Boron atom-labeled compound for targeting fibroblast activating protein as well as preparation method and application of boron atom-labeled compound

Abstract

The invention relates to the technical field of compound preparation and medicine, in particular to a boron atom-labeled compound for targeting fibroblast activating protein as well as a preparation method and application of the boron atom-labeled compound. The preparation method comprises the following steps: carrying out acid amine condensation on (4-(((2, 5-dioxopyrrolidine-1-yl) oxy) carbonyl) phenyl) boric acid and an FAP targeting ligand to obtain a boron atom-labeled compound targeting the fibroblast activating protein; and the FAP targeting ligand is FAPI-46 or FAP-2286 (Fibroblast Amplified Polymorphism). The compound is easy to prepare, high in purity and good in stability, the content of boron atoms in tumor tissue can be greatly increased, and a treatment basis is provided for boron neutron capture therapy.

Description

Technical Field

[0001] This invention relates to the fields of compound preparation and pharmaceutical technology, and in particular to a boron-labeled compound targeting fibroblast activation proteins, its preparation method, and its applications. Background Technology

[0002] Boron neutron capture therapy (BNCT) is a binary targeted radiotherapy technique that utilizes the nuclide boron-10 to capture neutrons, triggering a nuclear reaction that releases high-energy rays to kill cancer cells. To achieve good therapeutic effects, cancer patients often receive high doses of boron drugs to increase the boron content in tumor tissue. Currently used boron drugs are mainly mercaptododecanoate and (L)-4-dihydroxyboronylphenylalanine, but these drugs suffer from weak targeting ability in tumor tissue and uneven distribution, limiting their therapeutic efficacy. Therefore, there is an urgent need to develop a boron compound with high targeting and stability to enhance its selective enrichment ability at tumor sites. Summary of the Invention

[0003] In view of this, the present invention provides a boron-labeled compound targeting fibroblast activating protein, its preparation method, and its application. The present invention achieves targeted recognition and binding of fibroblast activating protein (FAP) by efficiently linking a FAP-targeting ligand (FAPI-46 or FAP-2286) to a boron-containing reagent. The boron-labeled compound targeting fibroblast activating protein of the present invention not only possesses excellent tumor targeting properties but also significantly increases the concentration of boron atoms in tumor tissue, thereby enhancing the therapeutic effect of BNCT and showing promising clinical application prospects.

[0004] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a compound with a structural formula as shown in Formula I or Formula II: Formula I; Formula II.

[0005] The compounds of this invention can target tumor tissue, increase the boron content in tumor tissue, and be further used for boron neutron capture therapy.

[0006] The second technical solution of the present invention is a method for preparing the above-mentioned compound, wherein (4-(((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)phenyl)boronic acid and FAP targeting ligand (a compound targeting fibroblast activation protein) are subjected to an acid-amine condensation reaction to obtain the compound; The FAP targeting ligands are FAPI-46 or FAP-2286.

[0007] In a preferred embodiment of the present invention, the molar ratio of (4-(((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)phenyl)boronic acid and the FAP targeting ligand is 1-10.

[0008] In a preferred embodiment of the present invention, the acid-amine condensation reaction is carried out under solvent and catalyst conditions; the solvent is an aqueous solution (salt water) or an organic solvent (N,N-dimethylformamide); the catalyst is N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 4-N,N-dimethylpyridine, 4-pyrrolidinylpyridine or 1-hydroxybenzotriazole.

[0009] In a preferred embodiment of the present invention, the reaction temperature of the acid-amine condensation reaction is -20°C to 80°C, and the reaction time is 0.5 to 48 hours.

[0010] The reaction endpoint monitoring methods used were thin-layer chromatography, high-performance liquid chromatography, or nuclear magnetic resonance analysis.

[0011] The yield of compounds of formula I or II (boron-labeled compounds targeting fibroblast activating proteins) prepared by the method of the present invention is 40%-95%, and the purity is 85%-100%.

