Tetrafunctional boron drug compound, preparation method, application and pharmaceutical composition

Abstract

The invention relates to a tetrafunctional boron drug compound, a preparation method, application and a pharmaceutical composition. The tetrafunctional boron drug compound comprises a tetrafunctional compound and a racemate, a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a solvate thereof. The tetrafunctional compound has an albumin binding agent for prolonging the blood half-life period of the drug, an isotope chelating agent, a 10B carrying agent for BNCT treatment, a targeted fibroblast activating protein ligand and a linker. The invention also provides a preparation method of the tetrafunctional boron drug compound. The invention also provides a tetrafunctional boron medicine compound composition for tumor nuclear medicine diagnosis and radioisotope therapy. The invention further provides a pharmaceutical composition for treating tumors through BNCT. The pharmaceutical composition comprises the tetrafunctional boron drug compound and at least one pharmaceutically acceptable auxiliary material. The problems of poor targeting property, poor water solubility, short blood half-life period, short tumor tissue drug residence time and the like of the existing boron drug are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical drugs, and in particular to a tetrafunctional boron drug compound, a preparation method, an application and a pharmaceutical composition. Background Art

[0002] The incidence and mortality of cancer have remained at a high level and have shown an upward trend year by year. At present, the methods for treating malignant tumors include surgery, targeted drug therapy, radiotherapy and immunotherapy. Since the 21st century, with the rapid development of precision medicine and nuclear medicine molecular imaging technology, integrated diagnosis and treatment has become a hot spot in precision medicine research at home and abroad. This integrated diagnosis and treatment system has been proven to be accurate and effective in long-term clinical practice by using molecular probes labeled with diagnostic radionuclides for clear diagnosis and precise quantification, and then using the same or similar molecular probes labeled with therapeutic radionuclides for targeted precision treatment. Integrated radionuclides diagnosis and treatment represented by targets such as prostate-specific membrane antigen (PSMA) and somatostatin receptor (SSTR) have made major breakthroughs at home and abroad. The approval of Lu-DOTA-TATE and Lu-PSMA-617 has opened a new era of precision diagnosis and treatment of neuroendocrine tumors and prostate cancer.

[0003] However, small molecule peptides such as DOTA-TATE and PSMA-617 are cleared from the blood quickly, and most of the drugs are excreted from the body through the urinary system, resulting in the inability of the target lesions to fully absorb the radioactive drugs and a low effective dose. This requires the use of higher doses of radionuclide therapy based on radioactive drugs, which brings certain toxic side effects. Therefore, optimizing the pharmacokinetics of molecular probes, improving the bioavailability of radionuclides, reducing the dose of radionuclides used, and increasing the stable residence time of radionuclide molecular probes in tumor cells, so that the therapeutic radionuclides can fully kill tumor cells within a limited decay time, and increasing the metabolic excretion rate of radionuclides in normal organs to reduce treatment side effects, is one of the important directions for the clinical development of integrated radionuclide diagnosis and treatment.

[0004] Albumin binding is an effective method to optimize the pharmacokinetics and pharmacodynamics of a series of drugs (including small molecules, peptides, etc.). A series of clinical (pre-) studies have shown that the binding of EB to albumin prolongs the residence time of drugs in the body and increases the uptake and absorption of radioactive drugs by tumors, so that a relatively ideal therapeutic effect can be achieved with a smaller dose of radioactive drugs. This will help reduce medical costs and improve efficacy in future clinical practice. However, the first-generation probes such as EB-modified PSMA still have obvious blood system toxicity in clinical studies due to their prolonged blood retention time. Therefore, how to determine the best albumin binder and molecular stabilizer to achieve the best balance of plasma protein binding properties, so as to achieve high tumor uptake and effective and rapid clearance from the blood pool, minimize the radioactivity in normal organs and tissues, and significantly enhance the retention time and uptake within the disease, is a key scientific issue currently being studied.

[0005] In addition, the emerging boron neutron capture therapy (BNCT) is a binary, cell-scale, highly targeted and high energy transfer line density radiotherapy. Compared with simple radiotherapy and chemotherapy, it has a dual screening function and significantly improves the accuracy of disease treatment. 10 B captures thermal neutrons to produce nuclear fission reactions, producing alpha particles and recoil Li nuclei, which selectively kill tumor cells. 10 The selective delivery of boron into tumor cells is the key to the success of this treatment. Therefore, boron-containing drugs need to have the characteristics of high tumor tissue uptake, low normal tissue uptake, rapid clearance after treatment and low self-toxicity. Since the initial use of boric acid as a boron delivery carrier for BNCT research, new boron drugs have been continuously studied and developed. At present, the drugs approved for BNCT clinical trials include 4-dihydroxyboronylphenylalanine (p-boronophenylala-nine, BPA) and disodium thiododecaborane (sodium borocaptate, BSH), but BPA has low boron content, high therapeutic dose, low adaptability to heterogeneous tumors, uneven distribution in tumors, and short biological half-life; BSH lacks active transport capacity, low tumor-specific accumulation ability and high cytotoxicity. At the same time, it is easily oxidized in the air and has poor chemical stability, which limits its application. In addition, how to prolong the blood half-life of boron drugs, improve the uptake of boron in tumor tissues and long-term retention are also clinical problems faced by BNCT. How to conduct real-time dynamic quantitative non-invasive analysis of the boron concentration in the lesion and surrounding tissues, and then select the appropriate treatment time window for neutron irradiation and propose accurate treatment plans for different patients is a key scientific issue in the development of neutron capture therapy and also a major challenge in its clinical application. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a tetrafunctional boron drug compound, a preparation method, an application and a pharmaceutical composition to solve at least one problem existing in the background technology, so as to provide a boron drug with affinity and high boron-10 content, as well as strong targeting, greater stability and longer retention time.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A tetrafunctional boron drug compound, including a tetrafunctional compound and its racemate, stereoisomer, tautomer, pharmaceutically acceptable salt or solvate; The tetrafunctional compound has an albumin binder part for extending the blood half-life of the drug, a chelating part, and a 10 B carrier part, fibroblast activation protein (FAP) targeting part and connecting part.

