Covalent polypeptide coupling drug compound as well as preparation and application thereof

Abstract

The invention relates to the technical field of polypeptide drug modification, and discloses a covalent polypeptide coupling drug compound as well as preparation and application thereof. According to the present invention, the polypeptide coupling drug SuFEx-RGD-VC-DOX is constructed, the cyclopeptide 9h is converted into the intermediate SuFEx-RGD-Alkyne, the adriamycin is converted into the adriamycin pre-linking body N3-VC-DOX through the VC dipeptide and the azide reagent, the connection of the cyclopeptide 9h and the adriamycin is achieved through the SuFEx-RGD-Alkyne and the N3-VC-DOX, and the SuFEx-RGD-VC-DOX is successfully prepared. Experiments prove that the SuFEx-RGD-VC-DOX significantly improves the binding stability of adriamycin and a tumor cell receptor, reduces the off-target rate, has good targeting ability to integrin, realizes precise killing of tumor cells, and has better anti-tumor performance compared with a traditional chemotherapeutic drug DOX.

Description

Technical Field

[0001] This invention relates to the field of peptide drug modification technology, and in particular to a covalently coupled peptide drug compound and its preparation and application. Background Technology

[0002] Malignant tumors pose a major global public health challenge. Currently, the most common clinical treatments for tumors include surgical resection, radiotherapy, and chemotherapy. However, traditional radiotherapy and chemotherapy methods generally suffer from insufficient targeting, and the cytotoxic drugs and ionizing radiation used often damage normal tissues, leading to severe toxic side effects and limiting further improvements in treatment efficacy.

[0003] Chemotherapy, short for chemical drug therapy, is a treatment that uses chemotherapeutic drugs to kill cancer cells. It is one of the most effective treatments for cancer. Chemotherapy is a systemic treatment; regardless of the route of administration (oral, intravenous, or intracavitary), the chemotherapy drugs circulate throughout most organs and tissues of the body via the bloodstream.

[0004] Doxorubicin (DOX), a classic anthracycline antitumor antibiotic, is a commonly used cytotoxic drug in clinical practice. It has a killing effect on a variety of tumors, but it has obvious defects: it lacks selectivity and kills both normal cells and tumor cells, causing toxic side effects such as nausea, vomiting, and bone marrow suppression; some tumor cells have developed drug resistance, resulting in poor treatment effects; and the drug is not stable enough when circulating in the body, affecting its bioavailability.

[0005] Peptide-conjugate drugs (PDCs) are targeted drug delivery systems composed of a targeting peptide, a linker, and a cytotoxic drug payload. They have attracted widespread attention due to their advantages such as small molecular weight, excellent tissue penetration, and low immunogenicity. However, existing PDCs generally suffer from off-target effects. The targeting peptide may weakly bind to receptors on the surface of non-target cells, or due to differences in receptor expression, the drug may be released into healthy tissues, causing toxic side effects. Therefore, developing peptide-conjugate drugs with higher targeting specificity and lower off-target rates is of significant clinical importance. Summary of the Invention

[0006] To address the issues of insufficient in vivo circulation stability, low bioavailability, and lack of selectivity of doxorubicin, this invention provides a covalently coupled peptide drug compound and its preparation and application. Based on thioyl fluorination modification and RGD cyclic peptide modification, the binding stability of doxorubicin to tumor cell receptors is improved, the off-target rate is reduced, and precise killing of tumor cells is achieved, thereby improving the bioavailability of doxorubicin.

[0007] The specific technical solution of this invention is as follows:

[0008] As a first aspect of the present invention, a covalently coupled peptide-conjugated drug compound is provided. The chemical structural formula of the covalently coupled peptide-conjugated drug compound is as follows:

[0009] .

[0010] This invention provides a peptide-conjugated drug, SuFEx-RGD-VC-DOX. Experimental verification shows that SuFEx-RGD-VC-DOX has good targeting ability for integrins and a lower off-target probability. The construction of SuFEx-RGD-VC-DOX significantly improves the binding stability of doxorubicin to tumor cell receptors, reduces the off-target rate, achieves precise killing of tumor cells, and improves the bioavailability of doxorubicin. Compared with the traditional chemotherapy drug DOX (doxorubicin), it has better anti-tumor performance.

[0011] As a second aspect of the present invention, a method for preparing a covalently coupled polypeptide drug compound is provided, comprising the following steps:

[0012] Step S1: Dissolve the cyclic peptide 9h in a solvent and react it with SO2F2 gas in the solvent. After the reaction, remove the protecting group from the product to obtain the thioyl fluorinated cyclic peptide, denoted as 10g'.

