Preparation and Application of Bifunctional Molecular Compounds Based on RSL3-induced Degradation of GPX4 Protein

Abstract

The present invention belongs to the technical field of chemical biology, and particularly relates to the preparation and application of a bifunctional molecular compound based on RSL3-induced degradation of GPX4 protein. The present invention provides a bifunctional molecular compound that can degrade GPX4 in HT1080 cells, promote lipid ROS accumulation and induce ferroptosis in cells. Compared with the prior art, the bifunctional molecular compound provided by the present invention has the advantages of low drug effective dose and does not require long-term and high-intensity binding to the target protein. The present invention also provides a preparation method of the bifunctional molecular compound, and the preparation method has the advantages of being simple and easy to operate, low cost, and suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical biology, and particularly relates to the preparation and application of a bifunctional molecular compound based on the degradation of GPX4 protein induced by RSL3. Background Art

[0002] Glutathione Peroxidase 4 (GPX4) is a member of the GPX family and one of the key negative regulatory factors in the ferroptosis pathway. GPX4 can reduce toxic lipid peroxides to non-toxic substances and maintain the intracellular redox balance. Most tumor cells contain relatively high levels of free Fe 2+ , and the mitochondrial activity is abnormally increased. These manifestations indicate that tumor cells have a high sensitivity to ferroptosis. At present, the main treatments for tumors are immunotherapy, chemotherapy, and radiotherapy, and their principles are all apoptosis. Therefore, when cancer cells evade apoptosis, resulting in drug resistance and disease recurrence, major challenges will arise. Ferroptosis has an inhibitory effect on the occurrence and development of tumors, and inducing ferroptosis can overcome the drug resistance of tumors and activate the immune response of tumors. Therefore, treating tumors by regulating ferroptosis is a new treatment method.

[0003] RSL3 is a small molecule inhibitor of GPX4. By binding to the active site of the selenocysteine of GPX4 and inhibiting the enzymatic activity of GPX4, it promotes the accumulation of lipid ROS and induces cellular ferroptosis. However, RSL3 has problems such as toxic side effects and drug resistance. Since the PROTAC technology was proposed, it has been widely used to target a variety of cancer targets, and using PROTAC to degrade tumor-related proteins has become a very promising cancer treatment strategy. PROTAC molecules can solve the drug resistance problem of small molecule inhibitors, and because PROTAC mainly plays a catalytic role, the dosing concentration and dosing frequency are both relatively low, which can reduce the possibility of toxic side effects and off-target effects. RSL3-PROTACs provide a new and effective method for inhibiting GPX4 from the degradation level and provide a new treatment strategy for diseases such as tumors. Summary of the Invention

[0004] The object of the present invention is to address the deficiencies of the prior art and provide a bifunctional molecular compound based on RSL3-induced degradation of GPX4 protein, as well as its preparation and application. By degrading GPX4 in cells, it promotes the accumulation of lipid ROS and induces ferroptosis. Research has found that RSL3-1 and RSL3-3 derived from the compound RSL3 select cIAP as the E3 ligase ligand and both have a certain degradation effect on GPX4; moreover, this phenomenon can be reversed by the proteasome inhibitors MG132 and ML4924, indicating that this type of compound exerts its effect through the ubiquitin-proteasome pathway. In addition, the research results show that the degradation of GPX4 promotes the accumulation of lipid ROS and induces ferroptosis. Therefore, designing a GPX4 protein degrader based on RSL3 will provide an effective strategy with high safety, strong anti-drug resistance, and broad application prospects for cancer treatment.

[0005] The specific solutions of the present invention are as follows:

[0006] The present invention provides two bifunctional molecular compounds based on RSL3-induced degradation of GPX4 protein as shown in Formula I and Formula II below, or their pharmaceutically acceptable salts, hydrates or prodrugs;

[0007]

[0008] Wherein, B is an E3 ligase ligand of ubiquitin, more preferably one of CRBN, VHL, MDM2, cIAP, UBR7, RNF114, CBLB, KEAP1.

[0009] The CRBN described above is one of the following structural formulas:

[0010]

[0011] Wherein:

[0012] W is selected from one of CH2, C=O, SO2, NH and N-alkyl; the alkyl is C1-C4 alkyl;

[0013] X is arbitrarily selected from one or both of O and S;

[0014] Z is selected from one of hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl and halogen;

[0015] G and G' are each independently selected from H, C1-C4 alkyl, -OH and a 5-10 membered heterocyclic group substituted by C1-C4 alkyl, and the heterocyclic group contains 1-3 heteroatoms of N, O or S;

[0016] R 1Selected from one of H, D, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halogenated C1-C4 alkyl, and deuterated C1-C4 alkyl;

[0017] The structural formula of the said VHL is:

[0018]

[0019] Wherein: R 2 Selected from one of CH3 and H;

[0020] The structural formula of the said MDM2 is:

[0021]

[0022] Wherein:

[0023] R 3 Is one of a piperazinyl group, a piperidinyl group, a heterocyclic group, or a linking group with the following structure:

[0024]

[0025] In the above linking group, n is an integer from 0 to 3;

[0026] Wherein, R 3 The said heterocyclic group is one of a piperazinone group, a pyrrolyl group, a pyrazolyl group, a furyl group, a thienyl group, an oxazolyl group, an isoxazolyl group, a thiazolyl group, an isothiazolyl group, a pyridyl group, a pyrimidinyl group, a pyrazinyl group, or a pyridazinyl group;

[0027] The structure of the said cIAP is:

[0028]

[0029] R 4 Is H or Boc.

[0030] The said L is any one of the following structures:

[0031]

[0032] Wherein: m is selected from integers between 1 and 10.

[0033] The present invention preferably relates to a bifunctional molecular compound based on RSL3-induced GPX4 protein degradation or a pharmaceutically acceptable salt, hydrate, or prodrug thereof as shown in Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX, Formula X, Formula XI, Formula XII, and Formula XIII below:

[0034]

[0035] Among the bifunctional molecular compounds based on RSL3-induced GPX4 protein degradation shown in the above formulas III, IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII, L is preferably any one of the following structures:

[0036]

[0037] m is an integer between 1 and 7.

[0038] A bifunctional molecular compound based on RSL3-induced GPX4 protein degradation, specifically selected from the following compounds: RSL3-1-1P-C, RSL3-1-3P-C, RSL3-1-5P-C, RSL3-1-3C-V1, RSL3-1-5C-V1, RSL3-1-7C-V1, RSL3-1-3C-V2, RSL3-1-5C-V2, RSL3-1-7C-V2, RSL3-1-1P-M, RSL3-1-3P-M, RSL3-1-5P-M, RSL3-1-3C-B4, RSL3-1-5C-B4, RSL3-1-7C-B4, RSL3-1-1P-B5, RSL3-1-3P-B5, RSL3-1-5P-B5, RSL3-3-1P-C, RSL3-3-3P-C, RSL3-3-5P-C, RSL3-3-3C-V1, RSL3-3-5C-V1, RSL3-3-7C-V1, RSL3-3-3C-V2, RSL3-3-5C-V2, RSL3-3-7C-V2, RSL3-3-1P-M, RSL3-3-3P-M, RSL3-3-5P-M, RSL3-3-3C-B4, RSL3-3-5C-B4, RSL3-3-7C-B4, RSL3-3-1P-B5, RSL3-3-3P-B5, and RSL3-3-5P-B5.

