.9 (t, J = 26.3 Hz), 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 solution of TFA (0.5 mL) in dichloromethane was added thereto, and the reaction was stirred at room temperature for 2 h. After the reaction was completed, the reaction liquid was spin-dried to obtain a brownish 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 rotary evaporated, washed with DCM three times, washed with saturated NaCl aqueous solution three times, dried with anhydrous sodium sulfate, concentrated the organic phase, dry loaded, column chromatography separation and purification of the sample, the ratio of the mobile phase dichloromethane and anhydrous methanol is 20:1, and add one thousandth of triethylamine. Yellow solid CC-151.21 mg, yield 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)glycinate (CC-1)

[0229]

[0230] 1 H NMR (400 MHz, DMSO) δ 9.83 (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 = 8.7 Hz, 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 = 7.2 Hz, 2H), 2.97 (p, J = 6.5 Hz, 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 = 6.8 Hz, 6H).

[0231] 13C NMR (101 MHz, DMSO) δ 169.0, 167.4, 165.5 (dd, J = 255.5, 13.1 Hz), 159.8 (dd, J = 258.5, 13.1 Hz), 160.9, 159.0, 158.0, 157.2, 155.3, 149.5, 142.9, 139.0, 136.4, 136.2, 132.3 (d, J = 7.1 Hz), 129.6, 129.3, 128.9, 128.3, 126.9, 126.1, 125.7 (dd, J = 15.1, 3.0 Hz), 125.3, 125.3, 122.1, 121.6, 121.5, 115.8, 115.5, 112.4 (dd, J = 23.2, 3.0 Hz), 106.3 (t, J = 25.3 Hz), 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 of compounds CC-P-7 to 9 was performed as for CC-P-2

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

[0237]

[0238] 1HNMR (400 MHz, CDC13) δ 8.77-8.71 (m, 2H), 8.51-8.45 (m, 2H), 8.14-8.10 (m, IH), 7.87-7.82 (m, IH), 7.55-7.50 (m, IH), 6.98-6.91 (m, 2H), 5.16 (s, IH), 4.89 (s, IH), 4.66 (t, J = 5.7 Hz, 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).

[0239] 13 C NMR (101 MHz, CDC13) δ 169.9, 166.2 (dd, J = 260.6, 11.1 Hz), 159.9 (dd, J = 259.6, 13.1 Hz), 156.0, 155.8, 148.5, 140.5, 138.8, 136.9, 133.0, 132.1 (d, J = 10.1 Hz), 126.3, 126.0, 125.5, 123.4 (dd, J = 14.1, 5.1 Hz), 121.8, 121.2, 121.2, 113.7, 112.2 (dd, J = 22.2, 4.0 Hz), 105.9 (t, J = 26.3 Hz), 79.3, 70.8, 68.3, 54.8, 54.5, 50.4, 48.6, 45.8, 40.4, 30.3, 28.4, 27.6.

[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] Example 11

[0242] 1-(3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)imidazo[l,2- b]pyridazin-3-yl)-lH-l,2,3-triazol-l-yl)propyl)piperidin-4-yl 2-(2-(5-(2,4-dihydroxy-5- isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamido)ethoxy)ethanoate (CC-3)

[0243]

[0244] The synthesis of compound CC-P-7 was carried out in the same manner 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[l,2- b]pyridazin-3-yl)-lH-l,2,3-triazol-l-yl)propyl)piperidin-4-yl 2-(2-(5-(2,4-dihydroxy-5- isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamido)ethoxy)ethanoate (CC-3):

[0252]

