Bone-targeted boron conjugate for BNCT as well as preparation method and application of bone-targeted boron conjugate

Abstract

The invention relates to the technical field of medicines, in particular to a bone-targeted boron conjugate for BNCT and a preparation method and application of the bone-targeted boron conjugate. Wherein the boron conjugate has a structure represented by formula I or a pharmaceutically acceptable salt thereof, wherein La, Lb and Lc are linking chains, and La, Lb and Lc can be independently deleted, n1 is 0 to 6, n2 and n3 are independently 0 to 20, and n2 + n3 is greater than 0 and less than or equal to 20, X1, X2, X3 and X4 are independently C or O, and X1 is not equal to X2, X3 is not equal to X4, R1 is selected from H, OH and halogen, R2 is selected from H and C1-6 alkyl, R3 is selected from H, halogen and carboxyl, and n1 is 0 to 6.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a bone-targeting boron conjugate for BNCT, its preparation method, and its uses. Background Technology

[0002] In recent years, boron neutron capture therapy (BNCT) has attracted attention as a novel treatment for glioblastoma multiforme and various malignant tumors (Kawabata S, Miyatake S, Kuroiwa T, Yokoyama K, Doi A, Iida K, et al. Boron neutron capture therapy for newly diagnosed glioblastoma. J Radiat Res 2009; 50(1):51-60.). BNCT is a particle radiation therapy method that selectively destroys malignant cells while preserving normal cells (Coderre JA, Morris GM. The radiation biology of boron neutroncapture therapy. Radiat Res 1999; 151(1):1-18.). BNCT is based on... 10 B atoms undergo nuclear trapping and fission reactions with low-energy thermal neutrons / hyperthermal neutrons to produce high-linear-energy-transfer alpha particles and... 7 The recoil of the Li nucleus. Because the trajectories of these particles are very short (9-10 micrometers; about the diameter of a cell), radiation damage is limited to particles containing... 10 B cells. Specifically, BNCT is triggered by a low-energy thermal neutron beam, which, unlike conventional high-energy X-rays or gamma particles commonly used in ionizing radiation therapy, has regional selectivity for radiation therapy and causes less damage to adjacent healthy tissue (RF Barth, AHS Loway, JH Goodman, RAGahbauer, N. Gupta, TE Blue, W. Yang, W. Tjarks, Neurosurgery 44 (1999) 433–450.). Furthermore, because the neutron beam is non-ionizing, only the absorbed cells... 10 Only boron in its tissues can trigger nuclear fission and subsequent self-destruction. Furthermore, boron has a higher nucleus density than any other common atomic nucleus in the human body (e.g., boron). 12 C and 14The neutron capture cross-section is three orders of magnitude larger than that of the tumor. Since the neutron beam itself does not cause major cell death, its reach can be extended to irradiate tissues surrounding the tumor to destroy smaller residual lesions caused by tumor recurrence or metastasis. Because BNCT employs a binary treatment modality, consisting of two independent components—neutron irradiation and boron reagent—the efficacy of BNCT depends primarily on the number of tumor cells reached by the boron-containing drug, assuming the neutron beam can adequately penetrate body tissue (MFHawthorne, MWLee, J. Neurooncol. 62 (2003) 33–45). For a successful BNCT treatment, it is necessary to deliver approximately 20–50 μg of boron per gram of tumor. 10 B and a sufficient number of neutrons must penetrate and be absorbed by the cell to trigger a fatal attack. 10 B(n,α) 7 Li fission reaction. Therefore, if 10 B Drugs can selectively target tumor cells, thus avoiding the side effects usually associated with ionizing radiation (Barth RF. Boron neutroncapture therapy at the crossroads: challenges and opportunities. Appl Radiat Isot 2009; 67(Suppl. 7-8). S3-6.).