[0013] Step S2: The thioyl fluorinated cyclic peptide is reacted with 5-hexynyl succinimide ester to obtain the modified molecule, denoted as SuFEx-RGD-Alkyne;

[0014] Step S3: Take the VC dipeptide containing the protecting group and react it with doxorubicin hydrochloride. After the reaction is completed, remove the protecting group of the VC dipeptide, and then add an azide reagent and a coupling agent to carry out an azide reaction to obtain the doxorubicin pre-linked polymer, denoted as N3-VC-DOX.

[0015] Step S4: The modified molecule SuFEx-RGD-Alkyne is reacted with the doxorubicin prelinker N3-VC-DOX to obtain a covalently coupled peptide drug compound, denoted as SuFEx-RGD-VC-DOX;

[0016] The structural formula of the cyclic peptide 9h is as follows:

[0017] ;

[0018] The structural formula of the modified molecule SuFEx-RGD-Alkyne is: ;

[0019] The structural formula of the VC dipeptide containing the Fmoc protecting group is as follows:

[0020] ;

[0021] The structural formula of N3-VC-DOX is:

[0022]

[0023] The structural formula of the covalently coupled polypeptide drug compound is:

[0024] .

[0025] As a preferred embodiment of the above method, in step S1, the solvent is acetonitrile.

[0026] As a preferred embodiment of the above method, in step S1, the reaction is carried out under the condition of adding an alkali, wherein the alkali is selected from one or more of DIEA, Et3N, and NaHCO3.

[0027] As a preferred embodiment of the above method, in step S2, the reaction is carried out under the condition of adding an alkali, wherein the alkali is selected from one or more of DIEA, Et3N, and NaHCO3.

[0028] In step S2, the CAS number of 5-hexyneic acid succinimide ester is 906564-59-8, and its structural formula is:

[0029]

[0030] As a preferred embodiment of the above method, in step S3, the azide reagent is 3-azidopropionic acid, and the coupling agent is HATU or / and HBTU.

[0031] As a preferred embodiment of the above method, in step S4, the reaction is carried out under the conditions of a reducing agent and a catalyst.

[0032] More preferably, the reducing agent is NaVc and / or TCEP.

[0033] More preferably, the catalyst is one or more of CuSO4∙5H2O, Cu(OAc)2, and CuI.

[0034] Based on the above-described covalently coupled peptide drug compound or the above-described preparation method, the present invention provides an application of the covalently coupled peptide drug compound in the preparation of tumor immunotherapy drugs.

[0035] Compared with the prior art, the present invention has the following technical effects:

[0036] (1) This invention provides a peptide-conjugated drug SuFEx-RGD-VC-DOX. Experimental verification shows that SuFEx-RGD-VC-DOX has a good targeting ability for integrins and a lower off-target probability. The construction of SuFEx-RGD-VC-DOX significantly improves the binding stability of doxorubicin to tumor cell receptors, reduces the off-target rate, achieves precise killing of tumor cells, and improves the bioavailability of doxorubicin. It has better anti-tumor performance than the traditional chemotherapy drug DOX.

[0037] (2) In this invention, the cyclic peptide 9h was converted into the intermediate SuFEx-RGD-Alkyne, and doxorubicin was converted into the doxorubicin prelinker N3-VC-DOX by VC dipeptide and azide reagent. The cyclic peptide 9h and doxorubicin were then linked by SuFEx-RGD-Alkyne and N3-VC-DOX, thus successfully preparing a polypeptide conjugate drug SuFEx-RGD-VC-DOX. Attached Figure Description

[0038] Figure 1 This is the liquid phase spectrum of compound N3-VC-DOX in this invention.

[0039] Figure 2 This is the mass spectrum of compound N3-VC-DOX in this invention.

[0040] Figure 3 This is the liquid phase spectrum of compound 10g' in this invention.

[0041] Figure 4 This is the liquid phase spectrum of the compound SuFEx-RGD-Alkyne used in this invention.

[0042] Figure 5 The liquid phase spectrum of SuFEx-RGD-VC-DOX obtained in Example 1 of this invention.

[0043] Figure 6 The mass spectra of SuFEx-RGD-VC-DOX obtained in Example 1 of this invention.

[0044] Figure 7 A comparison of the in vitro cytotoxicity of SuFEx-RGD-VC-DOX obtained in Example 1 of this invention with that of free doxorubicin.

