Evodiamine conjugate and application thereof

Abstract

The invention discloses an evodiamine conjugate or a pharmaceutical salt thereof. The structural general formula of the evodiamine conjugate or the pharmaceutical salt thereof is selected from one of the following structures, according to the invention, a compound 1a or a compound 1b with an extremely strong in-vitro inhibition effect on tumor cell growth is used as a pharmacophore to be fused with an Hsp90 inhibitor, and the Hsp90 inhibitor, namely the evodiamine conjugate, is designed and synthesized. According to the evodiamine conjugate disclosed by the invention, an Hsp90 inhibitor in the structure is partially combined with eHsp90 secreted into a microenvironment by tumor cells in a targeting manner, and the tumor targeting property of the evodiamine conjugate is remarkably improved compared with that of a compound 1a and a compound 1b under the cell endocytosis mediated by the eHsp90, so that a new strategy is expected to be provided for improving the tumor treatment effect of a natural product and a derivative thereof.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and in particular relates to a evodiamine conjugate and application thereof. BACKGROUND

[0002] For a long time, natural products (NPs) have been an important source of chemotherapeutic drugs and lead compounds. Specifically, NPs and their direct derivatives account for nearly 50% of the number of anti-tumor drugs. Compared with traditional synthetic chemical molecules, NPs exhibit more abundant structural diversity, higher system complexity and more extensive biological activity. Among them, the five-membered ring natural product molecule evodiamine has multiple biological activities, especially anti-tumor effect, and is considered a very promising lead compound.

[0003] Although evodiamine has unique advantages in biological activity, the poor anti-tumor effect in vivo limits its further development. The previously reported evodiamine derivatives, compounds 3-fluoro-10-hydroxy evodiamine and 10-hydroxy evodiamine, have significantly enhanced in vitro anti-tumor activity. However, due to the lack of tumor targeting, high doses of such derivatives are required to exert anti-tumor effect in vivo.

[0004] At present, due to the advantages of ligand-drug conjugates such as simple synthesis process, good stability and strong tumor penetration, they have become an effective strategy to improve the efficacy of anti-tumor drugs. In addition, selecting an ideal ligand with good tumor targeting is crucial to improve the efficacy of anti-tumor drugs. A large number of literature reports that heat shock protein 90 (Hsp90) inhibitors are often selected as ligands for the design of ligand-drug conjugates due to their excellent tumor targeting. Specifically, Hsp90 is overexpressed in tumor cells and is secreted into the tumor microenvironment in the form of extracellular Hsp90 (eHsp90), which regulates a variety of cellular proteins and is a promising drug target in cancer treatment. In addition, tumor cells can re-internalize eHsp90, thereby making Hsp90 inhibitors have excellent tumor targeting. At present, a large number of inhibitors have been developed around Hsp90, such as AUY922, C15 and EC144, and have shown effective anti-tumor activity.

[0005]

[0006] The prior art does not involve the introduction of the structure of the Hsp90 inhibitor into the structure of the evodiamine derivative provided by the present application. SUMMARY

[0007] The first object of the present application is to provide a evodiamine conjugate.

[0008] The second object of the present application is to provide the use of the evodiamine conjugate in the preparation of an antitumor drug.

[0009] In order to achieve the above object, the technical scheme adopted by the present application is as follows:

[0010] In the first aspect of the present application, an evodiamine conjugate or a pharmaceutically acceptable salt thereof is provided, and the structure general formula is selected from one of the following structures:

[0011]

[0012] wherein R1 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine); L1 is selected from -(CH2)n1-, n1 is selected from positive integers from 1 to 10 (preferably 1, 2, 3, 4, 5); R2 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine); L2 is selected from -(CH2)n2-; n2 is selected from 0-10 (preferably 0, 1, 2, 3, 4, 5); R3 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine); L3 is selected from -(CH2)n3-; n3 is selected from 0-10 (preferably 0, 1, 2, 3, 4, 5).

[0013] Most preferably, the structure of the evodiamine conjugate is selected from one of the following structures:

[0014]

[0015] The pharmaceutically acceptable salt is an acid addition salt of the evodiamine conjugate with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0016] In the second aspect of the present application, the use of the evodiamine conjugate or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug is provided.

[0017] The tumor is selected from colon cancer, breast cancer, pancreatic cancer, ovarian cancer, lung cancer, liver cancer, kidney cancer, prostate cancer, thyroid cancer, skin cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, osteosarcoma, central or peripheral nervous system tumor.

[0018] In the third aspect of the present application, the use of the evodiamine conjugate or a pharmaceutically acceptable salt thereof in the preparation of an Hsp90 inhibitor is provided.

[0019] The evodiamine conjugates prepared in this invention significantly enhance the tumor targeting and antitumor effects of evodiamine conjugates by targeting and binding to eHsp90, which mediates tumor cell endocytosis.

[0020] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0021] This invention fuses compound 1a or compound 1b, which exhibits strong in vitro inhibitory activity against tumor cell growth, with an Hsp90 inhibitor as a pharmacophore, thus designing and synthesizing a class of Hsp90 inhibitors, namely, evodiamine conjugates. By partially targeting and binding eHsp90 secreted into the tumor microenvironment by the Hsp90 inhibitor in the structure, and through eHsp90-mediated endocytosis, the tumor targeting of the evodiamine conjugates is significantly improved compared to compounds 1a and 1b, potentially providing a new strategy for enhancing the tumor therapeutic effects of natural products and their derivatives.

[0022] The compounds prepared in this invention generally exhibited good in vitro tumor cell inhibitory activity. Among them, compound 5a showed the best in vitro antitumor activity, demonstrating nanomolar-level antitumor cell proliferation activity, superior to compound 1a, and reducing the toxicity to normal HUVEC cells by approximately 6.9 times. Systematic bioactivity evaluation of compound 5a showed that it could arrest HCT116 cells in the G2 / M phase and induce apoptosis in a concentration-dependent manner. Its inhibitory activity against Hsp90 was comparable to that of the Hsp90 inhibitor AUY922, and it could achieve effective therapeutic effects in a nude mouse HCT116 xenograft tumor model by intraperitoneal injection or oral administration.

[0023] The compound prepared in this invention is a first-reported Hsp90 inhibitor and has further development and research value. Attached Figure Description

[0024] Figure 1 This is a schematic diagram showing the results of cell cycle and apoptosis assays of compound 5a in HCT116 cells.

[0025] Figure 2 This is a schematic diagram showing the Hsp90 inhibition activity of compound 5a.

[0026] Figure 3 This is a schematic diagram illustrating the in vivo antitumor activity of compound 5a in a nude mouse HCT116 xenograft tumor model. Detailed Implementation

[0027] In order to make the present application clearer, the following further describes the present application in conjunction with preferred embodiments. Those skilled in the art should understand that the specific descriptions below are illustrative rather than limiting, and the protection scope of the present application should not be limited thereby.

[0028] In the present application, Boc is tert-butyloxycarbonyl, CPT is camptothecin, DCM is dichloromethane, DIPEA is N,N-diisopropylethylamine, DMAP is 4-dimethylaminopyridine, DMF is N,N-dimethylformamide, EA is ethyl acetate, EDCI is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, HATU is 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, HRMS is high resolution mass spectrometry, IC 50 IC50 is half maximal inhibitory concentration, SD is standard deviation, TFA is trifluoroacetic acid, TGI is tumor growth inhibition rate.

[0029] Example 1

[0030] Synthesis of compound 5a

[0031]

[0032] First step, synthesis of compound 2a:

[0033] The synthesis method of compound 1a was prepared according to the literature (J. Med. Chem. 55, 2012, 7593-7613.).

[0034] Compound 1a (1000 mg, 2.97 mmol) was dissolved in 15 mL of anhydrous DMF, and N-Boc-glycine (624 mg, 3.56 mmol), DMAP (363 mg, 2.97 mmol) and EDCI (1707 mg, 8.91 mmol) were added in turn, and the reaction was stirred at room temperature for 6 h. The reaction solution was poured into 150 mL of ice water, and the white solid was obtained by suction filtration, which was compound 2a (954 mg, yield: 65%), without purification, and directly subjected to the next step reaction.

[0035] Second step, synthesis of compound 3a:

[0036] Compound 2a (800 mg, 1.62 mmol) was dissolved in 4 mL of anhydrous DCM, 1 mL of TFA was added, and the reaction was stirred at room temperature for 0.5 h. The reaction solution was poured into 20 mL of saturated NaHCO3 solution, extracted with DCM (3x10 mL), the organic phases were combined, and concentrated under reduced pressure to obtain compound 3a (574 mg, yield: 90%), without purification, and directly subjected to the next step reaction.

[0037] Step 3, synthesis of compound 5a:

[0038] Compound 4 was prepared according to literature (J. Med. Chem. 63 (2020) 5421-5441.).