[0003] Although boron-dependent neurotransmitter therapy (BNCT) has been clinically applied to treat malignant brain tumors, malignant melanoma, head and neck cancer, and liver cancer, scientists have encountered some problems with boron compounds in its clinical application. Many types of boron compounds, including amino acids, nucleic acids, and liposomes, have been reported as boron delivery carriers for BNCT, but only boron phenylalanine (BPA) and disodium mercaptodecahydroborate ([B...]) have been reported. 12 H n SH] 2- 2Na and BSH are two compounds used clinically in BNCT therapy to treat cancer. BPA is an essential amino acid analog that can be actively transported to brain tumors, via... 18 F-BPA, or BPA, can be detected in positron emission tomography (PET) imaging. However, it also accumulates in normal brain tissue but not in slowly proliferating malignant cells. In contrast, BSH contains abundant... 10BSH accumulates in tumor areas with enhanced permeability and retention (EPR), but less so in normal tissues. However, BSH exists only in the intercellular space and does not enter cells. Therefore, BSH is ineffective in treating GBM during BNCT (Kawabata S, Miyatake S, Kuroiwa T, Yokoyama K, Doi A, Iida K, et al Boron neutron capture therapy for newly diagnosed glioblastoma. J Radiat Res 2009; 50(1):51-60.). In addition, although carboranes such as BSH have excellent boron-containing capacity, their solubility in physiological media makes these compounds difficult to administer.

[0004] To overcome this limitation of BSH, several drug delivery systems incorporating therapeutic doses of BSH pharmacophores have been reported (Barth RF. Boron neutron capture therapy at the crossroads: challenges and opportunities. Appl Radiat Isot 2009; 67(Suppl. 7-8). S3-6). Boron carriers can be classified into three types: boron-containing small molecules, boron compound conjugates, and boron-delivered nanoparticles. Among these drugs, targeted boron delivery agents typically bind boron-containing drugs to tumor-targeting molecules such as nucleosides, porphyrins, peptides, proteins, or antibodies. Another type of targeted boron delivery agent is boron-delivered nanomaterials, which can utilize the enhanced permeation and retention (EPR) effect of nanomaterials and the active targeting effect mediated by tumor-targeting ligands grafted onto the material surface to transport various boron-containing compounds into tumor cells. However, these delivery methods still have certain problems. For example, regarding nucleosides, although the chemical interactions of boron incorporation into different components of DNA have been developed, there is currently no method to screen multiple boron-containing nucleosides for binding to DNA in living cells. Therefore, at this stage, manufacturing and testing individual boron-containing nucleosides is risky because it is difficult to predict which nucleosides will be recognized and integrated into DNA by cellular DNA biosynthesizers. For drug development, the understanding of the interactions between boron-containing nucleosides and cellular uptake, metabolism, replication, and repair systems remains unacceptably limited. In particular, more research is needed to understand the stability of boron-containing nucleotides incorporated into DNA, as DNA repair systems may effectively remove modified nucleotides from DNA (Am J Cancer Res, 2021 11(10):4668-4682). Protein transduction therapy, which involves penetrating the cell membrane through peptide and protein / peptide transduction domains, has significant advantages and is non-toxic in vitro and in vivo. Some researchers have successfully transduced BSH into cells using polyarginine peptides (11R). However, this BSH peptide has some limitations, such as the difficulty in synthesizing BSH-11R and the lack of pharmacokinetic imaging systems, which hinders its clinical application (Michiue H, Sakurai Y, Kondo N, Kitamatsu M, Bin F, Nakajima K, et al The acceleration of boron neutron capture therapy using multi-linked mecaptoundecahydrododecaborate (BSH)fused cell-penetrating peptide. Biomaterials 2014; 35(10):3396-405).

[0005] The effectiveness of BNCT largely depends on 10 B-cells specifically accumulate in tumor cells, thereby targeting and destroying cancer cells without affecting surrounding healthy tissues and blood. Therefore, developing novel tumor-selective drugs is crucial. 10 B-type delivery agents are undoubtedly one of the most important requirements for the success of BNCT. Based on extensive clinical experience and results during the development of BNCT, researchers have summarized some criteria for evaluating "ideal" delivery methods. 10 B. The "golden rule" of drug administration. The five most important principles are: (1) Each tumor cell needs at least 10 9 indivual 10 (1) Boron atoms; (2) T / N and T / B concentration ratios of boron ≥3; (3) Low intrinsic toxicity; (4) Rapid clearance from normal tissues and blood and enhanced tumor retention; (5) Balance of hydrophobic and hydrophilic tendencies, especially in the treatment of brain tumors. These principles guide the design of new BNCT drugs and the selection process for further in vitro and in vivo evaluation. However, achieving these elements is very difficult. In patients receiving BPA and BSH treatment, the tumor-to-blood boron concentration T / B and the tumor-to-normal tissue boron concentration ratio (T / N) are not ideal, which necessitates the development of more selective [BNCT drugs]. 10 B delivery agents. Significant human and financial resources have been dedicated to developing new boron delivery methods over the past few decades. Unfortunately, apart from BSH and BPA, no boron delivery molecules have been tested in clinical trials (K. Hu et al, oordination Chemistry Reviews, 405 (2020), 1-20).

