Boron-containing compound containing tumor targeting structure as well as preparation method and application of boron-containing compound

Abstract

The invention discloses a boron-containing compound containing a tumor targeting structure as shown in a general formula I and a preparation method and application of the boron-containing compound. The boron source carrier of the tumor targeting structure is provided on the basis of a monosaccharide analogue mother nucleus, and the boron-containing compound containing the tumor targeting structure is easy to modify and transform and has good biological adaptability and targeting capacity. The preparation method is simple and easy to modify and transform. The research on the uptake effect of the compound on tumor cells and normal cells finds that the compound has an obvious targeting effect on triple negative breast cancer cells. In addition, the compound provided by the invention has small damage to normal cells under a certain concentration, and provides a valuable reference for clinical potential boron drugs of BNCT.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a boron-containing compound containing a tumor targeting structure, and a preparation method and application thereof. Background Art

[0002] Boron Neutron Capture Therapy (BNCT) is an advanced radiotherapy technology that combines targeted therapy and heavy ion therapy. It was first proposed by Taylor and Goldhaber in 1935. Its basic principle is as follows: First, a neutron with tumor affinity is injected into the 10 Drug B is injected into the patient's body. After the boron drug selectively accumulates in the tumor cells, it is irradiated with thermal neutrons or epithermal neutrons from outside the patient's body. The neutrons are 10 B capture occurs 10 B(n,α) 7 Li reaction. Due to the product α particles and 7 The range of Li particles in biological tissues is 5μm-9μm, which is similar to the diameter of cells. They have the characteristics of high linear energy transfer (LET) and can effectively kill tumor cells. On the other hand, 10 B(n,α) 7 The Li reaction cross section is 3837 barns, which is much higher than the reaction cross section between neutrons and common elements in the human body. 10 Radiation damage to normal tissues of B can be controlled at a safe dose level.

[0003] Furthermore, because the path length of this reaction in water or biological tissue is limited to 4.5-10 μm, roughly equivalent to the diameter of a single cell, this property enables selective elimination of tumor cells while minimizing damage to normal cells. In theory, BNCT can achieve precise targeted therapy by ensuring that boron compounds are fully and accurately delivered to tumor tissue.

[0004] Currently, the drugs used in clinical BNCT treatment are 4-dihydroxyborylphenylalanine (BPA) and sodium mercaptoundec-ahydrododecaborate (BSH). 4-dihydroxyborylphenylalanine (BPA) has minimal cytotoxicity and enhanced tumor targeting, reducing neutron irradiation damage to normal tissues. However, it has poor solubility, contains only one boron atom per molecule, and its boronic acid group is unstable, susceptible to oxidation in the presence of reactive oxygen species in the body, resulting in a decrease in boron concentration at the tumor site. Another boron-containing drug, BSH, has a higher boron concentration and good water solubility, but lacks receptor-mediated tumor selectivity, resulting in a low tumor-to-blood ratio and significant side effects in clinical treatment. Furthermore, BSH is expensive, leading to high treatment costs.

[0005] In general, the new generation of boron-containing drugs should have the following characteristics: ① continuous high accumulation concentration in tumor sites; ② low concentration in normal tissues and rapid metabolism (T / N>3); ③ good biocompatibility and non-toxicity; ④ good thermal stability and chemical stability; ⑤ low cost, simple synthesis, and environmentally friendly preparation process.

[0006] Numerous studies have demonstrated that upregulation of endogenous lectin genes on the surface of tumor cells promotes their carbohydrate uptake. Furthermore, unlike the aerobic metabolism of normal cells, tumor cells metabolize via inefficient anoxic glycolysis, a fermentative form of glucose metabolism commonly known as the Warburg effect. This phenomenon promotes the massive uptake of glucose by tumor cells. Therefore, the synthesis of carbohydrate-containing boron-containing drugs to target tumor cells is of great significance. Summary of the Invention

[0007] Purpose of the Invention: This invention addresses the shortcomings of existing BNCT clinical drugs by providing a boron-containing compound with a tumor-targeting structure, its preparation method, and its application. Based on a monosaccharide analog nucleus, this invention provides a boron-source carrier for the tumor-targeting structure. The boron-containing compound with a tumor-targeting structure provided by this invention is easy to modify and engineer, and exhibits excellent biocompatibility and targeting capabilities.

