WO2023029935A1 - 肿瘤诊疗一体化的硼携带剂、其制备方法和用途 - Google Patents

肿瘤诊疗一体化的硼携带剂、其制备方法和用途 Download PDF

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WO2023029935A1
WO2023029935A1 PCT/CN2022/111728 CN2022111728W WO2023029935A1 WO 2023029935 A1 WO2023029935 A1 WO 2023029935A1 CN 2022111728 W CN2022111728 W CN 2022111728W WO 2023029935 A1 WO2023029935 A1 WO 2023029935A1
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cancer
compound
boron
liver
bbpa
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French (fr)
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刘志博
陈俊艺
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北京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds

Definitions

  • This application generally relates to the fields of radiopharmaceuticals and nuclear medicine, and in particular relates to a boron carrier for the integration of tumor diagnosis and treatment, which is used for boron neutron capture therapy and tumor diagnosis.
  • Boron neutron capture therapy is a binary targeted radiation precision medical technology.
  • the patient is first given boron carrier drugs that can be enriched in tumor cells, and then the patient is irradiated with thermal neutrons or epithermal neutrons.
  • the principle is to apply highly targeted boron-containing drugs to patients and generate enrichment in cancer cells.
  • the capture cross-section of 10B for thermal neutrons is very high compared to normal elements composed of human body. Using thermal neutrons and The capture reaction of the 10B atom makes the energy of the high-energy ⁇ particles and Li particles produced by it only act on the cancer cells of about 10 ⁇ m in size, causing irreversible damage to the structure of the cells, making them irreparable and resulting in apoptosis.
  • PET Positron Emission Tomography
  • positron-decaying isotopes such as 11C and 18F quickly collide with negative electrons widely distributed around them to annihilate, and convert the energy into two photons with an energy of 511keV in opposite directions.
  • the two photons were simultaneously detected by the instrument's two probes facing each other, indicating that an annihilation occurred along the line connecting the two probes.
  • PET can accurately localize and quantify the distribution of radioactivity in the body.
  • a three-dimensional PET image of the human body can be obtained. PET is one of the most effective means to study drug distribution in vivo.
  • 2-fluoro-4-boronic acid-L-phenylalanine (2-fluoro-4-L-boronophenylalanine, FBPA) is BPA substituted by the fluorine atom at the 2nd position on the benzene ring.
  • This fluorine atom can be radioactive 18F, at which point the molecule can be used as a PET probe to simulate the distribution of BPA in the body.
  • BNCT treatment must rely on the high enrichment of boron carrier in the tumor site, and customize the treatment plan according to its enrichment, which requires real-time monitoring of the concentration distribution of boron carrier in the body.
  • BPA is currently the only approved BNCT drug, so it is particularly important to study its distribution in the body.
  • the important method is to obtain [ 18 F]FBPA by introducing radioactive 18 F on the benzene ring of BPA by means of PET molecular imaging.
  • the distribution of BPA in the body is simulated by [ 18 F]FBPA, and then the BNCT is planned.
  • this application provides a new boron carrier for diagnosis and treatment, such as (S)-BBPA, which can be conveniently carried out
  • the radioactive labeling has the same chemical structure before and after labeling, which can accurately reflect its distribution in the body, so as to solve the problem of unclear real-time distribution of classic boron carrier agents in vivo, and provide more efficient BNCT planning; and through two intramolecular boron atoms to achieve a more efficient BNCT.
  • boronine is a class of amino acid derivatives developed by substituting the carboxyl group of natural amino acids with boron trifluoride. Through the 18F-19F isotope exchange reaction, it can use 18F for radiolabeling to construct a radioactive probe, and then realize PET imaging.
  • the inventors found in the research that the boron trifluoride group and the carboxylate group show a high degree of similarity in electrical properties, so boronine and its corresponding natural amino acid also show similarities in biological recognition and transport. Based on this, the applicant found that by replacing the carboxylate group in BPA with boron trifluoride group, BBPA can be constructed, wherein the chiral carbon atom has S configuration and R configuration.
  • the (R)-BBPA of the R configuration has the same chirality as the natural amino acid, while the (S)-BBPA of the S configuration is opposite to the configuration of the natural amino acid. Both of them can be highly enriched in the tumor area, and the (S)- BBPA showed a lower normal tissue background uptake, reflecting better clinical application value.
  • (S)-BBPA can be labeled with F-18 to realize molecular concentration distribution analysis in vivo; on the other hand, it can also be directly used as the boron carrier of BNCT.
  • the application provides a compound of formula I or a pharmaceutically acceptable salt thereof:
  • R group is hydrogen or an alkyl group.
  • the compound is an S-form or R-form compound.
  • the compound has the structure of Formula II:
  • R group is hydrogen or an alkyl group (eg C1-C10 alkyl, C1-C6 alkyl, such as methyl, ethyl, propyl, butyl, pentyl or hexyl).
  • alkyl group eg C1-C10 alkyl, C1-C6 alkyl, such as methyl, ethyl, propyl, butyl, pentyl or hexyl.
  • the boron atom attached to the phenyl ring is 10 B or natural boron.
  • at least one fluorine atom in -BF3- is a radioactive fluorine atom, eg18F .
  • alkyl is methyl
  • the boron atom in -BF3- is natural boron.
  • the application provides the preparation method of the compound of the application, which comprises the following steps: by compound 6 preferred KHF 2 solution was added, and hydrochloric acid was added to pH ⁇ 5, to produce compound 7-containing preferred Acid is then added to obtain the compound.
  • the method includes one or more of the following steps:
  • the present application provides a boron carrier composition, which comprises one or more compounds of formula I of the present application and a pharmaceutically acceptable carrier.
  • the boron carrier composition further comprises fructose, disodium undecahydromercaptododecaborate and dimers thereof, boronated dendrimer-EGF bioconjugate, EGFR monoclonal antibody-boron One or more of salt conjugates, FR-targeting boron-containing liposomes, FR-targeting boron-containing nanoparticles, and borate porphyrins.
  • the present application provides the use of the compound of the present application in the preparation of a medicament for treating or diagnosing cancer.
  • the compounds of the present application are highly enriched in tumors through the LAT-1 transporter, so as to achieve the purpose of treatment.
  • the cancer can be a cancer that expresses or overexpresses the LAT-1 transporter.
  • the drug is a boron carrier for integrated tumor therapy or boron neutron capture therapy.
  • the cancer is selected from squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, melanoma, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, cancer, breast cancer, colon cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, B cell lymphoma, leukemia or pituitary tumor.
  • This application uses the compound of formula I with the same chemical structure before and after 18 F labeling, such as (S)-BBPA and [ 18 F](S)-BBPA, which can be used for the integration of diagnosis and treatment of tumor diagnosis and BNCT treatment Drugs, through their identical chemical structures provide reliable drug distribution results in vivo.
  • This application provides a new type of boron carrier for diagnosis and treatment, such as (S)-BBPA, which is used to obtain the dynamic distribution of the boron carrier for treatment planning before BNCT, and can also be used directly for BNCT.
  • the present application provides a new method for preparing the compound of the present application.
  • (S)-BBPA has the same or better therapeutic effect, lower side effects and lower background uptake in vivo.
  • Figure 1 Radio-HPLC chart of [18F](S)-BBPA prepared.
  • Figure 2 Radio-HPLC profile of [18F](S)-BBPA, showing that [18F](S)-BBPA can maintain sufficient stability in a simulated in vivo environment within a 4-hour time scale (2 18F half-lives).
  • FIG. 3 PET/CT images of mice performed 15 minutes after tumor cell injection.
  • Figure 4 Graph comparing imaging contrast of (S)-BBPA and (R)-BBPA.
  • Figure 5 Graph of the therapeutic effect of (S)-BBPA and (R)-BBPA on mouse tumors.