[0006] Osteosarcoma is the most common type of primary bone cancer, classified as a malignant mesenchymal tumor in which the tumor directly produces defective osteoid (immature bone). It is a highly vascularized and destructive malignant tumor, most commonly found in the metaphysis of long bones. For the past two decades, radical surgical resection combined with aggressive chemotherapy has become the mainstream treatment for osteosarcoma, achieving a 5-year survival rate of 50-70% in patients without metastatic disease. Several regimens have been recommended, such as immunotherapy, tumor suppressors, or suicide gene therapy, or anticancer drugs not commonly used in osteosarcoma. However, one-third of patients still die from this devastating cancer, and for those with unresectable cancer, there is no curative systemic therapy.

[0007] Therefore, there is a need to develop new boron compounds that are easier to synthesize, have a long retention time in tumors, and selectively target and destroy tumor cells with minimal damage to normal tissues. Furthermore, more effective methods are needed for treating bone tissue tumors and related tumors; more specifically, more effective boron delivery compounds are needed for BNCT therapy. Summary of the Invention

[0008] In this invention, we have developed a boron conjugate for BNCT that can be readily synthesized, and experimental results demonstrate that the boron conjugate described herein is advantageous for the treatment of bone and bone-related conditions, particularly bone cancer, osteosarcoma, bone tumors, or bone metastases.

[0009] Some embodiments of the present invention provide, on one hand, a boron conjugate comprising a boron formulation portion having thereon attached and at least one bone-targeting portion, said bone-targeting portion being a bisphosphonate or a pharmaceutically acceptable salt thereof.

[0010] The boron preparation portion preferably contains... 10 B's carborane.

[0011] Preferably, the boron conjugate of the present invention has the structure of Formula I or a pharmaceutically acceptable salt thereof:

[0012]

[0013] Where La, Lb, and Lc are connecting chains, and La, Lb, and Lc can be independently missing. n1 is 0 to 6, n2 and n3 are independently 0 to 20, and 0 < n2 + n3 ≤ 20. X1, X2, X3, and X4 are independently C or O, and X1 ≠ X2, X3 ≠ X4. R1 is selected from H, OH, and halogens, and R2 is selected from H and C. 1-6 Alkyl group, R3 is selected from H, halogen and carboxyl group, n1 is 0 to 6.

[0014] Bisphosphonates bind to the bone hydroxyapatite matrix and negatively impact osteoclast activity; they were initially identified as potential bone-targeting components based on their structural similarity to pyrophosphate, a natural regulator of calcium homeostasis.

[0015] Suitable bisphosphonates include pharmaceutically acceptable salts of metal salts, such as aluminum salts, alkali metal salts, such as sodium or potassium salts, alkaline earth metal salts, such as calcium or magnesium salts, and ammonium or substituted ammonium salts, such as salts formed with the following amines: lower alkylamines, such as triethylamine, hydroxy-lower alkylamines, such as 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine, cycloalkylamines, such as dicyclohexylamine, or procaine, dibenzylamine, N,N-dibenzylethylenediamine, 1-cephalosporinamine, N-ethylpiperidine, N-benzyl-β-phenylethylamine, N,N′-didehydroalkylamine, ethylenediamine, or pyridine bases such as pyridine, chlorpheniramine or quinoline, or other amines that have been used to form salts with known penicillins or cephalosporins.