[0039] The present invention provides pharmaceutically acceptable salts of the bifunctional molecular compounds based on RSL3-induced GPX4 protein degradation, including addition salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, etc. Salts are formed with hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, succinic acid, and similar known acceptable acids.

[0040] In addition, the present invention also includes prodrugs of derivatives of the present invention. They may themselves have weak activity or even no activity, but after administration, they are converted into the corresponding bioactive form under physiological conditions (such as by metabolism, solvolysis or other means).

[0041] The present invention also provides a preparation method of the bifunctional molecular compound based on RSL3-induced GPX4 protein degradation, selected from Method A, Method B and Method C, specifically including the following steps:

[0042] Method A: The synthesis route of the GPX4 protein degrader shown in Formula I is as follows:

[0043] In this synthesis route, L is m is an integer between 1 and 7;

[0044]

[0045] Step 1-1: Synthesis of Compound 3

[0046] Dissolve p-formylbenzoic acid (1.0 equiv) in dichloromethane, cool to 0 °C, and successively add EDCI (2.0 equiv), HOBt (2.0 equiv), propargylamine (1.2 equiv) and DIPEA (4.0 equiv). Transfer the reactants to room temperature for reaction. After the reaction is complete, extract with H2O and dichloromethane. The organic layer is dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is purified by silica gel column chromatography and eluted with a gradient of petroleum ether / ethyl acetate from 2:1 to 1:1 to obtain Compound 3;

[0047] Step 1-2: Synthesis of Compound 5

[0048] Add D-tryptophan methyl ester hydrochloride (1.0 equiv) to dichloromethane, then add TEA (1.2 equiv), stir until completely dissolved, and react at room temperature. After the reaction is completed, concentrate the reaction system under reduced pressure to obtain Compound 5;

[0049] Step 1-3: Synthesis of Compound 6

[0050] Dissolve Compound 5 (1.0 equiv) in dichloromethane, then successively add Compound 3 (1.1 equiv) and TFA (1.5 equiv), and react at 45 °C. After the reaction is completed, concentrate the reaction solution under reduced pressure, add DMF to completely dissolve it, and extract with H2O and ethyl acetate; dry the organic layer over anhydrous sodium sulfate and concentrate under reduced pressure. The residue is purified by silica gel column chromatography with dichloromethane / methanol to obtain Compound 6;

[0051] Step 1-4: Synthesis of Compound 8

[0052] Dissolve compound 6 (1.0 equiv) in anhydrous dichloromethane and cool to 0 °C. Then add sodium bicarbonate (1.2 equiv), and dropwise add chloroacetyl chloride (1.5 equiv) in batches. Warm the reaction to room temperature and react. After the reaction is completed, quench with water and extract with H2O and DCM; dry the organic layer with anhydrous sodium sulfate and concentrate under reduced pressure. Purify the residue by silica gel column chromatography with a petroleum ether / ethyl acetate gradient elution to obtain compound 8;

[0053] Steps 1-5: Synthesis of compound of formula I

[0054] Dissolve compound 8 (1.2 equiv) and B-Linker (1 equiv) in THF, add H2O, then add CuSO4 (1.2 equiv) and sodium ascorbate (3.0 equiv), and stir at room temperature until the reaction is complete. Concentrate under reduced pressure to remove THF and then extract with H2O and DCM. Dry the organic layer with anhydrous sodium sulfate and concentrate under reduced pressure. Purify the residue by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the compound of formula I;

[0055] Method B: The synthesis route of the GPX4 protein degrader shown in formula II is as follows:

[0056] In this synthesis route, L is m is an integer between 1 and 7;

[0057]

[0058] Step 2-1: Synthesis of compound 10

[0059] Dissolve Boc-D-tryptophan (1.0 equiv) in anhydrous dichloromethane, cool to 0 °C, and then successively add EDCI (2.0 equiv), HOBt (2.0 equiv), propargylamine (1.2 equiv), DIPEA (4.0 equiv) with stirring, and transfer to room temperature for reaction for 3-4 hours. Then extract with H2O and DCM. Dry the organic layer with anhydrous sodium sulfate and then concentrate under reduced pressure. Purify the residue by silica gel column chromatography with petroleum ether / ethyl acetate = 2:1 to obtain compound 10;

[0060] Step 2-2: Synthesis of compound 11

[0061] Dissolve compound 10 (1.0 equiv) in ethyl acetate and add hydrogen chloride-ethyl acetate solution. Concentrate the reaction system under reduced pressure to obtain compound 11;

[0062] Step 2-3: Synthesis of compound 12

[0063] Compound 11 (1.0 equiv) was added to dichloromethane, followed by the addition of TEA (1.2 equiv). The mixture was stirred until completely dissolved and reacted at room temperature for 1 hour. The reaction system was concentrated under reduced pressure to obtain Compound 12;

[0064] Step 2-4: Synthesis of Compound 14

[0065] Compound 12 (1.0 equiv) was dissolved in dichloromethane, and then Compound 13 (1.1 equiv) and TFA (1.5 equiv) were successively added with stirring. After the reaction was complete at 45 °C, the reaction solution was concentrated under reduced pressure. DMF was added to make it completely dissolved, and it was extracted with H2O and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with petroleum ether / ethyl acetate to obtain Compound 14;

[0066] Step 2-5: Synthesis of Compound 15

[0067] Compound 14 (1.0 equiv) was dissolved in anhydrous dichloromethane and cooled to 0 °C. Then sodium bicarbonate (1.2 equiv) and chloroacetyl chloride (1.5 equiv) were added in portions. The reaction was carried out at room temperature; after the reaction was complete, the reaction was quenched with water and extracted with H2O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with petroleum ether / ethyl acetate to obtain Compound 15;

[0068] Step 2-6: Synthesis of the Compound of Formula II

[0069] Compound 15 (1.2 equiv) and B-Linker (1 equiv) were dissolved in THF, H2O was added, and then CuSO4 (1.2 equiv) and sodium ascorbate (3.0 equiv) were added. After stirring at room temperature until the reaction was complete; first, THF was removed by concentration under reduced pressure, and then it was extracted with H2O and DCM. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane and methanol as eluents to obtain the Compound of Formula II;

[0070] Method C: The synthesis route of the ubiquitin ligase E3 ligand and L (linker) is as follows:

[0071] (1) When the ubiquitin ligase E3 ligand is CRBN, preferably Tha (thalidomide derivative), its synthesis method is as follows:

[0072]

[0073] Dissolve the compound Tha (thalidomide derivative) in DMF, add DIPEA to the reaction system, add 1.2 equiv of Linker, react at 90 °C for 2 hours, then extract with water and ethyl acetate. The organic layer is dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue is purified by silica gel column chromatography to obtain the compound Tha-L;

[0074] (2) When the ubiquitin ligase E3 ligand is MDM2, its synthesis method is as follows:

[0075]

[0076] Dissolve MDM2 in DCM, then add 2 equiv of EDCI, 2 equiv of HOBt, 4 equiv of DIPEA under ice bath, then add 1.2 equiv of Linker. After the reaction is complete, dilute the reaction system with DCM, extract with water and DCM. The organic layer is dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the compound MDM2-L;

[0077] (3) When the ubiquitin ligase E3 ligand is VHL, its synthesis method is as follows:

[0078]

[0079] Its synthesis method is the same as that of MDM2-L, except that MDM2 is replaced by VHL, and correspondingly, VHL-L is prepared;

[0080] (4) When the ubiquitin ligase E3 ligand is cIAP, its synthesis method is as follows:

[0081]

[0082] Its synthesis method is the same as that of MDM2-L, except that VHL is replaced by cIAP, and correspondingly, cIAP-L is prepared.

[0083] The present invention provides a pharmaceutical composition, comprising a bifunctional molecular compound based on RSL3-induced GPX4 protein degradation or a pharmaceutically acceptable salt, hydrate or prodrug thereof, and a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle or a combination thereof.

[0084] Among them, the dosage form of the pharmaceutical composition is any one of injection, tablet or capsule.

[0085] The present invention also provides the above-mentioned bifunctional molecular compound based on RSL3-induced GPX4 protein degradation or a pharmaceutically acceptable salt, hydrate or prodrug thereof, or the above-mentioned pharmaceutical composition for use in the preparation of a medicament for treating cancer.

[0086] The cancer(s) described above is / are one or more of osteosarcoma, lung cancer, gastric cancer, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, brain cancer, ovarian cancer, bladder cancer, cervical cancer, testicular cancer, kidney cancer, head and neck cancer, lymphoma, leukemia, and skin cancer.

[0087] Advantages of the present invention:

[0088] Supported by PROTAC technology and using the GPX4 inhibitor RSL3 as a raw material, the present invention synthesizes bifunctional molecular compounds based on RSL3-induced GPX4 protein degradation with different Linker lengths.

[0089] The bifunctional molecular compounds based on RSL3-induced GPX4 protein degradation provided by the present invention effectively target and degrade GPX4; similar to a catalytic reaction, the drug has a low effective dose; it only provides binding activity and is event-driven, different from traditional occupancy-driven, and does not need to directly inhibit the functional activity of the target protein; the drug does not need to bind to the target protein for a long time and with high intensity. The bifunctional molecular compounds based on RSL3-induced GPX4 protein degradation provided by the present invention provide a new treatment method for treating tumors by inducing ferroptosis.

[0090] The preparation method of the bifunctional molecular compounds based on RSL3-induced GPX4 protein degradation provided by the present invention has the advantages of being simple and easy to operate, low cost, and suitable for large-scale industrial production. Brief Description of the Drawings

[0091] Figure 1 Shows the degradation effect of RSL3-1-B-L and RSL3-3-B-L of the present invention on GPX4;

[0092] Figure 2 Shows the cytotoxic activity of RSL3-1-7C-B4 of the present invention against HT1080 cells;

[0093] Figure 3 Shows the competitive experiment of B4 and RSL3 of the present invention on the degradation of GPX4 by RSL3-1-7C-B4 and the reversal experiments of MG132 and ML4924; Detailed Embodiments

[0094] The present invention is further illustrated by examples. However, it should not be understood that the scope of the present invention is limited to the following examples.

[0095] Example 1 Synthesis of Compound 3

[0096]

[0097] For the specific operation process, refer to Step 1-1. White solid, yield: 75%. 1¹H NMR (600 MHz, Chloroform-d) δ 10.09 (s, 1H), 7.99–7.93 (m, 4H), 6.40 (s, 1H), 4.29 (dd, J = 5.2, 2.6 Hz, 2H), 2.32 (t, J = 2.6 Hz, 1H).

[0098] Synthesis of Compound 5 in Example 2

[0099]

[0100] For the specific operation process, refer to Step 1-2. White solid, yield: 99%.

[0101] Synthesis of Compound 6 in Example 3

[0102]

[0103] For the specific operation process, refer to Step 1-3. Pale yellow solid, yield: 64%. 1 ¹H NMR (600 MHz, DMSO-d6) δ 10.34 (s, 1H), 8.93 (t, J = 5.6 Hz, 1H), 7.89–7.85 (m, 2H), 7.48–7.43 (m, 3H), 7.19 (dt, J = 8.0, 1.0 Hz, 1H), 7.01 (ddd, J = 8.1, 7.0, 1.3 Hz, 1H), 6.96 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 5.28 (d, J = 5.4 Hz, 1H), 4.06 (dd, J = 5.6, 2.5 Hz, 2H), 3.89 (dt, J = 11.2, 3.5 Hz, 1H), 3.71 (s, 3H), 3.12 (t, J = 2.5 Hz, 1H), 3.05 (ddd, J = 14.8, 4.1, 1.7 Hz, 1H), 2.91–2.82 (m, 2H).

[0104] Synthesis of Compound 8 in Example 4

[0105]

[0106] For the specific operation process, refer to Step 1-4. Pale yellow solid, yield: 81%. 11H NMR (600 MHz, Chloroform-d) δ 8.29 (s, 1H), 7.61 (d, J = 7.7 Hz, 3H), 7.31 (dd, J = 16.0, 7.7 Hz, 3H), 7.24–7.20 (m, 1H), 7.20–7.16 (m, 1H), 6.92 (s, 1H), 6.45 (s, 1H), 4.95 (s, 1H), 4.35 (d, J = 12.6 Hz, 1H), 4.25–4.18 (m, 3H), 3.70 (d, J = 15.7 Hz, 1H), 3.23 (dd, J = 15.7, 6.8 Hz, 1H), 3.03 (s, 3H), 2.28 (t, J = 2.3 Hz, 1H).

[0107] Synthesis of Compound 10 in Example 5

[0108]

[0109] For the specific operation process, see Step 2-1. White solid, yield: 82%. 1 1H NMR (600 MHz, Chloroform-d) δ 8.13 (s, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.37 (dt, J = 8.2, 0.9 Hz, 1H), 7.21 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.14 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 7.08–7.05 (m, 1H), 6.00 (s, 1H), 5.12 (s, 1H), 4.44 (s, 1H), 3.93 (s, 2H), 3.09–3.41 (m, 2H), 2.14 (t, J = 2.6 Hz, 1H), 1.42 (s, 9H).