  • Figure 6 Graph of the effect of (S)-BBPA and (R)-BBPA on mouse body weight.
  • Figure 7 Graph of measuring the radioactive intensity of cells using a gamma counter.
  • Figure 8A-B 1H-NMR and 19F-NMR spectra of (S)-BBPA.
  • the compounds of the present application can be L-form or S-form. These compounds can be used in positron emission tomography imaging or boron neutron capture therapy.
  • R is independently hydrogen or alkyl.
  • Alkyl refers to a saturated linear hydrocarbon group (eg C1-C10 alkyl, C1-C6 alkyl).
  • an alkyl group can be methyl, ethyl, propyl, butyl, pentyl, and the like.
  • the R group is methyl.
  • At least one fluorine atom in -BF 3 - is radiolabeled.
  • 1, 2 or 3 fluorine atoms are18F .
  • the boron atom in -BF 3 - may be natural boron.
  • the boron atom attached to the benzene ring is 10 B or natural boron.
  • the application also provides a compound of formula II or a pharmaceutically acceptable salt thereof:
  • the R group is hydrogen, or an alkyl group, preferably methyl.
  • the present application also provides a boron carrier composition, which comprises the compound of the present application and a pharmaceutically acceptable carrier.
  • the boron carrier composition may also contain fruit acids, or may contain other boron carriers such as disodium undecahydromercaptododecaborate and dimers thereof, boronated dendrimer-EGF bioconjugates, EGFR One or more of monoclonal antibody-borate conjugates, FR-targeting boron-containing liposomes, FR-targeting boron-containing nanoparticles, and borate porphyrins.
  • the present application also provides the preparation method of the compound.
  • the method may include one or more of the following steps:
  • step (1) p-bromophenylethanol can be dissolved in anhydrous tetrahydrofuran, cooled (for example -78°C), and n-butyllithium/hexane solution is added, the solution gradually turns yellow and then becomes off-white paste, Stir for 15 minutes (for example at -78°C); then add triisopropyl borate dropwise, the solution becomes clear and react for 20 minutes (for example -78°C). Then rise to room temperature, add hydrochloric acid, and stir at room temperature.
  • step (2) compound 1 can be dissolved in toluene, and compound 2 can be obtained by adding DMSO and N-methyliminoacetic acid to react.
  • step (3) compound 2 can be dissolved in a mixed solvent of anhydrous dichloromethane and anhydrous tetrahydrofuran, DMP oxidant is added, and sodium bicarbonate powder is added for reaction. Saturated sodium thiosulfate solution and saturated sodium bicarbonate solution were then added and stirred. The insoluble matter was removed by filtration, the organic phase was separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and the solvent was removed in vacuo. It can be separated by silica gel column chromatography to obtain compound 3 as a white solid.
  • step (4) compound 3 can be dissolved in anhydrous tetrahydrofuran, (S)-tert-butylsulfinamide is added, tetraisopropyl titanate is added dropwise, and stirred at room temperature. Add water to quench the reaction and stir, remove the insoluble matter by filtration and separate the organic phase, extract the aqueous phase with ethyl acetate, combine the organic phases, wash with saturated sodium chloride, dry over anhydrous sodium sulfate, and remove the solvent in vacuo. It can be separated by silica gel column chromatography to obtain compound 4 as yellow crystals.
  • PCy3 ⁇ HBF4 can be added to 1 mL of toluene, followed by CuSO4 ⁇ 5H2O and 110 ⁇ L of deionized water, and vigorously stirred at room temperature for 10 min. Subsequent addition of benzylamine under a nitrogen atmosphere immediately formed a dark blue catalyst. Keep the reaction system at a low temperature (eg 0-40°C, eg 0°C), and add compound 4 and B2pin2. Subsequently, the reaction system was returned to room temperature, and reacted until TLC showed that the reactant completely disappeared. The reaction mixture was washed with saturated EDTA and then with saturated brine. The organic phases were combined and then dried over Na2SO4 free, and the organic solvent was removed in vacuo to afford compound 5 as a yellow oily liquid.
  • step 6 compound 5 can be dissolved in methanol, NaOH solution is added, and stirred at room temperature.
  • HPLC analysis confirmed the disappearance of compound 5 and the production of compound 6.
  • KHF2 solution dropwise to the reaction mixture, measure the pH of the solution, and add an appropriate amount of hydrochloric acid (for example, to pH ⁇ 5), and stir at room temperature.
  • HPLC confirmed the disappearance of the starting material and the formation of compound 7.
  • step 7 HCl can be added dropwise to compound 7 powder, followed by stirring at room temperature (for example, for 1 hour).
  • HPLC confirmed the disappearance of the starting material and the generation of BBPA, and the white powder BBPA (for example, (S)-BBPA) was obtained using preparative HPLC.
  • step (8) BBPA (such as (S)-BBPA) can be dissolved in water, then pyridazine-HCl buffer is added, followed by water 18 F-water solution.
  • the reaction mixture was reacted (for example, at 85° C. for 15 min), then quenched with physiological saline, and free fluoride ions were removed with a Sep-Pak Alumina Light cartridge to obtain the final product.
  • the present application provides methods of treating or diagnosing cancer using boron carrier compositions.
  • a method may include the step of administering a boron carrier composition to a subject (eg, a human or mammal).
  • the application provides the use of the boron carrier composition in the preparation of medicines for treating or diagnosing cancer.
  • the drug can be a boron carrier used in the integration of tumor diagnosis and treatment or boron neutron capture therapy.
  • the present application also provides a boron carrier composition for treating or diagnosing cancer.
  • the cancer is a cancer that expresses or overexpresses the LAT-1 transporter.
  • the cancer may be selected from squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, melanoma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, Colon cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, B-cell lymphoma leukemia, or pituitary tumor.
  • a subject can be a human.
  • the cancer is a cancer that expresses or overexpresses the LAT-1 transporter.
  • the cancer is selected from squamous cell carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, melanoma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, cancer, breast cancer, colon cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, B cell lymphoma leukemia or pituitary tumor.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is often characterized by unregulated cell growth.
  • Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), Melanoma, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, Liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer and various types of head and neck cancer, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocyte (SL) NHL; Intermediate/follicular NHL; intermediate diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small anucleate NHL; storage disease NHL; mantle cell
  • Embodiment 1 the preparation of compound
  • Dissolve compound 3 (541mg, 1.97mmol) in 20ml of anhydrous tetrahydrofuran, add (S)-tert-butylsulfinamide (477mg, 3.94mmol, 2eq.), dropwise add tetraisopropyl titanate (whether it is related to synthetic Ti(OPri) 4 inconsistent in the scheme) (3.94mmol, 1165 ⁇ L, 1.2eq.), stirred at room temperature for 2 hours.
  • reaction system was returned to room temperature, and reacted for 1 hour until TLC showed that the reactant completely disappeared.
  • the reaction mixture was washed with saturated EDTA (2 ⁇ 5 mL) and then with saturated brine (5 mL).
  • the organic phases were combined and dried over Na2SO4 free, and the organic solvent was removed in vacuo to give compound 5 (49 mg, 75%) as a yellow oily liquid.
  • the crude product was directly used in the next reaction without purification.
  • Compound 2 to Compound 7 and (R)-BBPA were synthesized from commercially available p-bromophenylethanol (Compound 1) as a starting compound.
  • PCy 3 ⁇ HBF 4 (124 mg, 6 mol%) was added into 10 ml of toluene, followed by CuSO 4 ⁇ 5H 2 O solution (82 mg, 6 mol%) and deionized water (6 mL), and vigorously stirred at room temperature for 10 min.
  • Benzylamine (153 ⁇ L, 0.25 eq.) was then added under nitrogen, immediately forming a dark blue catalyst.