[0016] Bone is an attractive target for oncology drugs because systemic anticancer therapy involves high toxicity and a wide range of adverse side effects, especially for bone tumors, where higher doses may be required to achieve the concentrations needed at the disease site. Numerous studies, ranging from in vitro to animal models, have investigated bone-targeted chemotherapy for cancer. The anticancer properties of bisphosphonates alone could potentially provide bifunctional therapy through conjugation with further antitumor drugs, but due to the charged nature of bisphosphonates, most cells have limited uptake of them. Many studies have not adequately explored the effects of bisphosphonates beyond their bone-targeting effects on their conjugates, and many have also failed to investigate whether targeting can reduce toxicity. Numerous in vitro studies have shown that conjugating conventional cancer chemotherapy drugs with bisphosphonates can improve efficacy. However, compelling data on the function of many conjugates are still lacking. An earlier study demonstrated that bisphosphonates and methotrexate, linked by a peptide bond, successfully localized to bone (Hosainet et al., 1996; Sturtz et al., 1992). Later, another bisphosphonate-methotrexate conjugate was shown to induce apoptosis in OS (osteosarcoma) cells in vitro, but at a rate similar to standard methotrexate OS treatment (Yang et al., 2014a). Additionally, Roy's group demonstrated that bisphosphonates coupled with proteasome inhibitors are highly toxic to multiple myeloma cell lines (Agyine et al., 2013). However, in this invention, the bisphosphonate-boron conjugate is efficiently taken up, targets bone tissue, and is essentially non-toxic.

[0017] Preferably, the "carborane" in Formula I has the following structure:

[0018]

[0019] More preferably, the "carborane" is B-4.

[0020] Preferably, n2 and n3 are each independently 0-10, and 0 < n2 + n3 ≤ 10. More preferably, n2 or n3 is 0, and 0 < n2 + n3 ≤ 10.

[0021] Preferably, X1 = X3, X2 = X4.

[0022] Preferably, the La, Lb, and Lc, when present, each independently contain the following groups: -CH2-OC(O)-, -C(O)O-CH2-, -C(O)-, -C(O)-NH-, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-, -CH2-C(O)-NH-, -CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH 2-, -CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-CH2-, - CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-, -C(O)-O-CH2-, -C H2-C(O)-O-CH2-, -CH2-CH2-C(O)-O-CH2-, -C(O)-O-CH2-CH2-, -NH-C(O)-CH2-, -CH2-NH-C(O)-CH2-, -CH2-CH2-NH-C(O)-CH2-, -NH-C(O)-CH2-CH 2-, -CH2-NH-C(O)-CH2-CH2-, -CH2-CH2-NH-C(O)-CH2-CH2-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -NH-CH2-, -NH-CH2-CH2-, -CH2-NH-CH2-, -CH2 -CH2-NH-CH2-, -CH2-CH2-NH-CH2-CH2-, -C(O)-CH2-, -C(O)-CH2-CH2-, -CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2- CH2-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-,-CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-CH2-, -C(O)-NH-(CH2), 1-6 -NH-C(O)-、-NH-C(O)-NH-(CH2) 1-6 -NH-C(O)-, -N(R4)-, and combinations thereof, wherein R4 is H or selected from alkyl or substituted alkyl groups.

[0023] In a preferred embodiment, R1 is selected from H and OH, R2 is selected from H, R3 is selected from H, and n1 is 1 to 3.

[0024] In a preferred embodiment, the boron conjugate of the present invention has the following structure Ia or a pharmaceutically acceptable salt thereof:

[0025]

[0026] The definitions of carborane, Lb, Lc, X3, X4, n1, n2, R1, R2, and R3 are the same as those in the previous text.

[0027] In a preferred embodiment, the boron conjugate of the present invention has the following structure Ib or a pharmaceutically acceptable salt thereof:

[0028]

[0029] The definitions of carborane, Lb, Lc, n1, n2, R1, R2, and R3 are the same as those in the previous text.

[0030] Preferably, the Lb is selected from -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-C(O)-O-CH2-, -CH2-CH2-C(O)-O-CH2 -, -CH2-NH-C(O)-CH2-, -CH2-CH2-NH-C(O)-CH2-, -CH2-NH-C(O)-CH2-CH2-, -CH2-CH2-NH-C(O)-CH2-CH2-, -CH2-NH-CH2-, -CH2-CH2-NH-CH2-, -CH2-CH2-NH-CH2-CH2-, -C H2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2-CH2-CH2-C(O )-NH-CH2-CH2-NH-C(O)-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-CH2-.

[0031] More preferably, the Lb is selected from -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2-CH2-NH-CH2-, -CH2-CH2-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-.

