Intermediate for preparing targeted bone tissue boron conjugate and preparation method thereof

Abstract

The invention relates to the technical field of medicine, in particular to an intermediate for preparing a targeted bone tissue boron conjugate and a preparation method of the intermediate, the intermediate is a compound with a structure shown in a formula IIAX, La is # imgabs0 #, R5, R6, R8 and R9 are independently selected from H, C1-C6 alkyl, carboxyl and cyclohexyl, or R5 and R6 can be cyclized into a ring, the ring is a five-membered or six-membered ring, and R9 is selected from H, C1-C6 alkyl, carboxyl and cyclohexyl. R7 is H or C1-C6 alkyl, and m and p are each independently 0 to 12; r, t, q1, q2 and u are each independently 0 or 1, q1 and q2 are not 1 at the same time, and P is H or # imgabs1 #

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to providing a new boron conjugate targeting bone tissue for boron neutron capture therapy and its uses. Background Art

[0002] In recent years, boron neutron capture therapy (BNCT) has attracted attention as a new method for treating 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. JRadiat Res 2009;50(1):51 - 60.). BNCT is a particle radiotherapy method that selectively destroys malignant cells and preserves 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 the nuclear capture and fission reaction of B atoms with low - energy thermal neutrons / epithermal neutrons to generate high - linear energy transfer α particles and 7 the recoil of Li nuclei. Because the tracks of these particles are very short (9 - 10 microns; approximately one cell diameter), the radiation damage is limited to the cells containing 10 B. Specifically, BNCT is triggered by a low - energy thermal neutron beam, which is different from the conventional high - energy x - rays or gamma particles commonly used in ionizing radiation therapy, and thus has regional selectivity in radiation therapy and causes less damage to adjacent healthy tissues (R.F. Barth, A.H. Soloway, J.H. Goodman, R.A. Gahbauer, N. Gupta, T.E. Blue, W. Yang, W. Tjarks, Neurosurgery 44(1999)433–450.). In addition, since the neutron beam is non - ionizing, only the tissue that has absorbed 10 B will trigger nuclear fission and subsequent self - destruction. Moreover, boron has a higher neutron capture cross - section than any other common atomic nucleus in the human body (such as 12 C and 14N) The neutron capture cross section is three orders of magnitude larger. Since the neutron beam itself does not cause major cell death, the range of the neutron beam can be extended to irradiate the tissues around the tumor to destroy the smaller residual lesions caused by tumor recurrence or metastasis. Since BNCT uses a binary treatment mode that combines neutron irradiation and a boron agent to achieve the therapeutic effect, the efficacy of BNCT mainly depends on the number of boron-containing drugs that can reach the tumor cells, assuming that the neutron beam can fully penetrate the body tissues (M.F. Hawthorne, M.W.L. Lee, J. Neurooncol. 62 (2003) 33–45.). To achieve a successful BNCT treatment, it is necessary to deliver approximately 20 - 50 μg of 10 B per gram of tumor and a sufficient number of neutrons must penetrate and be absorbed by the cells to trigger the lethal 10 B(n,α) 7 Li fission reaction. Therefore, if the B drug can selectively target tumor cells, the side effects commonly associated with ionizing radiation can be avoided (Barth RF. Boron neutron capture therapy at the crossroads: challenges and opportunities. Appl Radiat Isot 2009;67(Suppl.7 - 8).S3 - 6.).

[0003] Although BNCT has been clinically applied to the treatment of malignant brain tumors, malignant melanoma, head and neck cancers, and liver cancers, however, in the clinical application of BNCT, scientists have encountered some problems with boron compounds. Many types of boron compounds, such as amino acids, nucleic acids, and liposomes, have been reported as boron delivery carriers for BNCT, but only two compounds, boronophenylalanine (BPA) and disodium mercaptoundecahydrododecaborate ([B 12 H n SH] 2- 2Na, BSH), are clinically used in BNCT therapy for cancer treatment. BPA is an essential amino acid analogue that can be actively transported into brain tumors and can be detected in positron emission tomography (PET) imaging by 18 F - BPA. However, it also accumulates in normal brain tissues and does not accumulate in slowly proliferating malignant cells. In contrast, BSH contains a rich 10B, and has enhanced permeability and retention (EPR) accumulation in the tumor region, but less in normal tissues. However, BSH only exists in the interstitial space and does not enter cells. Therefore, the effect of BSH in treating GBM during BNCT is insufficient (Kawabata S, Miyatake S, Kuroiwa T, Yokoyama K, Doi A, Iida K, et al Boron neutron capture therapy for newly diagnosed glioblastoma. JRadiat Res 2009;50(1):51-60.). Additionally, although carboranes such as BSH have excellent boron-holding ability, the solubility problem of these compounds in physiological media makes it difficult to administer these compounds.