  • An additional 50ml of toluene was added, the reaction system was maintained at 0°C, compound 5 (2.2g, 5.7mmol) and B 2 pin 2 (2.8g, 2.0eq.) were added. Subsequently, the reaction system was returned to room temperature, and reacted for 1 hour until TLC showed that the reactant completely disappeared.
  • the labeled [ 18 F](S)-BBPA was added to 50% fetal bovine serum and stored at 37°C. At a specific time, a small amount was taken out and analyzed by Radio-HPLC (C18, phase A water/0.1% TFA, phase B acetonitrile, 0-15 minutes: 80%+20% B) to determine the composition of radioactive substances therein.
  • the signal is represented by normalized intensity, that is, the difference between the maximum value and the minimum value of the signal is uniformly set to 1.
  • the results show that [ 18 F](S)-BBPA can maintain sufficient stability in the simulated in vivo environment within a time scale of 4 hours (two 18F half-lives). The results are shown in FIG. 2 .
  • Example 3 PET animal imaging
  • Tumor cells (BGC823 human gastric cancer cells, B16-F10 mouse melanoma and GH3 rat pituitary tumor) were cultivated to confluence in a 5% CO 2 incubator. Mice 4-6 weeks old were selected, the right shoulder was shaved, about 10 6 tumor cells were suspended in 50 ⁇ L PBS, and injected on the right shoulder of the mouse. They were raised in a sterile environment for 1-2 weeks, and the tumor model was established when the volume of the tumor reached 200-400 mm 3 .
  • BPA, (S)-BBPA, and (R)-BBPA containing natural boron were dissolved in normal saline at a dose of 100 mg/kg, and injected into the above-mentioned mice with B16-F10 and melanoma implanted in the shoulder through the tail vein , BNCT treatment was carried out at 1 hour, and the tumor size was measured in the following 21 days, and compared with the groups injected with related drugs alone and neutron beam irradiation alone.
  • both (S)-BBPA and (R)-BBPA with two boron atoms in the molecular structure can be used as efficient boron carriers, but the lower background uptake of (S)-BBPA can bring better results.
  • the difference in boron concentration can reduce the occurrence of side effects. This is of great significance for shortening the treatment cycle, reducing drug dosage, reducing radiation dose and patient expenses, and reducing complications and side effects.
  • the B16-F10 mouse melanoma cells under standard culture conditions were evenly transferred to 1.5ml EP tubes (4 ⁇ 10 5 /tube), centrifuged at 150 ⁇ g for 2 minutes, and the supernatant was removed.
  • Ala is the substrate of the amino acid transport system ASC system and A system, Glu is the substrate of the X c -transporter , neither of which can inhibit the uptake of [ 18 F]BBPA; while BCH is the LAT-1 transporter (Large-neutral A specific inhibitor of Amino Acid Transporter 1), which significantly inhibits the uptake of [ 18 F]BBPA, indicating that (S)-BBPA is transported into tumor cells through LAT-1; GPNA is a specific inhibitor of ASCT2 transporter (S)-BBPA is a boronine derivative of BPA, and BPA significantly inhibits the uptake of (S)-BBPA, and the inhibitory effect is even better than that of BCH, indicating that although ( S)-BBPA is different from BPA in chirality, but it still has a very high biological similarity in the transport pathway, and it has a completely consistent biological pathway. It can be concluded that (S)-BBPA is highly enriched in tumors based on the LAT-1 transporter, which is the
  • (S)-BBPA has high biological similarity with BPA, and it is mainly transported into cells through LAT-1.