[0032] Preferably, the Lc is missing or selected from -CH2-OC(O)-, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-, -CH2-C(O)-NH-, -CH2-CH2-C(O)-NH-, -CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C(O)-NH- CH2-, -CH2-CH2-CH2-C(O)-NH-, -CH2-C(O)-NH-CH2-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2- C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-, -CH2-CH2-C(O)-O-CH2-, -C H2-NH-C(O)-CH2-, -CH2-CH2-NH-C(O)-CH2-, -CH2-NH-C(O)-CH2-CH2-, -CH2-CH2-NH-C(O)-CH2-CH2-, -CH2-N H-CH2-, -CH2-CH2-NH-CH2-, -CH2-CH2-NH-CH2-CH2-, -CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O) -CH2-CH2-, -CH2-CH2-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C( O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-CH2-.

[0033] More preferably, the Lc is missing or selected from -CH2-OC(O)-, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-C(O)-NH-, -CH2-CH2-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-.

[0034] In a preferred embodiment, the boron conjugate of the present invention has the following structure Ic or a pharmaceutically acceptable salt thereof:

[0035]

[0036] The definitions of carborane, Lb, n1, n2, R1, R2, and R3 are the same as those in the previous text.

[0037] Preferably, Lb is selected from -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2-CH2-NH-CH2-, -CH2-CH2-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-; n2 is 1-10, R1 is selected from H and OH, R2 is selected from H, R3 is selected from H, and n1 is 1 to 3.

[0038] As an example, the compounds of formula I described in this invention are selected from:

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] In another aspect, the present invention provides a compound having the structure of Formula II or a salt thereof:

[0045]

[0046] The definitions of La, Lb, Lc, X1, X2, X3, X4, n1, n2, n3, R1, R2, and R3 are the same as those in the previous text.

[0047] Preferably, Formula II has the following structure:

[0048]

[0049] The definitions of Lb, Lc, X3, X4, n1, n2, R1, R2, and R3 are the same as those in the previous text.

[0050] Preferably, Formula II has the following structure:

[0051]

[0052] The definitions of Lb, Lc, n1, n2, R1, R2, and R3 are the same as those in the previous text.

[0053] Preferably, Formula II has the following structure:

[0054]

[0055] The definitions of Lb, n1, n2, R1, R2, and R3 are the same as those in the previous text.

[0056] For example, the compound of formula II is selected with the following structure:

[0057]

[0058]

[0059]

[0060] Another aspect of the present invention provides a compound having a structure of Formula III or an active ester thereof, such as N-hydroxysuccinimide, N-hydroxyphthalimide, N-hydroxythiophthalimide, o-nitrobenzene, p-nitrobenzene, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrobenzene, 3-carboxyl-4-nitrobenzene, pentafluorophenyl, and sulfonyltetrafluorophenyl:

[0061]

[0062] The definitions of carborane, La, Lb, Lc, X1, X2, X3, X4, n2, and n3 are the same as those in the previous text.

[0063] Preferably, the compound of formula III has the following structure:

[0064]

[0065] The definitions of carborane, Lb, Lc, X3, X4, and n2 are the same as those in the previous text.

[0066] Preferably, the compound of formula III has the following structure:

[0067]

[0068] The definitions of carborane, Lb, Lc, and n2 are the same as those in the previous text.

[0069] Preferably, the compound of formula III has the following structure:

[0070]

[0071] The definitions of carborane, Lb, and n2 are the same as those in the previous text.

[0072] Preferably, the compound of formula III has the following structure:

[0073]

[0074] The definitions of Lb and n2 are the same as those in the previous text.

[0075] For example, the compound of formula III of the present invention has the following structure or its active ester:

[0076]

[0077]

[0078] In addition, the present invention provides a method for preparing the conjugate of formula I, which is obtained by Michael addition reaction of a compound of formula II with a carborane thiol, and the reaction equation is as follows:

[0079]

[0080] The definitions of La, Lb, Lc, X1, X2, X3, X4, n1, n2, n3, R1, R2, and R3 are the same as those in the previous text.

[0081] The conjugate can be purified by column chromatography on silica gel or by HPLC.

[0082] Furthermore, the compound of formula II is prepared by condensation of a compound of formula IV or its active ester with a compound of formula V.

[0083]

[0084] The definitions of La, Lb, Lc, X1, X2, X3, X4, n1, n2, n3, R1, R2, and R3 are the same as those in the previous text.

[0085] Alternatively, the compound of formula I may be prepared by condensation of compound III or its active ester with compound V, and the reaction equation is as follows:

[0086]

[0087] The compound of formula III is prepared by Michael addition of the compound of formula IV with carborane thiols:

[0088]

[0089] The definitions of La, Lb, Lc, X1, X2, X3, X4, n1, n2, n3, R1, R2, and R3 appearing in the above structures and the concept of active esters are the same as those in the previous text.

