RGD (arginine-glycine-aspartic acid) peptide modified mercaptododecane derivative and synthesis method thereof

Abstract

The invention relates to an RGD (arginine-glycine-aspartic acid) peptide modified mercaptododeborane derivative and a synthesis method, the derivative utilizes RGD peptide to perform chemical coupling modification on mercaptododeborane anion salt (BSH), and the BSH and the RGD peptide are connected through a hydrophilic intermediate connector with a double bond at one end and carboxyl at the other end. A hydrophilic chain is selectively introduced to serve as an intermediate connector, a series of compounds are synthesized through a convenient and efficient sulfydryl-alkene click reaction and an amino-carboxyl amidation condensation reaction and are purified, various cationic BSH salts and RGD peptide are connected, and targeted modification of the RGD peptide on an existing boron carrying agent BSH is achieved. The derivative obtained by the invention is not reported before, has the potential to improve the tumor targeting ability of the existing boron carrying agent, and is expected to be used as a drug compound for BNCT treatment.

Description

Technical Field

[0001] The present invention relates to the fields of chemical synthesis and biomedicine, and particularly to a class of RGD peptide-modified mercapto dodecaborane derivatives and a synthesis method thereof. Background Art

[0002] Cancer treatment is an important field related to human survival and health. Currently, the best tumor treatment method is to reduce the damage to normal tissues while killing tumor cells. As a precise tumor treatment method, Boron Neutron Capture Therapy (BNCT) has the following advantages compared with the commonly used radiotherapy methods in clinics: (1) 10 B used in BNCT is non-radioactive and safe; (2) BNCT causes little damage to normal tissues; (3) BNCT does not require an oxygen enhancement effect, and can kill both oxygen-rich cells and oxygen-deficient cells; (4) BNCT requires fewer treatment sessions compared with other radiotherapy means. Currently, BNCT has been used for the treatment of glioblastoma multiforme, meningioma, head and neck cancer, lung cancer, breast cancer, hepatocellular carcinoma, skin malignancies, metastatic diseases, etc.

[0003] Boron carriers (compounds containing 10 B), as an important factor affecting the development and clinical application of BNCT, have currently gone through three stages. Among them, the targeted modification of the second-generation boron carriers (4-dihydroxyboron-L-phenylalanine (L-BPA) and undecahydromercapto dodecaborate anion salt (BSH)) alone or in combination is a promising direction for BNCT boron carriers, and BSH has a higher boron content compared with a single molecule of BPA.

[0004] Chinese invention patent CN115806567A (Publication date: March 17, 2023) discloses a method for modifying dodecaborane derivatives with heptamethine cyanine fluorescent dye IR-780. IR-780 has both fluorescence and photothermal conversion functions, and the modified boron carrier is expected to combine photothermal therapy (PTT) and BNCT. Chinese invention patent CN109125739B (Publication date: January 4, 2019) discloses a multifunctional polymer micelle targeted drug delivery system. Mercapto groups are introduced by chemically modifying some side chains of polyethylene glycol-polylysine block copolymer with dimethyl 3,3'-dithiobis(propionimidate) dihydrochloride, and then BSH is reacted with the side chains by disulfide bonds to form polymer micelles in aqueous solution, which can increase the drug delivery content and improve the targeted therapeutic effect. Chinese invention patent CN116655675A (Publication date: August 29, 2023) discloses a porphine-BSH conjugate and its application in boron neutron capture therapy, and porphine is used to modify BSH to achieve targeting glioma cells by penetrating the blood-brain barrier. Chinese invention patent CN116874515A (Publication date: October 13, 2023) discloses an amino acid-BSH targeted hybrid compound, its preparation method and application, and amino acid is used to modify BSH to increase the accumulation amount and selectivity of boron-containing compounds in tumor cells.

[0005] RGD peptides are a class of polypeptides with an arginine-glycine-aspartic acid structure, including linear peptides and cyclic peptides, and can be applied to the field of tumor targeting because RGD peptides have a binding effect with αvβ3 integrin highly expressed by most tumor cells. Integrin receptors are a group of important cell surface adhesion proteins and play a key role in tumor angiogenesis and tumor metastasis. Integrin αvβ3 mainly exists in the initial stage of angiogenesis, has little expression on normal epithelial cells and endothelial cells, and is overexpressed on endothelial cells stimulated by neovascularization of tumor cells such as melanoma, glioblastoma, breast cancer, and lung cancer.