  • LAT-1 is an amino acid transporter highly expressed in tumor cells, so (S)-BBPA can achieve high uptake in tumor tissue cells, which is helpful for high-contrast PET imaging and high-concentration boron enrichment to achieve efficient BNCT.

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Abstract

本申请涉及肿瘤诊疗一体化的硼携带剂、其制备方法和用途。提供了式I的化合物：其中R基团为氢或烷基。与苯环连接的硼原子可以是10B或天然硼。-BF3 -中的至少一个氟原子可以是放射性标记的。本申请一般涉及放射性药物及核医学领域。本申请的化合物可以用于肿瘤诊断与BNCT治疗的诊疗一体化药物，通过其相同的化学结构提供可靠的药物体内分布结果。

Description

肿瘤诊疗一体化的硼携带剂、其制备方法和用途
本申请要求申请日为2021年9月2日，申请号：202111028455.0，发明名称为“肿瘤诊疗一体化的硼携带剂、其制备方法和用途”的中国专利申请的优先权权益，该专利申请通过引用并入本文。
技术领域
本申请一般涉及放射性药物及核医学领域，具体地涉及肿瘤诊疗一体化的硼携带剂，用于硼中子捕获治疗和肿瘤诊断。
背景技术
硼中子俘获疗法(Boron neutron capture therapy,BNCT)是一种二元靶向放射精准医疗技术。先给患者施用能够在肿瘤细胞内富集的硼携带剂药物，再对病患进行热中子或者超热中子的照射。其原理是将强靶向性的含硼药物施于病患并在癌细胞中产生富集，10B对于热中子的捕获截面相对于人体组成的正常元素而言非常高，利用热中子与10B原子的俘获反应，使其产生的高能α粒子和Li粒子的能量仅作用于约10μm的癌细胞内，对细胞的结构造成不可逆的损伤，使其不能修复而凋亡。使用该疗法进行恶性肿瘤治疗可在杀灭肿瘤组织同时，最大程度保留患部周围正常组织及功能，以提高患者治疗后生活质量与生存时期。4-硼酸-L-苯丙氨酸(4-L-boronophenylalanine，BPA)是FDA批准的临床最常用的应用于BNCT的硼携带剂药物。但是，由于BNCT需要根据硼携带剂的药代动力学结果规划治疗策略，如何有效获取BPA在肿瘤及其他组织器官中的浓度则成为各项研究中的关键问题之一。
正电子发射断层扫描成像(Positron Emission Tomography,PET)是一种依托于放射性分子探针的分子影像技术，其结合了分子探针和医学影像技术，能对活体内病理、生理过程在细胞及分子水平进行定性和定量研究。从11C，18F等正电子衰变的同位素中发射出来的带正电子，很快与周围广泛分布的带负电子碰撞发生湮灭，并将能量转化为两个方向相反的能量为511keV的光子。两个光子被仪器相对的两个探头同时检测到，表明两个探头连线上发生了一次湮灭。这样，PET就能够对体内放射性的分布进行准确 的定位和定量。再经过计算机重建，即可获得三维的人体PET图像。PET是对活体内药物分布进行研究的最为有效的手段之一。
在PET成像的基础上结合治疗组件，实现肿瘤成像与治疗一体化是技术发展的方向。2-氟-4-硼酸-L-苯丙氨酸(2-fluoro-4-L-boronophenylalanine，FBPA)是苯环上2号位氟原子取代的BPA。这个氟原子可以是放射性的18F，此时该分子可以作为PET探针，以模拟BPA在体内的分布情况。以[ 18F]-FBPA为探针的PET影像技术现在已经应用于BNCT的疗前诊断，帮助了解BPA在病患体内的动态分布信息(Ishiwata K,et al.Melanoma Research,1992.PMID:1450671)，然而，FBPA和BPA在分子结构上存在差异，探针剂量的FBPA不能代表治疗剂量的BPA在体内的分布情况，FBPA-PET技术并不成熟，因此发展一种全新的能够实现诊疗一体化药物分子刻不容缓。
发明内容
BNCT治疗必须依赖于硼携带剂在肿瘤部位的高度富集，并根据其富集的情况，定制治疗方案，这要求对硼携带剂的体内浓度分布需要做实时监测。BPA作为目前唯一获批的BNCT药物，对其在体内的分布情况进行研究就尤为重要。其中重要方法即借助PET分子影像的方法，通过在BPA苯环上引入放射性的 18F得到[ 18F]FBPA。通过[ 18F]FBPA模拟BPA在体内的分布，进而对BNCT进行规划。但是，从化学结构上，以F原子取代苯环上的H原子，难以保证其化学性质及生物学性质与BPA仍然相同，这种差异将为BNCT带来较大的不确定性。
Figure PCTCN2022111728-appb-000001
针对现有的FBPA--PET不能准确有效反应BPA在体内的分布情况，精准高效实现BNCT的问题，本申请提供一种新的诊疗一体硼携带剂，例如(S)-BBPA，其可以进行便捷的放射性标记，标记前后具有相同的化学结构，可以准确反映其在体内的分布情况，以解决经典硼携带剂体内实时分布不清晰预测的问题，提供更加高效的BNCT规划；并且通过分子内的两个硼原子，实现更加高效的BNCT。
Figure PCTCN2022111728-appb-000002
本申请的构思如下：硼氨酸是一类将天然氨基酸的羧基用三氟化硼取代进而发展的一类氨基酸衍生物。通过18F-19F同位素交换反应，其可以使用18F进行放射性标记构建放射性探针，进而实现PET成像。发明人在研究中发现，三氟化硼基团和羧酸根在电性上表现出高度的相似性，故而硼氨酸与其相应的天然氨基酸在生物识别、转运上亦表现出相似性。基于此，申请人发现，通过替换BPA中的羧酸根为三氟化硼基团，构建BBPA，其中手性碳原子有S构型与R构型。R构型的(R)-BBPA与天然氨基酸手性相同，而S构型的(S)-BBPA与天然氨基酸构型相反，二者均可以在肿瘤区域有高富集，其中(S)-BBPA表现出较低的正常组织背景摄取，体现出较好的临床应用价值。一方面，(S)-BBPA可以进行F-18标示实现体内的分子浓度分布分析；另一方面，其也可以直接作为BNCT的硼携带剂。由于(S)-BBPA放射性标记前后具有完全一致的化学结构，通过[ 18F](S)-BBPA可以准确的反映(S)-BBPA在体内的分布情况，为BNCT治疗方案的实施提供重要依据。更重要的是，(S)-BBPA中具有2个硼原子，具有更强的硼元素载带能力，实现更高效的硼元素递送。
在一方面，本申请提供了式I的化合物或其药学可接受的盐：
Figure PCTCN2022111728-appb-000003
其中R基团为氢或烷基。
在一个实施方案中，化合物为S型或R型的化合物。
在一个实施方案中，化合物具有式II的结构：
Figure PCTCN2022111728-appb-000004
其中R基团为氢或烷基(例如C1-C10烷基，C1-C6烷基，如甲基、乙基、丙基、丁基、戊基或己基)。
在一个实施方案中，与苯环连接的硼原子是 10B或天然硼。在一个实施方案中，-BF 3 -中的至少一个氟原子是放射性氟原子，例如 18F。
在一个实施方案中，烷基是甲基。
在一个实施方案中，-BF 3 -中的硼原子是天然硼。
在另一个方面，本申请提供了本申请的化合物的制备方法，其包括以下步骤：通过对化合物6
Figure PCTCN2022111728-appb-000005
优选
Figure PCTCN2022111728-appb-000006
添加KHF 2溶液，并且添加盐酸至pH<5，以产生含化合物7
Figure PCTCN2022111728-appb-000007
优选
Figure PCTCN2022111728-appb-000008
然后加酸以得到化合物。
在一个实施方案中，方法包括以下步骤中的一个或多个：
(1)将对溴苯乙醇
Figure PCTCN2022111728-appb-000009
转化成化合物1
Figure PCTCN2022111728-appb-000010
(2)在添加N-甲基亚氨基乙酸的情况下将化合物1转化成化合物2
Figure PCTCN2022111728-appb-000011
(3)将化合物2转化成化合物3
Figure PCTCN2022111728-appb-000012
(4)将化合物3转化成化合物4
Figure PCTCN2022111728-appb-000013
(5)将化合物4转化成化合物5
Figure PCTCN2022111728-appb-000014
优选