[0008] According to the above technical means, the albumin binder part for extending the blood half-life of the drug, the chelating part, and the BNCT treatment part are connected by using a linking group. 10 The B carrier part and the targeting fibroblast activation protein (FAP) part are connected together to make a tetrafunctional compound with a high content of boron-10, which improves the affinity for tumor cells and prolongs the blood half-life. At the same time, it has the advantages of targeting, long residence time in tumor tissue, good stability and low biological toxicity, and has good application prospects in BNCT treatment.

[0009] Preferably, the structural formula of the tetrafunctional boron drug compound is as shown in formula (I) and its racemate, stereoisomer, tautomer, pharmaceutically acceptable salt or solvate; (I) In formula (I), R1 is selected from the group consisting of Evans blue (EB), ibuprofen, 4-(p-iodophenyl)butyric acid (IPBA) and sulfonamides; R2 is selected from , , , , , and The group composed of; R3 is selected from ; R4 is selected from ; L1 is selected from , where n is an integer between 1 and 8; L2 is an integer between or ; L3 is selected from .

[0010] Among them, R3 is selected from a polyhedral borane anion with a cage structure, which has a stable structure. Introducing it into the boron carrier helps to improve the stability of the boron drug and prolong the retention time. Each BSH molecule contains 12 10 B atoms can accumulate near brain tumors when the blood-brain barrier (BBB) ​​is damaged. Therefore, a higher content of boron-10 can be provided to the boron delivery agent for BNCT treatment.

[0011] R4 is selected from FAPI, which can target fibroblast activation protein (FAP) (FAP is not expressed or lowly expressed in normal tissues, but is overexpressed in more than 90% of epithelial tumors). FAPI can visualize the location and size of the tumor in PET-CT scans by combining with FAP, thereby helping to determine the type and location of the tumor and guide the treatment of the tumor. This part exists in the boron carrier and can realize the visualization treatment of BNCT.

[0012] In the L1 linker, the size of n determines the length of the PEG chain. Different PEG chain lengths affect the ester solubility of the drug, thereby affecting the retention time of the drug. The L2 and L3 parts realize the connection of the functional parts of the tetrafunctional compound.

[0013] Preferably, the R1 is selected from , , , , , and Composed of groups.

[0014] By utilizing the high affinity of the R1 part to albumin (the two are reversibly bound through covalent interaction, electrostatic interaction or hydrophobic interaction), the peptide boron drug is modified, which effectively prolongs the half-life of the tetrafunctional boron drug compound, increases the drug uptake by the target lesions and the retention of the drug in the target lesions, thereby improving the efficacy and reducing the drug dosage.

[0015] Preferably, n is selected from an integer between 1 and 8.

[0016] Preferably, the structural formula of the tetrafunctional compound is any one of the following structural formulas: , , , and ; Wherein, n is selected from an integer between 1 and 8.

[0017] Preferably, the R2 portion is chelated with a radionuclide or a natural isotope of a radionuclide. By chelating the radionuclide in the R2 portion, combined with diagnostic radionuclide labeling, a clear diagnosis and precise quantification are achieved, and labeling with therapeutic nuclides can achieve targeted therapy, thereby contributing to the development of integrated diagnosis and treatment and precision medicine.

[0018] Preferably, the radionuclide is capable of emitting beta particles, gamma photons or alpha particles.

[0019] Preferably, the radionuclide is selected from 18 F. 67 Ga, 68 Ga, 99m Tc, 86 Y. 90 Y. 64 Cu, 67 Cu, 55 Co. 149 Tb, 152 Tb, 155 Tb, 161 Tb, 177 Lu, 188 Re, 166 Ho, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 226 Th, 227 Th, 223 Ra and 230 At least one of U.

[0020] Preferably, the radionuclide is selected from 18 F. 68 Ga, 99m Tc, 89 Y. 64 Cu, 90 Y. 59 Co. 161 Tb, 177 Lu, 188 Re, 166 Ho, 213 Bi, 225 Ac and 223 At least one of Ra.

[0021] The present invention also provides a method for preparing the tetrafunctional boron drug compound according to the present invention, comprising the following steps: Compound A is dissolved in an organic solvent, and then EDCI and HOBt are added, mixed and reacted, and then N-(9-fluorenylmethoxycarbonyl)-D-lysine hydrochloride and NMM are added, and the reaction is carried out at room temperature overnight to obtain a crude product; the crude product is dissolved in a DMF solution containing N, N-diethylaniline (DEA), and the reaction is carried out at room temperature to obtain a first compound; The compound A is selected from at least one of Evans blue (EB), ibuprofen, 4-(p-iodophenyl)butyric acid (IPBA) and sulfonamides; The first compound is dissolved in an organic solvent, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) are added, mixed and reacted, and then (N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine and N-methylmorpholine (NMM) are added, and reacted at room temperature to obtain an intermediate product; the intermediate product is dissolved in a solution of an organic solvent containing N, N-diethylaniline (DEA), and reacted at room temperature to obtain a second compound; Compound B is dissolved in an organic solvent, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIEA) and (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-(3-(piperazine-1-yl)propoxy)quinoline-4-carboxamide (FAPI) are added, reacted at room temperature, and trifluoroacetic acid (TFA) is added and reacted at room temperature to obtain a third compound; the compound B is selected from , where n is selected from a positive integer; Dissolve compound C in an organic solvent, then add EDCI and HOBt, mix and react, then add the second compound and NMM, react at room temperature overnight, to obtain a fourth compound; The compound C is selected from , , , , , and The group composed of; The fourth compound is dissolved in an organic solvent, EDCI and HOBt are added, mixed and reacted, and then the third compound and NMM are added, reacted at room temperature overnight, TFA is added and reacted at room temperature to obtain the fifth compound; the fifth compound and 3-maleimidopropionic acid hydroxysuccinimide ester are dissolved in an organic solvent, DIEA is added, and reacted at room temperature to obtain the sixth compound; the sixth compound and BSH are dissolved in PBS solution, TCEP is added, and reacted at room temperature under nitrogen protection to obtain a tetrafunctional boron drug compound.