[0039] Compound 3a (200 mg, 0.51 mmol) was dissolved in 5 mL of anhydrous DMF, and compound 4 (266 mg, 0.61 mmol), HATU (289 mg, 0.76 mmol) and DIPEA (79 mg, 0.61 mmol) were added in turn, and the reaction was stirred at room temperature for 12 h. The reaction solution was poured into 50 mL of water, extracted with EA (3x20 mL). The organic phase was combined and concentrated under reduced pressure, and the remaining residue was purified by silica gel column chromatography (H2O:MeOH=30:70, V / V) to obtain white solid, which was compound 5a (157 mg, yield: 38%). 1 HNMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 9.78 (s, 1H), 9.69 (s, 1H), 9.41 (t, J = 5.9 Hz, 1H), 7.54 (dd, J = 8.9, 3.13 Hz, 1H), 7.41-7.36 (m, 2H), 7.23-7.20 (m, 4H), 7.19-7.15 (m, 2H), 6.86 (dd, J = 8.7, 2.3 Hz, 1H), 6.73 (s, 1H), 6.44 (s, 1H), 6.12 (s, 1H), 4.63 (dt, J = 10.8, 2.9 Hz, 1H), 4.27 (d, J = 5.9 Hz, 2H), 3.52 (t, J = 4.6 Hz, 4H), 3.37 (s, 2H), 3.24-3.18 (m, 1H), 3.00-2.93 (m, 1H), 2.89-2.83 (m, 2H), 2.70 (s, 3H), 2.29 (t, J = 4.6 Hz, 4H), 0.89 (d, J = 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 169.31, 167.37, 163.53, 161.12, 158.00, 157.68 (d, J C-F= 7.0 Hz), 122.31 (d, J C-F = 7.6 Hz), 121.12 (d, J C-F = 7.6 Hz), 121.12 (d, J C-F = 23.5Hz), 116.58, 115.59, 113.91 (d, J C-F = 23.3 Hz), 112.63, 112.37, 110.96,104.90, 103.25, 69.71, 66.64, 62.64, 53.63, 41.84, 40.48, 37.27, 25.89,22.86, 20.01. HRMS (ESI) m / z calcd for C 45 H 44 FN6O8 [M + H] + = 815.3199, found [M + H] + = 815.3106.

[0040] Example 2

[0041] Synthesis of compound 5b

[0042] Following the synthetic procedure of Reference Example 1, replacing the intermediate N-Boc-glycine in Example 1 with 3-((tert-butoxycarbonyl)amino)propanoic acid, afforded compound 5b as a white solid in 44% yield. 1H NMR (600 MHz, DMSO-d6) δ 11.30 (s, 1H), 9.75 (s, 2H), 9.05 (t, J = 5.6 Hz, 1H), 7.54 (dd, J = 8.9, 3.1 Hz, 1H), 7.38 (td, J = 8.6, 3.1 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.25 (d, J= 2.3 Hz, 1H), 7.22-7.19 (m, 5H), 6.89 (dd, J = 8.7, 2.3 Hz, 1H), 6.73 (s,1H), 6.45 (s, 1H), 6.10 (s, 1H), 4.62 (ddd, J = 13.0, 5.2, 2.0 Hz, 1H), 3.60(q, J = 6.5 Hz, 2H), 3.52 (t, J = 4.6 Hz, 4H), 3.38 (s, 2H), 3.24-3.17 (m,1H), 2.98 (p, J = 6.9 Hz, 1H), 2.87-2.77 (m, 4H), 2.69 (s, 3H), 2.30 (s, 4H),0.90 (d, J = 6.9 Hz, 6H). 13 C NMR (150 MHz, DMSO-d6) δ 171.08, 167.02, 163.49,160.69, 157.89,156.88, 155.30, 146.48, 144.13, 137.39, 134.90, 131.58,129.36, 129.12, 128.82, 128.25, 126.31, 126.10, 122.57, 122.25, 121.06 (d,J C-F = 23.3 Hz), 116.85, 115.29, 113.89 (d, J C-F = 23.4 Hz), 112.38, 112.32,111.15, 104.94, 103.28, 69.67, 66.62, 62.61, 53.60, 37.19, 35.72, 34.14,25.86, 22.84, 20.00. HRMS (ESI) m / z calcd for C 46 H 46 FN6O8: [M + H] += 829.3356, found [M + H] + = 829.3319.

[0043] Example 3

[0044] Synthesis of compound 5c

[0045] The synthesis of compound 5c was carried out according to the synthetic procedure of Reference Example 1, replacing the intermediate N-Boc-glycine in Example 1 with 4-((tert-butoxycarbonyl)amino)butyric acid, to give compound 5c as a white solid in 43% yield. 1 H NMR (600 MHz, DMSO-d6) δ 11.30 (s, 1H), 9.72 (d, J = 57.2 Hz, 2H), 8.93 (t, J = 5.8 Hz, 1H), 7.54 (dd, J = 8.9, 3.1 Hz, 1H), 7.40-7.35 (m, 2H), 7.23-7.19 (m, 6H), 6.87 (dd, J = 8.7, 2.3 Hz, 1H), 6.74 (s, 1H), 6.44 (s, 1H), 6.10 (s, 1H), 4.63-4.60 (m, 1H), 3.63-3.60 (m, 1H), 3.51 (t, J = 4.6 Hz, 4H), 3.37 (s, 2H), 3.23-3.18 (m, 1H), 2.97 (t, J = 6.9 Hz, 1H), 2.84-2.82 (m, 2H), 2.79 (t, J = 6.6 Hz, 1H), 2.69 (s, 3H), 2.58 (t, J = 7.4 Hz, 2H), 2.28 (s, 4H), 1.87-1.83 (m, 2H), 0.90 (d, J = 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 172.60, 166.83, 163.54, 160.77, 158.88, 158.30, 157.96, 156.51, 155.25, 146.51, 144.21, 137.46, 134.89, 131.61, 129.35, 129.21, 128.93, 128.28, 126.37, 126.15, 122.60 (d, J C-F = 7.3 Hz), 122.32 (d, J C-F= 7.8 Hz), 121.11 (d, J C-F = 23.5 Hz), 116.90, 115.23, 113.92 (d, J C-F = 23.5 Hz), 112.46, 112.36, 111.16,105.02, 103.26, 69.70, 66.64, 62.65, 53.63, 38.48, 37.21, 31.30, 25.89,24.72, 22.87, 20.03. HRMS (ESI) m / z calcd for C 47 H 48 FN6O8: [M + H] + = 843.3512, found [M + H] + = 843.3415.

[0046] Example 4

[0047] Synthesis of compound 5d

[0048] The synthesis of compound 1b was performed according to the literature (J. Med. Chem. 55, 2012, 7593-7613.).

[0049] The synthesis of Reference Example 1 was performed according to the literature, replacing compound 1a in Example 1 with compound 1b to give compound 5d as a white solid in 41% yield. 1H NMR (600 MHz, DMSO-d6) δ 11.23 (s, 1H), 9.76 (s, 1H), 9.67 (s, 1H), 9.41 (t, J = 5.9 Hz, 1H), 7.80 (dd, J = 7.7, 1.7 Hz, 1H), 7.50-7.47 (m, 1H), 7.37 (d, J = 8.7 Hz, 1H), 7.22-7.21 (m, 2H), 7.20 (d, J = 2.2 Hz, 1H), 7.16 (d, J = 8.2 Hz, 2H), 7.06 (d, J = 8.3 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 6.84 (dd, J = 8.7, 2.3 Hz, 1H), 6.73 (s, 1H), 6.44 (s, 1H), 6.15 (s, 1H), 4.65-4.62 (m, 1H), 4.27 (d, J = 5.9 Hz, 2H), 3.52 (t, J = 4.6 Hz, 4H), 3.37 (s, 2H), 3.24-3.19 (m, 1H), 2.97 (p, J = 6.9 Hz, 1H), 2.90 (s, 3H), 2.89 (s, 1H), 2.77 (dd, J = 15.4, 4.4 Hz, 1H), 2.29 (s, 4H), 0.89 (d, J = 6.9 Hz, 6H). 13 C NMR (150 MHz, DMSO-d6) δ 169.24, 167.34, 164.65, 161.07, 157.96, 157.18, 155.24, 149.20, 143.95, 137.39, 134.77, 133.95, 132.81, 129.50, 129.05, 128.61, 128.48, 128.29, 126.48, 126.12, 120.83, 119.72, 118.00, 116.42, 115.56, 112.58, 112.21, 110.80, 104.87, 103.22, 70.18, 66.61, 62.60, 53.59, 41.78, 41.29, 37.04, 25.84, 22.81, 19.89. HRMS (ESI) m / z calcd for C 45 H 45N6O8: [M + H] + = 797.3293, found [M + H] + = 797.3285.