Figure PCTCN2022111728-appb-000015
(6)将化合物5转化成化合物6
Figure PCTCN2022111728-appb-000016
优选
Figure PCTCN2022111728-appb-000017
以及
(7)将化合物6转化成化合物7
Figure PCTCN2022111728-appb-000018
优选
Figure PCTCN2022111728-appb-000019
(8)将化合物7转化成化合物8
Figure PCTCN2022111728-appb-000020
优选
Figure PCTCN2022111728-appb-000021
以及
(9)将化合物8转化为
Figure PCTCN2022111728-appb-000022
优选
Figure PCTCN2022111728-appb-000023
在另一个方面，本申请提供了硼携带剂组合物，其包含一种或多种本申请的式I的化合物和药学可接受的载体。
在一个实施方案中，硼携带剂组合物还包含果糖、十一氢巯基十二硼化二钠及其二聚体、硼化树枝状聚合物-EGF生物缀合物、EGFR单克隆抗体-硼酸盐缀合物、FR靶向性含硼脂质体、FR靶向性含硼纳米颗粒和硼酸盐卟啉中的一种或多种。
在又一个方面，本申请提供了本申请的化合物在制备药物中的用途，所述药物用于治疗或诊断癌症。本申请的化合物通过LAT-1转运体在肿瘤中高富集，从而实现治疗的目的。癌症可以是表达或过表达LAT-1转运体的癌症。
在一个实施方案中，药物是用于肿瘤诊疗一体化或硼中子俘获疗法的硼携带剂。
在一个实施方案中，癌症选自鳞状细胞癌、肺癌、腹膜癌、肝细胞癌、胃癌、黑素瘤、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝瘤、乳腺癌、结肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、肝癌、前列腺癌、外阴癌、甲状腺癌、肝癌、头颈癌、B细胞淋巴瘤、白血病或脑垂体瘤。
本申请的优点包括：
(1)本申请使用了 18F标记前后具有相同化学结构的式I的化合物，例如(S)-BBPA与[ 18F](S)-BBPA，可以用于肿瘤诊断与BNCT治疗的诊疗一体化药物，通过其相同的化学结构提供可靠的药物体内分布结果。
Figure PCTCN2022111728-appb-000024
(2)结构中具有两个B原子，具有更高的B递送效率。
(3)本申请的方法实现单个硼的氟化。
(4)本申请的化合物在肿瘤中具有高摄取。
(5)本申请提供了一种新型诊疗一体化的硼携带剂，例如(S)-BBPA，用于获取硼携带剂的动态分布，进行BNCT前的治疗规划，亦可以直接用于BNCT。
(6)本申请提供了制备本申请的化合物的新方法。
(7)与(R)-BBPA相比，(S)-BBPA在体内具有相同或更好的治疗效果，更低的副作用以及更低的背景摄取。
附图简述
图1：制备的[18F](S)-BBPA的Radio-HPLC图。
图2：[18F](S)-BBPA的Radio-HPLC图，表明在4小时时间尺度内(2个18F半衰期)，[18F](S)-BBPA在模拟体内环境中可以保持充分的稳定。
图3：在注射肿瘤细胞后15分钟对小鼠进行PET/CT得到的图。
图4：(S)-BBPA和(R)-BBPA的成像对比度比较的图。
图5：(S)-BBPA和(R)-BBPA对小鼠肿瘤的治疗效果的图。
图6：(S)-BBPA和(R)-BBPA对小鼠体重的影响的图。
图7：使用γ计数器测量细胞的放射性强度的图。
图8A-B：(S)-BBPA的1H-NMR和19F-NMR的谱图。
图9：(R)-BBPA的HPLC-MS结果。
具体实施方式
本申请提供了式I的化合物或其药学可接受的盐：
Figure PCTCN2022111728-appb-000025
本申请的化合物可以是L型或S型。这些化合物可以用于正电子发射断层扫描成像或者硼中子俘获疗法。
在式(I)中，R独立是氢或烷基。“烷基”是指饱和的线性烃基(例如C1-C10烷基，C1-C6烷基)。例如，烷基可以是甲基、乙基、丙基、丁基、戊基等。优选地，R基团是甲基。
在式(I)中，-BF 3 -中的至少一个氟原子是放射性标记的。例如，1、2或3个氟原子是 18F。-BF 3 -中的硼原子可以是天然硼。
在式(I)中，与苯环连接的硼原子是 10B或天然硼。
本申请还提供了式II的化合物或其药学可接受的盐：
Figure PCTCN2022111728-appb-000026
R基团为氢，或烷基，优选甲基。
本申请还提供了硼携带剂组合物，其包含本申请的化合物和药学可接受的载体。硼携带剂组合物还可以包含果酸，或者可以包含其它硼携带剂，例如十一氢巯基十二硼化二钠及其二聚体、硼化树枝状聚合物-EGF生物缀合物、EGFR单克隆抗体-硼酸盐缀合物、FR靶向性含硼脂质体、FR靶向性含硼纳米颗粒和硼酸盐卟啉中的一种或多种。
本申请还提供了化合物的制备方法。方法可以包括以下步骤中的一个或多个：
(1)将对溴苯乙醇
Figure PCTCN2022111728-appb-000027
转化成化合物1
Figure PCTCN2022111728-appb-000028
(2)在添加N-甲基亚氨基乙酸的情况下将化合物1转化成化合物2
Figure PCTCN2022111728-appb-000029
(3)将化合物2转化成化合物3
Figure PCTCN2022111728-appb-000030
(4)将化合物3转化成化合物4
Figure PCTCN2022111728-appb-000031
(5)将化合物4转化成化合物5
Figure PCTCN2022111728-appb-000032
优选
Figure PCTCN2022111728-appb-000033
(6)将化合物5转化成化合物6
Figure PCTCN2022111728-appb-000034
优选
Figure PCTCN2022111728-appb-000035
以及
(7)将化合物6转化成化合物7
Figure PCTCN2022111728-appb-000036
优选
Figure PCTCN2022111728-appb-000037
(8)将化合物7转化成化合物8
Figure PCTCN2022111728-appb-000038
优选
Figure PCTCN2022111728-appb-000039
以及
(9)将化合物8转化为
Figure PCTCN2022111728-appb-000040
优选
Figure PCTCN2022111728-appb-000041
在步骤(1)中，可以将对溴苯乙醇溶解于无水四氢呋喃中，冷却(例如-78℃)，加入正丁基锂/己烷溶液，溶液逐渐变黄后变为米白色糊状，搅拌15min(例如在-78℃)；随后滴加硼酸三异丙酯，溶液变澄清，反应20min(例如-78℃)。随后升至室温，加入盐酸，室温搅拌。分液，水相使用乙酸乙酯萃取，将有机相合并，饱和氯化钠洗涤，无水硫酸钠干燥，真空除去溶剂，得到白色固体化合物1。
在步骤(2)中，可以将化合物1溶解于甲苯，加入DMSO和N-甲基亚氨基乙酸反应获得化合物2。
在步骤(3)中，可以将化合物2溶解于无水二氯甲烷无水四氢呋喃的混合溶剂中，加入DMP氧化剂，再加入碳酸氢钠粉末反应。然后添加饱和硫代硫 酸钠溶液与饱和碳酸氢钠溶液，并且搅拌。过滤除去不溶物，分出有机相，水相使用乙酸乙酯萃取，将有机相合并，饱和氯化钠洗涤，无水硫酸钠干燥，真空除去溶剂。可以通过硅胶柱层析分离，得到白色固体化合物3。
在步骤(4)中，可以将化合物3溶解于无水四氢呋喃中，加入(S)-叔丁基亚磺酰胺，滴加钛酸四异丙酯，室温搅拌。加入水淬灭反应并搅拌，过滤除去不溶物分出有机相，水相使用乙酸乙酯萃取，将有机相合并，饱和氯化钠洗涤，无水硫酸钠干燥，真空除去溶剂。可以通过硅胶柱层析分离，得到黄色晶体化合物4。
在步骤(5)中，可以将PCy3·HBF4加入1mL甲苯中，随后加入CuSO4·5H2O和110μL去离子水，并在室温下剧烈搅拌10分钟。随后在氮气保护下加入苄胺，立即形成深蓝色催化剂。将反应体系维持低温度(例如0-40℃，例如0℃)，加入化合物4和B2pin2。随后，将反应体系恢复至室温，反应至TLC显示反应物完全消失。加入饱和EDTA洗涤反应混合物，再用饱和食盐水洗涤。将有机相合并随后用无Na 2SO 4干燥，并在真空中除去有机溶剂，得到黄色油状液体化合物5。