[0008] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0009] The present invention provides a boron-containing compound having a tumor-targeting structure and having a general formula I:

[0010]

[0011] in,

[0012] R is selected from any one of the following structures:

[0013]

[0014] A preferred embodiment of the present invention is that the R is a propane chain.

[0015] The compounds of the general formula I of the present invention are preferably the following compounds:

[0016]

[0017] The present invention also provides a method for preparing a boron-containing compound containing a tumor-targeting structure, comprising introducing an alkynyl group into L-ascorbic acid via propyne bromide, introducing an azide group containing different R groups into mercapto-o-carborane via a bromination reaction, and then subjecting the two to an azide-alkyne cycloaddition reaction catalyzed by active copper to produce the boron-containing compound containing a tumor-targeting structure.

[0018] wherein R is as defined in Formula I.

[0019] The present invention provides a method for preparing a boron-containing compound preferably containing a tumor-targeting structure, comprising the following steps:

[0020] (1) reacting L-ascorbic acid with 3-bromopropyne under heating conditions to obtain compound 2;

[0021]

[0022] (2) reacting compound 3 with an azide group containing different R groups at room temperature to obtain compound 4;

[0023]

[0024] (3) reacting compound 2 with compound 4 at room temperature to obtain compound 5 of formula I;

[0025]

[0026] wherein R is as defined in Formula I.

[0027] The compounds of the general formula I of the present invention can be prepared using the above or similar preparation methods, and the corresponding starting materials can be selected according to the different substituents and the different positions of the substituents. It should be appreciated by those skilled in the art that the above routes are helpful for understanding the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the variables are defined as mentioned in the general formula I.

[0028] Specifically, the present invention also provides a method for preparing a boron-containing compound CSH-DHA (abbreviated as CND) containing a tumor targeting structure, wherein the monosaccharide analog DHA is introduced into propyne bromide to obtain a targeting group compound that is easy to modify.

[0029] Glucose transporter isoform 1 (GLUT1) is responsible for glucose transport in the body. Tumors consume large amounts of glucose and highly express the GLUT1 transporter protein. Therefore, structural modification of boron drugs using carbohydrate derivatives can effectively improve the efficacy of BNCT through active tumor uptake. Dehydroascorbic acid (DHA) has been reported in the literature as a substrate for GLUT1 protein, with a structure similar to glucose. Through preliminary screening, the present invention selected DHA as a carrier with the best tumor-targeting effect, and 1-mercapto-o-carborane as a boron source, to produce the target product through a click reaction. The present invention synthesizes boron-containing targeting compounds (CND) with excellent tumor-targeting effects using a simple and efficient method, which has great potential for clinical application. Therefore, the boron-containing compounds of the present invention with tumor-targeting structures can serve as candidates for boron neutron capture therapy.

[0030] Through preliminary cell experiments, the optimal glucose-targeting moiety, DHA, was identified. This moiety was then coupled to a high-boron-loading thiol-o-carborane via click chemistry, yielding a novel boron-containing drug structure targeting the GLUT1 protein. Preliminary biological studies have demonstrated low cytotoxicity and significantly higher boron uptake in tumor cells than the currently available clinical drugs, BPA and BSH. In mice with 4T1 subcutaneous tumors, 12 hours after intravenous injection, the tumor-to-blood boron concentration ratio approached 3, demonstrating broad potential for BNCT-based breast cancer treatment. This provides a valuable reference for potential clinical boron-based drugs in BNCT.

[0031] The present invention not only provides a new approach to solving the problem of precise targeting of clinical boron-rich drugs, but also achieves tumor targeting of boron-rich drugs through the introduction of monosaccharide targeting groups. The compound is easy to modify and transform, has good biological adaptability, can achieve more efficient boron drug accumulation effects, more precise and efficient treatment, and can further promote the application of the compound of the present invention in the field of BNCT.

[0032] The present invention also provides a pharmaceutical composition comprising a boron-containing compound having a tumor-targeting structure and having a structure of general formula I and a pharmaceutically acceptable carrier or excipient.

[0033] The compounds of the present invention can be combined with pharmaceutically acceptable carriers or excipients to form formulations for administration. For example, solvents, diluents, sprays, etc. Various dosage forms of the pharmaceutical compositions of the present invention can be prepared according to methods well known in the pharmaceutical art. These pharmaceutical formulations can contain, for example, 0.05% to 90% by weight of the active ingredient, more typically about 15% to 60% by weight of the active ingredient, in combination with the carrier. Doses of the compounds of the present invention can range from 0.005 to 5000 mg / kg / day, although doses outside this range may be used depending on the severity of the disease or the dosage form.