[0012] The third technical solution of the present invention is a BNCT boron drug, the active ingredient of which includes the compound shown in Formula I or Formula II above.

[0013] In a preferred embodiment of the present invention, the BNCT boron drug further includes pharmaceutically acceptable excipients.

[0014] The present invention discloses the following technical effects: The compounds synthesized in this invention exhibit better biocompatibility and in vivo stability compared to the original drug, and can remain in the bloodstream for a longer period, further enhancing their accumulation efficiency at tumor sites. Furthermore, the compounds of this invention can significantly increase the proportion of boron in tumor tissue. This novel boron labeling method provides a more efficient and precise solution for BNCT treatment and is expected to play an important role in future targeted cancer therapy. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1This is the HPLC-MS result of compound A.

[0017] Figure 2 For compound A 1 1H NMR results.

[0018] Figure 3 The figure shows the in vivo drug concentration versus time gradient curve for compound A (the legend shows the half-life n=4).

[0019] Figure 4 This is the HPLC-MS result of compound B.

[0020] Figure 5 For compound B 1 1H NMR results.

[0021] Figure 6 The figure shows the in vivo drug concentration versus time gradient curve for compound B (the legend shows the half-life n=4).

[0022] Figure 7 The results of H&E staining imaging of tissue sections from various organs of mice.

[0023] Figure 8 The analysis included routine blood tests and blood biochemical indicators for mice (the P values ​​shown in the figure were obtained through one-way ANOVA, and P < 0.05 was considered statistically significant). Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] This invention provides a method for preparing a boron-labeled compound targeting fibroblast activation proteins (FAPs): First, the target FAP ligand is mixed with a boron-containing reagent in a suitable solvent system, and an appropriate catalyst is added to ensure a homogeneous and stable reaction system. Second, by controlling the reaction temperature and time, the efficient formation of amide bonds is promoted, achieving precise introduction of boron atoms. Finally, the product is purified using column chromatography, high-performance liquid chromatography, or recrystallization to ensure that the obtained amide condensation product has high purity and high yield. Details are as follows: The general reaction formula for the boron-labeled compound targeting fibroblast activation proteins in this invention is as follows:

[0030] FAPI-46 or FAP-2286 was dissolved in DMF, and (4-(((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)phenyl)boronic acid and triethylamine were added. The mixture was reacted at 40°C for 5 h, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by column chromatography and washed with ice-cold diethyl ether. The obtained product was a white solid with a yield of 82%-86% and a purity of over 90% as determined by HPLC. The obtained compound significantly improved the pharmacokinetic characteristics and targeting ability of the original drug, achieving higher boron atom enrichment efficiency at tumor sites.

[0031] Experimental results show that this method exhibits good applicability and reproducibility in all FAP-targeting ligands, and the reaction conditions are mild, making it suitable for large-scale preparation. The obtained product can be used for boron neutron capture therapy targeting tumors, exhibiting significantly enhanced boron atom enrichment capacity and therapeutic potential in tumor tissues. This invention utilizes boron-labeled compounds targeting fibroblast activating protein (FAP) obtained through specific binding, enabling highly directional boron atom aggregation in the tumor microenvironment. Fibroblast activating protein, a target highly expressed in fibroblasts associated with various malignant tumors, can effectively guide the compound to concentrate at the tumor site. Through this specific recognition mechanism, boron atoms can achieve efficient enrichment within tumor tissues, greatly enhancing the local radiation effect in BNCT therapy while reducing damage to surrounding normal tissues.

[0032] The boron-labeled compound targeting fibroblast activating proteins of this invention is not only simple to prepare, but also has high purity and good stability. It exhibits high uptake in tumor models, significantly increasing the boron content in tumor tissues and providing a therapeutic basis for boron neutron capture therapy. This is a novel non-radioactive chemical drug with significant clinical application value.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0035] The (4-(((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)phenyl)boronic acid used in the embodiments of the present invention was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., and FAPI-46 and FAP-2286 were purchased from Nantong Quanyi Biotechnology Co., Ltd.