[0004] To overcome this limitation of BSH, several drug delivery systems incorporating the pharmacophore of BSH at therapeutic doses 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 delivery nanoparticles. Among these drugs, targeted boron delivery agents usually combine boron-containing drugs with tumor-targeting molecules such as nucleosides, porphyrins, peptides, proteins, or antibodies. Another type of targeted boron delivery agent is boron delivery nanomaterials, which can transport various boron-containing compounds into tumor cells by utilizing the enhanced permeability and retention (EPR) effect of nanomaterials and the active targeting effect mediated by tumor-targeting ligands grafted on the material surface. However, there are still certain problems with these delivery methods. For example, for nucleosides, although the chemistry of boron incorporation into different components of DNA has been developed, there is currently no method to screen the binding of various boron-containing nucleosides 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 by cellular DNA biosynthetic enzymes and integrated into DNA. For drug development, the understanding of the interactions between boron-containing nucleosides and cellular uptake, metabolism, replication, and repair systems remains unacceptably scarce. In particular, more research is needed to understand the stability of boron-containing nucleotides incorporated into DNA because the DNA repair system may effectively remove modified nucleotides from DNA (Am J Cancer Res, 2021 11(10):4668-4682). Protein transduction therapy using cell-penetrating peptides and protein / peptide transduction domains to deliver multiple molecules (such as proteins, peptides, small interfering RNAs) has significant advantages and is non-toxic in vitro and in vivo. BSH has been successfully transduced into cells using polyarginine peptide (11R). However, this BSH peptide has some limitations, such as the difficulty in synthesizing BSH-11R and the lack of a pharmacokinetic imaging system, 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 mercaptoundecahydrododecaborate (BSH) fused cell-penetrating peptide. Biomaterials 2014;35(10):3396-405).

[0005] The effectiveness of BNCT largely depends on 10 the specific accumulation of boron in tumor cells, thereby targeting and destroying cancer cells without affecting surrounding healthy tissues and blood. Therefore, developing new 10 boron delivery agents with high tumor selectivity is undoubtedly one of the most important requirements for the success of BNCT. Based on a large amount of clinical experience and results during the development of BNCT, researchers have summarized some criteria for evaluating an "ideal" 10 boron dosing agent. The five most important criteria are: (1) Each tumor cell requires at least 10 9 boron 10 atoms; (2) The T / N and T / B concentration ratios of boron ≥ 3; (3) Low inherent toxicity; (4) Rapid clearance from normal tissues and blood and enhanced tumor retention; (5) A balance of hydrophobic and hydrophilic tendencies, especially in the treatment of brain tumors. These criteria have guided the design of new BNCT drugs and the selection process for further in vitro and in vivo evaluations. However, it is very difficult to achieve these elements. In patients receiving BPA and BSH treatments, the boron concentration ratios of tumor to blood (T / B) and tumor to normal tissue (T / N) are not ideal, which requires the development of more selective 10 boron delivery agents. In the past few decades, a large amount of human and financial resources have been dedicated to developing new boron delivery methods. Unfortunately, except for BSH and BPA, no boron delivery molecule has been tested in clinical trials (K. Hu et al, Coordination Chemistry Reviews, 405 (2020), 1 - 20).

[0006] Osteosarcoma is the most common type of primary bone cancer, which is classified as a malignant mesenchymal tumor in which the tumor directly produces defective osteoid (immature bone). It is a highly vascular and extremely destructive malignant tumor, most commonly occurring in the metaphysis of long bones. Over the past two decades, due to the fact that various forms of therapy consisting of radical surgical resection combined with aggressive chemotherapy have become the mainstream for treating osteosarcoma, the 5-year survival rate in patients without metastatic disease can reach 50 - 70%. Several regimens have been recommended, such as immunotherapy-based, tumor inhibitor, 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 these patients with unresectable cancer, there is no curative systemic therapy.