[0050] Example 5

[0051] Synthesis of compound 5e

[0052] Referring to the synthesis method of Example 1, compound 1a in Example 1 was replaced with compound 1b, and N-Boc-glycine was replaced with 3-((tert-butoxycarbonyl)amino)propanoic acid to obtain compound 5e as a white solid in a yield of 39%. 1 H NMR (600MHz, DMSO-d6) δ 11.18 (s, 1H), 9.72 (d, J = 53.0 Hz, 2H), 9.06 (t, J = 5.6Hz, 1H), 7.80 (dd, J = 7.8, 1.7 Hz, 1H), 7.49-7.46 (m, 1H), 7.34 (d, J = 8.7Hz, 1H), 7.22 (d, J = 2.3 Hz, 1H), 7.20 (s, 4H), 7.06 (dd, J = 8.3, 1.1 Hz,1H), 6.98-6.96 (m, 1H), 6.87 (dd, J = 8.7, 2.3 Hz, 1H), 6.74 (s, 1H), 6.45(s, 1H), 6.13 (s, 1H), 4.64-4.61 (m, 1H), 3.59 (q, J = 6.5 Hz, 2H), 3.52 (t,J = 4.6 Hz, 4H), 3.37 (s, 2H), 3.22-3.17 (m, 1H), 3.00-2.95 (m, 1H), 2.89 (s,3H), 2.87-2.85 (m, 1H), 2.82 (t, J = 6.8 Hz, 2H), 2.75-2.72 (m, 1H), 2.30 (s,4H), 0.90 (d, J = 6.9 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ 171.05, 166.97,164.64, 160.67, 157.96, 157.89, 155.25, 149.23, 144.10, 137.38, 134.71,133.93, 132.59, 129.34, 129.12, 128.80, 128.48, 128.25, 126.45, 126.11,120.84, 119.76, 118.02, 116.73, 115.29, 112.37, 112.18, 111.02, 104.94,103.24, 70.15, 66.61, 62.60, 53.59, 41.26, 36.99, 35.70, 34.10, 25.85, 22.83,19.91. HRMS (ESI) m / z calcd for C 46 H 47 N6O8: [M + H] + = 811.3450, found [M + H] + = 811.3452。

[0053] Example 6

[0054] Synthesis of compound 5f

[0055] The synthesis method of Reference Example 1 was referred to, and compound 1a in Example 1 was replaced with compound 1b, and N-Boc-glycine was replaced with 4-((tert-butoxycarbonyl)amino)butyric acid to obtain compound 5f as a white solid in a yield of 38%. 1H NMR (400MHz, DMSO-d6) δ 11.17 (s, 1H), 9.76 (s, 1H), 9.65 (s, 1H), 8.93 (t, J = 5.8Hz, 1H), 7.80 (dd, J = 7.8, 1.6 Hz, 1H), 7.50-7.46 (m, 1H), 7.35 (d, J = 8.7Hz, 1H), 7.24-7.20 (m, 5H), 7.06 (d, J = 8.1 Hz, 1H), 6.99-6.95 (m, 1H), 6.85(dd, J = 8.7 Hz, 2.3 Hz, 1H), 6.74 (s, 1H), 6.45 (s, 1H), 6.13 (s, 1H), 4.62(dd, J = 12.1 Hz, 5.6 Hz, 1H), 3.51 (t, J = 4.5 Hz, 4H), 3.38 (s, 2H), 3.32-3.30 (m, 1H), 3.23-3.16 (m, 1H), 2.97 (p, J = 6.9 Hz, 1H), 2.89 (s, 4H), 2.81(dd, J = 32.8, 5.2 Hz, 1H), 2.71 (d, J = 14.7 Hz, 1H), 2.58 (t, J = 7.3 Hz,2H), 2.30 (s, 4H), 1.88-1.81 (m, 2H), 0.90 (d, J = 6.9 Hz, 6H). 13 C NMR (100MHz, DMSO-d6) δ 172.58, 166.84, 164.71, 160.77, 158.30, 157.97, 155.25,149.25, 144.20, 134.72, 133.99, 132.66, 129.36, 129.24, 128.52, 128.28,126.53, 126.16, 120.86, 119.76, 118.03, 116.79, 112.46, 112.23, 111.04,105.02, 103.26, 70.22, 66.61, 62.61, 53.60, 41.35, 38.49, 37.03, 31.30,25.89, 24.72, 22.88, 19.96. HRMS (ESI) m / z calcd for C 47 H 49N6O8: [M + H] + = 825.3606, found [M + H] + = 825.3505.

[0056] Example 7

[0057] Synthesis of compound 5g

[0058]

[0059] First step, synthesis of compound 2g:

[0060] Compound la (1000 mg, 2.97 mmol) was dissolved in 15 mL of anhydrous DMF, 4-nitrophenyl chloroformate (718 mg, 3.56 mmol) and 4-methylmorpholine (360 mg, 3.56 mmol) were added successively, and the reaction was stirred at room temperature for 12 h. The reaction solution was poured into 150 mL of water and extracted with EA (3 x 60 mL). The organic phases were combined and concentrated under reduced pressure to 100 mL, and successively washed with 10% citric acid solution (100 mL x 3), saturated Na2CO3 solution (100 mL x 3), and saturated NaCl solution (100 mL x 1). The organic phase was concentrated under reduced pressure to obtain white solid, compound 2g (893 mg, yield: 60%), which was directly used in the next step without purification.

[0061] Second step, synthesis of compound 3g:

[0062] Compound 2g (800 mg, 1.59 mmol) was dissolved in 12 mL of anhydrous DCM, N-Boc-4-aminopiperidine (382 mg, 1.90 mmol) and 4-methylmorpholine (192 mg, 1.90 mmol) were added successively, and the reaction was stirred at room temperature for 4 h. The reaction solution was made up to 100 mL with DCM, and successively washed with 10% citric acid solution (100 mL x 3), saturated Na2CO3 solution (100 mL x 3), and saturated NaCl solution (100 mL x 1). The organic phase was concentrated under reduced pressure to obtain white solid, compound 3g (592 mg, yield: 66%), which was directly used in the next step without purification.

[0063] Third step, synthesis of compound 5g:

[0064] Compound 3g (100 mg, 0.18 mmol) was dissolved in 4 mL of dry DCM, 1 mL of TFA was added, and the reaction was stirred at room temperature for 0.5 h. The reaction was poured into 20 mL of saturated NaHC03solution and extracted with DCM (3 x 10 mL). The organic phases were combined and concentrated under reduced pressure to give a yellow solid, which was dissolved in 5 mL of dry DMF, and compound 4 (93 mg, 0.21 mmol), HATU (103 mg, 0.27 mmol), and DIPEA (28 mg, 0.22 mmol) were added in turn, and the reaction was stirred at room temperature for 12 h. The reaction was poured into 50 mL of water and extracted with EA (3 x 20 mL). The organic phases were combined and concentrated under reduced pressure, and the remaining residue was purified by silica gel column chromatography (H20:MeOH = 30:70, V / V) to give a white solid, which was compound 5g (60 mg, yield: 38%). 1 H NMR (400 MHz, DMSO-d6) δ 11.25 (s, 1H), 9.77 (s, 1H), 9.66 (s, 1H), 8.89 (d, J = 7.8 Hz, 1H), 7.54 (dd, J = 8.9, 3.2 Hz, 1H), 7.38 (td, J = 8.6, 3.1 Hz, 1H), 7.33 (d, J = 8.7 Hz, 1H), 7.25 (d, J = 8.3 Hz, 2H), 7.22-7.18 (m, 4H), 6.86 (dd, J = 8.7, 2.3 Hz, 1H), 6.76 (s, 1H), 6.45 (s, 1H), 6.10 (s, 1H), 4.63-4.60 (m, 1H), 4.10-4.00 (m, 3H), 3.55 (t, J = 4.6 Hz, 4H), 3.42 (s, 2H), 3.24-3.16 (m, 2H), 2.98 (p, J = 6.9 Hz, 2H), 2.83 (d, J = 7.7 Hz, 2H), 2.68 (s, 3H), 2.33 (t, J = 4.5 Hz, 4H), 1.85 (s, 2H), 1.50 (s, 2H), 0.92 (d, J = 6.9 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 166.58, 163.55, 160.11, 158.50,156.51, 155.23, 154.33, 146.52, 144.93, 137.47, 134.67, 131.49, 129.28,129.21, 128.92, 128.24, 126.32, 126.18, 122.64, 122.35, 121.11 (d, J C-F = 23.3Hz), 117.28, 115.14, 113.92 (d, J C-F = 23.8 Hz), 112.32, 112.26, 111.33,105.03, 103.27, 69.71, 66.67, 62.63, 53.64, 46.56, 37.21, 25.92, 22.90,20.03. HRMS (ESI) m / z calcd for C 49 H 51 FN7O8: [M + H] + = 884.3778.