[0006] Chinese Invention Patent CN110368501A (Publication Date: October 25, 2019) discloses an RGD peptide-modified boron drug delivery system and its preparation and application. Polyethylene glycol and RGD peptide are used to modify B nanosheets and effectively load doxorubicin and heat shock protein inhibitors to achieve the synergistic effect of combined cryogenic photothermal therapy and chemotherapy. Chinese Invention Patent CN103169664B (Publication Date: June 26, 2013) discloses an RGD peptide-modified double-layer drug-loaded nanoparticle and its preparation method, using RGD peptide to target-modify double-layer drug-loaded cationic nanoparticles. Chinese Invention Patent CN115120739A (Publication Date: September 30, 2022) discloses an RGD peptide-modified tumor-targeting polymer and its preparation method and application, providing an RGD peptide-polyethylene glycol-fluorenylmethoxycarbonyl drug release system for active tumor targeting. Chinese Invention Patent CN110522923A (Publication Date: December 3, 2019) provides a method for using fructose and RGD peptide together to modify lipid materials to endow them with dual targeting to triple-negative breast cancer.

[0007] In the prior art, although there have been relevant studies on using some RGD peptides to target-modify BSH, Jiejian Chen et al. in "Remarkable boron delivery of iRGD-modified polymeric nanoparticles for boron neutron capture therapy" tried to improve the selectivity of BSH to tumors by covalently grafting BSH into polymers and then surface-modifying with iRGD, but they delivered boron in the form of nanoparticles. Sadaaki Kimura et al. in "Synthesis and evaluation of cyclic RGD-boron cluster conjugates to develop tumor-selective boron carriers for boron neutron capture therapy" also tried to use RGD peptides to modify BSH, but there are problems such as differences in the selection of intermediate linkers, limited water solubility of the products, and complex reaction and purification methods. Based on this, the present invention provides a class of RGD peptide-modified mercaptododecaborane derivatives and synthesis methods. Summary of the Invention

[0008] The object of the present invention is to overcome the problems in existing related studies that attempt to modify BSH with RGD peptides, such as differences in the selection of intermediate linkers, limited water solubility of products, and complex reaction and purification methods. A class of RGD peptide-modified mercaptododecaborane derivatives and their synthesis methods are provided. An intermediate linker with good hydrophilicity is designed according to the compound structure, and the modification of the BSH structure by RGD peptides is achieved through a convenient and efficient reaction, and it is hoped that this boron carrier can be used in BNCT treatment.

[0009] A class of RGD peptide-modified mercaptododecaborane derivatives, which chemically couple and modify mercaptododecaborane anion salt (BSH) with RGD peptides. The BSH and RGD peptides are connected by a hydrophilic intermediate linker with a double bond at one end and a carboxyl group at the other end; this class of derivatives has the potential to improve the tumor targeting ability of existing boron carriers and is expected to be used as a pharmaceutical compound for BNCT treatment.

[0010] Furthermore, the dodecaborane structure is derived from mercaptododecaborane anion salt; among them, undecahydro-mercaptododecaborane exists in the form of a divalent anion, and the corresponding cation is an alkali metal cation, including lithium (Li+), sodium (Na+), potassium (K+), rubidium (Rb+), cesium (Cs+), or quaternary ammonium salt (NH4+), and their mixtures.

[0011] Furthermore, the RGD peptide has a binding effect with integrin αvβ3.

[0012] Furthermore, the RGD peptide structure is the remaining part after the RGD peptide participates in the reaction. Among them, the primary amine in the RGD peptide undergoes a carboxyl-amino amide condensation reaction with the carboxyl terminal of the intermediate linker. The RGD peptide needs to have a primary amine group to participate in the reaction. Possible RGD peptides include but are not limited to iRGD, cRGD fK, cRGDyK, cRGDfC, etc.

[0013] A synthesis method of a class of RGD peptide-modified mercaptododecaborane derivatives, and its synthesis reaction includes the following two steps:

[0014] Step S1: The mercapto group on BSH undergoes a thiol-ene click reaction with one end of the double bond under ultraviolet irradiation, and BSH and the intermediate linker are connected through the click reaction, where the molar amount of the intermediate linker is in excess of BSH, and the excess ratio is 10-100%, and the preferred excess ratio is 10-50%;

[0015] Step S2: First, activate the carboxyl group in the product of Step S1 with N,N'-carbonyldiimidazole (CDI), and then add the RGD peptide containing a primary amine to complete all reactions;

[0016] The molar amount of the product of Step S1 is in excess of CDI and the RGD peptide, and the preferred excess ratio is 10-30%.