[0022] Preferably, the method for preparing the tetrafunctional boron drug compound comprises the following steps: S1, dissolving 4-(p-iodophenyl)butyric acid (partially related compound of R1) in an organic solvent, then adding EDCI and HOBt at 0°C, reacting for 5-20 minutes, then adding N-(9-fluorenylmethoxycarbonyl)-D-lysine hydrochloride and NMM, moving to room temperature to react overnight, spinning off the solvent, separating and purifying to obtain a crude product; dissolving the crude product in a DMF solution containing 20% ​​N, N-diethylaniline (DEA), reacting at room temperature for 1-4 hours, spinning off the solvent, precipitating with ether, naturally evaporating the residual solvent, separating and purifying to obtain compound 1 / 1' / 1"; S2, dissolving compound 1 / 1' / 1" in an organic solvent, then adding EDCI and HOBt at 0°C, reacting for 5-20 minutes, then adding (N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine and NMM, moving to room temperature for reaction for 10-24 hours, spinning the solvent to dryness, HPLC purification, dissolving the purified compound in a solution of an organic solvent containing 20% ​​N,N-diethylaniline (DEA), reacting at room temperature for 1-4 hours, spinning the solvent to dryness, precipitating with ether, naturally evaporating the residual solvent, and separating by HPLC to obtain compound 2 / 2' / 2"; S3, dissolving 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid (partially related compounds of L1, n=1-8) in an organic solvent, adding HATU, DIEA and FAPI in sequence, reacting at room temperature for 1-4 hours, monitoring the reaction completion by LC-MS, removing DMF by rotation, adding TFA, reacting at room temperature for 1-4 hours, adding ether, a large amount of solid precipitated, centrifuging to dryness, and purifying by reverse phase preparative liquid phase to obtain compound 3; S4, dissolving tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (partially related compound of R2) in an organic solvent, then adding EDCI and HOBt at 0°C, reacting for 5-20 minutes, then adding compound 2 / 2' / 2" and NMM, moving to room temperature to react overnight, spinning the solvent to dryness, and HPLC purification to obtain compound 4 / 4' / 4"; S5. Dissolve compound 4 / 4' / 4" in an organic solvent, cool to 0°C, add EDCI and HOBt, react at 0°C for 5-20 minutes, then add compound 3 and NMM, transfer to room temperature and react overnight, monitor the reaction completion by LC-MS, concentrate to dryness, add TFA, react at room temperature for 1-4h, add ether, a large amount of solid precipitates, centrifuge to dryness, and purify by reverse phase preparative liquid phase to obtain compound 5 / 5' / 5"; S6, dissolving compound 5 / 5' / 5" and 3-maleimidopropionic acid hydroxysuccinimide ester in an organic solvent, adding DIEA, reacting at room temperature for 1-4 hours, monitoring the completion of the reaction by LC-MS, removing the organic solvent, and purifying by reverse phase preparative liquid phase to obtain compound 6 / 6' / 6"; S7. Compound 6 / 6' / 6" and BSH were dissolved in PBS solution, TCEP was added, and the mixture was reacted at room temperature for 24-48 hours under nitrogen protection. The reaction was completed after monitoring by LC-MS. The mixture was purified by reverse phase preparative liquid phase to obtain DOTA-IPBA-FAPI-BSH (n=1-8) / DOTA-BSA-FAPI-BSH (n=1-8) / L804-EB-FAPI-BSH (n=1-8), i.e., a tetrafunctional boron drug compound.

[0023] Preferably, the organic solvent is selected from DMF. Preferably, in S1, S2, S4 and S5, EDCI and HOBt are added, and the reaction time is 10 minutes at a temperature of 0°C. Preferably, in S1, S2, S3, S5 and S6, the reaction time at room temperature is 2 hours.

[0024] Preferably, the equivalent ratio of the 4-(p-iodophenyl)butyric acid (R1 related compound), EDCI, HOBt, N-(9-fluorenylmethoxycarbonyl)-D-lysine hydrochloride and NMM is 1: 1.1~1.5: 1.1~2: 1.1~2: 3~8. Preferably, the equivalent ratio of the first compound (compound 1 / 1' / 1"), EDCI, HOBt, (N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine and NMM is: 1: 1.1~1.5: 1.1~2: 1.1~2: 3~8. Preferably, the equivalent ratio of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid, HATU, DIEA and FAPI is 1.8~2.5: 1.1~1.5: 3~5: 1. Preferably, the equivalent ratio of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (R2 part related compound), EDCI, HOBt, the second compound (compound 2 / 2' / 2") and NMM is: 1: 1.1~1.5: 1.1~2: 1.1~2:3~8. Preferably, the equivalents of the fourth compound (compound 4 / 4' / 4"), EDCI, HOBt, the third compound (compound 3) and NMM are: 1: 1.1~1.5: 1.1~2: 1.1~2: 3~8. Preferably, the equivalent ratio of the fifth compound (compound 5 / 5' / 5"), 3-maleimidopropionic acid hydroxysuccinimide ester and DIEA is: 1: 1.1~1.5: 3~5. Preferably, the equivalent ratio of the sixth compound (compound 6 / 6' / 6"), BSH and TCEP is: 1: 1.1~1.5: 2~5.

[0025] Preferably, in S7, BSH is 10 The abundance of B is higher than 99%. Preferably, in S7, the reaction time at room temperature under nitrogen protection is 36 hours.