[0045] Figure 8 Comparison of the in vitro cell selective killing effect of SuFEx-RGD-VC-DOX obtained in Example 1 of this invention. Detailed Implementation

[0046] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0047] In one embodiment, a covalently coupled peptide-drug compound is provided. The chemical structural formula of the covalently coupled peptide-drug compound is as follows:

[0048] .

[0049] This embodiment constructed the aforementioned peptide-conjugated drug SuFEx-RGD-VC-DOX. Experimental verification showed that SuFEx-RGD-VC-DOX has good targeting ability for integrins and a low off-target probability. The construction of SuFEx-RGD-VC-DOX in this embodiment significantly improved the binding stability of doxorubicin to tumor cell receptors, reduced the off-target rate, achieved precise killing of tumor cells, and improved the bioavailability of doxorubicin. Compared with the traditional chemotherapy drug DOX, it has better anti-tumor performance.

[0050] In one embodiment, a method for preparing a covalently coupled peptide drug compound is provided, comprising the following steps:

[0051] Step S1: Dissolve the cyclic peptide 9h in a solvent and react it with SO2F2 gas in the solvent. After the reaction, remove the protecting group from the product to obtain the thioyl fluorinated cyclic peptide, denoted as 10g'.

[0052] Step S2: The thioyl fluorinated cyclic peptide is reacted with 5-hexynyl succinimide ester to obtain the modified molecule, denoted as SuFEx-RGD-Alkyne;

[0053] Step S3: Take the VC dipeptide containing the protecting group and react it with doxorubicin hydrochloride. After the reaction is completed, remove the protecting group of the VC dipeptide, and then add an azide reagent and a coupling agent to carry out an azide reaction to obtain the doxorubicin pre-linked polymer, denoted as N3-VC-DOX.

[0054] Step S4: The modified molecule SuFEx-RGD-Alkyne is reacted with the doxorubicin prelinker N3-VC-DOX to obtain a covalently coupled peptide drug compound, denoted as SuFEx-RGD-VC-DOX;

[0055] The structural formula of the cyclic peptide 9h is as follows:

[0056] ;

[0057] The structural formula of the modified molecule SuFEx-RGD-Alkyne is: ;

[0058] The structural formula of the VC dipeptide containing the Fmoc protecting group is as follows:

[0059] ;

[0060] The structural formula of N3-VC-DOX is:

[0061]

[0062] The structural formula of the covalently coupled polypeptide drug compound is:

[0063] .

[0064] In this embodiment, a peptide-conjugated drug, SuFEx-RGD-VC-DOX, was successfully prepared by converting cyclic peptide 9h into the intermediate SuFEx-RGD-Alkyne and doxorubicin into the doxorubicin pre-linker N3-VC-DOX via VC dipeptide and azide reagent. The cyclic peptide 9h and doxorubicin were then linked via SuFEx-RGD-Alkyne and N3-VC-DOX. In one embodiment, experimental verification showed that SuFEx-RGD-VC-DOX significantly improved the binding stability of doxorubicin to tumor cell receptors, reduced off-target rate, and exhibited good targeting ability against integrins, achieving precise killing of tumor cells. Compared with the traditional chemotherapy drug DOX, it has better anti-tumor performance.

[0065] In one embodiment, in step S1, the solvent is acetonitrile.

[0066] In one embodiment, in step S1, the reaction is carried out under the condition of adding a base. For example, one or more of DIEA (N,N-diisopropylethylamine), Et3N (triethylamine), and NaHCO3 (sodium bicarbonate). The role of the base is to promote the phenolic modification of the cyclic peptide precursor to aryl fluorosulfate after 9 hours to obtain the cyclic peptide precursor, denoted as 10g, which is a thioyl fluorinated cyclic peptide containing a protecting group.

[0067] In step S1, an alkaline catalyst promotes the reaction, and SO2F2 gas serves as the core reactant. Its sulfuryl fluoride group (-SO2F) specifically undergoes an electrophilic substitution reaction with the phenolic hydroxyl groups of the tyrosine residues of the RGD cyclic peptide, forming a stable Ar-O-SO2F structure, thus achieving the sulfuryl fluoride modification of the RGD cyclic peptide.

[0068] In step S1, the cyclic peptide 9h is reacted with SO2F2 gas, with acetonitrile as solvent and DIEA as base protectant. The reaction has a good modification effect on the SuFEx group, and the cyclic peptide precursor, i.e., compound 10g, is obtained.

[0069] In another embodiment, step S1 is as follows: Base is added to the stirred solution of the cyclic peptide 9h in acetonitrile; the color immediately changes from colorless to yellow. The reaction flask is then evacuated and backfilled three times with SO2F2 gas. The reaction mixture is stirred at 15-30°C, during which time the solution rapidly changes from yellow to colorless. After 3 minutes, volatiles are removed under vacuum. The crude product is purified by PTLC to obtain 10g of a colorless cyclic peptide precursor.