[0065] Example 8

[0066] Synthesis of compound 5h

[0067] Referring to the synthesis method of Reference Example 7, the compound 1a in Example 7 was replaced with compound 1b to obtain a light yellow solid, which was compound 5h, in a yield of 40%. 1H NMR (600 MHz, DMSO-d6) δ 11.12 (s, 1H), 9.76 (s,1H), 9.65 (s, 1H), 8.89 (d, J = 7.8 Hz, 1H), 7.80 (dd, J = 7.8, 1.6 Hz, 1H),7.49-7.46 (m, 1H), 7.32 (d, J = 8.7 Hz, 1H), 7.25 (d, J = 8.2 Hz, 2H), 7.21-7.19 (m, 3H), 7.06 (d, J = 8.2 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 6.85 (dd, J= 8.7, 2.3 Hz, 1H), 6.76 (s, 1H), 6.45 (s, 1H), 6.13 (s, 1H), 4.64-4.61 (m,1H), 4.10-3.93 (m, 3H), 3.55 (t, J = 4.6 Hz, 4H), 3.44 (s, 2H), 3.23-3.17 (m,2H), 3.01-2.96 (m, 2H), 2.91-2.86 (m, 4H), 2.76 (dd, J = 15.7, 4.5 Hz, 1H),2.34 (s, 4H), 1.84 (s, 2H), 1.50 (s, 2H), 0.92 (d, J = 6.9 Hz, 6H). 13 C NMR(150 MHz, DMSO-d6) δ 166.55, 164.67, 160.07, 158.45, 157.95, 155.19, 154.27,149.22, 144.89, 134.46, 133.94, 132.49, 129.26, 129.18, 128.92, 128.48,128.21, 126.44, 126.16, 120.81, 119.73, 117.98, 117.11, 115.11, 112.22,112.15, 111.14, 105.00, 103.25, 70.20, 66.59, 62.55, 53.57, 46.52, 41.31,36.98, 25.88, 22.86, 19.92. HRMS (ESI) m / z calcd for C 49 H 52 N7O8: [M + H] += 866.3872, found [M + H] + = 866.3877.

[0068] Example 9

[0069] Synthesis of compound 9a

[0070]

[0071] First step, synthesis of compound 6a:

[0072] Compound 1a (1000 mg, 2.97 mmol) was dissolved in 15 mL of anhydrous DMF, and 4-(benzyloxy)-4-oxobutanoic acid (741 mg, 3.56 mmol), DMAP (363 mg, 2.97 mmol) and EDCI (1708 mg, 8.91 mmol) were added in turn, and the reaction was stirred at room temperature for 6 h. The reaction solution was poured into 150 mL of water, extracted with EA (3 x 60 mL). The organic phases were combined and concentrated under reduced pressure to obtain white solid, compound 6a (969 mg, yield: 62%), which was used directly in the next step without purification.

[0073] Second step, synthesis of compound 7a:

[0074] Compound 6a (800 mg, 1.52 mmol) was dissolved in 5 mL of anhydrous DMF, and 10% Pd / C (220 mg) was added under H2environment, and the reaction was stirred at room temperature for 6 h. The reaction solution was poured into 50 mL of water, extracted with EA (3 x 20 mL). The organic phases were combined and concentrated under reduced pressure to obtain compound 7a (560 mg, yield: 84%), which was used directly in the next step without purification.

[0075] Third step, synthesis of compound 9a:

[0076] The synthesis method of compound 8 refers to the literature (CN113121505A).

[0077] Compound 7a (219 mg, 0.50 mmol) was dissolved in 5 mL of anhydrous DMF, and compound 8 (196 mg, 0.60 mmol), HATU (285 mg, 0.75 mmol) and DIPEA (78 mg, 0.60 mmol) were added in turn, and the reaction was stirred at room temperature for 12 h. The reaction solution was poured into 50 mL of water, extracted with EA (3 x 20 mL). The organic phases were combined and concentrated under reduced pressure, and the remaining residue was purified by silica gel column chromatography (H2O:MeOH=30:70, V / V) to obtain white solid, compound 9a (139 mg, yield: 37%).1 HNMR (400 MHz, DMSO-d6) δ 11.95 (s, 1H), 10.81 (s, 1H), 9.58 (s, 1H), 9.43 (s,1H), 8.70 (s, 1H), 7.53 (dd, J = 9.0, 3.1 Hz, 1H), 7.37 (td, J = 8.7, 3.1 Hz,1H), 7.27 (s, 4H), 7.18-7.14 (m, 2H), 6.90 (s, 1H), 6.78 (d, J = 2.3 Hz, 1H),6.64 (dd, J = 8.6, 2.3 Hz, 1H), 6.26 (s, 1H), 6.04 (s, 1H), 4.60 (ddd, J =12.9, 5.2, 1.9 Hz, 1H), 3.21-3.16 (m, 1H), 3.04-2.97 (m, 1H), 2.84-2.79 (m,1H), 2.76 (s, 4H), 2.75-2.71 (m, 1H), 2.69 (s, 3H), 1.02 (d, J = 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 177.21, 163.57, 158.70, 157.61, 156.33, 154.97,154.59, 151.20, 146.46, 145.61, 134.29, 132.16, 131.58, 130.54, 128.20,127.72, 127.13, 127.02, 126.09, 122.34 (d, J C-F = 7.1 Hz), 121.89 (d, J C-F =7.6 Hz), 121.05 (d, J C-F = 23.6 Hz), 113.87 (d, J C-F = 23.3 Hz), 112.71,112.52, 111.24, 105.23, 102.83, 102.70, 69.89, 37.07, 28.94, 26.13, 23.04,20.09. HRMS (ESI) m / z calcd for C 40 H 37 FN7O7: [M + H] += 746.2733, found [M + H] + = 746.2735.

[0078] Example 10

[0079] Synthesis of compound 9b

[0080] Following the procedure of Reference Example 9, replacing intermediate 4-(benzyloxy)-4- oxobutanoic acid in Example 9 with 5-(benzyloxy)-5-oxopentanoic acid, afforded compound 9b as a light yellow solid in 38% yield. 1 H NMR (400 MHz, DMSO-d6) δ 11.87 (s, 1H), 11.29 (s, 1H), 10.05 (s, 1H), 9.56 (s, 1H), 9.36 (s, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.54 (dd, J = 8.9, 3.1 Hz, 1H), 7.41 - 7.35 (m, 2H), 7.24 (d, J = 2.2 Hz, 1H), 7.20 (dd, J = 8.9, 4.5 Hz, 1H), 7.12 - 7.08 (m, 2H), 6.88 (dd, J = 8.7, 2.3 Hz, 1H), 6.85 (s, 1H), 6.25 (s, 1H), 6.10 (s, 1H), 4.65 - 4.60 (m, 1H), 3.25 - 3.17 (m, 1H), 2.99 (p, J = 6.9 Hz, 1H), 2.86 - 2.82 (m, 2H), 2.69 (s, 3H), 2.64 (t, J = 7.3 Hz, 2H), 2.46 (t, J = 7.3 Hz, 2H), 1.96 (p, J = 7.3 Hz, 2H), 1.00 (d, J = 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 172.68, 171.22, 163.54, 157.68 (d, J C-F = 238.7 Hz), 157.46, 155.07, 154.74, 146.50, 145.93, 144.17, 139.08, 134.88, 131.64, 129.51, 128.04, 127.55, 126.38, 125.90, 122.58 (d, J C-F= 7.3 Hz), 122.29 (d, J C-F = 7.5 Hz), 121.10 (d, J C-F = 23.0 Hz), 119.50, 116.90, 113.91 (d, J C-F = 23.3Hz), 112.48, 112.36, 111.17, 105.28, 102.81, 69.70, 37.22, 35.70, 33.41,26.04, 23.01, 20.86, 20.02. HRMS (ESI) m / z calcd for C 41 H 39 FN7O7: [M + H] + = 760.2890, found [M + H] + = 760.2804.