[0026] The present invention also provides a use of the tetrafunctional boron drug compound as described in the present invention in a drug for treating tumors. The tetrafunctional boron drug compound of the present invention can be directly dissolved in physiological saline or phosphate buffer solution or sterile water or glucose solution to prepare a boron drug preparation for infusion, which has the advantages of strong targeting, high stability and long retention time. According to experimental measurements, the tetrafunctional boron drug compound of the present invention can retain tumors for up to 120 hours.

[0027] Preferably, the tumor includes at least one of malignant melanoma, head and neck squamous cell carcinoma, glioma, thymic carcinoma, cervical cancer, endometrial cancer, neuroendocrine tumors, thyroid cancer, intestinal cancer and bone metastasis, nasopharyngeal carcinoma and breast cancer. Preferably, the tumor includes at least one of metastatic melanoma, recurrent melanoma, advanced melanoma, metastatic head and neck squamous cell carcinoma, recurrent head and neck squamous cell carcinoma, advanced head and neck squamous cell carcinoma, metastatic glioma, recurrent glioma and advanced glioma.

[0028] The present invention also provides a pharmaceutical composition for BNCT treatment of tumors, comprising the tetrafunctional boron drug compound as described in the present invention, and at least one pharmaceutically acceptable excipient.

[0029] Beneficial effects of the present invention: The tetrafunctional boron drug compound of the present invention connects together an albumin binder part for extending the blood half-life of the drug, a chelating part, a 10B carrier part for BNCT treatment and a targeted fibroblast activation protein (FAP) part by using a linking group, so that the tetrafunctional compound has a high content of boron-10, improves the affinity for tumor cells and extends the blood half-life, and at the same time has the advantages of targeting, long retention time in tumor tissue, good stability and low biological toxicity, overcomes the problems of poor targeting, poor water solubility, short blood half-life and short drug retention time in tumor tissue of existing boron drugs, and shows better effect in BNCT treatment of tumors than BPA currently approved for clinical use, and has promotion and application value in the technical field of development of new boron drugs for BNCT. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the MS graph of the compound DOTA-IPBA-FAPI-BSH (n=2) prepared in Example 1; Figure 2 It is the MS graph of the compound DOTA-IPBA-FAPI-BSH (n=3) prepared in Example 1; Figure 3 This is the MS graph of the compound DOTA-IPBA-FAPI-BSH (n=4) prepared in Example 1; Figure 4 It is the MS graph of the compound DOTA-IPBA-FAPI-BSH (n=6) prepared in Example 1; Figure 5 This is a purity test result diagram of the compound DOTA-IPBA-FAPI-BSH (n=2) prepared in Example 1; Figure 6 This is a graph showing the purity test results of the compound DOTA-IPBA-FAPI-BSH (n=3) prepared in Example 1; Figure 7 This is a graph showing the purity test results of the compound DOTA-IPBA-FAPI-BSH (n=4) prepared in Example 1; Figure 8 This is a graph showing the purity test results of the compound DOTA-IPBA-FAPI-BSH (n=6) prepared in Example 1; Fig. 9 This is a graph showing the radioactive labeling purity results of the compound DOTA-IPBA-FAPI-BSH (n=2) prepared in Example 1; Fig.10 This is a diagram showing the cellular uptake results of the compound DOTA-IPBA-FAPI-BSH (n=2) prepared in Example 1; Fig.11 This is a graph showing the test results of the cell binding affinity of the compound DOTA-IPBA-FAPI-BSH (n=2) prepared in Example 1; Fig.12 For intravenous injection 177 In vivo SPECT / CT images of Lu-DOTA-IPBA-FAPI-BSH (n=2); Fig.13 For intravenous injection 177 In vivo SPECT / CT images of Lu-DOTA-IPBA-FAPI-BSH (n=6); Fig.14 This is a diagram showing the BNCT killing effect of the compound DOTA-IPBA-FAPI-BSH (n=2) prepared in Example 1 in osteosarcoma cells carrying human FAP; Fig.15 This is a graph showing the results of a cell clone formation experiment of the compound DOTA-IPBA-FAPI-BSH (n=2) prepared in Example 1 in osteosarcoma cells carrying human FAP after BNCT irradiation. DETAILED DESCRIPTION