[0070] In another embodiment, the deprotection treatment in step S1 is as follows: an acid solution is added to the system after the reaction to remove the protecting groups -Pbf, -Boc, and -tBu.

[0071] Specifically, the deprotection treatment in step S1 is as follows: 10g of the cyclic peptide precursor is dissolved in TFA solution and stirred at 15-30°C. After the reaction is complete, the reaction mixture is added to ice-cold diethyl ether, a solid precipitates, is filtered, washed three times with diethyl ether, and dried to obtain 10g of the compound.

[0072] The role of diethyl ether is to promote the precipitation of 10g of the intermediate compound solid. The addition of trifluoroacetic acid can promote the removal of the acid-sensitive protecting group and the release of the side chain protecting group of the cyclic peptide precursor.

[0073] In one embodiment, in step S2, the reaction is carried out under the condition of adding a base, wherein the base is selected from one or more of DIEA, Et3N, and NaHCO3. The purpose of adding the base is to neutralize trifluoroacetic acid and promote the linkage of 5-hexyneic acid to the cyclic peptide precursor 10g'.

[0074] Optionally, step S2 can be as follows: dissolve 10 g of the cyclic peptide precursor in DMF, then add DIEA, and stir the reaction at 15–30 °C. Alternatively, dissolve 5-hexynyl succinimide ester in a certain volume of DMF, then add it to the reaction mixture, and stir at 15–30 °C for 0.5–10 hours. After the reaction is complete, purify the crude mixture to obtain a white solid, SuFEx-RGD-Alkyne.

[0075] In one embodiment, in step S3, the azide reagent is 3-azidopropionic acid, with the following structural formula: .

[0076] In one embodiment, in step S3, the coupling agent is HATU or / and HBTU.

[0077] In one embodiment, in step S3, the VC dipeptide refers to a dipeptide (Val-Cit) formed by linking Val amino acids and Cit amino acids. The protecting group in the VC dipeptide containing the protecting group can be understood as a conventional protecting group used for amino group protection in polypeptides, the purpose of which is to protect the amino groups in the VC dipeptide from degradation or reaction. For example, the VC dipeptide containing the protecting group is Fmoc-Val-Cit-PAB-PNP, purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., catalog number BD221977, with the following chemical structural formula:

[0078] .

[0079] In this embodiment of the invention, the VC dipeptide containing the above-mentioned protective group is denoted as Fmoc-Val-Cit-PAB-PNP, which is used as an example to illustrate the preparation method of the present invention.

[0080] In one embodiment, step S3 is as follows: Fmoc-Val-Cit-PAB-PNP and doxorubicin hydrochloride (DOX·HCl) are dissolved in DMF solvent, triethylamine is added as a catalyst, and the reaction is stirred; then DIEA is added and stirred to remove the Fmoc protecting group; then azide reagent and HBTU coupling agent are added, and after the reaction is completed, diethyl ether is added to precipitate, centrifuged, and purified by thin-layer chromatography to obtain the prodrug N3-VC-DOX.

[0081] As a preferred embodiment of the above method, in step S4, the reaction is carried out under the conditions of a reducing agent and a catalyst.

[0082] More preferably, the reducing agent is NaVc and / or TCEP.

[0083] More preferably, the catalyst is one or more of CuSO4∙5H2O, Cu(OAc)2, and CuI.

[0084] Optionally, step S4 can be: dissolving SuFEx-RGD-Alkyne and N3-VC-DOX in DMF solvent, adding copper catalyst and reducing agent, stirring and reacting, purifying by semi-preparative reversed-phase high-performance liquid chromatography gradient elution, and freeze-drying to obtain the reddish-brown solid target compound SuFEx-RGD-VC-DOX.

[0085] In this embodiment of the invention, the cyclic peptide 9h, i.e., the starting material, is a prior art. The preparation of cyclic peptide 9h can be carried out according to the method described in the specification of Chinese patent application CN2024106559643, as detailed in Example 3 of the specification.

[0086] In this embodiment of the invention, the procedure for purification using a semi-preparative reversed-phase high-performance liquid chromatography gradient elution is as follows: a C18 column is used, with 0.1% trifluoroacetic acid aqueous solution and acetonitrile as the mobile phase. The acetonitrile ratio is linearly reduced from 75% to 55% over 60 minutes at a flow rate of 3-5 mL / min, and detection is performed at a wavelength of 220 nm. After collecting the main fraction, it is freeze-dried to obtain a reddish-brown solid product with a purity ≥95%.