[0017] Furthermore, the synthesis reaction is carried out at room temperature and atmospheric pressure, and the specific temperature range is 10-30°C, and the preferred temperature range is 15-25°C.

[0018] Furthermore, the thiol-ene click chemical reaction time ranges from 2 to 6 hours depending on the specific intermediate linker, and the preferred time is 3 to 4 hours; the time for CDI to activate the carboxyl group ranges from 2 to 6 hours depending on the specific chemical structure, and preferably 3 to 4 hours; the time for the condensation reaction with the addition of RGD peptide ranges from 2 to 6 hours depending on the specific intermediate linker and RGD peptide, and preferably 3 to 5 hours.

[0019] Furthermore, the synthesis reactions are all carried out under magnetic stirring conditions. Step S1 is completed in an aqueous solvent, and the volume of pure water used for dissolution is as small as possible; Step S2 reacts in an anhydrous dimethyl sulfoxide solution, and other available solvents include but are not limited to anhydrous tetrahydrofuran, anhydrous dichloromethane, etc.

[0020] Furthermore, Step S1 and Step S2 are purified by the methods of poor solvent precipitation and dialysis respectively;

[0021] In Step S1, after the click chemical reaction between BSH and the intermediate linker is completed, the product and the intermediate linker are dissolved, and the product is precipitated from the system with the poor solvent anhydrous ethanol. The product is collected by centrifugation and washing multiple times. Available poor solvents include but are not limited to ethanol, ether, etc.;

[0022] In Step S2, according to the different molecular weights of the separated product and impurities, a dialysis bag with an appropriate cut-off molecular weight is selected, and the product is purified by dialysis. The final product is obtained as a solid after vacuum drying or freeze drying.

[0023] Compared with the prior art, the advantages and effects of this application are as follows:

[0024] 1. The synthesis method of a class of RGD peptide-modified thiol dodecaborane derivatives provided by the present invention has the potential to improve the targeting ability of the existing boron carrier BSH, and the present invention designs and upgrades the reactants, reaction conditions, reaction steps, and purification methods on the basis of the previous work in related fields.

[0025] 2. A class of RGD peptide-modified thiol dodecaborane derivatives provided by the present invention, after targeting and modifying BSH with RGD peptide by introducing a hydrophilic intermediate linker, not only maintains the good water solubility of the compound, but also shows low toxicity and better tumor-targeting performance in cell and animal experiments. Such compounds have not been reported before and are expected to be used in the clinical treatment of BNCT in the future.

[0026] The above description is only an overview of the technical solution of the present application. In order to understand the technical means of the present application more clearly, so that it can be implemented in accordance with the content of the specification, and in order to make the above and other purposes, features and advantages of the present application more obvious and understandable, the following will be described in detail with reference to the preferred embodiments of the present application and the accompanying drawings.

[0027] From the following detailed description of the specific embodiments of the present application in conjunction with the accompanying drawings, those skilled in the art will become more clear about the above and other purposes, advantages and features of the present application. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to actual scale.

[0029] Wherein:

[0030] Figure 1 is a schematic structural diagram of a thiol dodecaborane derivative modified with an RGD peptide (taking cRGDfK as an example of the RGD peptide);

[0031] Figure 2 is the chemical structure of some RGD peptides that can be used for targeted modification of thiol dodecaborane derivatives;

[0032] Figure 3 is a schematic diagram of the synthesis reaction of a thiol dodecaborane derivative modified with an RGD peptide (taking cRGDfK as an example of the RGD peptide);

[0033] Figure 4 is the nuclear magnetic resonance hydrogen spectrum characterization of the synthesis product;

[0034] Figure 5 is the infrared spectrum characterization of the synthesis product;

[0035] Figure 6 is the result of the cytotoxicity experiment;

[0036] Figure 7 is the cell uptake result of A549 cells;

[0037] Figure 8 is the tissue distribution result of the B content in the tumor-bearing nude mouse model after injecting the drug. Detailed Embodiments

[0038] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the following will clearly and completely describe the technical solutions in the embodiments of this application with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, rather than all of them. In the following description, providing specific details such as specific configurations and components is only to help comprehensively understand the embodiments of this application. Therefore, those skilled in the art should clearly understand that various changes and modifications can be made to the embodiments described here without departing from the scope and spirit of this application. Additionally, for the sake of clarity and conciseness, descriptions of known functions and structures are omitted in the embodiments.