[0031] The following will describe the embodiments of the present invention with reference to the preferred embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0032] If no specific techniques or conditions are specified in the specific embodiments, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0033] Example 1 A method for preparing a tetrafunctional boron drug compound comprises the following steps: S1. Dissolve 1 mmol of 4-(p-iodophenyl)butyric acid in 10 ml of DMF, then add 1.3 mmol of EDCI and 1.2 mmol of HOBt at 0°C, mix and react for 10 minutes, then add 1.2 mmol of N-(9-fluorenylmethoxycarbonyl)-D-lysine hydrochloride and 5 mmol of NMM, move to room temperature to react overnight, spin dry the solvent, purify by HPLC to obtain a crude product; dissolve the crude product in a DMF solution containing 20% ​​N,N-diethylaniline (DEA), react at room temperature for 2 h, spin dry the solvent, precipitate with ether, evaporate the residual solvent naturally, and separate by HPLC to obtain compound 1; S2, 1 mmol of compound 1 was dissolved in 10 ml of DMF, and then 1.3 mmol of EDCI and 1.2 mmol of HOBt were added at 0°C, mixed and reacted for 10 minutes, and then 1.2 mmol of (N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine and 5 mmol of NMM were added, and the mixture was moved to room temperature for reaction overnight, the solvent was dried by spin-drying, and purified by HPLC. The purified compound was dissolved in a DMF solution containing 20% ​​N,N-diethylaniline (DEA), reacted at room temperature for 2 h, the solvent was dried by spin-drying, precipitated with ether, and the residual solvent was naturally evaporated, and then separated by HPLC to obtain compound 2; S3, 1.8 mmol of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid was dissolved in 10 ml of DMF, and 1.2 mmol of HATU, 3 mmol of DIEA and 1 mmol of FAPI were added in sequence, and the mixture was reacted at room temperature for 2 h. The reaction was completed after LC-MS monitoring, and DMF was removed by rotation. 10 ml of TFA was added, and the mixture was reacted at room temperature for 2 h. 100 ml of ether was added, and a large amount of solid was precipitated. The mixture was centrifuged and dried, and purified by reverse phase preparative liquid phase to obtain compound 3; S4, 1 mmol of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid was dissolved in 10 ml of DMF, and then 1.3 mmol of EDCI and 1.2 mmol of HOBt were added at 0°C, and the reaction was carried out for 10 minutes. Subsequently, 1.2 mmol of compound 2 and 5 mmol of NMM were added, and the reaction was carried out at room temperature overnight. The solvent was dried and purified by HPLC to obtain compound 4; S5, 1 mmol of compound 4 was dissolved in 10 ml of DMF, cooled to 0°C, and then 1.3 mmol of EDCI and 1.2 mmol of HOBt were added, and the mixture was reacted at 0°C for 10 minutes, and then 1.2 mmol of compound 3 and 3 mmol of NMM were added, and the mixture was transferred to room temperature for overnight reaction. The reaction was complete after LC-MS monitoring, and the mixture was concentrated to dryness, and then 9.5 ml of TFA and 0.5 ml of DMF were added, and the mixture was reacted at room temperature for 2 h. 100 ml of ether was added, and a large amount of solid precipitated. The mixture was centrifuged to dryness, and then purified by reverse phase preparative liquid chromatography to obtain compound 5; S6, 1 mmol of compound 5 and 1.2 mmol of 3-maleimidopropionic acid hydroxysuccinimide ester were dissolved in 10 ml of DMF, and 3 mmol of DIEA was added, and the reaction was carried out at room temperature for 1 h. The reaction was completed after LC-MS monitoring, and DMF was removed by rotation. The compound was purified by reverse phase preparative liquid phase to obtain compound 6; S7. 1 mmol of compound 6 and 1.2 mmol of BSH were dissolved in 6 ml of PBS buffer solution, and then 2 mmol of TCEP was added. The mixture was reacted at room temperature for 36 h under nitrogen protection. The reaction was completed after monitoring by LC-MS. The mixture was purified by reverse phase preparative liquid phase to obtain DOTA-IPBA-FAPI-BSH, i.e., a tetrafunctional boron drug compound; wherein the structural formula of compound 1 is: ; The structural formula of compound 2 is: ; The structural formula of compound 3 is: ; The structural formula of compound 4 is: ; The structural formula of compound 5 is: ; The structural formula of compound 6 is: ; The structural formula of DOTA-IPBA-FAPI-BSH is: .

[0034] Example 2 A method for preparing a tetrafunctional boron drug compound comprises the following steps: S1. Dissolve 1 mmol of ibuprofen (BSA) in 10 ml of DMF, then add 1.3 mmol of EDCI and 1.2 mmol of HOBt at 0°C, mix and react for 10 minutes, then add 1.2 mmol of N-(9-fluorenylmethoxycarbonyl)-D-lysine hydrochloride and 5 mmol of NMM, move to room temperature to react overnight, spin dry the solvent, purify by HPLC to obtain a crude product; dissolve the crude product in a DMF solution containing 20% ​​N, N-diethylaniline (DEA), react at room temperature for 2 h, spin dry the solvent, precipitate with ether, evaporate the residual solvent naturally, and separate by HPLC to obtain compound 1'; S2, 1 mmol of compound 1' was dissolved in 10 ml of DMF, and then 1.3 mmol of EDCI and 1.2 mmol of HOBt were added at 0°C, mixed and reacted for 10 minutes, and then 1.2 mmol of (N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine and 5 mmol of NMM were added, and the mixture was moved to room temperature for reaction overnight, the solvent was dried by spin drying, and purified by HPLC. The purified compound was dissolved in a DMF solution containing 20% ​​N,N-diethylaniline (DEA), reacted at room temperature for 2 h, the solvent was dried by spin drying, precipitated with ether, and the residual solvent was naturally evaporated, and separated by HPLC to obtain compound 2'; S3, 1.8 mmol of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid was dissolved in 10 ml of DMF, and 1.2 mmol of HATU, 3 mmol of DIEA and 1 mmol of FAPI were added in sequence, and the mixture was reacted at room temperature for 2 h. The reaction was completed after LC-MS monitoring, and DMF was removed by rotation. 10 ml of TFA was added, and the mixture was reacted at room temperature for 2 h. 100 ml of ether was added, and a large amount of solid was precipitated. The mixture was centrifuged and dried, and purified by reverse phase preparative liquid phase to obtain compound 3; S4, dissolve 1 mmol of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid in 10 ml of DMF, then add 1.3 mmol of EDCI and 1.2 mmol of HOBt at 0°C, react for 10 minutes, then add 1.2 mmol of compound 2' and 5 mmol of NMM, move to room temperature to react overnight, spin dry the solvent, and purify by HPLC to obtain compound 4'; S5, 1 mmol of compound 4' was dissolved in 10 ml of DMF, cooled to 0°C, and then 1.3 mmol of EDCI and 1.2 mmol of HOBt were added, and the mixture was reacted at 0°C for 10 minutes. Then 1.2 mmol of compound 3 and 3 mmol of NMM were added, and the mixture was transferred to room temperature for overnight reaction. The reaction was complete after LC-MS monitoring, and the mixture was concentrated to dryness. Then 9.5 ml of TFA and 0.5 ml of DMF were added, and the mixture was reacted at room temperature for 2 h. Then 100 ml of ether was added, and a large amount of solid precipitated. The mixture was centrifuged to dryness, and then purified by reverse phase preparative liquid chromatography to obtain compound 5'; S6, 1 mmol of compound 5' and 1.2 mmol of 3-maleimidopropionic acid hydroxysuccinimide ester were dissolved in 10 ml of DMF, and 3 mmol of DIEA was added, and the reaction was carried out at room temperature for 1 hour. The reaction was completed after LC-MS monitoring, and DMF was removed by rotation. The compound was purified by reverse phase preparative liquid phase to obtain compound 6'; S7. Dissolve 1 mmol of compound 6' and 1.2 mmol of BSH in 6 ml of PBS buffer solution, add 2 mmol of TCEP, and react at room temperature for 36 h under nitrogen protection. The reaction is complete when monitored by LC-MS. Purify by reverse phase preparative liquid phase to obtain DOTA-BSA-FAPI-BSH, a tetrafunctional boron drug compound.