[0087] To illustrate the invention in more detail, the following embodiments are provided.

[0088] Example 1

[0089] The preparation of compound SuFEx-RGD-VC-DOX is provided, and the specific reaction process includes steps S1 to S4, as follows:

[0090] The reaction flow from step S1 to step S2 is shown below:

[0091]

[0092] S1: Under stirring at 400 rpm, DIEA (9.7 mg, 13 μL, 0.075 mmol) was added to a MeCN (1 mL) solution containing 9 h of cyclic peptide (0.025 mmol). The solution color immediately changed from colorless to yellow. The reaction flask was evacuated and purged three times with SO2F2 gas. The reaction mixture was stirred at room temperature, during which the solution rapidly changed from yellow back to colorless. After 3 minutes, volatiles were removed under reduced pressure. The crude product was purified by PTLC to obtain 10 g of colorless SuFEx-modified cyclic peptide. 10 g (0.01 mmol) of cyclic peptide was treated with a 0.5 mL TFA / H2O (95 / 5) deprotection system at room temperature for 1.5 h. After dilution with ice-cold diethyl ether, a solid was formed, filtered, and further purified by RP-HPLC to obtain 10 g' of white solid (7.1 mg, 95% yield).

[0093] S2: 10 g' (20.0 mg, 26.8 μmol) of the cyclic peptide was dissolved in DMF (400 μL), followed by the addition of DIEA (14.0 μL, 80.6 μmol, 3.0 equivalents), and the reaction was stirred at room temperature. Separately, 5-hexynyl NHS ester (5.6 mg, 26.8 μmol, 1.0 equivalent) was dissolved in DMF (100 μL) and added to the above reaction mixture, and the mixture was stirred for another 1 hour at room temperature. After the reaction was complete, the crude product was purified by RP-HPLC to give 19 mg of SuFEx-RGD-Alkyne white solid, with a yield of 85%.

[0094] The reaction flow from step S3 to step S4 is shown below:

[0095]

[0096]

[0097]

[0098] S3: Includes steps S3-1, S3-2, and S3-3:

[0099] S3-1: Dissolve 0.05 mmol Fmoc-Val-Cit-PAB-PNP and 0.05 mmol doxorubicin hydrochloride (DOX·HCl) in 400 μL of DMF, add 0.06 mmol triethylamine, and stir at 40 °C for 12 hours.

[0100] S3-2: Add DIEA (14.0 μL, 0.08 mmol) to the system after the S3-1 reaction is completed to remove the Fmoc protecting group, and stir at room temperature for 12 hours.

[0101] S3-3: Add 0.05 mmol of 3-azidopropionic acid and 0.05 mmol of HBTU coupling agent to the system after the S3-2 reaction. After reacting at room temperature for 8 hours, ether precipitation and centrifugation were performed. The product was purified by thin-layer chromatography (developing solvent: dichloromethane / methanol = 9:1, v / v) to obtain 35.2 mg of the product, i.e., the prodrug N3-VC-DOX, with a yield of 67%. This yield was calculated as the ratio of the actual molar amount of N3-VC-DOX obtained to the theoretical molar amount of N3-VC-DOX corresponding to the molar amount of DOX·HCl drug raw material. The product N3-VC-DOX was analyzed by HPLC and mass spectrometry. Results are shown below. Figure 1 , Figure 2 .like Figure 1 The image shown is an HPLC chromatogram. Figure 2 The image shown is a mass spectrum.

[0102] S4: N3-VC-DOX (15.7 mg, 15 μmol, 1.0 equivalent) and SuFEx-RGD-Alkyne (15.1 mg, 18 μmol, 1.2 equivalent) were dissolved in DMF (1.5 mL), and the mixture was stirred at room temperature. Separately, CuSO4·5H2O (2.25 mg, 9 μmol, 0.6 equivalent) and NaVc (11.9 mg, 60 μmol, 4.0 equivalent) were dissolved in H2O (750 μL), and then added to the reaction mixture. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the crude product was purified by RP-HPLC and freeze-dried to give 24.6 mg of SuFEx-RGD-VC-DOX as a reddish-brown solid, with a yield of 86%.

[0103] The obtained SuFEx-RGD-VC-DOX had an overall yield of 57.6%, calculated using DOX·HCl as the starting material. Specifically, the overall yield was calculated as the ratio of the actual molar amount of SuFEx-RGD-VC-DOX obtained to the theoretical molar amount of SuFEx-RGD-VC-DOX corresponding to the molar amount of DOX·HCl drug raw material. The overall yield calculation method was the same for other embodiments.