[0110] Synthesis of Compound 11 in Example 6

[0111]

[0112] For the specific operation process, see Step 2-2. White solid, yield: 99%.

[0113] Synthesis of Compound 12 in Example 7

[0114]

[0115] For the specific operation process, see Step 2-3. White solid, yield: 99%.

[0116] Synthesis of Compound 14 in Example 8

[0117]

[0118] For the specific operation process, refer to Steps 2-4. Pale yellow solid, yield: 80%.

[0119] Synthesis of Compound 15 in Example 9

[0120]

[0121] For the specific operation process, refer to Steps 2-5. Pale yellow solid, yield: 48%. 1 H NMR(600MHz,Chloroform-d)δ8.05(s,1H),7.93(s,2H),7.61(d,J=7.8Hz,1H),7.39(d,J=8.1Hz,2H),7.30(d,J=7.9Hz,1H),7.24–7.20(m,1H),7.17(t,J=7.5Hz,1H),7.02(s,1H),5.53(s,1H),4.90(s,1H),4.38–4.23(m,2H),3.90(s,3H),3.81–3.55(m,2H),3.15(s,1H),3.07(s,1H).

[0122] Synthesis of Compound RSL3-1-nP-C in Example 10 (n = 1, 3, 5, taking n = 1 as an example)

[0123]

[0124] For the specific operation process, refer to Steps 1-5. Pale yellow solid, yield: 86%. 1 H NMR(400MHz,DMSO-d6)δ11.10(s,1H),10.92(s,1H),8.89(s,1H),7.88(s,2H),7.73(d,J=7.9Hz,1H),7.56–7.51(m,1H),7.47(d,J=7.8Hz,3H),7.21(d,J=7.5Hz,1H),7.02(td,J=23.4,22.3,8.1Hz,5H),6.58(t,J=5.9Hz,1H),6.01(s,1H),5.39(s,1H),5.06(dd,J=12.9,5.4Hz,1H),4.73(d,J=13.8Hz,1H),4.46(s,4H),3.81(t,J=5.2Hz,2H),3.57(s,2H),3.53(d,J=13.0Hz,4H),3.41(d,J=5.7Hz,2H),2.92–2.81(m,1H),2.65–2.53(m,2H),2.01(t,J=7.9Hz,2H).

[0125] Synthesis of Compound RSL3-1-nC-V1 in Example 11 (n = 3, 5, 7, taking n = 3 as an example)

[0126]

[0127] For the specific operation process, refer to Steps 1 - 5. Pale yellow solid, yield: 96%. 1 H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 8.98 (d, J = 6.2 Hz, 2H), 8.57 (t, J = 6.1 Hz, 1H), 8.00–7.91 (m, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.69 (ddd, J = 13.5, 6.1, 3.3 Hz, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.39 (t, J = 7.6 Hz, 4H), 7.29 (d, J = 7.9 Hz, 1H), 7.17–7.09 (m, 3H), 7.05 (t, J = 7.4 Hz, 1H), 6.88 (s, 1H), 5.23 (d, J = 6.7 Hz, 1H), 5.16 (d, J = 3.5 Hz, 1H), 4.85 (d, J = 13.8 Hz, 1H), 4.53 (d, J = 9.2 Hz, 1H), 4.50–4.38 (m, 5H), 4.35 (s, 1H), 4.31 (t, J = 7.1 Hz, 2H), 4.25–4.22 (m, 1H), 4.22–4.12 (m, 1H), 3.66 (s, 2H), 2.88 (s, 3H), 2.44 (s, 3H), 2.00 (dd, J = 6.8, 3.3 Hz, 2H), 1.27 (d, J = 6.3 Hz, 2H), 1.24 (s, 2H), 0.93 (s, 9H). 13 C NMR (101 MHz, DMSO-d6) δ 172.43, 171.63, 170.66, 170.08, 167.38, 166.26, 151.97, 143.00, 139.93, 136.89, 134.03, 130.09, 129.86, 129.11, 129.00, 127.88, 127.40, 123.40, 122.15, 119.25, 118.64, 111.76, 106.90, 69.34, 59.16, 56.97, 56.83, 52.96, 52.10, 51.71, 49.36, 43.58, 42.12, 38.53, 38.37, 35.64, 35.32, 32.10, 26.81, 26.68, 16.38, 14.35. HRMS (ESI + ): m / z calculated for C 51H 57 ClN 10 NaO8S[M+Na] + , 1027.3668; found, 1027.3685.

[0128] Synthesis of the compound RSL3-1-nC-V2 in Example 12 (n = 3, 5, 7, taking n = 3 as an example)

[0129]

[0130] For the specific operation process, see Steps 1-5. Pale yellow solid, yield: 63.7%. 1 H NMR (600 MHz, DMSO-d6) δ 10.90 (d, J = 4.5 Hz, 1H), 8.98 (d, J = 4.6 Hz, 1H), 8.39 (dd, J = 7.9, 4.3 Hz, 1H), 7.96–7.90 (m, 2H), 7.79 (dd, J = 8.3, 4.3 Hz, 2H), 7.74–7.63 (m, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.45–7.41 (m, 2H), 7.37 (ddd, J = 8.6, 4.5, 2.3 Hz, 2H), 7.29 (dd, J = 8.4, 3.8 Hz, 1H), 7.15–7.11 (m, 2H), 7.07–7.02 (m, 1H), 6.88 (d, J = 4.3 Hz, 1H), 5.23 (d, J = 6.3 Hz, 1H), 5.13 (d, J = 3.6 Hz, 1H), 4.90 (t, J = 7.2 Hz, 1H), 4.85 (dd, J = 13.9, 4.4 Hz, 1H), 4.49 (dt, J = 8.4, 4.9 Hz, 3H), 4.41 (td, J = 8.1, 4.4 Hz, 1H), 4.30 (dd, J = 7.3, 4.5 Hz, 2H), 4.27 (s, 1H), 4.15 (q, J = 5.7 Hz, 1H), 3.61–3.58 (m, 2H), 3.16 (d, J = 4.8 Hz, 2H), 3.12 (d, J = 14.7 Hz, 1H), 2.88 (d, J = 4.2 Hz, 2H), 2.45 (d, J = 4.4 Hz, 2H), 2.23 (dd, J = 13.6, 6.2 Hz, 1H), 2.15 (d, J = 16.3 Hz, 1H), 2.02–1.96 (m, 3H), 1.78 (td, J = 8.6, 4.5 Hz, 1H), 1.63 (d, J = 8.2 Hz, 1H), 1.46 (d, J = 6.8 Hz, 1H), 1.36 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 4.3 Hz, 9H). 1313C NMR(151MHz, DMSO-d6) δ 171.56, 171.08, 170.68, 169.98, 167.37, 151.97, 148.18, 145.34, 145.11, 136.89, 130.13, 129.86, 129.28, 129.12, 126.84, 123.38, 122.14, 119.23, 118.62, 69.23, 59.02, 57.03, 56.72, 52.10, 49.35, 48.17, 40.57, 38.16, 35.61, 32.12, 31.61, 29.45, 26.88, 26.67, 22.86, 16.41, 14.35. HRMS(ESI + ): m / z calculated for C 52 H 59 ClN 10 NaO8S [M+Na] + , 1041.3824; found, 1041.3883.