[0035] Wherein, the structural formula of compound 1' is: ; The structural formula of compound 2' is: ; The structural formula of compound 3 is: ; The structural formula of compound 4' is: ; The structural formula of compound 5' is: ; The structural formula of compound 6' is: ; The structural formula of DOTA-BSA-FAPI-BSH is: .

[0036] Example 3 A method for preparing a tetrafunctional boron drug compound comprises the following steps: S1. Dissolve 1 mmol of EB in 10 ml of DMF, then add 1.3 mmol of EDCI and 1.2 mmol of HOBt at 0°C, mix and react for 10 minutes, then add 1.2 mmol of N-(9-fluorenylmethoxycarbonyl)-D-glutamic acid-1-tert-butyl ester and 5 mmol of NMM, move to room temperature to react overnight, spin dry the solvent, and separate by HPLC to obtain compound 1"; S2, 1 mmol of compound 1" was dissolved in 10 ml of DMF, and then 1.3 mmol of EDCI and 1.2 mmol of HOBt were added at 0°C, mixed and reacted for 10 minutes, and then 1.2 mmol of (N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine and 5 mmol of NMM were added, and the mixture was moved to room temperature for reaction overnight, the solvent was spin-dried, and HPLC was used for purification. The purified compound was dissolved in a DMF solution containing 20% ​​N, N-diethylaniline (DEA), reacted at room temperature for 2 h, the solvent was spin-dried, precipitated with ether, and the residual solvent was naturally evaporated, and then separated by HPLC to obtain compound 2"; S3, 1.8 mmol of 2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxyacetic acid (partially related compounds of L1, n=1-8) was dissolved in 10 ml of DMF, 1.2 mmol of HATU and 3 mmol of DIEA were added, and finally 1 mmol of FAPI was added, and the mixture was reacted at room temperature for 2 h. The reaction was completed after LC-MS monitoring, and DMF was removed by rotation, and 10 ml of TFA was added, and the mixture was reacted at room temperature for 2 h. 100 ml of ether was added, and a large amount of solid was precipitated. The mixture was centrifuged and dried, and compound 3 was obtained after purification by reverse phase preparative liquid chromatography; S4, dissolve 1 mmol of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid in 10 ml of DMF, then add 1.3 mmol of EDCI and 1.2 mmol of HOBt at 0°C, react for 10 minutes, then add 1.2 mmol of compound 2" and 5 mmol of NMM, move to room temperature to react overnight, spin dry the solvent, add 10 ml of 3 M sodium hydroxide aqueous solution, react at room temperature for 2 hours, spin dry, and purify by reverse phase preparative liquid phase to obtain compound 4"; S5, 1 mmol of compound 4" was dissolved in 10 ml of DMF, cooled to 0°C, and then 1.3 mmol of EDCI and 1.2 mmol of HOBt were added, and the mixture was reacted at 0°C for 10 minutes. Then 1.2 mmol of compound 3 and 3 mmol of NMM were added, and the mixture was transferred to room temperature to react overnight. The reaction was complete after LC-MS monitoring, and the mixture was concentrated to dryness. Then 9.5 ml of TFA and 0.5 ml of DMF were added, and the mixture was reacted at room temperature for 2 h. Then 100 ml of ether was added, and a large amount of solid precipitated. The mixture was centrifuged to dryness, and then purified by reverse phase preparative liquid phase to obtain compound 5". S6, 1 mmol of compound 5" and 1.2 mmol of 3-maleimidopropionic acid hydroxysuccinimide ester were dissolved in 10 ml of DMF, and 3 mmol of DIEA was added, and the reaction was carried out at room temperature for 1 hour. The reaction was completed after LC-MS monitoring, and DMF was removed by rotation, and the compound was purified by reverse phase preparative liquid phase to obtain compound 6"; S7. Dissolve 1 mmol of compound 6 and 1.2 mmol of BSH in 6 ml of PBS buffer solution, add 2 mmol of TCEP, and react at room temperature for 36 h under nitrogen protection. The reaction is complete after LC-MS monitoring. Purify by reverse phase preparative liquid phase to obtain L804-EB-FAPI-BSH, a tetrafunctional boron drug compound.

[0037] Wherein, the structural formula of compound 1" is: ; The structural formula of compound 2" is ; The structural formula of compound 3 is: ; The structural formula of compound 4" is: ; The structural formula of compound 5" is: ; The structural formula of compound 6" is: ; The structural formula of L804-EB-FAPI-BSH is: .

[0038] Detection and analysis 1) Mass spectrometry analysis The compound DOTA-IPBA-FAPI-BSH (n=2, 3, 4, 6) prepared in Example 1 was subjected to mass spectrometry analysis. The results are as follows: Figures 1 to 4 shown.

[0039] from Figures 1 to 4 From the analysis, we can see that the mass spectra of the compounds corresponding to n=2, 3, 4, and 6 all have target peaks and have been marked in the figure.

[0040] 2) Purity analysis The compound DOTA-IPBA-FAPI-BSH (n=2, 3, 4, 6) prepared in Example 1 was analyzed for purity using a high performance liquid chromatograph. The results are as follows: Figures 5 to 8 shown.

[0041] from Figures 5 to 8 From the analysis, we can see that the purity of the compounds corresponding to n=2, 3, 4, 6 are 97%, 97%, 96%, 99% respectively.