[0253] 1 H NMR (400 MHz, DMSO) δ 9.82 (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 = 9.5 Hz, 1H), 7.81 (q, J = 7.9 Hz, 1H), 7.46 (t, J = 10.0 Hz, 1H), 7.24 - 7.13 (m, 5H), 6.72 (s, 1H), 6.49 (s, 1H), 4.75 (s, 1H), 4.60 (t, J = 2.0 Hz, 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 = 7.0 Hz, 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 first tests the HSP90 enzyme binding activity by fluorescence polarization, which is a key verification of whether such compounds can specifically bind to extracellular HSP90, thereby achieving the design purpose of molecular conjugates that can specifically bind to extracellular HSP90 to transport drugs to tumor sites. 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] Table 1 shows that alkane chains as linkers have slightly better binding activity to HSP90 than PEG chains, and that binding activity decreases with increasing chain length. Most small molecule drug conjugates maintain a certain level of binding activity compared to the positive compounds, ensuring that the final compounds bind 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] Table 2 shows that at a concentration of 1 nM, the PI3K inhibitor CC-M-1 exhibited an inhibition rate of 86.4% against PI3Kα kinase, ensuring its inhibitory effect. Using CC-M-1 as a PI3K inhibitor, the cleavable small molecule drug conjugate exhibited a lower inhibition rate against PI3Kα kinase than the PI3K inhibitor alone, effectively masking the active site of the PI3K inhibitor alone.

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

[0268] The present invention uses the sulforhodamine B (SRB) assay to evaluate the anti-proliferative 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, we can see 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 its inhibitory effect on HCT-116 and HT-29 is IC 50 The results were 0.20 μM and 0.89 μM, respectively, which were superior to the PI3K inhibitor CC-M-1 alone.

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

[0273] The present invention uses the sulforhodamine B (SRB) assay to evaluate the anti-proliferative 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)]]> 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 on colorectal cancer cells HCT-116 / HT-29 was IC 50 were 0.20 μM and 0.89 μM, while the inhibitory IC 50 The IC value for HCT-116 and HT-29 inhibition is 8.60 μM. 50 The results showed that CC-11 has excellent selectivity, which is 50 times and 10 times of 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 highly expressed extracellular HSP90 protein in tumors and enters tumor cells via endocytosis. The cleavable small molecule drug conjugate is hydrolyzed in the presence of esterases to release the active drug. Therefore, it is crucial to investigate whether the cleavable small molecule conjugate is successfully cleaved by esterases and its half-life. The present invention monitored the hydrolysis of compound CC-11 using HPLC and LC-MS.

[0279] like Figure 1 The HPLC results for the plasma stability test of compound CC-11 are shown. Note: ***p<0.001, nsp>0.05. HPLC results show that CC-11 has a half-life of less than 0.5 hours and is nearly completely hydrolyzed within 1 hour. LC-MS also confirms that CC-11 is 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 show 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 test of compounds CC-M-1 and CC-11

[0281] The present application evaluates the anti-tumor activity of compounds CC-M-1 and CC-11 in HCT116 xenograft tumor Balb / c nude mice model. First, HCT-116 cells are inoculated subcutaneously at 5 million / 100ul / each in the right axillary of 5-6 weeks old Balb / c nude mice. After waiting for the tumor to grow to 100mm 3 around, the nude mice are randomly grouped, 6 in each group, and CC-M-1 (15mg / kg) and CC-11 (25.875mg / kg) are administered by gavage, after which the tumor volume of the mice is recorded every two days (as Figure 31 A) and the body weight (as Figure 31 B), Figure 31 C) are given.

[0282] As Figure 31 D) shows that the TGI of CC-M-1 (15mg / kg) group and CC-11 (25.875mg / kg) group are 32.95% and 63.12% respectively. This indicates that CC-11 can effectively inhibit the growth of tumors in mice, and compounds CC-M-1 and CC-11 have little effect on the body weight of mice.

[0283] The present application is not limited to the above examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application and are protected by the appended 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): The Linker includes Here, 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 pharmaceutical composition, characterized in that It comprises the small molecule drug conjugate according to any one of claims 1 to 2; the pharmaceutical composition is formulated into an injectable fluid, aerosol, cream, gel, pill, capsule, syrup, or transdermal patch.

4. Use of the small molecule drug conjugate according to any one of claims 1 to 2 or the pharmaceutical composition according to claim 3 in the preparation of a PI3K inhibitor or an anti-colorectal cancer drug.

5. The use according to claim 4, 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.

6. The method for synthesizing the small molecule drug conjugate CC-1 as claimed in claim 2, 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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