[0131] Synthesis of Compound RSL3-1-nP-M in Example 13 (n = 1, 3, 5, taking n = 1 as an example)

[0132]

[0133] For the specific operation process, refer to Steps 1-5. Pale yellow solid, yield: 92%. 11H NMR (600 MHz, DMSO-d6) δ 10.89 (s, 1H), 9.00 (t, J = 5.7 Hz, 1H), 7.92 (d, J = 6.7 Hz, 2H), 7.78 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 7.8 Hz, 2H), 7.29 (d, J = 8.1 Hz, 1H), 7.15 (dd, J = 8.2, 6.0 Hz, 4H), 7.13–7.09 (m, 3H), 7.04 (d, J = 7.6 Hz, 3H), 6.97 (d, J = 8.0 Hz, 2H), 6.88 (s, 1H), 6.61 (d, J = 9.1 Hz, 2H), 5.66 (d, J = 9.4 Hz, 1H), 5.60 (s, 1H), 5.24 (d, J = 6.8 Hz, 1H), 4.86 (d, J = 13.9 Hz, 1H), 4.72 (p, J = 6.0 Hz, 1H), 4.47 (q, J = 5.9, 5.4 Hz, 5H), 3.83 (s, 3H), 3.75 (t, J = 5.3 Hz, 2H), 3.74–3.66 (m, 2H), 3.59 (d, J = 16.8 Hz, 1H), 3.48 (d, J = 15.8 Hz, 1H), 3.43–3.37 (m, 4H), 3.23–3.18 (m, 1H), 3.16–3.08 (m, 3H), 2.98 (s, 2H), 2.87 (s, 3H), 1.26 (d, J = 5.9 Hz, 3H), 1.21 (d, J = 5.9 Hz, 3H). 13 13C NMR (151 MHz, DMSO-d6) δ 170.67, 167.83, 167.37, 166.24, 164.86, 162.90, 156.98, 143.02, 136.89, 134.05, 132.40, 131.76, 131.62, 130.13, 129.88, 129.21, 129.13, 127.94, 127.91, 127.38, 126.35, 122.13, 119.23, 118.64, 111.75, 106.90, 105.51, 99.80, 70.36, 69.06, 68.92, 55.91, 52.96, 52.07, 51.71, 49.66, 49.01, 46.94, 43.61, 42.48, 38.77, 35.31, 22.18, 22.11, 21.56. HRMS (ESI + ): m / z calculated for C 61 H 62 Cl3N 11 NaO 10 [M + Na] +,1238.3629; found,1238.3636.

[0134] Synthesis of the compound RSL3-1-nC-B4 in Example 14 (n = 3, 5, 7, taking n = 3 as an example)

[0135]

[0136] For the specific operation process, see Steps 1-5. Pale yellow solid, yield: 50.3%. 1 H NMR(600MHz, DMSO-d6) δ10.89(s, 1H), 8.97(t, J = 5.8Hz, 1H), 7.88(s, 1H), 7.84(d, J = 8.5Hz, 1H), 7.79(d, J = 8.1Hz, 2H), 7.61(d, J = 9.0Hz, 1H), 7.57(d, J = 7.8Hz, 1H), 7.29(d, J = 8.0Hz, 1H), 7.25(t, J = 7.5Hz, 2H), 7.22–7.20(m, 2H), 7.17–7.12(m, 3H), 7.11(d, J = 8.3Hz, 1H), 7.04(t, J = 7.5Hz, 1H), 6.88(s, 1H), 6.18(d, J = 6.1Hz, 1H), 5.23(d, J = 6.8Hz, 1H), 4.85(d, J = 13.9Hz, 1H), 4.48(d, J = 5.6Hz, 2H), 4.46(d, J = 8.5Hz, 1H), 4.33(ddd, J = 10.3, 8.4, 4.6Hz, 1H), 4.21(d, J = 7.1Hz, 1H), 4.17(td, J = 6.9, 3.9Hz, 2H), 3.87(dd, J = 6.1, 2.7Hz, 1H), 3.60(s, 3H), 3.48(d, J = 15.8Hz, 1H), 3.14–3.09(m, 1H), 2.87(s, 3H), 2.81(dd, J = 13.4, 6.9Hz, 1H), 2.65(dd, J = 13.4, 8.1Hz, 1H), 2.07–2.02(m, 1H), 1.98(dt, J = 14.6, 7.1Hz, 1H), 1.87(dq, J = 18.7, 7.1Hz, 2H), 1.61(ddd, J = 13.1, 10.3, 4.7Hz, 1H), 1.52(dq, J = 12.3, 6.2Hz, 1H), 1.44(ddd, J = 13.5, 9.2, 4.7Hz, 1H), 0.81(d, J = 6.5Hz, 3H), 0.75(d, J = 6.4Hz, 3H). 1313C NMR(151MHz, DMSO-d6) δ 173.18, 172.41, 171.10, 170.67, 167.36, 166.20, 145.33, 143.01, 139.23, 136.89, 134.06, 129.87, 129.66, 129.11, 128.61, 127.40, 126.54, 126.35, 123.36, 122.13, 119.22, 118.64, 111.75, 106.90, 71.67, 53.61, 52.95, 52.37, 52.07, 51.69, 50.04, 49.22, 49.06, 43.61, 37.34, 35.31, 32.42, 26.54, 24.48, 23.24, 21.57. HRMS(ESI + ): m / z calculated for C 46 H 53 ClN8NaO9 [M+Na] + , 919.3522; found, 919.3547.

[0137] Synthesis of Compound RSL3-1-nP-B5 in Example 15 (n = 1, 3, 5, taking n = 1 as an example)

[0138]