在步骤6中，可以将化合物5溶于甲醇中，加入NaOH溶液，室温搅拌。HPLC分析确认化合物5消失及化合物6产生。随后向反应混合物中滴加KHF2溶液，测定溶液pH，并加入适量的盐酸(例如至pH<5)，室温搅拌。HPLC确认原料的消失与化合物7的生成。可以使用制备HPLC纯化，得到白色粉末化合物7。
在步骤7中，可以将HCl滴加入化合物7粉末中，随后室温下搅拌(例如1小时)。HPLC确认原料的消失和BBPA的生成，使用制备HPLC得到白色粉末BBPA(例如(S)-BBPA)。
在步骤(8)中，可以将BBPA(例如(S)-BBPA)溶解于水中，再加入哒嗪-HCl缓冲液，随后加入水 18F-水溶液。将反应混合物反应(例如在85℃反应15min)，随后用生理盐水淬灭反应，并用Sep-Pak Alumina Light cartridge除去游离的氟离子得到最终产物。
本申请提供了使用硼携带剂组合物治疗或诊断癌症的方法。方法可以包括对受试者(例如人或哺乳动物)施用硼携带剂组合物的步骤。本申请提供了硼携带剂组合物在制备药物中的用途，所述药物用于治疗或诊断癌症。药物可以是用于肿瘤诊疗一体化或硼中子俘获疗法的硼携带剂。本申请还提供了 硼携带剂组合物，用于治疗或诊断癌症。
本申请提供了治疗和/或诊断癌症的方法，其包括对受试者施用本文所述的化合物或硼携带剂组合物。在一个实施方案中，癌症是表达或过表达LAT-1转运体的癌症。癌症可以选自鳞状细胞癌、肺癌、腹膜癌、肝细胞癌、胃癌、胰腺癌、黑素瘤、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝瘤、乳腺癌、结肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、肝癌、前列腺癌、外阴癌、甲状腺癌、肝癌、头颈癌、B细胞淋巴瘤白血病或脑垂体瘤。受试者可以是人。
本申请提供了本文所述的化合物或硼携带剂组合物，用于治疗和/或诊断癌症。在一个实施方案中，癌症是表达或过表达LAT-1转运体的癌症。在一个实施方案中，癌症选自鳞状细胞癌、肺癌、腹膜癌、肝细胞癌、胃癌、胰腺癌、黑素瘤、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝瘤、乳腺癌、结肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、肝癌、前列腺癌、外阴癌、甲状腺癌、肝癌、头颈癌、B细胞淋巴瘤白血病或脑垂体瘤。
术语“癌症”和“癌性”指或描述哺乳动物中特征通常为细胞生长不受调节的生理学状况。癌症的例子包括但不限于癌瘤、淋巴瘤、母细胞瘤、肉瘤、和白血病。此类癌症的更具体例子包括鳞状细胞癌、肺癌(包括小细胞肺癌、非小细胞肺癌、肺腺癌、和肺鳞癌)、腹膜癌、肝细胞癌、胃癌(包括胃肠癌)、黑素瘤、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝瘤、乳腺癌、结肠癌、结肠直肠癌、子宫内膜或子宫癌、唾液腺癌、肾癌、肝癌、前列腺癌、外阴癌、甲状腺癌、肝癌和各种类型的头颈癌，及B细胞淋巴瘤(包括低级/滤泡非何杰金氏淋巴瘤(NHL)；小淋巴细胞(SL)NHL；中级/滤泡NHL；中级弥漫性NHL；高级成免疫细胞NHL；高级成淋巴细胞NHL；高级小无核裂细胞NHL；贮积病NHL；套细胞淋巴瘤；AIDS相关淋巴瘤；和瓦尔登斯特伦氏巨球蛋白血症)；慢性淋巴细胞性白血病(CLL)；急性成淋巴细胞性白血病(ALL)；毛细胞白血病；慢性成髓细胞性白血病；和移植后淋巴增殖性病症(PTLD)、以及与瘢痣病、水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖。
提供以下示例性的实施方案以进一步说明本申请。应当理解这些实施例仅仅是示例性的而非限制性的，本申请仅由所附权利要求书限定。
实施例
实施例1：化合物的制备
(S)-BBPA的制备
合成方案
Figure PCTCN2022111728-appb-000042
将对溴苯乙醇(5mmol，0.7ml)溶解于50ml无水四氢呋喃中，冷却至-78℃。逐滴加入正丁基锂/己烷溶液(12mmol，2.4eq)，溶液逐渐变黄后变为米白色糊状，保持在-78℃搅拌15min。随后滴加硼酸三异丙酯(15mol，3.5ml，3eq.)，溶液变澄清，-78℃反应20min。随后升至室温，加入50ml 10％盐酸，室温搅拌15min。分液，水相使用乙酸乙酯萃取(20ml×3)，将有机相合并，饱和氯化钠洗涤(30ml×2)，无水硫酸钠干燥，真空除去溶剂，得到白色固体化合物1(0.66g，80％)。化合物无须进一步纯化，直接用于下步反应。1H NMR(400MHz,Methanol-d4)δ7.60–6.94(m,4H),3.75(t,J＝7.1Hz,2H),2.82(t,J＝7.1Hz,2H)。
化合物1(0.66g，4mmol)溶于16ml甲苯中，加入1.6ml DMSO，再加入N-甲基亚氨基乙酸(647mg，4.4mmol，1.1eq.)。回流，并安装加满甲苯的分水器，约2小时后结束反应。在真空中除去有机溶剂，使用硅胶柱层析分离，得到白色晶体化合物2(650mg，59％)。1H NMR(400MHz,Methanol-d4)δ7.53–7.10(m,4H),4.35–3.96(m,4H),3.75(t,J＝7.0Hz,2H),3.31(s,3H),2.82(t,J＝7.1Hz,2H)。
将化合物2(1.1g，4mmol)溶于40ml无水二氯甲烷与40ml无水四氢呋喃的混合溶剂中，加入DMP氧化剂(1.7g，4mmol，1eq.)，再加入碳酸氢钠粉末(3.36g，40mmol，10eq.)。室温反应1小时。加入40ml饱和硫代硫酸钠溶液与40ml饱和碳酸氢钠溶液，搅拌15分钟。过滤除去不溶物，分出有机相，水相使用乙酸乙酯萃取(20ml×3)，将有机相合并，饱和氯化钠洗涤(30ml×2)，无水硫酸钠干燥，真空除去溶剂。硅胶柱层析分离，得到白色固体化合物3(541mg，49％)。1H NMR(400MHz,Acetone-d6)δ9.74(s,1H),7.41(dd,J＝100.7,7.9Hz,4H),4.48–4.07(m,4H),2.75(s,3H)。
将化合物3(541mg，1.97mmol)溶解于20ml无水四氢呋喃中，加入(S)-叔丁基亚磺酰胺(477mg，3.94mmol，2eq.)，滴加钛酸四异丙酯(是否与合成方案中的Ti(OPri) 4不一致)(3.94mmol，1165μL，l.2eq.)，室温搅拌2小时。加入20ml水淬灭反应并搅拌10min，过滤除去不溶物分出有机相，水相使用乙酸乙酯萃取(20ml×3)，将有机相合并，饱和氯化钠洗涤(30ml×2)，无水硫酸钠干燥，真空除去溶剂。硅胶柱层析分离，得到黄色晶体化合物4(490mg，66％)。1H NMR(400MHz,Acetone-d6)δ8.07(s,1H),7.43(dd,J＝83.2,7.9Hz,4H),4.51–4.03(m,4H),3.91(d,J＝5.1Hz,2H),2.73(s,3H),1.12(s,9H)。
将PCy 3·HBF 4(2.4mg，6.6μmol，5mol％)加入1mL甲苯中，随后加入CuSO 4·5H 2O(1.65mg，6.6μmol，5mol％)和去110μL离子水，并在室温下剧烈搅拌10分钟。随后在氮气保护下加入苄胺(3μL，27μmol，20mol％)，立即形成深蓝色催化剂。将反应体系维持在0℃，加入化合物4(50mg，132μmol，1.0eq.)和B 2pin 2(67mg，264μmol，2.0eq.)。随后，将反应体系恢复至室温，反应1小时至TLC显示反应物完全消失。加入饱和EDTA(2×5mL)洗涤反应混合物，再用饱和食盐水(5mL)洗涤。将有机相合并随后用无Na 2SO 4干燥，并在真空中除去有机溶剂，得到黄色油状液体化合物5(49mg， 75％)。粗产品不用纯化直接用于下一步反应。
将化合物5(49mg，99μmol，1.0eq.)溶于1ml甲醇中，加入1ml 1M NaOH溶液(1mmol，10eq.)，室温搅拌一小时。HPLC分析确认化合物5消失及化合物6产生。随后向反应混合物中滴加0.33mL 3M KHF 2溶液(10eq.)，测定溶液pH，并加入适量的盐酸至pH<5，室温搅拌4小时。HPLC(C18，A相水/0.1％TFA，B相乙腈，0-15分钟：95％A-5％A，其余为B)确认原料的消失与化合物7的生成。使用制备HPLC纯化，得到白色粉末化合物7(24mg，55％)。