[0039] It should be understood that the "one embodiment" or "this embodiment" mentioned throughout the specification means that the specific features, structures, or characteristics related to the embodiment are included in at least one embodiment of this application. Therefore, the "one embodiment" or "this embodiment" that appears throughout the specification does not necessarily refer to the same embodiment. In addition, these specific features, structures, or characteristics can be combined in one or more embodiments in any suitable manner.

[0040] In addition, this application may repeat reference numerals and / or letters in different examples. This repetition is for the purpose of simplification and clarity, and does not itself indicate the relationship between the various embodiments and / or settings discussed.

[0041] The term "and / or" in this document is merely a description of the association relationship of the associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist simultaneously. The term " / and" in this document is a description of another association object relationship, indicating that two relationships can exist. For example, A / and B can represent: A exists alone, and A and B exist. Additionally, the character " / " in this document generally indicates that the associated objects before and after are in an "or" relationship.

[0042] The term "at least one" in this document is merely a description of the association relationship of the associated objects, indicating that three relationships can exist. For example, at least one of A and B can represent: A exists alone, A and B exist simultaneously, and B exists alone.

[0043] It should also be noted that in this document, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise", or any other variant thereof are intended to cover non-exclusive inclusion.

[0044] Embodiment 1

[0045] This example introduces a class of RGD peptide-modified mercaptododecaborane derivatives. This class of derivatives chemically couples and modifies mercaptododecaborane anion salt (BSH) with RGD peptide. The BSH and RGD peptide are connected by a hydrophilic intermediate linker with a double bond at one end and a carboxyl group at the other end. This class of derivatives has the potential to improve the tumor targeting ability of existing boron carriers and is promising as a pharmaceutical compound for BNCT treatment.

[0046] For the structural schematic diagram of RGD peptide-targeted modified mercaptododecaborane derivatives (taking cRGDfK as an example of RGD peptide), please refer to the appendix Figure 1 . When n = 1, this intermediate linker is 3-(2-propen-1-yloxy)propionic acid. When n = 2, this intermediate linker is 3-[2-(2-propen-1-yloxy)ethoxy]-propionic acid. When n = 3, this intermediate linker is 3-[2-[2-(2-propen-1-yloxy)ethoxy]ethoxy]-propionic acid. As n gradually increases, the intermediate linker is mainly a polyethylene glycol (PEG) segment.

[0047] Preferably, the dodecaborane structure is derived from mercaptododecaborane anion salt; among them, undecahydro-mercaptododecaborane exists in the form of a divalent anion, and the corresponding cation is an alkali metal cation, including lithium (Li+), sodium (Na+), potassium (K+), rubidium (Rb+), cesium (Cs+), or quaternary ammonium salt (NH4+), and mixtures thereof.

[0048] Preferably, the RGD peptide has a binding effect with integrin αvβ3;

[0049] Preferably, the RGD peptide structure is the remaining part after the RGD peptide participates in the reaction. Among them, the primary amine in the RGD peptide undergoes a carboxyl-amino amidation condensation reaction with the carboxyl terminal of the intermediate linker. The RGD peptide needs to have a primary amine group to participate in the reaction. Possible RGD peptides include, but are not limited to, iRGD, cRGD fK, cRGDyK, cRGDfC, etc. For the chemical structures of some RGD peptides that can be used for the targeted modification of mercaptododecaborane derivatives, please refer to the appendix Figure 2 .

[0050] The technical effects achieved by this example are as follows: After targeting and modifying BSH with RGD peptide by introducing a hydrophilic intermediate linker in this example, not only the good water solubility of the compound is maintained, but also low toxicity and better tumor-targeting performance are demonstrated in cell and animal experiments. This class of compounds has not been reported before and is promising for future clinical treatment of BNCT.

[0051] Example 2

[0052] Based on Example 1, this example introduces a synthesis method for a class of RGD peptide-modified mercaptododecaborane derivatives. Its synthesis reaction includes the following two steps:

[0053] Step S1: The thiol group on BSH and one end of the double bond undergo a thiol-ene click reaction under ultraviolet irradiation, and BSH and the intermediate linker are connected through the click reaction. The molar amount of the intermediate linker is in excess of that of BSH, and the excess ratio is 10-100%, preferably 10-50%;

[0054] Step S2: First, activate the carboxyl group in the product of Step S1 using N,N'-carbonyldiimidazole (CDI), and then add the RGD peptide containing a primary amine to complete the entire reaction;

[0055] The molar amount of the product of Step S1 is in excess of that of CDI and the RGD peptide, and the preferred excess ratio is 10-30%;

[0056] For the overall synthesis reaction schematic diagram of the RGD peptide-targeted modified thiol dodecaborane derivative (taking cRGDfK as an example of the RGD peptide), please refer to the appendix Figure 3 。

[0057] Preferably, the synthesis reaction is carried out at room temperature and normal pressure, and the specific temperature range is 10-30 °C, and the preferred temperature range is 15-25 °C.