[0139] For the specific operation process, refer to Steps 1 - 5. Pale yellow solid, yield: 73%. 11H NMR(600MHz,DMSO-d6)δ10.93(s,1H),9.05(t,J=5.8Hz,1H),8.19(t,J=5.7Hz,1H),8.03(d,J=8.0Hz,4H),7.93(s,1H),7.80(d,J=8.1Hz,2H),7.57(d,J=7.8Hz,1H),7.33(t,J=7.4Hz,2H),7.31–7.29(m,3H),7.28–7.24(m,1H),7.13(dd,J=20.1,7.9Hz,3H),7.05(t,J=7.5Hz,1H),6.88(s,1H),5.24(d,J=6.8Hz,1H),4.87(d,J=13.9Hz,1H),4.48(d,J=4.2Hz,2H),4.46–4.44(m,2H),4.26(td,J=8.5,5.9Hz,1H),4.00(d,J=3.3Hz,1H),3.96–3.94(m,1H),3.93–3.90(m,1H),3.57–3.53(m,1H),3.48(d,J=15.8Hz,1H),3.37(t,J=6.0Hz,2H),3.19–3.15(m,1H),3.14–3.10(m,2H),2.93(dd,J=13.7,8.0Hz,1H),2.87(s,3H),1.61–1.55(m,1H),1.54–1.49(m,1H),1.48–1.43(m,1H),1.25–1.22(m,2H),0.87(d,J=6.5Hz,3H),0.85(d,J=6.5Hz,3H). 13 13C NMR(151MHz,DMSO-d6)δ172.24,171.05,170.68,167.38,166.22,145.34,143.03,139.72,136.90,136.78,134.04,130.12,129.90,129.11,129.08,127.39,126.48,123.79,122.12,119.22,118.64,111.76,106.89,69.01,68.73,60.22,54.71,52.07,51.75,49.66,43.63,41.43,38.84,35.30,35.13,24.68,23.29,22.43,21.57,21.23.HRMS(ESI + ):m / z calculated for C 45 H 55 ClN9O8[M+H]+ ,884.3862; found,884.3787.

[0140] Synthesis of Compound RSL3-3-nP-C in Example 16 (n = 1, 3, 5, taking n = 1 as an example)

[0141]

[0142] For the specific operation process, see Steps 2-6. Pale yellow solid, yield: 71%. 1 H NMR(600MHz,DMSO-d6)δ11.09(s,1H),10.89(s,1H),8.11(s,1H),7.85(d,J = 7.9Hz,2H),7.66(s,1H),7.55(t,J = 7.8Hz,1H),7.50(d,J = 7.9Hz,1H),7.38–7.26(m,3H),7.11–7.06(m,2H),7.02(dd,J = 9.9,7.0Hz,2H),6.94(s,1H),6.57(t,J = 6.0Hz,1H),5.05(dd,J = 12.9,5.4Hz,1H),4.96(d,J = 6.2Hz,1H),4.79(s,1H),4.53(d,J = 13.4Hz,1H),4.44(s,2H),3.84(s,3H),3.79(t,J = 5.4Hz,3H),3.57(t,J = 5.5Hz,2H),3.41(q,J = 5.7Hz,3H),3.13–2.95(m,2H),2.90–2.82(m,1H),2.62–2.51(m,2H),2.04–1.98(m,1H).

[0143] Synthesis of Compound RSL3-3-nC-V1 in Example 17 (n = 3, 5, 7, taking n = 3 as an example)

[0144]

[0145] For the specific operation process, see Steps 2-6. Pale yellow solid, yield: 82%. 11H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 8.98 (s, 1H), 8.56 (t, J = 6.1 Hz, 1H), 8.11 (s, 1H), 7.96 (d, J = 9.3 Hz, 1H), 7.85 (d, J = 7.9 Hz, 2H), 7.74–7.61 (m, 2H), 7.52 (d, J = 7.8 Hz, 1H), 7.42–7.36 (m, 4H), 7.31 (d, J = 8.1 Hz, 2H), 7.14–7.01 (m, 2H), 6.95 (s, 1H), 5.15 (s, 1H), 4.99 (d, J = 6.5 Hz, 1H), 4.80 (s, 1H), 4.53 (d, J = 9.2 Hz, 1H), 4.47–4.38 (m, 2H), 4.35 (s, 1H), 4.23 (d, J = 6.6 Hz, 2H), 4.17 (d, J = 5.3 Hz, 1H), 4.13 (d, J = 5.0 Hz, 1H), 3.84 (s, 3H), 3.66 (s, 2H), 3.31 (d, J = 4.4 Hz, 3H), 3.01 (s, 1H), 2.44 (s, 3H), 1.96 (s, 2H), 1.27 (d, J = 6.4 Hz, 2H), 1.24 (s, 2H), 0.93 (s, 9H). 13 13C NMR (101 MHz, DMSO-d6) δ 172.43, 171.57, 170.08, 167.96, 166.53, 151.96, 148.15, 139.95, 136.79, 130.07, 129.12, 129.09, 127.86, 123.25, 121.93, 119.11, 118.59, 111.71, 69.34, 65.50, 59.16, 56.95, 56.84, 52.62, 49.28, 49.06, 42.06, 38.54, 38.39, 35.64, 34.71, 32.02, 30.46, 30.25, 26.81, 26.59, 25.21, 23.71, 22.85, 22.48, 19.11, 16.37, 14.35, 14.01, 11.26.

[0146] Synthesis of Compound RSL3-3-nC-V2 in Example 18 (n = 3, 5, 7, taking n = 3 as an example)

[0147]

[0148] For the specific operation process, refer to Steps 2-6. Pale yellow solid, yield: 89%. 11H NMR (600 MHz, DMSO-d6) δ 10.90 (s, 1H), 8.99 (s, 1H), 8.39 (d, J = 7.8 Hz, 1H), 8.11 (s, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.86 (d, J = 8.1 Hz, 2H), 7.68–7.65 (m, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.45–7.41 (m, 2H), 7.37 (d, J = 8.3 Hz, 2H), 7.30 (q, J = 10.1, 8.9 Hz, 3H), 7.13–7.07 (m, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.96 (s, 1H), 5.12 (d, J = 3.5 Hz, 1H), 5.00 (d, J = 6.4 Hz, 1H), 4.91 (t, J = 7.3 Hz, 1H), 4.81 (s, 1H), 4.50 (d, J = 9.2 Hz, 1H), 4.41 (t, J = 8.1 Hz, 1H), 4.30–4.24 (m, 3H), 4.24–4.20 (m, 1H), 3.85 (s, 3H), 3.60 (d, J = 5.2 Hz, 2H), 3.00 (s, 1H), 2.45 (s, 3H), 2.22 (q, J = 7.5 Hz, 1H), 2.15 (q, J = 7.4 Hz, 1H), 2.01 (t, J = 7.4 Hz, 2H), 1.98–1.94 (m, 2H), 1.80–1.76 (m, 1H), 1.64 (q, J = 6.9 Hz, 1H), 1.46 (d, J = 7.1 Hz, 1H), 1.37 (d, J = 7.0 Hz, 3H), 0.93 (s, 9H). 13 13C NMR (151 MHz, DMSO-d6) δ 171.51, 171.07, 169.97, 166.53, 152.00, 148.13, 145.14, 130.11, 129.29, 129.12, 129.01, 126.85, 123.10, 69.23, 65.49, 59.02, 57.03, 56.74, 52.60, 49.31, 48.16, 35.63, 32.08, 29.47, 29.03, 26.89, 26.61, 23.72, 22.87, 22.55, 19.11, 16.40, 14.35, 11.27. HRMS (ESI + ): m / z calculated for C 52 H 59 ClN 10 NaO8S [M+Na] + , 1041.3824; found, 1041.4177.