1.1mL 1M HCl(1.1mmol，20eq.)滴加入化合物7粉末中(24mg.54μmol，1.0eq.)，随后室温下搅拌1小时。HPLC确认原料的消失和BBPA的生成，使用制备HPLC(C18，A相水/0.1％TFA，B相乙腈，0-15分钟：95％A-5％A，其余为B)得到白色粉末(S)-BBPA(9.3mg，74％)。 19FNMR(400MHz,D 2O,ppm):147.65.LC-MS:Found:232.1(MS -),Calculated:232.1。
放射化学标记
将(S)-BBPA(0.3mg)溶解与50μL水中，再加入5μl哒嗪-HCl缓冲液(1.0M,pH～2.0)，随后加入水 18F-水溶液(30μL,370MBq)。反应混合物在85℃反应15min，随后用0.5ml生理盐水淬灭反应，并用Sep-Pak Alumina Light cartridge除去游离的氟离子得到最终产物[ 18F](S)-BBPA(～148MBq,RCY40％)。Radio-HPLC结果如图1(HPLC方法，A相：乙腈，B相：水/0.1％TFA；20％A，余为B)。
(R)-BBPA的制备
合成方案
Figure PCTCN2022111728-appb-000043
如上所示，化合物2至化合物7及(R)-BBPA由可商业购买的对溴苯乙醇(化合物1)作为起始化合物合成。
将对溴苯乙醇(5mmol,0.7ml,化合物1)溶解于50ml无水四氢呋喃中，冷却至-78℃。逐滴加入正丁基锂/己烷溶液(12mmol,2.4eq)，溶液逐渐变黄后变为米白色糊状，保持在-78℃搅拌15min。随后滴加硼酸三异丙酯(15mol,3.5ml,3eq.)，溶液变澄清，-78℃反应20min。随后升至室温，加入50ml 10％盐酸，室温搅拌15min。分液，水相使用乙酸乙酯萃取(20ml x 3)，将有机相合并，饱和氯化钠洗涤(30ml x 2)，无水硫酸钠干燥，真空除去溶剂，得到白色固体化合物2(0.66g,80％)。化合物无须进一步纯化，直接用于下步反应。化合物2：1H NMR(400MHz,Methanol-d4)δ7.60–6.94(m,4H),3.75(t,J＝7.1Hz,2H),2.82(t,J＝7.1Hz,2H)。
化合物2(0.66g,4mmol)溶于16ml甲苯中，加入1.6ml DMSO，再加入N-甲基亚氨基乙酸(647mg,4.4mmol,1.1eq.)。回流，并安装加满甲苯的分水器，约2小时后结束反应。在真空中除去有机溶剂，使用硅胶柱层析分离，得到白色晶体化合物3(650mg,59％)。化合物3：1H NMR(400MHz,Methanol-d4)δ7.53–7.10(m,4H),4.35–3.96(m,4H),3.75(t,J＝7.0Hz,2H),3.31(s,3H),2.82(t,J＝7.1Hz,2H)。
将化合物3(1.1g,4mmol)溶于40ml无水二氯甲烷与40ml无水四氢呋喃 的混合溶剂中，加入DMP氧化剂(1.7g,4mmol,1eq.)，再加入碳酸氢钠粉末(3.36g,40mmol,10eq.)。室温反应1小时。加入40ml饱和硫代硫酸钠溶液与40ml饱和碳酸氢钠溶液，搅拌15分钟。过滤除去不溶物，分出有机相，水相使用乙酸乙酯萃取(20ml x 3)，将有机相合并，饱和氯化钠洗涤(30ml x 2)，无水硫酸钠干燥，真空除去溶剂。硅胶柱层析分离，得到白色固体化合物4(541mg,49％)。化合物4：1H NMR(400MHz,Acetone-d6)δ9.74(s,1H),7.41(dd,J＝100.7,7.9Hz,4H),4.48–4.07(m,4H),2.75(s,3H)。
将化合物4(541mg,1.97mmol)溶解于20ml无水四氢呋喃中，加入叔丁基亚磺酰胺(477mg,3.94mmol,2eq.)，滴加钛酸四异丙酯(3.94mmol,1165μl,2eq.)，室温搅拌2小时。加入20ml水淬灭反应并搅拌10min，过滤除去不溶物分出有机相，水相使用乙酸乙酯萃取(20ml x 3)，将有机相合并，饱和氯化钠洗涤(30ml x 2)，无水硫酸钠干燥，真空除去溶剂。硅胶柱层析分离，得到黄色晶体化合物5(490mg,66％)。化合物5：1H NMR(400MHz,Acetone-d6)δ8.07(s,1H),7.43(dd,J＝83.2,7.9Hz,4H),4.51–4.03(m,4H),3.91(d,J＝5.1Hz,2H),2.73(s,3H),1.12(s,9H)。
将PCy 3·HBF 4(124mg,6mol％)加入10ml甲苯中，随后加入CuSO 4·5H 2O溶液(82mg,6mol％)和去离子水(6mL)，并在室温下剧烈搅拌10min。随后在氮气保护下加入苄胺(153μL,0.25eq.)，立即形成深蓝色催化剂。加入额外的50ml甲苯，将反应体系维持在0℃，加入化合物5(2.2g,5.7mmol)和B 2pin 2(2.8g,2.0eq.)。随后，将反应体系恢复至室温，反应1小时至TLC显示反应物完全消失。加入饱和EDTA(2x 50mL)洗涤反应混合物，再用饱和食盐水(50mL)洗涤。将有机相合并随后用无Na 2SO 4干燥，并在真空中除去有机溶剂，得到黄色油状液体化合物6(3.0g)。粗产品不用纯化直接用于下一步反应。
将化合物6(3.0g,5.7mmol)溶于50ml乙腈中，加入57ml 1M NaOH溶液，室温搅拌半小时。随后加入4.7ml浓盐酸中和反应，得到含化合物7的反应混合物，直接用于下步反应。
向含化合物7的反应混合物中加入14.25ml 3M的KHF 2溶液(10eq.)，再逐滴加入4.7ml浓盐酸至pH<0，室温反应2小时。在30℃减压蒸发除去HCl降低酸度至pH～2，反应30min。加入乙腈共沸干燥，使用乙腈洗涤固体， 弃去不溶物，乙腈相使用pre-HPLC纯化(A相：乙腈，B相：水/0.1％TFA；0-2min：5％A，2-11min：5％A-50％A，其余为B)，得到(R)-BBPA。
实施例2：放射性稳定性研究
将被标记的[ 18F](S)-BBPA加入50％胎牛血清，并保存于37℃中。在特定时刻，取出少量，使用Radio-HPLC分析(C18，A相水/0.1％TFA，B相乙腈，0-15分钟：80％+20％B)，确定其中的放射性物质组成。信号以归一化强度表示，即信号最大值与最小值的差值统一设定为1。结果表明在4小时时间尺度内(2个18F半衰期)，[ 18F](S)-BBPA在模拟体内环境中可以保持充分的稳定，结果如图2所示。
实施例3：PET动物成像
肿瘤模型动物的建立：将肿瘤细胞(BGC823人源胃癌细胞、B16-F10小鼠黑色素瘤和GH3大鼠脑垂体瘤)于5％CO 2培养箱中培育至汇合。小鼠选取4-6周龄小鼠，右肩剃毛，将约10 6个肿瘤细胞悬浮于50μL PBS中，注射于小鼠右肩上。在无菌环境中饲养1-2周，待肿瘤体积至200～400mm 3，肿瘤模型建立完成。
将标记的化合物[ 18F](S)-BBPA或[ 18F](R)-BBPA取200μCi溶于0.2ml生理盐水中，通过尾静脉注射进入肩部种植有肿瘤的小鼠体内，并在注射后1小时进行15分钟的PET/CT采集并作图像重建，结果如图3所示。[ 18F](S)-BBPA或[ 18F](R)-BBPA均可以指示出肿瘤位置，[ 18F](S)-BBPA虽然肿瘤摄取较低，但是其成像背景、肌肉摄取较低，因此实际的成像对比度较高，如定量分析结果图4所示。
实施例4：低剂量硼中子捕获治疗实验
将含有天然硼的BPA、(S)-BBPA和(R)-BBPA以100mg/kg的剂量溶解于生理盐水中，并通过尾静脉注射至上述肩部种植B16-F10和黑色素瘤的小鼠体内，在1h时进行BNCT处理，在后续21天测量肿瘤大小，并与单独注射相关药物及单纯进行中子束照射的组别作对比。
从图5和图6中可以看出，由于(S)-BBPA和(R)-BBPA具有两个硼原子，联合中子束治疗效果显著，而BPA硼浓度较低，在低剂量下治疗效果低于 BBPA。同时注意到，虽然(S)-BBPA和(R)-BBPA都较好地抑制了肿瘤生长，但是(R)-BBPA较显著的减轻了体重，这可能是由于(R)-BBPA在肌肉中有较高的背景摄取，引起了较强的副作用。综上所述，分子结构内具有两个硼原子的(S)-BBPA和(R)-BBPA均可以作为高效的硼载剂，但是(S)-BBPA较低的背景摄取可以带来更好的硼浓度差异，减少副作用的发生。这对缩短治疗周期、减少药物用量、降低辐照剂量与患者费用支出，减少并发症与副作用具有重要意义。
实施例5：(S)-BBPA摄取机制研究