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

[0008] In the present invention, we have developed a boron conjugate that can be easily synthesized, and the test results demonstrate that the boron conjugate described herein can be advantageously used for treating bone and bone-related disorders, particularly bone cancer, osteosarcoma.

[0009] Certain embodiments of the present invention provide, on the one hand, a boron conjugate comprising a boron agent moiety attached thereto and at least one bone targeting moiety, wherein the bone targeting moiety is a bisphosphonate or a pharmaceutically acceptable salt thereof.

[0010] Wherein the boron agent moiety preferably contains 10 a carborane containing

[0011] Preferably, the boron conjugate of the present invention has the following structure of Formula I:

[0012] 10 [[B]]-A-L-T

[0013] I

[0014] Wherein 10 [[B]] is a carborane, A is selected from N, S, O or C, L is a linking chain, and T is a bisphosphonate or a pharmaceutically acceptable salt thereof.

[0015] Preferably, the A is S.

[0016] Preferably, the 10 [[B]] has the following structure:

[0017]

[0018] Linking chains L useful in the present invention include those linkers having one or more different reactive functional groups that covalently link to the bisphosphonate moiety and the 10 [[B]] moiety. The linking moiety has two or more different reactive functional groups. In some cases, a multivalent linker can be used. In a preferred embodiment of the present invention, the linking chain provides for linking A and the moiety to the bisphosphonate. For example, when A is a thiol group, the linking chain has a maleimide or haloacetyl group moiety. However, those skilled in the art will understand that any reactive functional group can be present on the linker as long as it is compatible with the functional group on the moiety to be covalently linked.

[0019] ​Preferably, L has the following structure:

[0020] or -CO-

[0021] where La is

[0022] where R 5 , R 6 , R 8 , R 9 are each independently selected from H, C 1 -C 6 alkyl, carboxyl, cyclohexyl, or R 5 and R 6 can form a ring, and the ring is a five - or six - membered ring. R 7 , R 10 , R 11 are each independently selected from H or C 1 -C 6 alkyl. Exemplary C 1 -C 6 alkyls are methyl, ethyl, n - propyl, isopropyl, n - butyl, isobutyl, sec - butyl, tert - butyl, n - pentyl, isopentyl, sec - pentyl, neopentyl, hexyl. m, p, and v are each independently 0 to 12; r, t, q 1 , q 2 and u are each independently 0 or 1, where q 1 , q 2 are not both 1.

[0023] Preferably, L has the following structure:

[0024]

[0025] where R 5 , R 6 , R 7 , R 8 , R 9 , m, q 1 , q 2 , p, and t are defined as above.

[0026] Preferably, L has the following structure:

[0027]

[0028] where R 7 , R 8 , R 9 , q 1 , q 2 , p, and t are defined as above. R 12 , and R13 Each independently selected from H or C 1 -C 6 alkyl, exemplary of said C 1 -C 6 alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl. m is from 0 to 12.

[0029] Exemplarily, the linking chain has the following structure:

[0030]

[0031]

[0032] Preferably, the bisphosphonic acid is a class of molecules composed of a phosphorus-carbon-phosphorus backbone and has the following structural formula T1:

[0033]

[0034] wherein R 1 is selected from H, OH and halogen, R 2 is selected from H and C 1-6 alkyl, R 3 is selected from H, halogen and carboxyl, R 4 is selected from H and C 1-6 alkyl, and n is from 0 to 6.

[0035] Exemplarily, the T1 is selected from compound fragments having the following structures:

[0036]

[0037] Bisphosphonic acids bind to the bone hydroxyapatite matrix and have a negative impact on osteoclast activity; they were first identified as potential bone-targeting moieties based on their structural similarity to pyrophosphate, a natural regulator of calcium homeostasis.

[0038] Suitably, the pharmaceutically acceptable salts of the bisphosphonic acid include 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 salts or substituted ammonium salts, for example salts formed with the following amines: lower alkylamines such as triethylamine, hydroxy-lower alkylamines such as 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine or tris-(2-hydroxyethyl)amine, cycloalkylamines such as dicyclohexylamine, or procaine, dibenzylamine, N,N-dibenzylethylenediamine, 1-cephaleneamine, N-ethylpiperidine, N-benzyl-β-phenethylamine, N,N′-didehydroamino, ethylenediamine, or pyridine bases such as pyridine, collidine or quinoline, or other amines that have been used to salt known penicillins or cephalosporins.