[0104] The product SuFEx-RGD-VC-DOX was analyzed by HPLC and mass spectrometry. Results are shown below. Figure 5 , Figure 6 .like Figure 5 The image shown is an HPLC chromatogram. Figure 6 The mass spectrum is shown. The mass spectrometry data of compound SuFEx-RGD-VC-DOX is: HRMS(ESI) m / z calcd for C83H102FN17O31S / 1883.6600, found 1884..7900.

[0105] The 10g of cyclic peptide obtained in step S1 was analyzed by NMR, and the data are as follows:

[0106] The 1H NMR spectrum data of 10g of cyclic peptide are as follows:

[0107] 1HNMR(500MHz,DMSO-d6)δ8.43(d,J=8.0Hz,1H),8.13(d,J=5.9Hz,1H),8.01(d,J=7.9Hz,1H),7.76(d,J=2.1Hz,2H),7.62-7.44(m,3H),7.19(d,J=7 .9Hz,1H),6.75(d,J=16.0Hz,1H),6.38(s,2H),4.82-4.76(m,1H),4.69( dd,J=11.3,3.1Hz,1H),4.62(q,J=7.4Hz,1H),4.29(td,J=11.0,5.5Hz,2H ),4.20-4.14(m,1H),4.09(dd,J=16.8,6.7Hz,1H),3.73(s,3H),3.52(dd ,J=16.9,3.4Hz,1H),3.04(dd,J=13.4,5.4Hz,1H),3.01-2.90(m,5H),2.7 3(dd,J=16.3,7.0Hz,1H),2.49-2.44(m,4H),2.42(s,3H),2.00(s,3H),1 .64-1.54(m,1H),1.41(s,8H),1.37(d,J=6.6Hz,18H),1.34-1.30(m,2H).

[0108] The carbon NMR spectrum data of 10g of cyclic peptide are as follows:

[0109] 13 CNMR(126MHz,DMSO)δ172.10,170.75,170.38,169.70,169.62,169.24,165.52,157.89,15 6.45,155.29,146.82,139.54,137.71,135.41,134.68,134.24,131.87,129.83,126.35,1 24.74,122.27,122.12,116.69,86.73,80.74,78.96,63.09,57.34,55.34,52.91,52.11,51.81,49.44,42.95,42.31,37.78,36.53,30.38,28.74,28.53,28.13,19.38,18.03,12.71.

[0110] Cyclic peptide 10g' is the substance obtained by removing the acid-sensitive protecting group from cyclic peptide 10g. Figure 3 The liquid chromatography spectrum of the cyclic peptide 10g' prepared in this embodiment is shown below. The structural formula of the cyclic peptide 10g' is as follows:

[0111]

[0112] The white solid SuFEx-RGD-Alkyne obtained in step S2 was analyzed by HPLC and mass spectrometry. The HPLC chromatogram results are shown below. Figure 4 The mass spectrometry results are as follows: HRMS (ESI) m / z calcd for C 34 H 44 FN8O 14 S (M + H + ) 839.2676, found 839.2656.

[0113] The N3-VC-DOX obtained in step S3 was analyzed by HPLC and mass spectrometry. The results are shown below. Figure 1 , Figure 2 .like Figure 1 The image shown is an HPLC chromatogram. Figure 2 The mass spectrum is shown. The mass spectrometry data of compound N3-VC-DOX is HRMS(ESI) m / z calcd for C49H59N9O17 / 1045.4000, found 1046.0000.

[0114] Example 2

[0115] This embodiment provides the preparation of compound SuFEx-RGD-VC-DOX. Compared with Example 1, the difference is that in step S3-1, the condensation reaction temperature of Fmoc-Val-Cit-PAB-PNP with DOX·HCl is changed from 40℃ in Example 1 to 25℃ (room temperature), 35℃, 50℃, and 60℃ for four sets of experiments. Other steps are the same as in Example 1.

[0116] The results showed that the reaction at 35°C yielded 34.1 mg of product N3-VC-DOX, which was comparable to the yield in Example 1; the reaction at 50°C yielded 37.6 mg of product N3-VC-DOX; the reaction at 25°C resulted in a slower reaction rate and a lower yield of only 24.1 mg of product N3-VC-DOX; and the reaction at 60°C resulted in a decrease in yield instead of an increase, yielding 26.0 mg of product N3-VC-DOX. Furthermore, TLC monitoring revealed a significant increase in decomposition byproducts of the doxorubicin component.