[0036] Example 1 Drug synthesis: FAPI-46 (100 mg) was dissolved in 5 mL DMF, and 2 molar concentrations of (4-(((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)phenyl)boronic acid and 3 molar concentrations of triethylamine were added. The reaction system was stirred at 40 °C for 5 hours. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure. The residue was purified by column chromatography (eluting agent: ethanol-water (7:3)), and then washed with ice-cold ether to obtain a white solid target product (compound A) with a yield of 84% and a purity of 98.5%. The correct structure was confirmed by HPLC-MS and NMR analysis. Figure 1 and Figure 2 ).

[0037] In vivo pharmacokinetic studies: The obtained product was injected intravenously into Balb / C mice (5 weeks old, male, provided by Jiangsu Jicui Pharmaceutical Co., Ltd.) at a dose of 30 mg / kg. Venous blood was collected at fixed time points, and serum drug concentrations were detected by HPLC-MS (sample size n=4). Results showed that the half-life of compound A in plasma was extended to more than 1.5 times that of the parent drug (FAPI-46). Figure 3 ).

[0038] Tumor model construction: HT1080-FAP cells (purchased from Hunan Fenghui Biotechnology Co., Ltd.) were subcutaneously injected into the right axilla of Balb / c nude mice (5 weeks old, male, provided by Jiangsu Jicui Yaokang) at an inoculation rate of 2 million cells / mouse, resulting in HT1080-FAP tumor model mice (sample size: 4, tumor volume exceeding 50 mm). 3 This indicates that the model was successfully built.

[0039] ICP-MS Detection: Compound A was injected into the mouse model via tail vein at a dose of 30 mg / kg. Three hours later, various tissues and organs were harvested, weighed, and chopped, then digested overnight in nitric acid. The nitric acid was diluted with deionized water to a concentration of 5%, filtered through a 0.22 µm polyethersulfone syringe filter (DiKMA Technologies, Lake Forest, CA), and analyzed using ICP-MS (NexION1000, Perkin Elmer). Boron content (λ=249.677 nm) was analyzed based on a boron standard calibration curve prepared from a 0-250 ppb boron standard solution (Merk). Normalization was performed using the weight of each tissue sample. Results showed that compound A exhibited excellent tumor targeting in the mouse xenograft model, with boron atom enrichment in tumor tissues 5-50 times higher than in other tissues (see Table 1 for boron content analysis results of compound A in various tissues and organs). This finding provides a novel molecular tool for BNCT therapy and has broad clinical application prospects.

[0040] Toxicological parameters were determined: Compound A was administered via tail vein injection to Balb / C mice (5 weeks old, male, provided by Jiangsu Jicui Pharmaceutical Co., Ltd., n=4) at a dose of 100 mg / kg, every two days for one week. After isoflurane anesthesia, blood was collected from the heart and divided into two portions: one without anticoagulant for blood biochemistry analysis; the other anticoagulant with EDTA for routine blood tests. Simultaneously, the liver, kidneys, lungs, and heart of the mice were dissected, prepared into paraffin blocks, and stained with H&E. Histological results showed that compound A did not produce significant tissue toxicity to the liver, kidneys, lungs, and heart of the mice. Figure 7 Blood biochemistry and routine blood tests also showed that, compared with the control group, compound A did not cause toxicity to the above organs or significant blood toxicity. Figure 8 ).

[0041] Example 2: Using FAP-2286 as a prodrug, the product was synthesized under the same reaction conditions as in Example 1. The resulting target product was also a white solid (compound B), with a yield of 86% and a purity of 93%. The correct structure was confirmed by HPLC-MS and NMR analysis. Figure 4 and Figure 5).

[0042] In vivo pharmacokinetic studies: The obtained product was injected intravenously into Balb / C mice (5 weeks old, male, provided by Jiangsu Jicui Pharmaceutical Co., Ltd.) at a dose of 50 mg / kg. Venous blood was collected at fixed time points, and serum drug concentrations were detected by HPLC-MS (sample size n=4). Results showed that the half-life of compound B in plasma was extended to approximately 1.2 times that of the parent drug (FAP-2286). Figure 6 ).