[0034] The present invention also provides the use of the boron-containing compound containing a tumor targeting structure in the preparation of a drug for treating targeted tumor cells.

[0035] The boron-containing compound is an integrated compound of monosaccharide analogue DHA and a boron source.

[0036] The present invention also provides the use of the boron-containing compound containing a tumor targeting structure in the preparation of a BNCT drug, wherein the drug has good tumor targeting properties.

[0037] The boron-containing compound containing a tumor-targeting structure of the present invention has low cytotoxicity in both tumor cells 4T1 and normal cells HUVEC.

[0038] The boron-containing compound containing a tumor-targeting structure of the present invention can be efficiently taken up after being incubated with tumor cells and normal cells.

[0039] Beneficial effects:

[0040] This invention leverages the results of previous screening of boron-containing compounds with tumor-targeting structures, selects the monosaccharide analog DHA, which has the best targeting effect, as the tumor-targeting group, and 1-mercapto-o-carborane as the boron source, to construct a boron-rich compound CND containing a DHA targeting group with tumor-targeting effect. The preparation method of the present invention is simple and easy to modify and transform. Studies of its uptake effects on tumor cells and normal cells revealed that this class of compounds has a significant targeting effect on triple-negative breast cancer cells. Furthermore, at certain concentrations, the compounds of the present invention cause minimal damage to normal cells, providing a valuable reference for potential clinical boron drugs for BNCT. This may further advance the clinical application of the compounds of the present invention in BNCTs for skin cancer, breast cancer, osteosarcoma, and other cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the compound DHA-1 in Example 1.

[0042] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the compound CSH-N3 in Example 1.

[0043] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of compound CND in Example 1.

[0044] Figure 4 This is the carbon NMR spectrum of compound CND in Example 1.

[0045] Figure 5 This is the high-resolution mass spectrum of compound CND in Example 1.

[0046] Figure 6 Cell viability after incubation of the compound CND of the present invention with normal cells HUVEC and triple-negative breast cancer cells 4T1 for 24 hours at different concentrations.

[0047] Figure 7 It is the boron uptake amount after the compound CND of the present invention is incubated with normal cells HUVEC and triple-negative breast cancer cells 4T1 at a certain concentration for 12 hours. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0049] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0050] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0051] Example 1 Synthesis of Boron-Containing Compound CSH-DHA Containing DHA Targeting Structure

[0052] (1) Synthesis of carbohydrate targeting group DHA-1

[0053]

[0054] KOH (0.953 g, 17 mmol) was weighed and dissolved in 60 ml of deionized water. LA ascorbic acid (3 g, 17 mmol) was added with stirring and allowed to react at room temperature under nitrogen for 1 h. Subsequently, 3.03 ml of propyne bromide was dissolved in 120 ml of acetone and reacted at 40°C for 48 h. After completion of the reaction, the crude product was purified by silica gel column chromatography (DCM:MeOH = 30:1, v / v) to obtain the white viscous compound DHA-1 (3.2 g, 88% yield).

[0055] Its H NMR spectrum is shown in Figure 1 .

[0056] 1 H NMR(400MHz,DMSO-d6)δ(ppm)5.10(dd,J=15.5,2.4Hz,1H),4.98(dd,J=15.5,2.5Hz,1H),4.53(s,1H),4.27(dd,J=6.2,4.2Hz,1H),4.12(dd,J=9 .5,6.2Hz,1H),3.85(dd,J=9.4,4.3Hz,1H),3.68-3.61(m,1H),2.93(t,J=2.7Hz,1H),2.66(dd,J=16.4,2.7Hz,1H),2.47(dd,J=16.4,2.8Hz,1H).

[0057] (2) Synthesis of compound CSH-N3

[0058]

[0059] 1-Mercapto-o-carborane CSH (100 mg, 0.57 mmol), 1-azido-3-bromopropane (94 mg, 0.57 mmol), potassium bicarbonate (57 mg, 0.57 mmol), and potassium iodide (141 mg, 0.85 mmol) were dissolved in 20 ml of MeCN. The mixture was refluxed at 80°C under nitrogen for 30 hours. The reaction flask was removed and filtered while hot to remove insoluble impurities. The filtrate was neutralized with dilute hydrochloric acid to a pH of approximately 7. The neutralized filtrate was extracted with dichloromethane and saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, and then spin-dried to dryness. The resulting compound, CSH-N3, was obtained as a pale yellow oil (100 mg, 70% yield).