[0081] Example 11

[0082] Synthesis of compound 9c

[0083] Following the synthetic procedure of Reference Example 9, replacing the intermediate 4-(benzyloxy)-4-oxobutanoic acid in Example 9 with 6-(benzyloxy)-6-oxohexanoic acid, afforded compound 9c as a light yellow solid in 39% yield. 1H NMR (600 MHz, DMSO-d6) δ 11.88 (s, 1H), 11.30 (s, 1H), 10.03 (s, 1H), 9.58 (s, 1H), 9.36 (s, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.54 (dd, J = 8.9, 3.1 Hz, 1H), 7.39 (td, J = 8.6, 3.1 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.22 (d, J = 2.2 Hz, 1H), 7.20 (dd, J = 8.9, 4.5 Hz, 1H), 7.09 (d, J = 8.8 Hz, 2H), 6.85 (dd, J = 8.7, 2.3 Hz, 1H), 6.83 (s, 1H), 6.24 (s, 1H), 6.10 (s, 1H), 4.61 (dt, J = 11.1, 3.3 Hz, 1H), 3.23-3.16 (m, 1H), 2.98 (p, J = 6.9 Hz, 1H), 2.89 (s, 1H), 2.84-2.82 (m, 2H), 2.73 (s, 1H), 2.69 (s, 1H), 2.61 (t, J = 6.8 Hz, 2H), 2.37 (t, J = 6.7 Hz, 2H), 1.69 (t, J = 3.62 Hz, 4H), 0.99 (d, J = 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 172.87, 171.64, 163.55, 162.83, 157.68 (d, J C-F = 238.8 Hz), 157.47, 155.08, 154.75, 146.50, 145.93, 144.19, 139.15, 134.88, 131.64, 129.47, 128.02, 127.56, 126.39, 125.90, 122.53, 122.28 (d, J C-F = 8.0 Hz), 121.11 (d, J C-F = 23.0 Hz), 119.47, 116.89, 113.91 (d, J C-F= 23.2 Hz), 112.51, 112.36, 111.15, 105.26, 102.81, 69.71, 37.22, 36.53, 33.83, 26.03, 25.01, 24.60, 23.00, 20.02. HRMS (ESI) m / z calcd for C 42 H 41 FN7O7: [M + H] + = 774.3046, found [M + H] + = 774.3044.

[0084] Example 12

[0085] Synthesis of compound 9d

[0086] Referring to the synthesis method of Reference Example 9, the compound 1a in Example 9 was replaced with compound 1b to obtain a light yellow solid, which is compound 9d, yield: 40%. 1 H NMR (400 MHz, DMSO-d6) δ 11.95 (s, 1H), 10.70 (s, 1H), 9.58 (s, 1H), 9.43 (s, 1H), 8.68 (s, 1H), 7.78 (dd, J = 7.7, 1.6 Hz, 1H), 7.48-7.44 (m, 1H), 7.27 (s, 4H), 7.14 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.1 Hz, 1H), 6.94 (t, J = 7.0 Hz, 1H), 6.90 (s, 1H), 6.75 (d, J = 2.3 Hz, 1H), 6.62 (dd, J = 8.6, 2.4 Hz, 1H), 6.27 (s, 1H), 6.07 (s, 1H), 4.61 (dd, J = 13.1, 5.0 Hz, 1H), 3.18 (td, J = 12.2, 4.7 Hz, 1H), 3.00 (p, J = 6.9 Hz, 1H), 2.89 (s, 3H), 2.87-2.80 (m, 1H), 2.76 (s, 4H), 2.66 (dd = 15.3, 4.6 Hz, 1H), 1.03 (d, J = 6.9 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 177.22, 164.76,157.62, 154.98, 154.60, 151.19, 149.16, 145.62, 134.30, 133.95, 132.16,131.63, 131.41, 128.48, 128.21, 127.73, 127.19, 127.13, 126.09, 120.51,119.50, 117.59, 112.59, 112.53, 111.11, 105.23, 102.84, 102.61, 70.45, 41.52,36.89, 28.95, 26.14, 23.05, 20.02. HRMS (ESI) m / z calcd for C 40 H 38 N7O7: [M + H] + =728.2827, found [M + H] + = 728.2825。

[0087] Example 13

[0088] Synthesis of compound 9e

[0089] The synthesis method of Reference Example 9 was referred to, compound 1a in Example 9 was replaced by compound 1b, 4-(benzyloxy)-4-oxobutanoic acid was replaced by 5-(benzyloxy)-5-oxopentanoic acid, to obtain a light yellow solid, i.e. compound 9e, yield: 40%. 1HNMR (400 MHz, DMSO-d6) δ 11.87 (s, 1H), 11.17 (s, 1H), 10.05 (s, 1H), 9.56(s, 1H), 9.35 (s, 1H), 7.79 (dd, J = 7.8, 1.7 Hz, 1H), 7.60-7.56 (m, 2H),7.50-7.46 (m, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.21 (d, J = 2.3 Hz, 1H), 7.11-7.08 (m, 2H), 7.05 (d, J = 8.2 Hz, 1H), 6.99-6.95 (m, 1H), 6.87-6.84 (m, 2H),6.25 (s, 1H), 6.14 (s, 1H), 4.62 (dd, J = 12.76, 4.98 Hz, 1H), 3.24-3.17 (m,1H), 2.99 (p, J = 6.9 Hz, 1H), 2.89 (d, J = 2.48 Hz, 4H), 2.73 (d, J = 0.6Hz, 1H), 2.63 (t, J = 7.3 Hz, 2H), 2.45 (t, J = 7.3 Hz, 2H), 1.99-1.92 (m,2H), 1.00 (d, J = 6.9 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6) δ 172.66, 171.23,164.72, 157.47, 155.07, 154.75, 149.23, 145.94, 144.16, 139.08, 134.71,134.00, 132.72, 129.51, 128.51, 128.05, 127.55, 126.54, 125.91, 120.84,119.73, 119.50, 117.99, 116.79, 112.49, 112.23, 111.05, 105.29, 102.81,70.24, 41.37, 37.05, 35.71, 33.41, 26.05, 23.02, 20.86, 19.95. HRMS (ESI) m / z calcd for C 41 H 40 N7O7: [M + H] + = 742.2984, found [M + H] += 742.2913.

[0090] Example 14

[0091] Synthesis of compound 9f

[0092] Following the procedure of Reference Example 9, replacing compound la in Example 9 with compound lb, 4-(benzyloxy)-4-oxobutanoic acid with 6-(benzyloxy)-6- oxohexanoic acid, resulted in compound 9f as a light yellow solid in 39% yield. 1 HNMR (400 MHz, DMSO-d6) δ 11.87 (s, 1H), 11.17 (s, 1H), 10.02 (s, 1H), 9.57(s, 1H), 9.36 (s, 1H), 7.79 (dd, J = 7.8, 1.6 Hz, 1H), 7.59-7.56 (m, 2H),7.50-7.45 (m, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.19 (d, J = 2.3 Hz, 1H), 7.10-7.08 (m, 2H), 7.05 (d, J = 8.1 Hz, 1H), 6.98-6.94 (m, 1H), 6.85-6.82 (m, 2H),6.25 (s, 1H), 6.13 (s, 1H), 4.62 (dd, J = 12.4, 5.2 Hz, 1H), 3.24-3.16 (m,1H), 2.99 (p, J = 6.9 Hz, 1H), 2.89 (d, J = 2.6 Hz, 4H), 2.73 (s, 1H), 2.60(t, J = 6.7 Hz, 2H), 2.37 (t, J = 5.4 Hz, 2H), 1.69 (p, J = 3.7 Hz, 4H), 0.99(d, J = 6.9 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 172.85, 171.63, 164.72,157.47, 155.08, 154.75, 149.23, 145.94, 144.18, 139.15, 134.70, 134.00,132.71, 129.47, 128.51, 128.02, 127.55, 126.55, 125.91, 120.84, 119.72,119.47, 117.99, 116.77, 112.51, 112.23, 111.02, 105.27, 102.82, 70.24, 41.37,37.04, 36.53, 33.83, 26.04, 25.01, 24.59, 23.01, 19.94. HRMS (ESI) m / z calcdfor C 42 H 42 N7O7: [M + H] + = 756.3140, found [M + H] + = 756.3138。

[0093] Example 15

[0094] Synthesis of compound 11a

[0095] The synthesis method of compound 10 was referred to the literature (J. Med. Chem. 66 (2023) 733-751.). The synthesis method of Reference Example 9 was referred to, and compound 8 in Example 9 was replaced with compound 10 to obtain a white solid, which was compound 11a, in a yield of 39%. 1H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H), 8.14 (t, J = 5.5 Hz, 1H), 8.05 (s, 1H), 7.54 (dd, J = 8.9, 3.1 Hz, 1H), 7.41-7.34 (m, 2H), 7.25 (s, 1H), 7.21-7.18 (m, 2H), 6.85 (dd, J = 8.7, 2.3 Hz, 1H), 6.69 (s, 2H), 6.10 (s, 1H), 5.26 (s, 2H), 4.63-4.59 (m, 1H), 3.72 (s, 3H), 3.29 (q, J = 7.0 Hz, 3H), 3.24-3.16 (m, 1H), 2.82 (s, 2H), 2.77 (t, J = 6.9 Hz, 2H), 2.69 (s, 3H), 2.57-2.52 (m, 3H), 2.24 (s, 3H), 2.15 (s, 3H). 13 C NMR (100 MHz, DMSO-d6) δ 172.47, 171.28, 163.85, 163.54, 160.02, 157.69 (d, J C-F = 238.8 Hz), 153.95,153.84, 152.16, 149.30, 146.50, 144.27, 134.86, 131.61, 126.34, 125.58,123.93, 122.61, 122.32, 121.11 (d, J C-F = 23.4 Hz), 116.88, 113.91 (d, J C-F =23.0 Hz), 112.48, 112.34, 111.04, 108.02, 95.63, 89.82, 74.68, 69.71, 60.33,55.42, 46.92, 40.81, 38.62, 37.22, 30.48, 29.75, 20.56, 20.00, 13.37, 10.76.HRMS (ESI) m / z calcd for C 42 H 40 ClFN9O5: [M + H] + = 804.2819, found [M + H] + =804.2842。