[0039] Bone is an attractive target for oncology drugs because systemic anti-cancer treatment involves high toxicity and extensive adverse side effects, especially for bone tumors, where higher doses may be required to achieve the desired concentration at the disease site. Multiple studies from in vitro research to animal models have investigated bone-targeted cancer chemotherapy. The anti-cancer properties of bisphosphonates alone may provide bifunctional therapy through further coupling with anti-tumor drugs, but due to the charged nature of bisphosphonates, most cells have limited uptake of bisphosphonates. Many studies have not fully explored the effects of bisphosphonates beyond their bone-targeting action on their conjugates, and many studies have not explored whether the targeting action can reduce toxicity. Many in vitro studies have shown that combining traditional cancer chemotherapy drugs with bisphosphonates can improve efficacy. However, there is still a lack of convincing data on the function of many conjugates. An early study showed that bisphosphonate and methotrexate were bound together through a peptide bond and successfully localized to bone (Hosain 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, the Roy group demonstrated that bisphosphonate-conjugated proteasome inhibitors were highly toxic to multiple myeloma cell lines (Agyin et al, 2013). However, in the present invention, the bisphosphonate boron conjugate can be effectively taken up, target bone tissue, and is substantially non-toxic.

[0040] Preferred crosslinking agents for the linker chain of the present invention include moieties based on maleimide or haloacetyl. According to the present invention, such non-cleavable linkers are represented as being derived from moieties based on maleimide or haloacetyl. Crosslinking agents including moieties based on maleimide include N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (CL-1), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxyl-(6-aminohexanoate) (CL-2), κ-maleimidoundecanoic acid N-succinimidyl ester (CL-3), γ-maleimidobutyric acid N-succinimidyl ester (CL-4), ε-maleimidohexanoic acid N-hydroxysuccinimide ester (CL-5), m-maleimidobenzoyl-N-hydroxysuccinimide ester (CL-6), N-(α-maleimidoacetoxy)-succinimide ester (CL-7), succinimidyl-6-(β-maleimidopropionamido)hexanoate (CL-8), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (CL-9), N-(p-maleimidophenyl) isocyanate (CL-10), CL-18, CL-19, CL-20, CL-21, CL-22, CL-23 and CL-24 or the crosslinking agent has an SO 3 - group on the N-succinimide ring of the aforementioned crosslinking agent.

[0041]

[0042]

[0043] Crosslinking agents including moieties based on haloacetyl include N-succinimidyl-4-(iodoacetyl)-aminobenzoate (CL-11), N-succinimidyl-4-(bromoacetyl)-aminobenzoate (CL-12), N-succinimidyl iodoacetate (CL-13), N-succinimidyl bromoacetate (CL-13), N-succinimidyl bromoacetate (CL-14), N-succinimidyl 3-(iodoacetamido)propionate (CL-15), N-succinimidyl 3-(bromoacetamido)propionate (CL-16) and N-succinimidyl 3-(chloroacetamido)propionate (CL-17). These crosslinking agents form non-cleavable linkers which are derived from moieties based on haloacetyl. Representative structures for crosslinking agents based on haloacetyl are as follows:

[0044]

[0045] Other active esters can also be applied, such as N-hydroxyphthalimido ester, N-hydroxy thiophthalimido ester, o-nitrophenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 3-sulfonyl-4-nitrophenyl ester, 3-carboxy-4-nitrophenyl ester, pentafluorophenyl ester, and sulfonyl tetrafluorophenyl ester.

[0046] Synthesis of the conjugate

[0047] The conjugate of carborane and bisphosphonic acid can be formed using any currently known or later developed techniques.

[0048] The method of linking carborane and bisphosphonic acid generally involves two reaction steps. In one method, the bisphosphonic acid can be modified with a crosslinker. Then, the modified bisphosphonic acid is reacted with one or more thiol-containing carboranes to generate the conjugate.

[0049] Exemplarily, the thiol-containing carborane can first be modified with a crosslinker, and subsequently the modified carborane and bisphosphonic acid are reacted. For example, the thiol-containing carborane can be reacted with a maleimide compound or a haloacetyl compound to obtain a carborane thioether with an active succinimidyl ester or thiosuccinimidyl ester. These carboranes containing the active linker moiety are reacted with bisphosphonic acid to generate the carborane and bisphosphonic acid conjugate.