[0117] Therefore, it can be concluded that the condensation reaction temperature in step S3 should be controlled between 35℃ and 50℃, which can ensure a sufficient reaction rate and effectively inhibit the degradation of doxorubicin. In this example, the total yields of SuFEx-RGD-VC-DOX prepared at 25℃ and 60℃ were 39% and 42%, respectively.

[0118] Example 3

[0119] This embodiment provides the preparation of compound SuFEx-RGD-VC-DOX. Compared with Example 1, the main difference is that in step S3-2, the 0.08 mmol of DIEA base reagent after removing the Fmoc protecting group is replaced with 0.08 mmol of triethylamine. The other steps are the same as in Example 1.

[0120] The results showed that the total yield of SuFEx-RGD-VC-DOX prepared in this example was 45% when triethylamine was used as the deprotection base reagent.

[0121] Example 4

[0122] This embodiment provides the preparation of compound SuFEx-RGD-VC-DOX. Compared with Example 1, the main difference is that in step S3-3, the addition of 0.05 mmol HBTU coupling agent is replaced with the addition of 0.05 mmol EDCI or 0.05 mmol HATU, and two sets of experiments are performed. The other steps are the same as in Example 1.

[0123] The results showed that the total yield of SuFEx-RGD-VC-DOX prepared in this example was 46% when EDCI was used as the coupling reagent, and the total yield of SuFEx-RGD-VC-DOX prepared in this example was 48% when HATU was used as the coupling reagent.

[0124] Example 5

[0125] This embodiment provides the preparation of compound SuFEx-RGD-VC-DOX. The main difference from Example 1 is that in step S4, 9 μmol of the catalyst CuSO4∙5H2O is replaced with 9 μmol of CuI. All other steps are the same as in Example 1.

[0126] The results showed that the total yield of SuFEx-RGD-VC-DOX prepared in this example was 37% when CuI was used as a catalyst.

[0127] Example 6

[0128] This embodiment investigates the targeted uptake mechanism of the peptide-conjugated drug SuFEx-RGD-VC-DOX obtained in Example 1 on integrin-overexpressing tumor cells. The specific process is as follows:

[0129] To investigate the targeted uptake mechanism of SuFEx-RGD-VC-DOX on integrin-overexpressing tumor cells, A549 cells were used as the research subject. Utilizing the autofluorescence properties of DOX, flow cytometry was used to examine the cellular drug uptake behavior. A PBS control group, a free DOX treatment group, and a SuFEx-RGD-VC-DOX treatment group were set up. Fluorescence intensity reflected the cumulative intracellular drug concentration, and the cellular uptake efficiency and mechanism results of different groups were compared and analyzed. Figure 7 As shown, compared to the PBS control group (red peak), the fluorescence signal in the free DOX treatment group (blue peak) shifted to the right, indicating that the drug entered the cells via passive diffusion. However, the SuFEx-RGD-VC-DOX treatment group (orange peak) exhibited the most significant fluorescence enhancement, with its peak position clearly located to the right of the free DOX. This significant "right shift" confirms the higher cellular uptake efficiency of the PDC compound. This is mainly attributed to the specific binding of integrins highly expressed on the surface of A549 cells to the RGD cyclic peptide in PDC, thereby initiating receptor-mediated endocytosis and "pumping" more drug into the cell. This is also the direct reason why the compound SuFEx-RGD-VC-DOX exhibits stronger cytotoxicity.

[0130] Example 7

[0131] This embodiment verifies the targeted delivery efficiency and safety of the peptide conjugate SuFEx-RGD-VC-DOX obtained in Example 1. The specific process is as follows:

[0132] (1) Material preparation: A549 (high expression of integrin) and HEK293 cells in logarithmic growth phase were cultured in DMEM containing 10% fetal bovine serum; SuFEx-RGD-VC-DOX and free DOX were prepared into working solutions of various concentrations with PBS; CCK-8 kit, 96-well plate and microplate reader were prepared.

[0133] (2) Cell treatment: Prepare 5×10⁻⁶ cells of each of the two cell types. 4 Inoculate 5 × 10³ cells / well of suspension into 96-well plates. After 24 h of culture and adhesion, discard the old culture medium and add fresh culture medium containing different concentrations of drug (0.1-20 μmol / L series concentrations, 6 replicates / group, including blank and negative control). Incubate in the dark for 48 h.

[0134] (3) Toxicity test: Add 10 μL of CCK-8 reagent to each well, incubate for 2 hours, and then measure the OD value at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0135] (4) Data processing: Calculate cell viability according to the formula [(experimental group OD - blank OD) / (negative control OD - blank OD) × 100%], plot and statistically analyze using GraphPad Prism 8.0, and compare the differences in drug toxicity.