在细胞摄取实验中，通过加入高浓度的氨基酸转运体底物或抑制剂，通过竞争抑制的方式，可以对(S)-BBPA进入细胞的机制进行研究。本研究中，选择丙氨酸(Alanine，Ala)、谷氨酸(Glutamate，Glu)、BCH(2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)、BPA和GPNA(L-γ-Glutamyl-p-nitroanilide)作为竞争底物或者抑制剂，与对照组相比较。
将标准培育条件下的B16-F10小鼠黑色素瘤细胞平均转移至1.5ml EP管中(4×10 5/管)，150xg离心2min，除去上清液。配制一系列含有天然氨基酸(25mM)或抑制剂(10mM)的PBS溶液，分别加入至上述含有细胞的EP管中(0.5ml,n＝6)，其中加入普通PBS的对照组。然后向每管中加入10μl含有740kBq的[ 18F](S)-BBPA的生理盐水。将上述细胞在37℃下孵育60min，然后150xg离心2min，弃去上清液，再用PBS(1ml×2)洗涤。最终使用γ计数器测量每管中细胞的放射性强度，计算各组与对照组之间的比值，结果如图7所示。
Ala是氨基酸转运系统ASC系统和A系统的底物，Glu是X c -转运体的底物，二者都不能抑制[ 18F]BBPA的摄取；而BCH是LAT-1转运体(Large-neutral Amino Acid Transporter 1)的特异性抑制剂，其显著地抑制了[ 18F]BBPA的摄取，说明(S)-BBPA是通过LAT-1的转运进入肿瘤细胞；GPNA是ASCT2转运体的特异性抑制剂，其不能抑制(S)-BBPA的摄取；(S)-BBPA是BPA的硼氨酸衍生物，BPA显著地抑制了(S)-BBPA的摄取，抑制效果甚至优于BCH，说明尽管(S)-BBPA与BPA手性不同，但其在转运通路上仍然具有极高的生物相似性，其具有完全一致的生物通路。由此可以得出基本结论(S)-BBPA基于LAT-1转运体在肿瘤中高富集，这与BPA的转运机制相同。
综上所述，(S)-BBPA与BPA具有较高的生物相似性，主要通过LAT-1转运进入细胞。LAT-1是肿瘤细胞高表达的氨基酸转运体，因此(S)-BBPA可以在肿瘤组织细胞中取得高摄取，有助于PET的高对比度成像和高浓度的硼富集实现高效BNCT。
虽然参考其具体的实施方案描述了本申请，但本领域技术人员应了解可以进行多种改变，可以替换等同内容而不偏离本申请的精神和范围。另外，可进行许多修改以使具体的情况、材料、组合物、方法、一个或多个方法步骤适应本申请的目的、精神和范围。所有这类修改意在随附权利要求书的范围内。

Claims (12)

  1. 式I的化合物或其药学可接受的盐
    Figure PCTCN2022111728-appb-100001
    其中R基团为氢，或烷基，优选C1-C6烷基，优选甲基。
  2. 权利要求1的化合物或其药学可接受的盐，其中所述化合物为S构型或R构型。
  3. 权利要求1或2的化合物或其药学可接受的盐，其中与苯环连接的硼原子是 10B或天然硼，
    优选地，其中在-BF 3 -中至少一个氟原子是放射性氟原子，优选 18F；
    优选地，其中在-BF 3 -中硼原子是天然硼。
  4. 硼携带剂组合物，其包含一种或多种权利要求1-3中任一项的化合物和药学可接受的载体。
  5. 权利要求4的硼携带剂组合物，其还包含果糖、十一氢巯基十二硼化二钠及其二聚体、硼化树枝状聚合物-EGF生物缀合物、EGFR单克隆抗体-硼酸盐缀合物、FR靶向性含硼脂质体、FR靶向性含硼纳米颗粒和硼酸盐卟啉中的一种或多种。
  6. 权利要求1-3中任一项的化合物的制备方法，其包括以下步骤：通过对化合物6
    Figure PCTCN2022111728-appb-100002
    优选
    Figure PCTCN2022111728-appb-100003
    添加KHF 2溶液，并且添加盐酸至pH<5，以产生含化合物7
    Figure PCTCN2022111728-appb-100004
    优选
    Figure PCTCN2022111728-appb-100005
    然后进一步加酸以得到权利要求1-3中任一项的化合物。
  7. 权利要求6的方法，其包括以下步骤中的一个或多个：
    (1)将对溴苯乙醇
    Figure PCTCN2022111728-appb-100006
    转化成化合物1
    Figure PCTCN2022111728-appb-100007
    (2)在添加N-甲基亚氨基乙酸的情况下将化合物1转化成化合物2
    Figure PCTCN2022111728-appb-100008
    (3)将化合物2转化成化合物3
    Figure PCTCN2022111728-appb-100009
    (4)将化合物3转化成化合物4
    Figure PCTCN2022111728-appb-100010
    (5)将化合物4转化成化合物5
    Figure PCTCN2022111728-appb-100011
    优选
    Figure PCTCN2022111728-appb-100012
    (6)将化合物5转化成化合物6
    Figure PCTCN2022111728-appb-100013
    优选
    Figure PCTCN2022111728-appb-100014
    以及
    (7)将化合物6转化成化合物7
    Figure PCTCN2022111728-appb-100015
    优选
    Figure PCTCN2022111728-appb-100016
    (8)将化合物7转化成化合物8
    Figure PCTCN2022111728-appb-100017
    优选
    Figure PCTCN2022111728-appb-100018
    以及
    (9)将化合物8转化为
    Figure PCTCN2022111728-appb-100019
    优选
    Figure PCTCN2022111728-appb-100020
  8. 权利要求1-3中任一项的化合物或权利要求4或5的硼携带剂组合物在制备药物中的用途，所述药物用于治疗或诊断癌症；优选地，癌症是表达或过表达LAT-1转运体的癌症。
  9. 权利要求8的用途，其中所述药物是用于肿瘤诊疗一体化或硼中子俘获疗法的硼携带剂。
  10. 权利要求8或9的用途，其中所述癌症选自鳞状细胞癌、肺癌、腹膜癌、肝细胞癌、胃癌、胰腺癌、黑素瘤、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝瘤、乳腺癌、结肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、肝癌、前列腺癌、外阴癌、甲状腺癌、肝癌、头颈癌、B细胞淋巴瘤白血病或脑垂体瘤。
  11. 治疗和/或诊断受试者中的癌症的方法，其包括对受试者施用权利要求1-3中任一项的化合物或权利要求4或5的硼携带剂组合物；优选地，癌症是表达或过表达LAT-1转运体的癌症；优选地，所述癌症选自鳞状细胞癌、肺癌、腹膜癌、肝细胞癌、胃癌、胰腺癌、黑素瘤、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝瘤、乳腺癌、结肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、肝癌、前列腺癌、外阴癌、甲状腺癌、肝癌、头颈癌、B细胞淋巴瘤白血病或脑垂体瘤；优选地受试者是人。
  12. 权利要求1-3中任一项的化合物或权利要求4或5的硼携带剂组合物，用于治疗和/或诊断癌症；优选地，癌症是表达或过表达LAT-1转运体的癌症；优选地，所述癌症选自鳞状细胞癌、肺癌、腹膜癌、肝细胞癌、胃癌、胰腺癌、黑素瘤、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝瘤、乳腺癌、结肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、肝癌、前列腺癌、外阴癌、甲状腺癌、肝癌、头颈癌、B细胞淋巴瘤白血病或脑垂体瘤。
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