[0050] Exemplarily, the thiol-containing carborane is modified with a crosslinker to obtain, for example, the following conjugate intermediate compounds:

[0051]

[0052]

[0053]

[0054] The crosslinking agents used for bisphosphonate modification are derived from moieties based on maleimide or haloacetyl groups. The crosslinking agents based on maleimide moieties include: N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (CL-1), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxyl-(6-aminohexanoate) (CL-2), κ-maleimidoundecanoic acid N-succinimidyl ester (CL-3), γ-maleimidobutyric acid N-succinimidyl ester (CL-4), ε-maleimidohexanoic acid N-hydroxysuccinimide ester (CL-5), m-maleimidobenzoyl-N-hydroxysuccinimide ester (CL-6), N-(α-maleimidoacetoxy)-succinimide ester (CL-7), succinimidyl-6-(β-maleimidopropionamido)hexanoate (CL-8), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (CL-9), and N-(p-maleimidophenyl) isocyanate (CL-10), or the crosslinking agent has an SO 3 - group on the N-succinimide ring of the aforementioned crosslinking agents. Exemplary ones include: thio-N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (CL-1-SO 3 - ), thio-N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxyl-(6-aminohexanoate) (CL-2-SO 3 - ), thio-κ-maleimidoundecanoic acid N-succinimidyl ester (CL-3-SO 3 - ), thio-γ-maleimidobutyric acid N-succinimidyl ester (CL-4-SO 3 - ), thio-ε-maleimidohexanoic acid N-hydroxysuccinimide ester (CL-5-SO 3 - ), thio-m-maleimidobenzoyl-N-hydroxysuccinimide ester (CL-6-SO 3 - ), thio-N-(α-maleimidoacetoxy)-succinimide ester (CL-7-SO 3 - ), thio-succinimidyl-6-(β-maleimidopropionamido)hexanoate (CL-8-SO 3 - ), thio-N-succinimidyl 4-(p-maleimidophenyl)-butyrate (CL-9-SO 3 -)。Exemplarily, the following structurally bisphosphonic acid-modified crosslinker intermediate compounds are obtained:

[0055]

[0056]

[0057]

[0058] The crosslinkers used for bisphosphonic acid modification include crosslinkers based on haloacetyl moieties, including: N-succinimidyl 4-(iodoacetyl)-aminobenzoate (CL-11), N-succinimidyl 4-(bromoacetyl)-aminobenzoate (CL-12), N-succinimidyl iodoacetate (CL-13), N-succinimidyl bromoacetate (CL-13), N-succinimidyl bromoacetate (CL-14), N-succinimidyl 3-(iodoacetamido)propionate (CL-15), N-succinimidyl 3-(bromoacetamido)propionate (CL-16), and N-succinimidyl 3-(chloroacetamido)propionate (CL-17). Exemplarily, the following structurally bisphosphonic acid-modified crosslinker intermediate compounds are obtained:

[0059]

[0060]

[0061] Then, the modified bisphosphonic acid is treated with thiol-containing carborane (1.25 molar equivalents / maleimidyl or iodoacetyl group) to produce a conjugate.

[0062] Exemplarily, a preferred method is to modify bisphosphonic acid with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) to introduce a maleimidyl group, and then react the modified bisphosphonic acid with thiol-containing carborane to obtain a thioether-linked conjugate. The conjugate can be purified on silica gel by column chromatography or by HPLC.

[0063] Preferably, the method for linking carborane and bisphosphonic acid can also be described as: Michael addition of thiol-containing carborane and maleimide compound to obtain intermediate IIA, and intermediate IIA reacts with bisphosphonic acid or its pharmaceutically acceptable salt to obtain a carborane conjugate. It is represented by the following reaction equation:

[0064]

[0065] Where La, R 1 , R 2 , R 3 , R 4 and the definitions of n are the same as those defined above.

[0066] Exemplarily, the carborane is selected from those having the following structures:

[0067]

[0068] More preferably, the carborane is of formula B-4.

[0069] Preferably, the present invention provides a compound having the structure of formula IIAX, which has the following structure:

[0070]

[0071] wherein the carborane and La are as defined above, and P is H or

[0072] The intermediate for preparing the boron diphosphate conjugate, which has the following structure of formula IIA:

[0073]

[0074] wherein the carborane and La are as defined above.