[0058] Preferably, the reaction time of the thiol-ene click chemical reaction is between 2 and 6 hours depending on the specific intermediate linker, and the preferred time is 3-4 hours; the time for CDI to activate the carboxyl group is between 2 and 6 hours depending on the specific chemical structure, preferably 3-4 hours; the time for adding the RGD peptide for the condensation reaction is between 2 and 6 hours depending on the specific intermediate linker and RGD peptide, preferably 3-5 hours.

[0059] Preferably, the synthesis reaction is carried out under magnetic stirring conditions. Step S1 is completed in an aqueous solvent, and the volume of pure water used for dissolution is as small as possible; Step S2 reacts in an anhydrous dimethyl sulfoxide solution, and other available solvents include but are not limited to anhydrous tetrahydrofuran, anhydrous dichloromethane, etc.

[0060] Preferably, Step S1 and Step S2 are purified by the methods of precipitation with a poor solvent and dialysis respectively;

[0061] In Step S1, after the click chemical reaction between BSH and the intermediate linker is completed, the product is dissolved with the intermediate linker, and the product is precipitated from the system using the poor solvent anhydrous ethanol. The product is collected through multiple centrifugations and washings. Available poor solvents include but are not limited to ethanol, ether, etc.;

[0062] In Step S2, according to the different molecular weights of the separated product and impurities, a dialysis bag with an appropriate cut-off molecular weight is selected, and the product is purified by dialysis. The final product is obtained as a solid after vacuum drying or freeze drying.

[0063] The technical effects achieved in this embodiment are as follows: This embodiment has the potential to improve the targeting ability of the existing boron carrier BSH, and on the basis of the previous foundation in the relevant field, the reactants, reaction conditions, reaction steps, and purification methods have all been designed and upgraded.

[0064] Example 3

[0065] On the basis of the above embodiment, this embodiment introduces the compound obtained when the intermediate linker is 3-(2-propen-1-yloxy)propionic acid and the determination of its structure.

[0066] Weigh 44.2 mg of Cs 2 [BSH] and 19.5 mg of 3-(2-propen-1-yloxy)propionic acid (APA) are respectively dissolved in pure water. The pure water used for dissolution should be as little as possible, about 2 - 5 mL. The reaction system is placed in a 20 mL quartz glass reaction flask and magnetically stirred for 3 hours under ultraviolet light irradiation (405 nm, 30 W). The product is precipitated with anhydrous ethanol more than 10 times the volume of the reaction system solution, and the product is collected by repeating the centrifugation and washing operations, denoted as BSH-APA. Weigh 28.6 mg of the product from step S1 and 7.4 mg of CDI and dissolve them in anhydrous DMSO respectively. The mixture is placed in a 20 mL glass reaction flask and magnetically stirred for 3 hours. Then add 27.4 mg of cRGDfK cyclic peptide and magnetically stir for 3 hours. After that, the mixed solution is dialyzed in pure water for 3 days, and the pure water is replaced regularly. The solution in the dialysis bag is freeze-dried to obtain the final solid product denoted as BSH-cRGD, corresponding to the product with Figure 1 n = 1 in the attachment.

[0067] After the synthesis, the chemical structure of the product is characterized by nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR), infrared spectrum (FT-IR), and the B content is determined by inductively coupled plasma optical emission spectrometer (ICP-OES) to determine the structure of BSH-cRGD.

[0068] Human lung cancer cells A549 cells (high expression of ανβ3 integrin) and mouse fibroblast cells L929 are used to detect the in vitro cytotoxicity and cell uptake of BSH-cRGD. The two kinds of cells are seeded in a 96-well plate at a density of 8000 cells / well. After adherent incubation for 24 hours, they are incubated with solutions containing BSH-cRGD at concentrations of 5, 10, 20, 40, 80, 160 μM for another 24 hours, and the cytotoxicity is detected by the CCK-8 method. BSH and BSH-cRGD are fluorescently labeled with FITC, the cell nucleus is stained with Hoechst33342 dye, and the cell membrane is stained with DiI dye. Then the cell uptake is observed under a laser confocal fluorescence microscope.

