Amino acid amine-borane compounds, their preparation methods and applications

Abstract

This invention discloses an amino acid amine-borane compound, its preparation method, and its applications, belonging to the field of tumor therapeutic drug technology. The amino acid amine-borane compound shown in formula (I) is obtained through the protection of functional groups in amino acids, borane complexation, and deprotection reactions. Cellular experiments show that tumor cells have a much higher uptake capacity for this compound than the boron neutron capture clinical therapeutic drug 4-dihydroxyboron-L-phenylalanine (BPA), and no signs of toxicity were observed, indicating its significant potential as a novel boron delivery agent for boron neutron capture therapy.

Description

Technical Field

[0001] This invention belongs to the field of tumor therapeutic drug technology, specifically relating to amino acid amine-borane compounds, their preparation methods, and applications. Background Technology

[0002] Malignant tumors are among the most serious diseases threatening human life and health. The development of highly efficient diagnostic and treatment technologies for cancer is in high demand, and significant progress has been made in the treatment of malignant tumors, including surgery, drug therapy, radiotherapy, and immunotherapy. Among these, approximately 70% of cancer patients choose radiotherapy each year. With the development of modern medical technology, radiotherapy techniques are constantly evolving, progressing from non-selective radiotherapy to proton and heavy ion radiotherapy with a certain degree of longitudinal energy selectivity. Despite this, high-grade gliomas and other malignant tumors still exhibit strong resistance to current treatments. As a cell-level precise binary targeted tumor therapy technology, boron neutron capture therapy (BNCT) combines biological targeting with the effect of heavy ions to selectively and precisely kill cancer cells at the cellular level, requiring only 1-2 irradiations. It has significant advantages for treating advanced malignant tumors that are difficult to operate on and cannot be treated with traditional radiotherapy and chemotherapy. BNCT will carry... 10 B-targeted drugs are injected into the patient's body. 10 B will specifically accumulate in tumor tissue, and the affected area will be irradiated with hyperthermic neutrons. The neutrons and 10 B occurred 10 B(n,α) 7 Li nuclear reactions form its isotopes. 11 B. Subsequently. 11 B-cell fission produces high energy. 7 Li ions and alpha particles have a range of only about one cell diameter. Cancer cells are precisely killed because their DNA undergoes irreversible breakage under the action of heavy ions, while normal cells are basically undamaged.

[0003] Currently, boron-dependent neuron-mediated neutron transplantation (BNCT) is experiencing explosive growth, with neutron sources shifting from reactors to more economical, safe, and reliable accelerator neutron sources, achieving a key technological breakthrough in neutron beam acquisition. However, the development of boron-targeted drugs for BNCT still faces challenges. Currently, only 4-dihydroxyboron-L-phenylalanine (BPA) and sodium undecylmercaptododecoboride (BSH) are used clinically, with BPA being the only marketed clinical treatment globally. However, BPA suffers from insufficient tumor specificity, short blood half-life and retention time, and extremely poor solubility, resulting in low boron atom enrichment in tumor tissue. Clinically, BPA must be excipiented with fructose to improve water solubility and requires large-volume intravenous injection, causing significant nephrotoxic side effects and severely impacting the efficacy of BNCT. Therefore, developing a class of small-molecule organoboron compounds with economical and efficient synthesis processes, readily available raw materials, good water solubility, and potentially broad tumor targeting as candidate drug molecules is crucial for improving the clinical efficacy of BNCT. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide amino acid amine-borane compounds, their preparation methods and applications, so as to solve the technical problems of existing BNCT drugs, such as difficulty in synthesis, poor water solubility and insufficient tumor targeting.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] The first aspect of the present invention discloses amino acid amine-borane compounds having compounds of formula (I) or pharmaceutically acceptable salts thereof, as well as stereoisomers, isotope products, and derivatives of said compound (I) or pharmaceutically acceptable salts thereof:

[0007]

[0008] Wherein, R is a hydrogen atom, alkyl, aryl, heteroaryl, alkyl containing a substituent, aryl containing a substituent, or heteroaryl containing a substituent; R′ is a hydrogen atom, alkyl, or alkyl containing a substituent.

[0009] A second aspect of the present invention discloses a method for preparing the above-mentioned amino acid amine-borane compound, comprising the following steps:

[0010] 1) Dissolve amino acids and N,O-bis(trimethylsilyl)acetamide in an organic solvent and react to obtain silyl-protected amino acids;

[0011] 2) Add the borane complex to the reaction mixture of step 1 to obtain an amino acid amine-borane compound.