[0075] Preferably, the intermediate IIA has the following structure:

[0076]

[0077] wherein the carborane, R 5 , R 6 , R 7 , R 8 , R 9 , m, q 1 , q 2 , p and t are as defined above.

[0078] Preferably, the intermediate IIA has the following structure:

[0079]

[0080] wherein R 7 , R 8 , R 9 , q 1 , q 2 , p and t are as defined above. R 12 and R 13 are each independently selected from H or C 1 -C 6 alkyl, exemplarily the C 1 -C 6Alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, m 1 is from 0 to 12.

[0081] Preferably, the present invention provides an active intermediate for preparing a boron diphosphate conjugate, and the intermediate has the following formula IIAP structure:

[0082]

[0083] wherein carborane and La have the same definitions as above.

[0084] Preferably, the intermediate IIAP has the following structure:

[0085]

[0086] wherein the carborane, R 5 , R 6 , R 7 , R 8 , R 9 , m, q 1 , q 2 , p and t have the same definitions as above.

[0087] Preferably, the intermediate IIAP has the following structure:

[0088]

[0089] where R 7 , R 8 , R 9 , q 1 , q 2 , p and t have the same definitions as above. R 12 and R 13 each independently selected from H or C 1 -C 6 alkyl group, and exemplary C 1 -C 6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl. m is from 0 to 12.

[0090] Exemplarily, the formula IIA structure is the following compound or its active ester, and the active ester such as N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, N-hydroxythiophthalimide ester, o-nitrophenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 3-sulfonyl-4-nitrophenyl ester, 3-carboxy-4-nitrophenyl ester, pentafluorophenyl ester, and sulfonyl tetrafluorophenyl ester:

[0091]

[0092]

[0093]

[0094] Compared with the preparation method in which the maleimide compound is first coupled with bisphosphonic acid to obtain an intermediate and then coupled with thiol-containing carborane to obtain a carborane bisphosphonate conjugate, the preparation method in which the thiol-containing carborane undergoes Michael addition with the maleimide compound to obtain intermediate IIA, and intermediate IIA reacts with bisphosphonate to obtain a carborane bisphosphonate conjugate has a higher yield.

[0095] Exemplarily, the following structure borane-bisphosphonic acid conjugate is obtained by using the above coupling method provided by the present invention:

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] The conjugate provided by the present invention shows excellent anti-tumor activity in vitro, while in terms of plasma clearance rate and toxicity, the conjugate of the present invention shows a significant reduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 11H NMR spectrum of conjugate GNS4117 in DMSO-d6 at 400 MHz.

[0111] Figure 2 1H NMR spectrum of intermediate IIA1.

[0112] Figure 3 Mass spectrum of intermediate IIA1.

[0113] Figure 4 Bar graph showing the HepG2 cell uptake ability of GNS4117.

[0114] Figure 5 Cell survival rate of HepG2 cells after neutron irradiation following uptake of GNS4117.

[0115] Figure 6 Images showing the results of the colony formation assay of HepG2 cells after neutron irradiation following uptake of GNS4117.

[0116] Figure 7 Bar graph showing the statistical results of the number of colonies of HepG2 cells after neutron irradiation following uptake of GNS4117. Detailed implementation methods

[0117] To better understand the technical solution of the present invention, the technical solution of the present invention will be further described below in conjunction with specific embodiments. The embodiments are only for helping to understand the present invention and should not be construed as specific limitations on the present invention.

[0118] Method 1: Exemplarily, taking the compound GNS4117 as an example, the present invention adopts the following synthesis method:

[0119]

[0120] Dissolve 4-(N-carboxycyclohexylmethyl) maleimide (100.00 mg, 0.42 mmol) and BSH (73.00 mg, 0.42 mmol) in N,N-dimethylformamide (3 mL), and slowly add triethylamine (42 mg, 0.42 mmol) dropwise to the above system. The reaction system was stirred overnight at room temperature. The obtained crude product was concentrated by distillation under reduced pressure, and the concentrated solution was purified by Prep-thin layer chromatography with MeOH / DCM (0-20%). The solvent was removed from the filtered filtrate under reduced pressure. After freeze-drying, the product BSM was obtained as a yellow solid. Dissolve BSM (15.00 mg, 36.06 μmol), N-hydroxysuccinimide (6.95 mg, 30.05 μmol) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (4.18 mg, 30.05 μmol) in DMSO (1 mL) and buffer solution PBS (1 mL) (PH = 7.2-7.4), and add sodium alendronate 9.80 mg, 30.05 μmol) to the above system. The reaction system was stirred at room temperature for 24 h. After the reaction was completed, the solid was obtained by centrifugation, washed 3 times by ultracentrifugation with ultrapure water, and the obtained solid was freeze-dried to obtain the product as a pale yellow solid.