[0090] The conjugates provided by this invention exhibit excellent antitumor activity in vitro, while showing significant reductions in plasma clearance and toxicity. The boron conjugates of this invention also exhibit better water solubility compared to the boron conjugates disclosed in patent application number 2023116205087. Attached Figure Description

[0091] Figure 1 The image shows the MALDI-TOF MS plot of the intermediate compound MP1-10-17.

[0092] Figure 2 The image shows the MALDI-TOF MS plot of the intermediate compound MP2-10-17.

[0093] Figure 3 The image shows the MALDI-TOF MS plot of the intermediate compound MP3-10-17.

[0094] Figure 4 The image shows the MALDI-TOF MS plot of the intermediate compound MP4-10-17.

[0095] Figure 5 The image shows the MALDI-TOF MS plot of the intermediate compound MP5-10-17.

[0096] Figure 6 The image shows the MALDI-TOF MS plot of the intermediate compound MP6-10-17.

[0097] Figure 7 The image shows the MALDI-TOF MS plot of the intermediate compound MP2-20-17.

[0098] Figure 8 The image shows the MALDI-TOF MS plot of the intermediate compound MP2-30-17.

[0099] Figure 9 The image shows the MALDI-TOF MS plot of the intermediate compound MP2-40-17.

[0100] Figure 10 The image shows the MALDI-TOF MS plot of the intermediate compound MP2-50-17. Figure 11 The image shows the MALDI-TOF MS plot of the intermediate compound MP2-12-17.

[0101] Figure 12The image shows the MALDI-TOF MS plot of the intermediate compound MP2-12-17.

[0102] Figure 13 The image shows the MALDI-TOF MS plot of the intermediate compound MP2-13-17.

[0103] Figure 14 The image shows the MALDI-TOF MS plot of the intermediate compound MP2-14-17.

[0104] Figure 15 The image shows the MALDI-TOF MS plot of the intermediate compound MP2-15-17.

[0105] Figure 16 The image shows the MALDI-TOF MS plot of the intermediate compound MP4-10-15.

[0106] Figure 17 The image shows the MALDI-TOF MS plot of the intermediate compound MP4-10-3.

[0107] Figure 18 The image shows the MALDI-TOF MS plot of the intermediate compound MP4-10-25.

[0108] Figure 19 The image shows the MALDI-TOF MS plot of the intermediate compound MP4-10-23. Detailed Implementation

[0109] To better understand the technical solution of the present invention, the technical solution of the present invention will be further described below with reference to specific embodiments. The embodiments are only for the purpose of helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0110] As an example, taking the C4P2-10-17 compound as an example, the present invention employs the following synthetic method:

[0111]

[0112] Maleimide-diethylene glycol-carboxylic acid (100.00 mg, 0.39 mmol), N-hydroxysuccinimide (53.70 mg, 0.47 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (89.18 mg, 0.47 mmol) were dissolved in N,N-dimethylformamide (2 mL). Sodium alenphosphate trihydrate (152.14 mg, 0.47 mmol) was added to the above system. The reaction system was stirred overnight at room temperature. The crude product was concentrated by vacuum distillation, and the concentrate was purified by MeOH / DCM (0-20%) Prep-TLC. The filtrate was filtered to remove the solvent under reduced pressure. After freeze-drying, the product was obtained as a pale yellow solid. The product obtained above (50.00 mg, 102.45 μmol), BSH (50.00 mg, 112.63 μmol), and [unspecified ingredient] were dissolved in DMSO (1 mL). Triethylamine (20.70 mg, 204.90 μmol) was added to the above system. The reaction system was stirred overnight at room temperature. The crude product was concentrated by vacuum distillation, and the concentrate was prepared using a reverse-phase C18 column with the mobile phase being: Phase A (0.8% water), Phase B (ACN), gradient (5%-100%). After preparation, the solvent was removed under reduced pressure. After freeze-drying, the product was obtained as a pale yellow solid.

[0113] Referring to the above preparation method, and combining it with the method disclosed in part of the technical solution of this invention, or through reasonable optimization, the intermediate compounds in Table 1 and the boron coupling compounds in Table 2 can be obtained.