[0060] The H NMR spectrum of compound CSH-N3 is shown in Figure 2 .

[0061] 1 H NMR (400MHz, Acetone-d6) δ (ppm) 4.84 (s, 1H), 3.51 (t, J = 6.5Hz, 2H), 3.13 (t, J = 7.3Hz, 2H), 1.96-1.90 (m, 2H).

[0062] (3) Synthesis of DHA-targeted boron drug CSH-DHA (abbreviated as CND)

[0063]

[0064] Compound CSH-N3 (100 mg, 0.4 mmol) was weighed and dissolved in 20 ml of a mixture of dichloromethane and methanol (DCM:MeOH = 1:1, v / v). TBTA (42 mg, 0.08 mmol) and Cu(CNCH3)4PF6 (30 mg, 0.08 mmol) were then added. The mixture was stirred under nitrogen for ten minutes, and DHA-1 (171 mg, 0.8 mmol) was added. The reaction was stirred at room temperature overnight. The next day, the starting material spot disappeared after plate scanning. The solvent was removed on a rotary evaporator, and the crude product was purified by silica gel column chromatography (DCM:MeOH = 30:1, v / v) to obtain CSH-DHA (81 mg, 50% yield) as a white solid powder.

[0065] The H NMR spectrum of compound CSH-DHA is shown in Figure 3 , see C NMR spectrum Figure 4 , mass spectrometry see Figure 5 .

[0066] ESI-MS (m / z): 496.25 [M+Na] + .

[0067] 1 H NMR (400MHz, Methanol-d4) δ (ppm) 8.08 (s, 1H), 5.64-5.55 (m, 2H), 4.76 (d, J = 1.6Hz, 1H), 4.67 (s, 1H), 4.49 (t, J = 6 .7Hz,2H),3.81(q,J=7.9,6.9Hz,1H),3.63-3.58(m,2H),3.32(s,3H),2.94(t,J=7.3Hz,2H),2.19(p,J=7.1Hz,3H).

[0068] 13 C NMR(126MHz,Methanol-d4)δ(ppm)149.56,124.69,120.18,75.44,69.09,68.45,63.45 ,62.06,48.43,48.16,47.99,47.82,47.65,47.48,47.31,47.14,33.34,29.37,28.87.

[0069] Example 3 Cytotoxicity test of compound CND of the present invention

[0070] To verify the biocompatibility of boron-containing compounds with tumor-targeting moieties at the cellular level, we evaluated in vitro cytotoxicity in two cell lines (breast cancer 4T1 cells and umbilical vein endothelial cells (HUVECs)) using the standard CCK-8 assay. Both cell lines were purchased from the American Type Culture Collection (ATCC).

[0071] The specific experimental steps are as follows:

[0072] 1. Collect cells in the logarithmic phase and adjust the concentration of the cell suspension. Add 100 μL to each well. When plating, adjust the cell density to 1000-10000 / well (fill the edge wells with sterile PBS). Incubate in a 5% CO2, 37°C normoxic incubator for 24 hours until the cell monolayer covers the bottom of the well (96-well plate). Then start adding drugs.

[0073] 2. Completely dissolve the boron-containing compound CND in DMSO and dilute it with DMEM medium containing 10% serum to a concentration gradient of 0, 5, 25, 50, 100, 250, and 500 μM. Add 100 μL to each well and incubate in a 5% CO2, 37°C normoxic incubator for 24 hours. Observe under an inverted microscope.

[0074] 3. Add 10 μL of CCK-8 solution (purchased from Biyuntian Biotechnology Co., Ltd.) to each well and continue incubation for 2 hours;

[0075] 4. Terminate the culture and measure the absorbance of each well at 450 nm using an enzyme-linked immunosorbent assay (ELISA) in the dark.

[0076] The following formula was used to calculate cell viability:

[0077] Viability (%)=(average absorbance of test wells-average absorbance of culture medium control wells) / (average absorbance of untreated wells-average absorbance of culture medium control wells)×100%.

[0078] Figure 6 The results of the cytotoxicity evaluation experiment of the compound of the present invention show that the compound CND has an effect on tumor cells ( Figure 6 A) and normal cells ( Figure 6 B) showed low toxicity. When the concentration of compound CND was lower than 150 μM, the survival rate of both cells was above 80%, and when the concentration of compound was as high as 500 μM, the cell survival rate was still above 75%. Figure 6 ). This indicates that the boron-containing compound with a tumor-targeting structure has good biological adaptability, and the potential of compound CND in the field of BNCT can be further explored.