[0043] Tumor model construction: HT1080-FAP cells (purchased from Hunan Fenghui Biotechnology Co., Ltd.) were subcutaneously injected into the right axilla of Balb / c nude mice (5 weeks old, male, provided by Jiangsu Jicui Yaokang) at an inoculation rate of 2 million cells / mouse, resulting in HT1080-FAP tumor model mice (sample size: 4, tumor volume exceeding 50 mm). 3 This indicates that the model was successfully built.

[0044] ICP-MS Detection: Compound B was injected into the mouse model via tail vein at a dose of 50 mg / kg. Three hours later, tissues and organs were harvested, weighed, and minced, then digested overnight in nitric acid. The nitric acid was diluted with deionized water to a concentration of 5%, filtered through a 0.22 µm polyethersulfone syringe filter (DiKMA Technologies, Lake Forest, CA), and analyzed using ICP-MS (NexION1000, Perkin Elmer). Boron content (λ = 249.677 nm) was analyzed based on a boron standard calibration curve prepared from a 0-250 ppb boron standard solution (Merk). Normalization was performed by the weight of each tissue sample. Results showed that compound B exhibited excellent tumor targeting in the mouse xenograft model, with boron enrichment in tumor tissues exceeding that in other tissues by 1-60 times (see Table 2 for boron content analysis results of compound B in various tissues and organs).

[0045] Toxicological parameters were determined: Compound B was administered via tail vein injection to Balb / C mice (5 weeks old, male, provided by Jiangsu Jicui Pharmaceutical Co., Ltd., n=4) at a dose of 100 mg / kg, every two days for one week. After isoflurane anesthesia, blood was collected from the heart and divided into two portions: one without anticoagulant for blood biochemistry analysis; the other anticoagulant with EDTA for routine blood tests. Simultaneously, the liver, kidneys, lungs, and heart of the mice were dissected, prepared into paraffin blocks, and stained with H&E. Histological results showed that compound A did not produce significant tissue toxicity to the liver, kidneys, lungs, and heart of the mice. Figure 7Blood biochemistry and routine blood tests also showed that, compared with the control group, compound A did not cause toxicity to the above organs or significant blood toxicity. Figure 8 ).

[0046] Table 1. Results of boron content analysis of compound A in various tissues and organs.

[0047] The data in Table 1 are in ng / mg and are presented as mean ± SD, n=4. Table 2. Results of boron content analysis of compound B in various tissues and organs.

[0048] The data in Table 2 are in ng / mg and are presented as mean ± SD, n=4. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound having the structural formula shown in Formula I or Formula II: Formula I; Formula II.

2. A method for preparing the compound according to claim 1, characterized in that, (4-(((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)phenyl)boronic acid and FAP targeting ligand were subjected to an acid-amine condensation reaction to obtain a boron-labeled compound targeting fibroblast activation protein. The FAP targeting ligands are FAPI-46 or FAP-2286.

3. The preparation method according to claim 2, characterized in that, The molar ratio of (4-(((2,5-dioxopyrrolidone-1-yl)oxy)carbonyl)phenyl)boronic acid to FAP targeting ligand is 1-10.

4. The preparation method according to claim 2, characterized in that, The acid-amine condensation reaction is carried out under solvent and catalyst conditions; the solvent is an aqueous solution or an organic solvent; the catalyst is N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 4-N,N-dimethylpyridine, 4-pyrrolidinylpyridine or 1-hydroxybenzotriazole.

5. The preparation method according to claim 2, characterized in that, The reaction temperature for acid-amine condensation is -20℃ to 80℃, and the reaction time is 0.5 to 48 hours.

6. A BNCT boron drug, characterized in that, The active ingredient includes the compound described in claim 1.

7. The BNCT boron drug according to claim 6, characterized in that, It also includes pharmaceutically acceptable excipients.

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