[0121] Method 2: Dissolve 4-(N-carboxycyclohexylmethyl) maleimide (100.00 mg, 0.42 mmol), N-hydroxysuccinimide (6.95 mg, 30.05 μmol) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (4.18 mg, 30.05 μmol) in DMSO (1 mL) and buffer solution PBS (1 mL) (PH = 7.2-7.4), and add sodium alendronate 9.80 mg, 30.05 μmol) to the above system. The reaction system was stirred at room temperature for 24 h. Add BSH (73.00 mg, 0.42 mmol) dissolved in N,N-dimethylformamide (3 mL), and slowly add triethylamine (42 mg, 0.42 mmol) dropwise to the above system. The reaction system was stirred overnight at room temperature), after the reaction was completed, the solid was obtained by centrifugation, washed 3 times by ultracentrifugation with ultrapure water, and the obtained solid was freeze-dried to obtain the product as a pale yellow solid.

[0122] Method 3: Add BSH (73.00 mg, 0.42 mmol) dissolved in N,N-dimethylformamide solvent to the N,N-dimethylformamide solution of a haloacetyl-based crosslinker (0.42-1.0 mmol). Then add N,N-diisopropylethylamine (0.5-1.5 mmol), after the reaction is completed, the solid is obtained by centrifugation, washed 3 times by ultracentrifugation with ultrapure water, and the obtained solid is freeze-dried to obtain the product of the boron bisphosphonate conjugate.

[0123] According to the above preparation method, combined with the methods disclosed in the technical solution part of the present invention or through reasonable optimization, the intermediate compounds in Table 1 below and the carborane bisphosphonate conjugate in Table 2 can be obtained.

[0124] Table 1

[0125]

[0126]

[0127]

[0128]

[0129] Table 2

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] Example 2. Test on the uptake ability of the conjugate by HepG2 cells

[0142] Repeat three batches. Seed HepG2 cells in a 6-well plate. After the cells adhere, add 100, 200, 300, 400, 500, 600, 700 μg / mL of the boron bisphosphonate conjugate respectively. Incubate for 24 h, collect the cells, centrifuge at 200 g for 3 min, discard the supernatant, collect the cell pellet and count. Digest the cells with concentrated nitric acid, and use ICP-MS to measure the boron content in every 10 6 cells, and calculate the average value and record it in Table 3 and Table 4.

[0143] Table 3

[0144]

[0145]

[0146] Table 4

[0147]

[0148] When the concentration of BSH changed by ten times from 3.75 μg / mL to 37.5 μg / mL during incubation, the cellular uptake was very low, indicating that the ability of BSH to enter cells was very limited. However, the uptake ability of the conjugate increased significantly with the increase in drug concentration, indicating that the problem of boron drug entering cells was well improved.

[0149] Example 3. Cell survival rate test after neutron irradiation

[0150] Repeat five batches. After HepG2 cells adhered, add boron diphosphate conjugate drugs at different concentrations for incubation. After 24 h of incubation, receive BNCT irradiation (2.57×10 8 cm -2 ·s -1 for 1 h). Seed the cells into 96-well plates. After adhesion, determine the cell survival rate according to the instructions using the CCK-8 method. Specifically, remove the cell culture supernatant, add the basal medium containing CCK-8, and incubate at 37 °C for 24 hours. Then, detect the absorbance at 562 nm using a microplate reader. Take the absorbance of the cells that received only BNCT without boron drug incubation as the normalization standard. The blank group is the absorbance value generated by the basal medium plus CCK-8. The survival rate calculation formula for other experimental groups is "Survival rate = (Absorbance value of the experimental group - Absorbance value of the blank group) / (Absorbance value of the single BNCT group - Absorbance value of the blank group)". Calculate the average value and record it in Table 5. When the BSH concentration is 15 μg / mL, the cell survival rate is 63.10%.