[0149] Synthesis of Compound RSL3-3-nP-M in Example 19 (n = 1, 3, 5, taking n = 1 as an example)

[0150]

[0151] For the specific operation process, refer to Steps 2-6. Pale yellow solid, yield: 70.3%. 1 H NMR (600 MHz, DMSO-d6) δ 10.90 (s, 1H), 8.12 (s, 1H), 7.90 (t, J = 5.7 Hz, 1H), 7.86 (d, J = 7.9 Hz, 2H), 7.65 (d, J = 5.1 Hz, 1H), 7.52 (d, J = 7.9 Hz, 2H), 7.30 (d, J = 9.1 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H), 7.13–7.07 (m, 3H), 7.03 (q, J = 7.2 Hz, 3H), 6.97 (d, J = 8.1 Hz, 3H), 6.61 (d, J = 8.5 Hz, 2H), 5.66 (d, J = 9.1 Hz, 1H), 5.61 (s, 1H), 4.98 (d, J = 6.3 Hz, 1H), 4.80 (s, 1H), 4.72 (p, J = 6.0 Hz, 1H), 4.54 (s, 1H), 4.40 (s, 2H), 3.84–3.81 (m, 6H), 3.74–3.66 (m, 4H), 3.62–3.56 (m, 1H), 3.45 (s, 2H), 3.34 (s, 4H), 3.23–3.08 (m, 4H), 3.07–2.89 (m, 4H), 1.27 (d, J = 6.0 Hz, 3H), 1.21 (d, J = 6.0 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 167.93, 167.83, 166.53, 164.78, 157.13, 136.79, 131.82, 130.11, 129.28, 129.12, 127.99, 121.91, 119.10, 118.58, 111.72, 105.67, 99.87, 70.53, 69.05, 68.88, 60.23, 55.97, 52.61, 49.63, 49.42, 49.01, 46.88, 42.48, 38.75, 38.55, 34.74, 29.48, 28.83, 22.18, 22.10, 14.55, 14.36. HRMS (ESI + ): m / z calculated for C 61 H 62 Cl3N 11 NaO10 [M+Na] + ,1238.3629; found,1238.3629.

[0152] Synthesis of Compound RSL3-3-nC-B4 in Example 20 (n = 3, 5, 7, taking n = 3 as an example)

[0153]

[0154] For the specific operation process, see Step 2-6. Pale yellow solid, yield: 71%. 1 H NMR(600MHz,DMSO-d6)δ10.90(s,1H),8.10(s,1H),7.86(d,J = 8.0Hz,2H),7.83(d,J = 8.5Hz,1H),7.60(d,J = 9.1Hz,2H),7.52(d,J = 7.8Hz,1H),7.30(d,J = 8.1Hz,2H),7.26(t,J = 7.4Hz,3H),7.21(d,J = 7.5Hz,2H),7.16(t,J = 7.3Hz,1H),7.10(t,J = 7.6Hz,1H),7.03(t,J = 7.5Hz,1H),6.96(s,1H),6.18(d,J = 6.1Hz,1H),4.98(d,J = 6.3Hz,1H),4.81(s,1H),4.54(s,1H),4.35–4.29(m,1H),4.20(q,J = 9.0,8.6Hz,1H),4.12(s,2H),3.88–3.85(m,1H),3.84(s,3H),3.76(s,1H),3.60(s,3H),3.49–3.43(m,1H),3.06–3.96(m,2H),2.80(dd,J = 13.4,7.0Hz,1H),2.64(dd,J = 13.4,8.0Hz,1H),2.07–1.99(m,1H),1.98–1.92(m,1H),1.88–1.79(m,2H),1.63–1.58(m,1H),1.50(d,J = 6.8Hz,1H),1.47–1.41(m,1H),0.80(d,J = 6.5Hz,3H),0.74(d,J = 6.4Hz,3H). 1313C NMR(151MHz, DMSO-d6) δ 173.18, 172.41, 171.06, 169.64, 167.91, 166.52, 145.66, 139.23, 136.78, 134.66, 131.57, 129.66, 129.08, 128.61, 126.55, 123.21, 121.91, 119.11, 118.59, 116.79, 115.74, 111.76, 71.62, 62.22, 54.48, 53.62, 52.61, 52.37, 50.04, 49.18, 49.06, 44.32, 37.35, 34.69, 32.33, 29.61, 29.31, 26.46, 24.47, 23.21, 21.56. HRMS(ESI + ): m / z calculated for C 46 H 53 ClN8NaO9 [M+Na] + , 919.3522; found, 919.3547.

[0155] Synthesis of Compound RSL3-3-nP-B5 in Example 21 (n = 1, 3, 5, taking n = 1 as an example)

[0156]

[0157] For the specific operation process, refer to Step 2-6. Pale yellow solid, yield: 61%. 11H NMR (600 MHz, DMSO-d6) δ 10.92 (s, 1H), 8.18–8.15 (m, 2H), 8.00 (t, J = 6.5 Hz, 4H), 7.92 (dd, J = 13.2, 8.4 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.5 Hz, 3H), 7.31–7.28 (m, 3H), 7.27–7.26 (m, 1H), 7.10 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.7 Hz, 1H), 6.97–6.94 (m, 1H), 4.99 (s, 1H), 4.82 (s, 1H), 4.55 (s, 1H), 4.38 (s, 2H), 4.27–4.21 (m, 2H), 3.99 (d, J = 3.4 Hz, 1H), 3.84 (s, 3H), 3.70 (s, 2H), 3.37–3.32 (m, 3H), 3.23 (dd, J = 13.9, 5.6 Hz, 1H), 3.11 (q, J = 6.5 Hz, 1H), 3.03 (s, 1H), 2.93 (dd, J = 13.7, 8.1 Hz, 1H), 2.86 (dd, J = 13.7, 6.3 Hz, 1H), 1.57 (dt, J = 13.4, 6.6 Hz, 1H), 1.53–1.48 (m, 1H), 1.46–1.42 (m, 1H), 1.23 (d, J = 2.9 Hz, 2H), 0.87–0.85 (m, 3H), 0.84 (d, J = 6.6 Hz, 3H). 13 13C NMR (151 MHz, DMSO-d6) δ 172.25, 171.04, 170.82, 169.18, 166.53, 144.65, 136.77, 132.57, 129.90, 129.46, 129.09, 127.39, 126.48, 123.58, 121.85, 120.58, 119.10, 116.06, 111.72, 108.87, 102.50, 71.49, 68.97, 68.70, 66.77, 60.22, 55.38, 54.70, 52.59, 51.74, 49.62, 49.05, 41.43, 38.84, 35.13, 24.67, 23.27, 22.42, 21.23, 14.55. HRMS (ESI + ): m / z calculated for C 45 H 55 ClN9O8 [M+H] +,884.3862; found,884.3787.

[0158] Example 22 Detection of cytotoxic activity by CCK8 method

[0159] (1) Preparation of complete medium: Pipette 7.5 mL of FBS and 500 μL of penicillin-streptomycin double-antibody mixture, and then add DMEM medium to 50 mL to make the final concentrations of FBS and double-antibody 15% and 1% respectively.