[0069] Example 4

[0070] Based on the above embodiments, this embodiment is similar to Embodiment 3, but the polyethylene glycol intermediate linker used in this embodiment is 283.5 mg, and other conditions are the same, corresponding to the product with n = 40 in the appendix Figure 1 which is denoted as BSH-PEG-cRGD.

[0071] Similar to Embodiment 3, chemical structure characterization was carried out to verify the chemical structure of the synthesized product. Please refer to the appendix Figure 4 , the proton nuclear magnetic resonance spectra of BSH, BSH-APA, BSH-cRGD, and BSH-PEG-cRGD. The broad peak around 1 ppm of chemical shift corresponds to the resonance absorption peak of B-H. Both BSH-cRGD and BSH-PEG-cRGD show this peak. At the same time, the resonance absorption peak around 7 ppm in the spectra of BSH-cRGD and BSH-PEG-cRGD corresponds to the H on the benzene ring in the cRGD structure, and the resonance absorption peak around 8 ppm is related to the N-H structure. Please refer to the appendix Figure 5 , the Fourier transform infrared spectra of BSH, APA, cRGD, BSH-APA, BSH-cRGD, and BSH-PEG-cRGD. The stretching vibration peak of the B-H bond is around 2500 cm -1 and is shown in all molecules containing the BSH structure. The vibration peak of BSH-PEG-cRGD around 2500 cm -1 is weakened in intensity compared with others, probably due to the influence of the PEG long chain. At the same time, the vibration peaks around 1100 cm -1 , 1600 cm -1 , and 1700 cm -1 may be related to the C=O, C=C, and C-O-C groups respectively. The analysis results of the B content by ICP-OES show that the actually measured B contents of BSH-cRGD and BSH-PEG-cRGD are 15.8% and 7.1% respectively, which are basically close to the theoretical calculated values of 13.8% and 4.5%.

[0072] Similar to Embodiment 3, further, in vitro cytotoxicity and cell uptake experiments were carried out on the synthesized product. Please refer to the appendix Figure 6, The effects of BSH-cRGD and BSH-PEG-cRGD on the survival rates of two cell lines, L929 and A549, are shown in the figure. It indicates that BSH has the lowest toxicity. In the drug concentration range of 5 - 160 μM, the survival rates of both cell lines are about 90%. In the range of 5 - 80 μM, the survival rates of both cell lines treated with BSH-cRGD and BSH-PEG-cRGD are above 80%. At 160 μM, for A549 cells, the survival rate of cells treated with BSH-PEG-cRGD is about 80%, which is higher than that of cells treated with BSH-cRGD (about 60%). For L929 cells, the survival rates under the treatment of both boron carriers are about 60%. L929 is a cell line with high expression of non-integrin αvβ3. Therefore, no obvious uptake of BSH, BSH-cRGD, and BSH-PEG-cRGD was observed under the laser confocal fluorescence microscope. Attached Figure 7 Instead, the uptake of three boron carriers by A549 cells was observed. In the figure, the blue color shows the cell nuclei stained with Hoechst 33342, the red color shows the cell membranes stained with DiI, and the green color shows the three boron carriers labeled with FITC. BSH-FITC shows the least green fluorescence, and BSH-PEG-cRGD-FITC shows the most green fluorescence. This indicates that A549 cells take up more BSH-cRGD-FITC than BSH-FITC, and more BSH-PEG-cRGD-FITC than BSH-cRGD-FITC. Moreover, the fluorescence of the boron carriers taken up by A549 cells is mainly concentrated in the cytoplasm, and there is basically no distribution in the cell nuclei. At the same time, the quantitative cell uptake experiment also shows that after A549 cells take up BSH-cRGD and BSH-PEG-cRGD, the B content in each cell reaches 10 9 B atoms.