[0079] Example 4 Boron uptake experiment of the compound CND of the present invention in different cells.

[0080] The boron uptake experiment of the present invention was conducted in breast cancer cells 4T1 and endothelial cells HUVEC. The concentration of compound CND was selected to be an appropriate concentration below the safety concentration in the previous cell evaluation, and was determined to be 150 μM.

[0081] Breast cancer cells 4T1 and endothelial cells HUVEC were seeded into six-well cell culture plates (1×10 6 / well) and then incubated in a 5% CO2, 37°C normoxic incubator for 24 hours. The original DMEM medium containing 10% fetal bovine serum (final concentration of 300 μM) was replaced with an equivalent culture medium containing each boron-containing derivative. The cells were kept in a 5% CO2, 37°C normoxic incubator for 24 hours, the culture medium was aspirated, and washed 3 times with PBS. They were then digested by trypsin and counted using a hemocytometer. All samples (in 1 mL of pure water) were added to a mixture of perchloric acid (60%, 0.6 mL) and hydrogen peroxide (31%, 0.3 mL) and ultrasonically crushed at 80°C for 4 hours. The mixture was then filtered through a 0.45 μm microporous filter membrane, and the filtrates were combined and added to a volumetric flask and accurately determined to 10 mL. The boron concentration was then measured by inductively coupled plasma emission spectrometry (ICP-MS).

[0082] The formula for calculating cellular boron uptake is as follows:

[0083] Intake (μg / 10 6 cells) = (average boron concentration of the test × volume of the test solution on the machine) / cell count value of the well

[0084] like Figure 7 As shown in A, both tumor cells and normal cells have a high boron uptake, that is, the boron-containing compound with tumor-targeting structure can effectively enter the cell through the sugar transporter on the cell membrane. The boron uptake values ​​of tumor cells and normal cells in 12 hours are both 100ng B / 10 6 cells, far higher than the 10 required for treatment 9 B atoms / single cell. And there is a significant difference in the boron uptake between tumor cells and normal cells at the same time ( Figure 7 B), indicating that tumor cells have significantly higher boron uptake than normal cells. This indicates that the carbohydrate-targeting compound CND exhibits good targeting ability in in vitro testing and is expected to become a new generation of boron-containing therapeutic drugs.

[0085] Since the boron-containing compound containing a tumor-targeting structure and having the general formula I provided by the present invention also contains a monosaccharide analogue core and a boron source, it also has tumor-targeting properties and boron accumulation capabilities.

[0086] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A boron-containing compound having a tumor-targeting structure and having the general formula I or a pharmaceutically acceptable salt thereof: in, R is selected from any one of the following structures:

2. The boron-containing compound containing a tumor targeting structure according to claim 1, characterized in that: The R is located at the connection between the boron source and the carbohydrate targeting group.

3. The compound according to claim 1, characterized in that Selected from:

4. A method for preparing the boron-containing compound containing a tumor targeting structure according to claim 1, characterized in that: An alkynyl group is introduced into L-ascorbic acid by propyne bromide, and an azide group containing different R groups is introduced into the mercapto-o-carborane through a bromination reaction. The two undergo an azide-alkyne cycloaddition reaction under the catalysis of active copper to generate the boron-containing compound containing the tumor-targeting structure; wherein R is as defined in Formula I.

5. The preparation method according to claim 4, characterized in that The steps include: (1) reacting L-ascorbic acid with 3-bromopropyne under heating conditions to obtain compound 2; (2) reacting compound 3 with an azide group containing different R groups at room temperature to obtain compound 4; (3) reacting compound 22 with compound 4 at room temperature to obtain compound 5 of formula I; wherein R is as defined in Formula I.

6. A pharmaceutical composition, characterized in that The invention comprises the boron-containing compound containing a tumor targeting structure according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier or excipient.

7. Use of the boron-containing compound containing a tumor targeting structure according to any one of claims 1 to 3 in the preparation of a drug for treating targeted tumor cells.

8. The use according to claim 7, characterized in that The boron-containing compound is an integrated compound of monosaccharide analogue DHA and a boron source.

9. Use of the boron-containing compound containing a tumor-targeting structure according to any one of claims 1 to 3 in the preparation of BNCT drugs.

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