[0042] 3) Stability studies of radiolabeled compounds DOTA-IPBA-FAPI-BSH (n=2) passed 177 Lu labeling, the results are as follows Fig. 9 shown.

[0043] from Fig. 9 From the analysis, we can see that 177 The radiolabeled purity of Lu-DOTA-IPBA-FAPI-BSH (n=2) can reach more than 99%.

[0044] 4) 177 Affinity of Lu-DOTA-IPBA-FAPI-BSH (n=2) in HT-1080-FAP cells The specific operation is: to study the cell binding ability of boron drug preparations to human FAP-positive HT-1080 tumor cells (HT-1080-FAP) as the research object. 177 After Lu-DOTA-IPBA-FAPI-BSH (n=2) was incubated in HT-1080-FAP cells for 0.25, 0.5, 1, 2, 4, 8, and 24 h, at 4 h, 177 The cell uptake of Lu-DOTA-IPBA-FAPI-BSH (n=2) exceeded 35%, and the uptake results after 4 h of incubation were as follows Fig.10 As shown. Fig.10 As shown in the analysis, only about 2% uptake was detected in HT-1080 cells. Saturation binding and competitive inhibition assays were used to assess binding affinity, and the results are shown in Fig.11 As shown. Fig.11 From the analysis, we can see that 177 Lu-DOTA-IPBA-FAPI-BSH (n=2) showed high affinity to HT-1080-FAP cells.

[0045] 5) 177 SPECT imaging of Lu-DOTA-IPBA-FAPI-BSH (n=2) in HT-1080-FAP tumor-bearing mice like Fig.12 As shown, intravenous injection 177 SPECT imaging was performed at 1, 4, 8, 24, 48, 72, 96 h, and 5, 6, 7, 8, 9, 10, 14, 21, and 28 days after Lu-DOTA-IPBA-FAPI-BSH (36 MBq); 1 h later 177Lu-DOTA-IPBA-FAPI-BSH was mainly concentrated in the liver and kidneys. After 4 hours, tumor uptake increased significantly, and the target to non-target ratio (T / N) increased significantly. Tumor uptake reached the highest value in 8-24 hours, and it was retained for a long time in the subsequent 28 days. The retention time in the tumor was significantly prolonged, exceeding 28 days, indicating that 177 Lu-DOTA-IPBA-FAPI-BSH has good in vivo stability, and its good tumor retention ability is conducive to long-term radionuclide therapy and long-term BNCT treatment of tumors, as well as a significant increase in the effective dose, which can enhance the anti-tumor efficacy of radionuclide therapy and BNCT; this provides DOTA-IPBA-FAPI-BSH with sufficient time window and BNCT irradiation times for its efficient tumor radionuclide therapy and BNCT treatment.

[0046] 6) 177 SPECT / CT imaging of Lu-DOTA-IPBA-FAPI-BSH (n=6) in HT-1080-FAP tumor-bearing mice like Fig.13 As shown, the radioactivity is 37 MBq 177 SPECT / CT imaging of Lu-DOTA-IPBA-FAPI-BSH (n=6) was performed at 1, 2, 4, 8, 24, 48, 72h and 4, 5, 6, 7, 8, 9, 10, 11, 14, 21, and 28 days after tail vein injection. In FAP-positive HT1080 tumor-bearing mice, the image quality was good, and the imaging agent uptake was uniform in the tumor site without abnormal concentration. 177 Lu-DOTA-IPBA-FAPI-BSH mainly illuminates the tumor in the tumor lesion area, with a high target to non-target ratio (T / N). Tumor uptake decreased significantly in 4-24 hours, which may be due to the high dose in the early stage, the tumor liquefaction edema caused the FAP protein to be inactivated, thus causing the imaging agent to fall off. However, from the 2nd to 28th day, as the tumor tissue repaired, the imaging agent was recombined and enriched in the tumor tissue, and the uptake was significantly improved. In the subsequent two weeks, it maintained an ultra-long retention time, and the retention time in the tumor was significantly extended to more than 15 days. The background value was significantly reduced, and the T / N ratio was significantly improved, indicating that 177Lu-DOTA-IPBA-FAPI-BSH (n=6) has good in vivo stability and ultra-long blood half-life to achieve rapid in vivo diagnosis and ultra-long-term radionuclide therapy, and its good tumor retention ability is conducive to long-term radionuclide therapy and long-term BNCT therapy of tumors, which can enhance the anti-tumor efficacy of radionuclides and BNCT. This provides DOTA-IPBA-FAPI-BSH (n=6) with sufficient time window and BNCT irradiation times for efficient tumor radionuclide therapy and BNCT treatment. Such a long-term tumor retention effect is far better than the currently publicly reported literature data. It is the first data report in the world and may become the century-old radioactive diagnostic and therapeutic probe in the field of nuclear medicine.

[0047] 7) BNCT killing effect of DOTA-IPBA-FAPI-BSH (n=2) on human FAP osteosarcoma cells like Fig.14 As shown, when the concentration of DOTA-IPBA-FAPI-BSH (n=2) was 500 μg / mL, it was in vitro and in vitro with human FAP-positive osteosarcoma cells (FAP + After incubation with HOS for 12 h, the cells were placed in the collimator of the RFQ BNCT device and bombarded with moderated thermal neutrons. After incubation for 24, 48, and 72 h, FAP + The survival rate of HOS was significantly higher than that of the PBS control group and the neutron bombardment group. Fig.15 As shown, the results of cell clone formation experiments showed that FAP + The cloning ability of HOS cells after being cultured with DOTA-IPBA-FAPI-BSH (n=2) and irradiated with BNCT was significantly lower than that of the non-neutron irradiated group and the simple neutron irradiated group. The above results show that DOTA-IPBA-FAPI-BSH (n=2) neutron bombardment can effectively inhibit the growth of FAP cells. + HOS has a significant killing effect.