[0096] Example 16

[0097] Synthesis of compound 11b

[0098] Following the procedure of Reference Example 9, replacing compound 8 in Example 9 with compound 10, 4-(benzyloxy)-4-oxobutanoic acid with 5-(benzyloxy)-5- oxopentanoic acid, resulted in compound 11b as a light brown solid in 43% yield. 1 HNMR (400 MHz, DMSO-d6) δ 11.29 (s, 1H), 8.08-8.05 (m, 2H), 7.54 (dd, J = 8.9,3.1 Hz, 1H), 7.41-7.37 (m, 1H), 7.35 (d, J = 8.9 Hz, 1H), 7.25 (s, 1H), 7.21-7.18 (m, 2H), 6.84 (dd, J = 8.7, 2.3 Hz, 1H), 6.68 (s, 2H), 6.10 (s, 1H),5.24 (s, 2H), 4.63-4.59 (m, 1H), 3.30-3.24 (m, 2H), 3.22-3.17 (m, 1H), 2.83(d, J = 8.3 Hz, 2H), 2.69 (s, 3H), 2.57 (q, J = 6.9 Hz, 4H), 2.22 (s, 5H),2.15 (s, 3H), 1.88 (p, J = 7.4 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 172.70,172.07, 163.85, 163.54, 160.02, 157.69 (d, J C-F = 238.6 Hz), 153.94, 152.16,149.28, 146.50, 144.16, 134.88, 131.54, 126.37, 125.57, 123.92, 122.59 (d,J C-F = 7.5 Hz), 122.30 (d, J C-F = 7.7 Hz), 121.11 (d, J C-F = 23.0 Hz), 116.89,113.91 (d, J C-F= 23.7 Hz), 112.46, 112.37, 111.14, 108.02, 95.66, 89.90,74.62, 69.71, 60.32, 56.54, 46.91, 38.49, 37.22, 34.81, 33.48, 29.52, 21.16,20.53, 20.02, 19.07, 13.37, 10.74. HRMS (ESI) m / z calcd for C 43 H 42 ClFN9O5: [M +H] + = 818.2976, found [M + H] + = 818.2973.

[0099] Example 17

[0100] Synthesis of compound 11c

[0101] Following the procedure of Reference Example 9, replacing compound 8 in Example 9 with compound 10, 4-(benzyloxy)-4-oxobutanoic acid with 6-(benzyloxy)-6- oxohexanoic acid, gave compound 11c as a light brown solid in 43% yield. 1 HNMR (400 MHz, DMSO-d6) δ 11.30 (s, 1H), 8.07 (s, 1H), 8.04 (t, J = 5.8 Hz,1H), 7.54 (dd, J = 8.9, 3.1 Hz, 1H), 7.40 (dd, J = 8.7, 3.1 Hz, 1H), 7.35 (d,J = 8.6 Hz, 1H), 7.25 (s, 1H), 7.21-7.18 (m, 2H), 6.84 (dd, J = 8.7, 2.3 Hz,1H), 6.70 (s, 2H), 6.10 (s, 1H), 5.26 (s, 2H), 4.64-4.59 (m, 1H), 3.72 (s,3H), 3.26 (q, J = 6.8 Hz, 2H), 3.22-3.16 (m, 1H), 2.82 (d, J = 5.7 Hz, 2H),2.68 (s, 3H), 2.55 (t, J = 7.0 Hz, 4H), 2.23 (s, 3H), 2.16 (s, 5H), 1.66-1.61(m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 172.84, 172.44, 164.02, 163.51, 159.99,157.65 (d, J C-F = 238.7 Hz), 153.80, 153.74, 152.13, 149.02, 146.47, 144.14,134.83, 131.58, 131.46, 126.34, 125.65, 124.04, 122.54 (d, J C-F = 7.1 Hz),122.26 (d, J C-F = 7.6 Hz), 121.07 (d, J C-F = 23.3 Hz), 116.85, 113.87 (d, J C-F =23.3 Hz), 112.45, 112.32, 111.09, 107.97, 95.67, 89.88, 74.54, 69.66, 60.33,46.78, 38.42, 37.17, 35.48, 33.77, 25.14, 24.58, 20.51, 19.97, 13.36, 10.73.HRMS (ESI) m / z calcd for C 44 H 44 ClFN9O5: [M + H] + = 832.3132, found [M + H] + =832.3157.

[0102] Example 18

[0103] Synthesis of compound 11d

[0104] The synthesis method of Reference Example 9 was referred to, and compound 8 in Example 9 was replaced with compound 10, and compound 1a was replaced with compound 1b, to obtain a light brown solid, i.e. compound 11d, in a yield of 46%. 1H NMR (400 MHz, DMSO-d6) δ 11.16 (s, 1H), 8.14 (t, J = 5.7 Hz, 1H), 8.05 (s, 1H), 7.79 (dd, J = 7.8, 1.6 Hz, 1H), 7.50-7.45 (m, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.25 (s, 1H), 7.16 (d, J = 2.3 Hz, 1H), 7.05 (d, J = 7.2 Hz, 1H), 6.96 (td, J = 7.5, 1.0 Hz, 1H), 6.84 (dd, J = 8.7, 2.3 Hz, 1H), 6.69 (s, 2H), 6.13 (s, 1H), 5.26 (s, 2H), 4.64-4.59 (m, 1H), 3.71 (s, 3H), 3.33-3.26 (m, 3H), 3.23-3.16 (m, 1H), 2.89 (s, 4H), 2.78-2.72 (m, 3H), 2.57-2.52 (m, 3H), 2.24 (s, 3H), 2.15 (s, 3H). 13 CNMR (100 MHz, DMSO-d6) δ 172.43, 171.28, 164.71, 163.84, 160.01, 153.94, 153.83, 152.15, 149.29, 149.21, 144.25, 134.67, 133.98, 132.66, 131.57, 128.50, 126.49, 125.57, 123.93, 120.82, 119.70, 117.97, 116.74, 112.47, 112.19, 110.90, 108.01, 95.62, 89.81, 74.67, 70.23, 60.32, 46.91, 41.35, 38.61, 37.03, 30.47, 29.74, 20.55, 19.92, 13.36, 10.75. HRMS (ESI) m / z calcdfor C 42 H 41 ClN9O5: [M + H] + = 786.2914, found [M + H] + = 786.2831。

[0105] Example 19

[0106] Synthesis of compound 11e

[0107] Following the procedure of Reference Example 9, replacing compound 8 in Example 9 with compound 10, compound la with compound lb, and 4-(benzyloxy)-4- oxobutanoic acid with 5-(benzyloxy)-5-oxopentanoic acid, resulted in compound 11e as a light brown solid in 45% yield. 1 H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 8.08 (t, J = 5.8 Hz, 1H), 8.05 (s, 1H), 7.79 (dd, J = 7.8, 1.6 Hz, 1H), 7.50-7.46 (m, 1H), 7.34 (d, J = 8.7 Hz, 1H), 7.25 (s, 1H), 7.17 (d, J = 2.3 Hz, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.96 (t, J = 7.0 Hz, 1H), 6.83 (dd, J = 8.7, 2.3 Hz, 1H), 6.69 (s, 2H), 6.13 (s, 1H), 5.24 (s, 2H), 4.64-4.59 (m, 1H), 3.71 (s, 3H), 3.30-3.25 (m, 2H), 3.23-3.16 (m, 1H), 2.89 (s, 4H), 2.74 (dd, J = 15.3, 4.4 Hz, 1H), 2.57 (td, J = 7.3, 4.1 Hz, 4H), 2.22 (s, 5H), 2.15 (s, 3H), 1.88 (p, J = 7.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 172.67, 172.07, 164.71, 163.84, 160.02, 153.94, 153.83, 152.16, 149.29, 149.23, 144.15, 134.70, 133.99, 132.67, 131.54, 128.51, 126.52, 125.57, 123.92, 120.82, 119.72, 117.98, 116.76, 112.46, 112.22, 111.01, 108.02, 95.66, 89.90, 74.62, 70.23, 60.32, 46.90, 41.37, 38.48, 37.03, 34.81, 33.48, 21.15, 20.53, 19.94, 13.37, 10.74. HRMS (ESI) m / z calcd for C 43 H 43 ClN9O5: [M + H] + = 800.3070, found [M + H] + =800.2978.