[0073] Furthermore, a subcutaneous xenograft nude mouse model of A549 lung cancer cells was constructed. When the tumor size was approximately 200 mm 3 , the nude mice were randomly grouped, and BSH and BSH-PEG-cRGD were injected into the tail vein at a drug concentration of 30 mg / kg (calculated according to the B content). At 6, 12, and 24 hours after injection, tissues such as the heart, liver, spleen, lung, kidney, brain, muscle, and tumor, as well as blood, were collected from the nude mice. They were dried in an 85°C oven for 48 hours, ground and weighed, 1 mL of concentrated nitric acid and 200 μL of concentrated hydrochloric acid were added, and acid digestion was carried out by heating in an 85°C water bath for 2 hours. Then it was diluted to 10 mL, and the B content was measured using ICP-OES. Please refer to the attached Figure 8, The tissue distribution results of the B content in the tumor-bearing nude mouse model after injecting drugs. Calculated according to the dry weight of B content in each organ, blood, and tumor, after injecting BSH, at 6, 12, and 24 hours, the B contents in the tumor were 5.0±1.1 μg / g, 2.8±1.7 μg / g, and 2.6±1.0 μg / g respectively, which were basically equivalent to the B contents in other normal organs (heart: 4.0±1.2 μg / g, 4.6±3.0 μg / g, 1.1±0.3 μg / g; brain: 0.4±0.1 μg / g, 0.7±0.2 μg / g, 1.1±0.3 μg / g; spleen: 2.4±0.9 μg / g, 2.1±1.8 μg / g, 0.9±0.1 μg / g; lung: 3.2±1.5 μg / g, 3.1±0.9 μg / g, 2.7±0.8 μg / g; muscle: 1.2±0.5 μg / g, 1.2±0.1 μg / g, 1.4±0.4 μg / g; blood: 0.4±0.2 μg / g, 0.7±0.2 μg / g, 0.6±0.3 μg / g; kidney: 1.9±0.2 μg / g, 1.5±0.2 μg / g, 1.3±0.2 μg / g; liver: 1.9±0.1 μg / g, 1.0±0.1 μg / g, 0.8±0.1 μg / g). This can also be seen from the generally lower tumor:normal tissue ratio (T / N) and tumor:blood ratio (T / B) of the B content. The T / B ratios at 6, 12, and 24 hours were 10.1±1.0, 3.6±1.5, and 4.0±2.5 respectively. The T / N ratio was the largest when compared with the brain tissue, and was 11.8±3.3, 4.1±3.3, and 3.7±0.8 at 6, 12, and 24 hours respectively. The other T / N ratios were all below 3. These indicate that the targeting ability of BSH to tumors is relatively limited.

[0074] After injection of BSH-PEG-cRGD, at 6, 12, and 24 hours, the B content in the tumor was 12.4 ± 1.9 μg / g, 9.3 ± 0.9 μg / g, and 7.0 ± 1.3 μg / g respectively, while the B content in other normal organs except the kidneys and liver and in the blood was relatively small (heart: 2.0 ± 0.4 μg / g, 0.9 ± 0.4 μg / g, 0.5 ± 0.4 μg / g; brain: 0.7 ± 0.2 μg / g, 0.5 ± 0.1 μg / g, 0.6 ± 0.4 μg / g; spleen: 0.6 ± 0.5 μg / g, 1 ± 0.8 μg / g, 1.5 ± 0.7 μg / g; lung: 3.5 ± 1.0 μg / g, 1.7 ± 0.6 μg / g, 0.3 ± 0.2 μg / g; muscle: 1.9 ± 0.7 μg / g, 1.3 ± 0.6 μg / g, 4.8 ± 1.5 μg / g; blood: 1.8 ± 1.5 μg / g, 0.5 ± 0.4 μg / g, 0.5 ± 0.05 μg / g). This indicates that modifying BSH with cRGD indeed increases the accumulation of B at the tumor site, and more obvious B metabolism can be seen from the change in B content from 6 hours to 24 hours after injection of the boron carrier. For the injection of BSH-PEG-cRGD, the T / B was 15.4 ± 5.1, 13.7 ± 4.8, and 13.9 ± 2.7 at 6, 12, and 24 hours respectively, and the T / N ratio was basically above 3 except for the liver and kidneys. The B content in the liver and kidneys increased more, indicating that BSH-PEG-cRGD may be metabolized through the liver and kidneys. Therefore, how to further extend the retention time of BSH-PEG-cRGD at the tumor site is also an issue that needs to be considered emphatically in the future.

[0075] Example 5

[0076] On the basis of the above embodiments, this example is similar to Example 3, but 49.5 mg of iRGD is used, and other conditions are the same, to obtain the product with n = 1 in the corresponding appendix Figure 1 where the RGD peptide part is iRGD.

[0077] Example 6

[0078] On the basis of the above embodiments, this example is similar to Example 3, but the thiol-ene click reaction time in step S1 is 2 hours, and other conditions are the same, to obtain the product with n = 1 in the corresponding appendix Figure 1 where n = 1.