[0114] Table 1

[0115]

[0116]

[0117]

[0118]

[0119] Table 2

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] Example 2. HepG2 cell uptake assay of the conjugate

[0128] Three batches were repeated. HepG2 cells were seeded in 6-well plates. After cell attachment, boron coupling agent at concentrations of 100, 200, 300, 400, 500, 600, and 700 μg / mL was added, respectively. After incubation for 24 h, cells were collected, centrifuged at 200g for 3 min, the supernatant was discarded, the cell pellet was collected and counted, and the cells were digested with concentrated nitric acid. Cell counts were determined by ICP-MS per 10^6 cells. 6 The boron content in each cell was calculated and the average value is recorded in Tables 3 and 4.

[0129] Table 3

[0130]

[0131] Table 4

[0132]

[0133]

[0134] When the concentration of BSH changed tenfold from 3.75 μg / mL to 37.5 μg / mL during incubation, the uptake by cells was still very low, indicating that the ability of BSH to enter cells is very limited. However, the uptake capacity of the conjugate increased significantly with increasing drug concentration, indicating that it effectively improved the problem of boron drug entry into cells.

[0135] Example 3. Cell viability assay after neutron irradiation

[0136] Five batches were repeated, with different concentrations of boron-coupled drugs added after HepG2 adherence and incubated for 24 hours, followed by BNCT irradiation (2.57 × 10⁻⁶). 8 cm -2 ·s -1For 1 hour, cells were seeded into 96-well plates. After adhesion, cell viability was determined using the CCK-8 assay according to the instructions. Specifically, the cell culture supernatant was removed, and medium containing CCK-8 was added. After incubation at 37°C for 24 hours, the absorbance at 562 nm was detected using a microplate reader. The absorbance of cells receiving BNCT alone without boron conjugate incubation was used as the normalization standard. The absorbance value of the blank group was the absorbance value of medium with CCK-8. The survival rate of other experimental groups was calculated using the formula: "Survival rate = (Absorbance value of experimental group - Absorbance value of blank group) / (Absorbance value of BNCT alone group - Absorbance value of blank group)". The average values ​​are recorded in Table 5. When the BSH concentration was 15 μg / mL, the cell viability was 63.10%.

[0137] Table 5

[0138]

[0139]

[0140] Example 4. Clonogenesis experiment after neutron irradiation

[0141] Three batches were repeated, with different concentrations of boron bisphosphonate-coupled drugs added after HepG2 adherence and incubated for 24 hours. After incubation, the cells were irradiated with BNCT (2.57 × 10⁻⁶). 8 cm -2 ·s -1 For 1 hour, cells were seeded into 6-well plates at 1000 cells / well, 2 mL of whole culture was added, and the plates were incubated at 37°C with 5% CO2 for 7-10 days. After observing the formation of more obvious cell clones in the PBS group, the culture medium was removed, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature for 2 hours, washed 3 times with PBS, stained with 0.5% crystal violet at room temperature for 2 hours, washed 3 times with PBS, and photographed. The obtained photos were analyzed using ImageJ software to count the number of clones formed, and the average value was calculated and recorded in Table 6.

[0142] Table 6

[0143]

[0144]

Claims

1. A bone-targeting boron conjugate for BNCT, having the structure of formula (I) or a pharmaceutically acceptable salt thereof: Where La, Lb, and Lc are connecting chains, and La, Lb, and Lc can be independently missing. n1 is 0 to 6, n2 and n3 are independently 0 to 20, and 0 < n2 + n3 ≤ 20. X1, X2, X3, and X4 are independently C or O, and X1 ≠ X2, X3 ≠ X4. R1 is selected from H, OH, and halogens, and R2 is selected from H and C. 1-6 Alkyl group, R3 is selected from H, halogen and carboxyl group, n1 is 0 to 6.