[0108] Example 20

[0109] Synthesis of compound 11f

[0110] The synthesis method of Reference Example 9 was referred to, the compound 8 in Example 9 was replaced by compound 10, the compound 1a was replaced by compound 1b, and the 4-(benzyloxy)-4-oxobutanoic acid was replaced by 6-(benzyloxy)-6-oxohexanoic acid, to obtain a light brown solid, i.e. compound 11f, in the yield of 44%. 1H NMR (400 MHz, DMSO-d6) δ 11.17 (s, 1H), 8.05 (s, 1H), 8.02 (d, J = 5.7 Hz, 1H), 7.80 (dd, J = 7.8, 1.6 Hz, 1H), 7.50-7.45 (m, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.25 (s, 1H), 7.18 (d, J = 2.2 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 6.96 (t, J = 7.5 Hz, 1H), 6.83 (dd, J = 8.7, 2.3 Hz, 1H), 6.69 (s, 2H), 6.13 (s, 1H), 5.25 (s, 2H), 4.62 (dd, J = 13.1, 4.7 Hz, 1H), 3.71 (s, 3H), 3.27 (q, J = 6.8 Hz, 2H), 3.22-3.16 (m, 1H), 2.89 (s, 4H), 2.75 (dd, J = 15.4, 4.3 Hz, 1H), 2.55 (t, J = 7.1 Hz, 4H), 2.23 (s, 3H), 2.15 (s, 5H), 1.65-1.61 (m, 4H). 13 C NMR (100 MHz, DMSO-d6) δ 172.83, 172.48, 164.70, 163.85, 160.01, 153.94, 153.82, 152.16, 149.29, 149.23, 144.17, 134.69, 133.97, 132.66, 131.50, 128.50, 126.53, 125.57, 123.94, 120.83, 119.73, 117.98, 116.75, 112.49, 112.22, 110.98, 108.02, 95.68, 89.89, 74.59, 70.22, 60.31, 46.90, 41.35, 38.47, 37.02, 35.52, 33.81, 25.18, 24.62, 20.54, 19.93, 13.35, 10.74. HRMS (ESI) m / z calcd for C 44 H 45 ClN9O5: [M + H] += 814.3227, found[M + H] + = 814.3132.

[0111] Example 21

[0112] In vitro antitumor activity and cytotoxicity test (IC50) of the compounds prepared in Examples 1-20 of this invention 50 Both antitumor activity and cytotoxicity can be measured using IC50. 50 This indicates that the IC50 of tumor cells... 50 Antitumor activity, IC50 in normal cells 50 : Cytotoxicity.

[0113] The inhibitory effects of the compounds of this invention on the proliferation of three types of tumor cells (human colon cancer cells HCT116, human breast cancer cells MCF-7, and human pancreatic cancer cells MIA PaCa-2) were tested. Cytotoxicity tests were also performed on the compounds using a normal cell line (human umbilical vein endothelial cells HUVEC). The CCK8 assay was used, and all culture media were mixed media: 90% basal medium, 10% fetal bovine serum (Gibco), and 1% penicillin-streptomycin solution (HyClone). Specifically, HCT116 cells used McCoy's 5A basal medium (Gibco), MCF-7 cells used MEM / NEAA basal medium (Gibco), MIA PaCa-2 cells used DMEM high-glucose basal medium (Gibco), and HUVEC cells used ECM basal medium (ScienCell). Cells were cultured at 37 °C in an incubator with 5% CO2 and sterile water evaporation. Cells were purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai. 50 The value represents the cell growth inhibition rate, and the drug concentration corresponding to a value of 50%.

[0114] Step a: Cells in the logarithmic growth phase are treated with 7 × 10⁻⁶ cells / day. 3 The cells / well were planted in 96-well plates at a density of 5% CO2 and cultured in a humid incubator for 24 h.

[0115] Step b: Prepare a stock solution of the test compound with a concentration of 10 mM using DMSO, and then dilute the stock solution of the compound three times with culture medium in a sterile EP tube (1.5 mL) to the required concentration.

[0116] Step c: Aspirate the culture medium from the 96-well plate and add the different concentrations of drug solution to be tested in sequence, repeating the process in triplicate, adding 100 µL of drug solution to each well;

[0117] Step d: Put the 96-well plate into the incubator for 72 h;

[0118] Step e: Aspirate the culture medium of the 96-well plate, and add 100 μL / well of CCK8 reagent diluted 10 times with DMEM high glucose medium;

[0119] Step f: Put the 96-well plate into the cell incubator for 0.5 h, and use the enzyme label instrument (Wellscan MK, Labsystems) to read the absorbance value (OD value) at 450 nm, and use the software Graphd Prism 9.0 to calculate the IC 50 value.

[0120] Experimental results: The IC 50 values of the compounds of the present application are shown in Table 1. Most of the compounds, except for compounds 5g and 5h, showed significant inhibitory activity (IC 50 = 7.7-237.6 nM) against the tumor cell lines tested. In addition, HCT116 cells were the most sensitive to the compounds of the present application, among which compound 5a showed the strongest anti-tumor activity (IC 50 = 7.7 nM), which was superior to compound 1a (IC 50 = 10.2 nM). Notably, the toxicity of compound 5a (IC 50 = 74.2 nM) to HUVEC cells was reduced by about 6.9 times compared to compound 1a (IC 50 = 10.7 nM). Therefore, compound 5a showed good in vitro anti-tumor activity against colon cancer cells HCT116 and significantly reduced cytotoxicity compared to compound 1a.

[0121] Table 1 In vitro anti-tumor activity and cytotoxicity test (IC 50 , unit nmoL / L) of compounds a

[0122]

[0123] a IC 50 values were the average of three independent experiments, expressed as mean ± standard deviation.

[0124] Example 22

[0125] Cycle and apoptosis test of compound 5a prepared by the present application in HCT116 cells

[0126] Step a: The cell culture conditions refer to Example 21; cells in the logarithmic growth phase were seeded at 2×10 5The density of 1 cell / hole is planted in a 6-hole plate (Corning) and placed in a cell incubator for overnight culture;

[0127] Step b: Compound 5a is configured into a 10 mM stock solution using DMSO, and then the compound stock solution is prepared into the required concentration with the culture medium in a 5.0 mL sterile EP tube, and a blank control group (culture medium containing 1 ‰ DMSO) is set;

[0128] Step c: The culture medium in the 6-hole plate is sucked out, and the test liquid is sequentially added to the 6-hole plate, 2 mL of the test liquid is added to each hole, and 3 times of parallel test are performed for each concentration;

[0129] Step d: The 6-hole plate is placed in an incubator for 48 h;

[0130] Step e: The culture medium in each hole of the 6-hole plate is sequentially collected in a 15 mL centrifuge tube, 1 mL of PBS is added to wash the floating cells, and then collected in the centrifuge tube;

[0131] Step f: 0.5 mL of trypsin (gibco) without EDTA is added to each hole for 0.5 min of digestion, 2 mL of complete culture medium is added to terminate the digestion, the cells are blown off and collected in the centrifuge tube, and then centrifuged at 2000 r / min for 5 min;

[0132] Step g: The upper culture medium is sucked and discarded, 1 mL of PBS is added to resuspend the cells, and then centrifuged at 2000 r / min for 5 min, and the PBS is repeatedly washed for 2 times;

[0133] Step h: The upper PBS is sucked and discarded, and cell cycle (MULTI SCIENCES) or apoptosis (BioLegend ® ) detection reagents are added, the cells are uniformly blown by using a pipette, then transferred to a flow cytometry tube, and incubated at room temperature for 15 min in the dark, and then detected by using a DAKEWE EXFLOW-206 flow cytometer, and the data is processed by using software GraphPad Prism 9.

[0134] Experimental results: The cycle and apoptosis test results of the prepared compound 5a in HCT116 cells are as shown in Figure 1 Figure 1 ​is a schematic diagram of the cycle and apoptosis test results of compound 5a in HCT116 cells. Wherein, A is a schematic diagram of the effect of compound 5a on the cell cycle of HCT116, B is a schematic diagram of the effect of compound 5a on the apoptosis of HCT116, C is a schematic diagram of the cell cycle of HCT116 in the blank control group, D is a schematic diagram of the apoptosis of HCT116 in the blank control group, E is a statistical analysis result schematic diagram of figures A and C, and F is a statistical analysis result schematic diagram of figures B and D.

[0135] As shown in A, C and E in the figure, with the increase of the concentration of compound 5a, the G2 phase proportion of HCT116 cells was 48.86% (20 nM), 83.06% (50 nM) and 88.34% (100 nM) respectively, and the G2 phase proportion of HCT116 cells in the blank control group was 19.20%, and compound 5a could inhibit HCT116 cells in G2 phase in a concentration-dependent manner. As shown in B, D and F in the figure, with the increase of the concentration of compound 5a, the apoptosis proportion of HCT116 cells was 18.45% (20 nM), 25.85% (50 nM) and 43.92% (100 nM) respectively, and the apoptosis proportion of HCT116 cells in the blank control group was only 0.44%, and compound 5a could promote the apoptosis of HCT116 cells in a concentration-dependent manner.