[0079] Example 7

[0080] On the basis of the above embodiments, this example is similar to Example 3, but the CDI activation carboxyl reaction time in step S2 is 4 hours, and the reaction is carried out for 4 hours after adding the RGD peptide, and other conditions are the same, to obtain the product in the corresponding appendix Figure 1The product with n = 1.

[0081] Example 8

[0082] Based on the above embodiments, this example is similar to Example 4, but 22.2 mg of Na2[BSH] is used, and other conditions are the same, obtaining the corresponding attachment Figure 1 The product with n = 40.

[0083] Example 9

[0084] Based on the above embodiments, this example is similar to Example 4, but 43 mg 10 B-enriched Cs 2 10 [BSH] is used, and other conditions are the same, obtaining the corresponding attachment Figure 1 with n = 40 but 10 B-enriched product.

[0085] The above are only the preferred embodiments of the present invention, and it does not limit the protection scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any changes, modifications, substitutions, integrations, and parameter changes made to these embodiments by conventional substitutions or by achieving the same functions without departing from the principle and spirit of the present invention fall within the protection scope of the present invention.​

Claims

1. A class of RGD peptide-modified mercapto dodecaborane derivatives, characterized in that: This type of derivative utilizes RGD peptide to chemically couple and modify mercapto dodecaborane anion salt (BSH), wherein the BSH and RGD peptide are connected via a hydrophilic intermediate linker having a double bond at one end and a carboxyl group at the other end.

2. The RGD peptide-modified mercapto dodecaborane derivative according to claim 1, characterized in that: The dodecaborane structure comes from mercapto dodecaborane anion salt; wherein the undecahydromercapto dodecaborane exists in the form of a divalent anion, and the corresponding cation is an alkali metal cation, including lithium (Li+) or sodium (Na+) or potassium (K+) or rubidium (Rb+) or cesium (Cs+) or quaternary ammonium salt (NH4+) and a mixture thereof.

3. The RGD peptide-modified mercapto dodecaborane derivative according to claim 2, characterized in that: RGD peptide has a binding effect with integrin αvβ3.

4. The RGD peptide-modified mercapto dodecaborane derivative according to claim 3, characterized in that: The RGD peptide structure is the remaining part after the RGD peptide participates in the reaction, wherein the primary amine in the RGD peptide undergoes a carboxyl-amino amidation condensation reaction with the carboxyl end of the intermediate connector.

5. A method for synthesizing a class of RGD peptide-modified mercapto dodecaborane derivatives according to any one of claims 1 to 4, characterized in that: The synthesis reaction includes the following two steps: Step S1: The thiol group on BSH and one end of the double bond undergo a thiol-ene click reaction under ultraviolet irradiation, and BSH and the intermediate linker are connected through the click reaction, wherein the molar amount of the intermediate linker is in excess of BSH, and the excess ratio is 10-100%; Step S2: first activate the carboxyl group in the product of step S1 using N,N-carbonyldiimidazole (CDI), and then add the RGD peptide containing primary amine to complete the entire reaction; The molar amount of the product of step S1 is in excess of CDI and RGD peptides.

6. The method for synthesizing a class of RGD peptide-modified mercapto dodecaborane derivatives according to claim 5, characterized in that: The synthesis reaction is carried out at room temperature and normal pressure, with a specific temperature range of 10 to 30°C.

7. The method for synthesizing a class of RGD peptide-modified mercapto dodecaborane derivatives according to claim 5, characterized in that: The reaction time of the thiol-ene click chemistry, the time of activating the carboxyl group by CDI and the time of adding the RGD peptide for the condensation reaction are all 2 to 6 hours.

8. The method for synthesizing a class of RGD peptide-modified mercapto dodecaborane derivatives according to claim 6 or 7, characterized in that: The synthesis reactions are all carried out under magnetic stirring conditions, the step S1 is completed in a water solvent, and the step S2 is carried out in an anhydrous dimethyl sulfoxide solution.

9. The method for synthesizing a class of RGD peptide-modified mercapto dodecaborane derivatives according to claim 8, characterized in that: Step S1 and step S2 are purified by precipitation with a poor solvent and dialysis respectively; In step S1, after the click chemistry reaction between BSH and the intermediate connector is completed, the product and the intermediate connector are dissolved, and the product is precipitated from the system using anhydrous ethanol, a poor solvent, and the product is collected by multiple centrifugation and washing; In step S2, the product is purified by dialysis, and the final product is vacuum dried or freeze-dried to obtain a solid product.

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