[0151] Table 5

[0152]

[0153]

[0154] Example 4. Colony formation test after neutron irradiation

[0155] Repeat three batches. Similar to the above, after HepG2 cells adhered, add boron diphosphate conjugate drugs at different concentrations for incubation. After 24 h of incubation, receive BNCT irradiation (2.57×10 8 cm -2 ·s -1(for 1 h), the cells were seeded into 6-well plates at a density of 1000 cells / well, and 2 mL of complete medium was added. The cells were cultured at 37 °C in a 5% CO 2 2 incubator for 7 - 10 days. After observing obvious cell clones in the PBS group, the medium was removed, and the cells were washed 3 times with PBS. Then, the cells were fixed with 4% paraformaldehyde at room temperature for 2 h, washed 3 times with PBS, stained with 0.5% crystal violet at room temperature for 2 h, washed 3 times with PBS, photographed with a camera, and the number of clones formed was analyzed and counted using ImageJ software. The average value was calculated and recorded in Table 6.

[0156] Table 6

[0157]

[0158]

Claims

1. A compound having the structure of formula IIAX: wherein the carborane is selected from the following structures: wherein the La is wherein R 5 ,R 6 ,R 8 ,R 9 are each independently selected from H, C 1 -C 6 alkyl, carboxyl, cyclohexyl, or R 5 and R 6 can form a ring, and the ring is a five- or six-membered ring, R 7 is H or C 1 -C 6 alkyl, m and p are each independently 0 to 12; r, t, q 1 ,q 2 and u are each independently 0 or 1, where q 1 ,q 2 are not both 1 P is H or 2. The compound according to claim 1, having the following structure of formula IIA: wherein the carborane and La are as defined in claim 1.

3. The compound according to claim 2, having the following structure: wherein, The carborane, R 5 , R 6 , R 7 , R 8 , R 9 , m, q 1 , q 2 , p and t are defined in the same way as those in claim 1.

4. The compound according to claim 2, having the following structure: wherein R 7 , R 8 , R 9 , q 1 , q 2 , p and t are defined in the same way as in claim 1. R 12 and R 13 are each independently selected from H or C 1 -C 6 alkyl, m 1 is from 0 to 12.

5. The compound according to claim 2, selected from the following compounds or their active esters:

6. The compound according to claim 5, wherein the active ester is N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, N-hydroxythiophthalimide ester, o-nitrophenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 3-sulfonyl-4-nitrophenyl ester, 3-carboxy-4-nitrophenyl ester, pentafluorophenyl ester or sulfonyltetrafluorophenyl ester.

7. The compound according to claim 1, having the following structure of formula IIAP: wherein the carborane and La are as defined in claim 1.

8. The compound according to claim 7, having the following structure of formula IIAA2: wherein, The carborane, R 5 , R 6 , R 7 , R 8 , R 9 , m, q 1 , q 2 , p and t are defined in the same manner as in claim 1.

9. The compound according to claim 7, having the following structure of formula IIAB2: wherein R 7 ,R 8 ,R 9 ,q 1 ,q 2 ,p and t are defined in the same manner as in claim 1. R 12 and R 13 are each independently selected from H or C 1 -C 6 alkyl, and m 1 is from 0 to 12.

10. The compound according to claim 7, selected from the following structures:

11. A method for preparing a compound of formula IIA, obtained by Michael addition of a carborane containing thiol and a maleimide compound: wherein the carborane and La are as defined in claim 1.

12. The preparation method according to claim 11, further comprising condensing the compound of formula IIA with an activator to obtain an active ester of the compound of formula IIA, and the active ester is N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, N-hydroxythiophthalimide ester, o-nitrophenyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, 3-sulfonyl-4-nitrophenyl ester, 3-carboxy-4-nitrophenyl ester, pentafluorophenyl ester or sulfonyltetrafluorophenyl ester.

13. Use of a compound of formula IIA or its active ester, coupling intermediate IIA or its active ester with a bisphosphonic acid or a pharmaceutically acceptable salt thereof to obtain a targeted bone tissue boron conjugate GNS, wherein the definitions of carborane and La are the same as those in claim 1, and R 1 is selected from H, OH, and halogen, and R 2 is selected from H and C 1-6 alkyl group, and R 3 is selected from H, halogen, and carboxyl group, and R 4 is selected from H and C 1-6 alkyl group, and n is from 0 to 6.

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