[0136] The in vitro cytotoxicity of compound SuFEx-RGD-VC-DOX and free doxorubicin (DOX) was compared with that of A549 cells (highly expressing integrins) and HEK293 cells (normal cells). The experimental results are as follows: Figure 8 As shown.

[0137] The results showed that both drugs exhibited dose-dependent cytotoxicity.

[0138] The results also showed that in the target A549 cells, the cell-inhibiting effect of SuFEx-RGD-VC-DOX was significantly stronger than that of free DOX. For example, at a concentration of 10 μmol / L, the cell survival rate in the SuFEx-RGD-VC-DOX treatment group decreased to about 30%, while the DOX group remained close to 70%. This indicates that the RGD peptide successfully mediated the targeted recognition and uptake of DOX on integrin-overexpressing cells, enhancing the drug's efficacy.

[0139] The results also showed that in untargeted normal HEK293 cells, free DOX exhibited severe toxicity, with cell survival decreasing sharply with increasing concentration; while the SuFEx-RGD-VC-DOX group showed extremely low toxicity to normal cells within the tested concentration range (survival rate remained above 80%). This result confirms that the strategy of constructing the SuFEx-RGD-VC-DOX drug can effectively reduce the systemic toxicity of DOX and significantly improve the therapeutic window.

[0140] As can be seen from the above results, the present invention significantly improves the binding stability of doxorubicin to tumor cell receptors, reduces off-target rate, achieves precise killing of tumor cells, and improves the bioavailability of doxorubicin through the construction of SuFEx-RGD-VC-DOX, thus exhibiting better anti-tumor performance compared to the traditional chemotherapy drug DOX.

[0141] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0142] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A covalently coupled polypeptide drug compound, characterized in that: The chemical structural formula of the covalently coupled polypeptide drug compound is as follows: 。 2. The method for preparing a covalently coupled polypeptide drug compound as described in claim 1, characterized in that: Includes the following steps: Step S1: Dissolve the cyclic peptide 9h in a solvent and react it with SO2F2 gas in the solvent. After the reaction, remove the protecting group from the product to obtain the thioyl fluorinated cyclic peptide, denoted as 10g'. Step S2: The thioyl fluorinated cyclic peptide is reacted with 5-hexynyl succinimide ester to obtain the modified molecule, denoted as SuFEx-RGD-Alkyne; Step S3: Take the VC dipeptide containing the protecting group and react it with doxorubicin hydrochloride. After the reaction is completed, remove the protecting group of the VC dipeptide, and then add an azide reagent and a coupling agent to carry out an azide reaction to obtain the doxorubicin pre-linked polymer, denoted as N3-VC-DOX. Step S4: The modified molecule SuFEx-RGD-Alkyne is reacted with the doxorubicin prelinker N3-VC-DOX to obtain a covalently coupled peptide drug compound, denoted as SuFEx-RGD-VC-DOX; The structural formula of the cyclic peptide 9h is as follows: ； The structural formula of the modified molecule SuFEx-RGD-Alkyne is: ; The structural formula of the VC dipeptide containing the Fmoc protecting group is as follows: ； The structural formula of N3-VC-DOX is: The structural formula of the covalently coupled polypeptide drug compound is:

3. The preparation method according to claim 2, characterized in that: In step S1, the solvent is acetonitrile.

4. The preparation method according to claim 2 or 3, characterized in that: In step S1, the reaction is carried out under the condition of adding an alkali, wherein the alkali is selected from one or more of DIEA, Et3N, and NaHCO3.

5. The preparation method according to claim 2, characterized in that: In step S2, the reaction is carried out under the condition of adding an alkali, wherein the alkali is selected from one or more of DIEA, Et3N, and NaHCO3.

6. The preparation method according to claim 2, characterized in that: In step S3, the azide reagent is 3-azidopropionic acid, and the coupling agent is HATU or / and HBTU.

7. The preparation method according to claim 2, characterized in that: In step S4, the reaction is carried out under the conditions of a reducing agent and a catalyst.

8. The preparation method according to claim 7, characterized in that: The reducing agent is NaVc and / or TCEP.

9. The preparation method according to claim 7, characterized in that: The catalyst is one or more of CuSO4∙5H2O, Cu(OAc)2, and CuI.

10. The use of a covalently coupled peptide drug compound as described in claim 1, or a covalently coupled peptide drug compound prepared by any one of claims 2 to 9, in the preparation of tumor immunotherapy drugs.

Images