2. The boron coupling compound according to claim 1, wherein "carborane" in formula (I) is selected from the following structures:

3. The boron coupling compound according to claim 1, wherein n2 and n3 are each independently 0-10, and 0 < n2 + n3 ≤ 10; X1 = X3, X2 = X4; La, Lb, and Lc, when present, each independently contain the following groups: -CH2-OC(O)-, -C(O)O-CH2-, -C(O)-, -C(O)-NH-, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-, -CH2-C(O)-NH-, -CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-, - CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-CH2-, -CH2 -CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-, -C(O)-O-CH2-, -CH2- C(O)-O-CH2-, -CH2-CH2-C(O)-O-CH2-, -C(O)-O-CH2-CH2-, -NH-C(O)-CH2-, -CH2-NH-C(O)-CH2-, -CH2-CH2-NH-C(O)-CH2-, -NH-C(O)-CH2-CH2- , -CH2-NH-C(O)-CH2-CH2-, -CH2-CH2-NH-C(O)-CH2-CH2-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -NH-CH2-, -NH-CH2-CH2-, -CH2-NH-CH2-, -CH2-C H2-NH-CH2-, -CH2-CH2-NH-CH2-CH2-, -C(O)-CH2-, -C(O)-CH2-CH2-, -CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2-C H2-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-,-CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-CH2-, -C(O)-NH-(CH2), 1-6 -NH-C(O)-、-NH-C(O)-NH-(CH2) 1-6 -NH-C(O)-, -N(R4)- and combinations thereof, wherein R4 is H or selected from C1-C3 alkyl or substituted C1-C3 alkyl; R1 is selected from H and OH, R2 is selected from H, R3 is selected from H, and n1 is 1 to 3.

4. The boron conjugate according to claim 1, having the structure of formula (Ia) or a pharmaceutically acceptable salt thereof: The definitions of carborane, Lb, Lc, X3, X4, n1, n2, R1, R2, and R3 are the same as those in claim 1.

5. The boron conjugate according to claim 1, having the structure of formula (Ib) or a pharmaceutically acceptable salt thereof: in: Carborane is of formula (B-4); The definitions of n1, n2, R1, R2, and R3 are the same as those in claim 1; Lb is selected from -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -C H2-CH2-NH-CH2-, -CH2-CH2-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-; Lc is missing or selected from -CH2-OC(O)-, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-C(O)-NH-, -CH2-CH2-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-.

6. The boron conjugate according to claim 1, having the structure of formula (Ic) or a pharmaceutically acceptable salt thereof: in: Lb is selected from -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -C H2-CH2-NH-CH2-, -CH2-CH2-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-; n² is 1-10; R1 is selected from H and OH; R2 is selected from H; R3 is selected from H; n1 is between 1 and 3.

7. The boron coupling compound according to claim 1, wherein the compound of formula (I) is selected from:

8. An intermediate compound for preparing BNCT boron couplings, having the structure of formula (II) or a salt thereof: The definitions of La, Lb, Lc, X1, X2, X3, X4, n1, n2, n3, R1, R2, and R3 are the same as those in any of claims 1-6.

9. The compound according to claim 8, wherein formula (II) has the following structure: The definitions of Lb, Lc, X3, X4, n1, n2, R1, R2, and R3 are the same as those in any of claims 1-6.

10. The compound according to claim 8, wherein formula (II) has the following structure: The definitions of Lb, Lc, n1, n2, R1, R2, and R3 are the same as those in any of claims 1-6.

11. The compound according to claim 8, wherein formula (II) has the following structure: The definitions of Lb, n1, n2, R1, R2, and R3 are the same as those in any of claims 1-6.

12. The compound according to claim 8, wherein the compound of formula II has the following structure:

13. A method for preparing the boron coupling compound of formula (I) according to claim 1, comprising Michael addition of a compound of formula (II) with a carborane thiol, wherein the reaction equation is as follows: The definitions of La, Lb, Lc, X1, X2, X3, X4, n1, n2, n3, R1, R2, and R3 are the same as those in any of claims 1-6.

14. The preparation method according to claim 13, wherein the compound of formula (II) is prepared by condensation of a compound of formula (IV) or its active ester with a compound of formula (V). The definitions of La, Lb, Lc, X1, X2, X3, X4, n1, n2, n3, R1, R2, and R3 are the same as those in any of claims 1-6.

15. A method for preparing the boron coupling compound of formula (I) according to claim 1, comprising the condensation of a compound of formula (III) or its active ester with a compound of formula (V), wherein the reaction equation is as follows: The definitions of La, Lb, Lc, X1, X2, X3, X4, n1, n2, n3, R1, R2, and R3 are the same as those in any of claims 1-6.

16. The preparation method according to claim 15, wherein the compound of formula (III) is prepared by Michael addition of the compound of formula (IV) with a carboranethiol: The definitions of La, Lb, Lc, X1, X2, X3, X4, n1, n2, n3, R1, R2, and R3 are the same as those in any of claims 1-6.

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