[0160] (2) Cell cryopreservation: Prepare cell cryopreservation solution according to the ratio of FBS:DMSO = 9:1. Use the gradient cryopreservation method to cryopreserve cells and finally store them in liquid nitrogen.

[0161] (3) Cell passage: Observe under the microscope. HT1080 cells grow adherently with complete morphology and clear edges. Passage can be carried out when the cell confluence reaches 80% - 90%. Use a pipette to discard the old medium in the culture flask / dish, gently blow down the cells with 4 mL of new medium, mix well and evenly disperse them into two new culture flasks containing 4 mL of medium. After shaking well, continue to culture in a 37°C, 5% CO2 incubator.

[0162] (4) Count the cells with good growth status using a hemocytometer. Before starting, clean the hemocytometer and coverslip with an alcohol cotton ball and let it dry. Align the coverslip with the edge of the H-shaped groove of the hemocytometer, and use a pipette to aspirate 10 μL of cell suspension and gently push it along the edge of the coverslip to let the suspension fill the counting area by the surface tension of the liquid without generating bubbles. Let it stand for a while, and count the cells in the upper grid of the hemocytometer under the microscope according to the rule of "counting the upper but not the lower, counting the left but not the right". Cells per milliliter = total number of cells / 4 × 10 4 .

[0163] (5) Centrifuge the cells with good growth status and in the logarithmic growth phase, resuspend them with complete medium, and count them using a hemocytometer. Inoculate them into a 96-well plate at a concentration of 3.0x10 4 cells / well, and place them in a 37°C, 5% CO2 incubator for incubation. When the cells are completely adherent and the confluence reaches 60%, start drug administration. After the drug acts for 24 h, add 10 μL of CCK8 reagent to each well (note that this reagent needs to be prepared and used immediately and stored in the dark), incubate in a 37°C incubator for 30 min, and then use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value at 450 nm.

[0164] (6) Calculate using the OD values of each group of samples:

[0165]

[0166] The results are shown in Table 1 and Figure 2as shown

[0167] Example 23 Detection of Changes in GPX4 Protein Expression by Western Blot

[0168] (1) Preparation of Cell Protein Samples:

[0169] Cells in the logarithmic growth phase were seeded in six-well plates. When the cells adhered and grew to a confluence of about 60%, the cells were treated with bifunctional molecular compounds based on RSL3 at different concentrations to induce GPX4 protein degradation. The cells in each group were blown down and collected. They were washed three times with normal saline and finally centrifuged at 4°C and 3500 rpm for 10 min. A certain amount of RIPA protein lysate containing 1 mM PMSF was added and the cells were lysed on ice for 30 min. They were centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was carefully aspirated and transferred to a new EP tube without aspirating the precipitate.

[0170] (2) Determination of Protein Concentration by BCA

[0171] The protein concentration was measured using a BCA kit. First, prepare the standard protein curve stock solution: Take 8 EP tubes, add 6 μL of protein standard (30 mg / mL) and 54 μL of normal saline to the first tube, and add 30 μL of normal saline to the remaining tubes. Use the method of half-dilution to prepare the standard curve stock solution. For the cell protein sample group: Take 3 μL of the protein sample and add it to 27 μL of normal saline to dilute the protein sample 10 times and mix well. Take 20 μL of each prepared stock solution and add it to a 96-well plate, and then add 200 μL of the mixed solution of reagent A: reagent B = 50:1, and incubate at 37°C in the dark for 30 min. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at 562 nm, and calculate the protein concentration of the corresponding sample according to the protein standard curve. To ensure the same loading volume, add the corresponding normal saline and 5x Loading Buffier for adjustment. Finally, heat in a thermostatic metal bath at 95 - 100°C for 5 - 10 min and store at -20°C for later use.

[0172] (3) SDS-PAGE Electrophoresis

[0173] Prepare separating gels and stacking gels with different concentrations according to the size of the protein bands to be detected. Place the gel plate steadily in the electrophoresis tank, add sufficient electrophoresis buffer, and horizontally and vertically remove the comb. Add Marker and samples successively according to the specific experiment. Run the stacking gel at a constant voltage of 70 V for 45 - 55 min to stack the samples on the same horizontal line, and run the separating gel at a constant voltage of 110 V for 60 - 70 min to separate proteins with different molecular weights.

[0174] (4) Transfer and Blocking

[0175] Transfer the membrane using the wet transfer method: Remove the SDS-PAGE gel, cut it into the required size and place it in the transfer buffer for later use. Cut a PVDF membrane of the same size as the gel and soak it in methanol for 15 s for activation. Take two layers of filter paper, fully soak them with the transfer buffer and squeeze out the air bubbles. Stack them neatly in the order of filter paper - gel - PVDF membrane - filter paper to form a "sandwich" and put it into the transfer apparatus. Then place the transfer apparatus in an ice box to avoid excessive temperature during the transfer process. Transfer at a constant current of 200 mA for 35 - 110 min. After the transfer is completed, take out the PVDF membrane and block it in a blocking solution containing 5% skim milk powder at room temperature for 1 h.

[0176] (5) Immunoblot hybridization and detection

[0177] After blocking, wash the PVDF membrane with TBST solution 1 - 2 times and then place it in the primary antibody solution and incubate overnight at 4°C. The next day, take out the PVDF membrane, wash it 3 times with TBST, 10 min each time. After washing, place it in the secondary antibody solution and incubate for 1 h at 4°C. Then wash it 6 times with TBST, 5 min each time. Finally, evenly drop the prepared developing solution on the membrane and put it into the gel imager for exposure. The results are as Figure 1 and Figure 3 shown.

[0178] Table 1 Compound names, corresponding structures and their cytotoxic activities in HT1080 cells

[0179]

[0180]

Claims

1. A bifunctional molecular compound based on RSL3-induced degradation of GPX4 protein, characterized in that, Specifically selected from the following compounds: RSL3-1-nC-B4: wherein, n = 5, 7, corresponding to the compounds RSL3-1-5C-B4, RSL3-1-7C-B4; RSL3-3-nC-B4: wherein, n = 3, 5, 7, corresponding to the compounds RSL3-3-3C-B4, RSL3-3-5C-B4, RSL3-3-7C-B4; RSL3-3-5P-B5: wherein, n = 5.

2. Use of the bifunctional molecular compound based on RSL3-induced GPX4 protein degradation or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating tumors.

3. A pharmaceutical composition comprising the bifunctional molecular compound based on RSL3-induced GPX4 protein degradation or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable excipient.

4. The pharmaceutical composition according to claim 3, characterized in that, The dosage form of the pharmaceutical composition is any one of injection, tablets and capsules.

5. Use of a pharmaceutical composition according to any one of claims 3-4 in the preparation of a medicament for treating tumors.

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