[0048] In summary, the tetrafunctional boron drug compound of the present invention is connected by a linker group to extend the blood half-life of the drug, the albumin binder part, the chelating part, and the 10 The B carrier part and the targeting fibroblast activation protein (FAP) part are connected together, making the tetrafunctional compound have a high content of 10B has improved affinity for tumor cells and prolonged blood half-life, and at the same time has the advantages of excellent targeting, long residence time in tumor tissue, good in vivo stability and low biological toxicity, overcoming the problems of existing radioactive drugs and boron drugs such as poor targeting, poor water solubility, short blood half-life and short drug residence time in tumor tissue. It shows better effect in BNCT treatment of tumors than BPA currently approved for clinical use, and has high clinical promotion and application value in the field of new boron drug development technology for BNCT.

[0049] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or change made by a person skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. A tetrafunctional boron drug compound, characterized in that: Including tetrafunctional compounds and racemates, stereoisomers, tautomers, pharmaceutically acceptable salts or solvates thereof; The tetrafunctional compound has an albumin binder part for extending the blood half-life of the drug, a chelating part, and a 10 B carrier part, fibroblast activation protein (FAP) targeting part and connecting part.

2. The tetrafunctional boron drug compound according to claim 1, characterized in that: Its structural formula is shown in formula (I), （I） In formula (I), R1 is selected from the group consisting of Evans blue (EB), ibuprofen, 4-(p-iodophenyl)butyric acid (IPBA) and sulfonamides; R2 is selected from , , , , , and The group composed of; R3 is selected from ; R4 is selected from ; L1 is selected from , where n is selected from an integer between 1 and 8; L2 is selected from or ; L3 is selected from .

3. The tetrafunctional boron drug compound according to claim 2, characterized in that: The R1 is selected from , , , , , and The group composed of; And / or, the n is selected from an integer between 1 and 8.

4. The tetrafunctional boron drug compound according to claim 1, characterized in that: The structural formula of the tetrafunctional compound is any one of the following structural formulas: 、 、 、 and ； Wherein, n is selected from an integer between 1 and 8.

5. The tetrafunctional boron drug compound according to claim 1, characterized in that: The R2 moiety chelates a radionuclide or a natural isotope of a radionuclide.

6. The tetrafunctional boron drug compound according to claim 5, characterized in that: The radionuclide is capable of emitting beta particles, gamma photons or alpha particles; And / or, the radionuclide is selected from 18 F. 67 Ga, 68 Ga, 99m Tc, 86 Y. 90 Y. 64 Cu, 67 Cu, 55 Co. 149 Tb, 152 Tb, 155 Tb, 161 Tb, 177 Lu, 188 Re, 166 Ho, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 226 Th, 227 Th, 223 Ra and 230 At least one of U.

7. A method for preparing a tetrafunctional boron drug compound according to any one of claims 1 to 6, characterized in that: The following steps are involved: Dissolving compound A in an organic solvent, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt), mixing and reacting, then adding N-(9-fluorenylmethoxycarbonyl)-D-lysine hydrochloride and N-methylmorpholine (NMM), reacting at room temperature overnight to obtain a crude product; dissolving the crude product in a DMF solution containing N, N-diethylaniline (DEA), reacting at room temperature to obtain a first compound; The compound A is selected from at least one of Evans blue (EB), ibuprofen, 4-(p-iodophenyl)butyric acid (IPBA) and sulfonamides; The first compound is dissolved in an organic solvent, and then EDCI and HOBt are added, mixed and reacted, and then (N2-(((9H-fluorene-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine and NMM are added, and reacted at room temperature to obtain an intermediate product; the intermediate product is dissolved in a solution of an organic solvent containing N,N-diethylaniline (DEA), and reacted at room temperature to obtain a second compound; Dissolving compound B in an organic solvent, adding 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIEA) and (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-(3-(piperazine-1-yl)propoxy)quinoline-4-carboxamide (FAPI), and reacting at room temperature to obtain an intermediate product; dissolving the intermediate product in a solution of an organic solvent containing trifluoroacetic acid (TFA), and reacting at room temperature to obtain a third compound; The compound B is selected from , where n is selected from a positive integer; Dissolve compound C in an organic solvent, then add EDCI and HOBt, mix and react, then add the second compound and NMM, react at room temperature overnight, to obtain a fourth compound; The compound C is selected from the group consisting of , , , , , and The group composed of; The fourth compound is dissolved in an organic solvent, EDCI and HOBt are added, mixed and reacted, and then the third compound and NMM are added, reacted at room temperature overnight, TFA is added and reacted at room temperature again to obtain the fifth compound; The fifth compound and 3-maleimidopropionic acid hydroxysuccinimide ester are dissolved in an organic solvent, DIEA is added, and the mixture is reacted at room temperature to obtain a sixth compound; The sixth compound and BSH are dissolved in a PBS solution, and then TCEP is added, and the mixture is reacted at room temperature under nitrogen protection to obtain a tetrafunctional boron drug compound.

8. Use of the tetrafunctional boron drug compound according to any one of claims 1 to 6 in drugs for treating tumors.

9. The use according to claim 8, characterized in that: The tumor includes at least one of malignant melanoma, head and neck squamous cell carcinoma, brain glioma, thymic carcinoma, cervical cancer, endometrial cancer, neuroendocrine tumor, thyroid cancer, intestinal cancer and bone metastasis, nasopharyngeal carcinoma and breast cancer; And / or, the tumor includes at least one of metastatic melanoma, recurrent melanoma, advanced melanoma, metastatic head and neck squamous cell carcinoma, recurrent head and neck squamous cell carcinoma, advanced head and neck squamous cell carcinoma, metastatic brain glioma, recurrent brain glioma and advanced brain glioma.

10. A pharmaceutical composition for BNCT treatment of tumors, characterized in that: The invention comprises a tetrafunctional boron drug compound as claimed in any one of claims 1 to 6, and at least one pharmaceutically acceptable excipient.

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