[0136] Example 23

[0137] Hsp90 inhibitory activity of compound 5a prepared by the present application

[0138] Step a: dilute the DMSO concentrated solution (10 mM) of the compound to different concentrations with buffer (buffer: 20 mM HEPES, pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 0.01% NP40, 2 mM DTT, 0.1 mg / mL BGG);

[0139] Step b: dilute GM4-FITC and purified Hsp90α in the same way N (GM4-FITC, 30 nM; Hsp90α N , 90 nM);

[0140] Step c: add equal volumes (20 μL) of compound diluent, GM4-FITC diluent and Hsp90α N diluent into a 384-well plate (Corning #3575) in sequence, and set control group 1: 20 μL GM4-FITC + 40 μL buffer, control group 2: 20 μL GM4-FITC + 20 μL Hsp90αN + 20 μL buffer.

[0141] Step d: After covering the 384-well plate, shake at room temperature, avoid light for 30 min, use SpectraMax iD3 Multi-Mode Microplate Reader to detect the fluorescence polarization (FP) value (excitation wavelength: 485 nm, emission wavelength: 535 nm).

[0142] Step e: Calculate the Hsp90 inhibition rate of the target compound at each concentration:

[0143] inhibition rate (%) = [1-(P test -P min ) / (P max -P min )] × 100%

[0144] Use software Graphd Prism 9.0 to calculate IC 50 value (P min : FP value of control group 1, P max : FP value of control group 2, P test : FP value of the well containing the target compound to be tested).

[0145] Experimental results: The Hsp90 inhibition activity of the compound 5a prepared by the present application is as shown in Figure 2 , Figure 2 is a schematic diagram of the Hsp90 inhibition activity results of compound 5a. Among them, A is a schematic diagram of the Hsp90 inhibition activity of Hsp90 inhibitor AUY922 on Hsp90, and B is a schematic diagram of the Hsp90 inhibition activity of compound 5a on Hsp90.

[0146] As can be seen from the figure, the Hsp90 inhibition activity of compound 5a is comparable to that of Hsp90 inhibitor AUY922 (5a, IC 50 = 19.4 nM; AUY922, IC 50 = 8.8 nM), and compound 5a can retain Hsp90 inhibition activity.

[0147] Example 24

[0148] The in vivo anti-tumor activity of the compound 5a prepared by the present application in a nude mouse HCT116 xenograft tumor model (all animal experiments were approved by the Experimental Animal Management and Use Committee of the Navy Medical University (Second Military Medical University), ethical approval number: SMMU22377145).

[0149] Step a: Female nude mice (body weight: 18 g-20 g) were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences. The experimental nude mice were bred under specific pathogen-free conditions, placed in a 12 / 12 h light / dark environment, and the temperature was controlled at 25±1 ℃. During the experiment, the nude mice could freely obtain water and food to ensure their health;

[0150] Step b: The cell suspension (about 5×10 6 HCT116 cells) was subcutaneously implanted into the right limb of the armpit of the nude mice, and when the implanted tumor volume reached 100 to 200 cubic millimeters, the administration was started;

[0151] Step c: The above nude mice were randomly divided into groups (5 in each group), and administered by intraperitoneal injection for 21 consecutive days. During the administration, the length (A) and width (B) of the tumor were measured every 2 days using a vernier caliper, the tumor volume (Tumor volume (TV)=(A×B 2 ) / 2) was calculated, and the body weight of the nude mice was measured;

[0152] Step d: After the end of the experiment, the nude mice were sacrificed, and the tumor was removed and weighed (for the nude mice administered orally: the intraperitoneal injection administration was changed to oral administration for 15 consecutive days, and the other conditions remained unchanged).

[0153] Grouping and administration:

[0154] Intraperitoneal injection: Compound 1a: 5 mg / kg; AUY922: 7 mg / kg; Compound 1a+AUY922: 5 mg / kg+7 mg / kg; Compound 5a: 6 mg / kg; Compound 5a: 12 mg / kg.

[0155] Oral administration: Compound 1a: 10 mg / kg; AUY922: 14 mg / kg; Compound 1a+AUY922: 10 mg / kg+14 mg / kg; Compound 5a: 12 mg / kg; Compound 5a: 24 mg / kg.

[0156] The in vivo anti-tumor activity of the compound 5a prepared in the present application in the nude mouse HCT116 xenograft tumor model is as shown in Figure 3 , Figure 3is a schematic diagram of the in vivo anti-tumor activity of compound 5a in a nude mouse HCT116 xenograft tumor model. Wherein, A is a schematic diagram of the tumor growth inhibition of compound 5a at a dose of 6 mg / kg and 12 mg / kg by intraperitoneal injection, B is a schematic diagram of the body weight change of mice after intraperitoneal injection, C is a schematic diagram of the tumor weight of nude mice dissected on the 21st day of intraperitoneal injection administration, D is a schematic diagram of the relative tumor volume (v / v) on the 21st day of intraperitoneal injection administration, E is a schematic diagram of the tumor growth inhibition of compound 5a at a dose of 12 mg / kg and 24 mg / kg by oral administration, F is a schematic diagram of the body weight change of mice after oral administration, G is a schematic diagram of the tumor weight of nude mice dissected on the 15th day of oral administration, H is a schematic diagram of the relative tumor volume (v / v) on the 15th day of oral administration. n = 5; * P < 0.05; ** P < 0.01; *** P < 0.001; ns: no significant difference (T test); v / v: tumor volume on the last day / tumor volume on the first day.

[0157] As shown in Figures A, C, and D, after 21 consecutive days of intraperitoneal injection, compound 5a exhibited dose-dependent in vivo antitumor effects (6 mg / kg, TGI = 63.1%; 12 mg / kg, TGI = 72.9%), superior to compound 1a (5 mg / kg (equivalent to 12 mg / kg of compound 5a), TGI = 19.0%), the Hsp90 inhibitor AUY922 (7 mg / kg (equivalent to 12 mg / kg of compound 5a), TGI = 47.0%), and the combination of compound 1a and AUY922 (5 mg / kg + 7 mg / kg, TGI = 41.8%). Furthermore, as shown in Figure B, after intraperitoneal injection of different doses of compound 5a, the body weight changes in nude mice were similar and did not decrease compared to day 1. However, the body weight of mice in the AUY922 group and the combination of compound 1a and AUY922 group decreased, indicating reduced toxicity of compound 5a. As shown in Figures E, G, and H, after 15 days of continuous oral administration, compound 5a exhibited dose-dependent in vivo antitumor effects (12 mg / kg, TGI = 47.3%; 24 mg / kg, TGI = 61.2%), significantly superior to compound 1a (10 mg / kg (equivalent to 24 mg / kg of compound 5a), TGI = 13.5%), the Hsp90 inhibitor AUY922 (14 mg / kg (equivalent to 24 mg / kg of compound 5a), TGI = 21.9%), and the combination of compound 1a and AUY922 (10 mg / kg + 14 mg / kg, TGI = 25.6%). Furthermore, as shown in Figure F, oral administration of compound 5a had almost no effect on the body weight changes in mice. These results indicate that compound 5a exhibits excellent in vivo antitumor activity after both intraperitoneal injection and oral administration.

[0158] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A evodiamine conjugate or a pharmaceutically acceptable salt thereof, characterized in that, The structural general formula is selected from one of the following structures: ； wherein R1is selected from hydrogen, halogen; L1is selected from -(CH2)n1-, ; n1is selected from a positive integer from 1 to 10; R2is selected from hydrogen, halogen; L2is selected from -(CH2)n2-; n2is selected from 0 to 10; R3is selected from hydrogen, halogen; L3is selected from -(CH2)n3-; n3is selected from 0 to 10.

2. The evodiamine conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, n1 is selected from 1, 2, 3, 4, 5.

3. The evodiamine conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein n2 is selected from 0, 1, 2, 3, 4, 5.

4. The evodiamine conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein n3 is selected from 0, 1, 2, 3, 4, 5.

5. The evodiamine conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein The structure of the evodiamine conjugate is selected from one of the following structures: 。 6. The evodiamine conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein The pharmaceutical salt is an acid addition salt of the evodiamine conjugate with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

7. Use of the evodiamine conjugate or the pharmaceutical salt thereof according to any one of claims 1 to 6 in the preparation of an antitumor drug.

8. Use according to claim 7, characterized in that, The tumor is selected from colon cancer, breast cancer, pancreatic cancer, ovarian cancer, lung cancer, liver cancer, kidney cancer, prostate cancer, thyroid cancer, skin cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, osteosarcoma, and tumors of the central or peripheral nervous system.

9. Use of the evodiamine conjugate or the pharmaceutical salt thereof according to any one of claims 1 to 6 in the preparation of an Hsp90 inhibitor.

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