[0012] A third aspect of the present invention discloses the use of the above-mentioned amino acid amine-borane compound in the preparation of pharmaceutical formulations for BNCT.

[0013] Preferably, the BNCT drug formulation is a drug or formulation for treating tumors, or a drug or formulation for inhibiting the progression of tumors.

[0014] More preferably, the drug or preparation that inhibits the progression of tumors is a drug that inhibits the progression of malignant or metastatic tumors.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The amino acid amine-borane compound provided by this invention has the structure shown in formula (I). In this structure, the amino group of the amino acid has a lone pair of electrons, and the boron atom on the borane has an empty orbital. The lone pair of electrons on the amino group readily coordinates with the boron atom to form an amine-borane product. By protecting the carboxyl functional group in the amino acid, a silicon-protected intermediate is obtained to reduce the polarity of the amino acid molecule. Then, by coordinating the amino group with the borane, an amine-borane derivative is obtained, and then the amino acid amine-borane product is successfully obtained after deprotection. Because this structure is similar to that of the amino acid, it can be transported into tumor cells through amino acid transport proteins on the cell membrane surface. The preparation method of the entire compound is simple and low-cost, and it is suitable for the synthesis of various amino acids and amino acid structural analogs. Cell experiments show that the uptake capacity of this compound by tumor cells is much higher than that of the boron neutron capture clinical therapeutic drug BPA, and no signs of toxicity were observed, indicating that it has important potential as a novel boron delivery agent for boron neutron capture therapy. Attached Figure Description

[0017] Figure 1 The phenylalanine amine-borane of Example 1 1 H NMR spectrum;

[0018] Figure 2 The phenylalanine amine-borane of Example 1 13 C NMR spectrum;

[0019] Figure 3 The phenylalanine amine-borane of Example 1 11 B NMR spectrum;

[0020] Figure 4 Tryptophanamine-borane, as described in Example 2 1 H NMR spectrum;

[0021] Figure 5 Tryptophanamine-borane, as described in Example 2 13 C NMR spectrum;

[0022] Figure 6 Tryptophanamine-borane, as described in Example 2 11 B NMR spectrum.

[0023] Figure 7 Methionine amine-borane, as described in Example 3 1 H NMR spectrum;

[0024] Figure 8 Methionine amine-borane, as described in Example 3 13 C NMR spectrum;

[0025] Figure 9 Methionine amine-borane, as described in Example 3 11 B NMR spectrum;

[0026] Figure 10 Threonine amine-borane, as described in Example 4 1 H NMR spectrum;

[0027] Figure 11 Threonine amine-borane, as described in Example 4 13 C NMR spectrum;

[0028] Figure 12 Threonine amine-borane, as described in Example 4 11 B NMR spectrum;

[0029] Figure 13 Isoleucine amine-borane, as described in Example 5 1 H NMR spectrum;

[0030] Figure 14 Isoleucine amine-borane, as described in Example 5 13 C NMR spectrum;

[0031] Figure 15 Isoleucine amine-borane, as described in Example 5 11 B NMR spectrum;

[0032] Figure 16 The figure shows the cytotoxicity test results of compound 1 of the present invention;

[0033] Figure 17 The figure shows the cytotoxicity test results of compound 2 of the present invention;

[0034] Figure 18 The figure shows the cytotoxicity test results of compound 3 of the present invention;

[0035] Figure 19 The figure shows the cytotoxicity test results of compound 4 of the present invention;

[0036] Figure 20 The figure shows the cytotoxicity test results of compound 5 of the present invention;

[0037] Figure 21The figure shows the cytotoxicity test results of compound 6 of the present invention;

[0038] Figure 22 The figure shows the cytotoxicity test results of compound 7 of the present invention;

[0039] Figure 23 The figure shows the cytotoxicity test results of compound 8 of the present invention;

[0040] Figure 24 The figure shows the experimental results of boron atom uptake of compounds 1-7 of the present invention in 4T1 cells. Detailed Implementation

[0041] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0042] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0043] In this text, the term "boron neutron capture therapy" (BNCT) refers to a tumor treatment method comprising the steps of administering a boron-containing compound to the subject requiring treatment and irradiating the subject with thermal neutrons. The term "tumor" refers to the uncontrolled and progressive growth of tissue with increasing cell proliferation. Uncontrolled proliferation is a state different from normal cell proliferation, such as a state of significantly increased cell proliferation rate. The term "progressive" refers to strong progression or increase. The term "tumor cell" refers to cells in histology. The term "tumor treatment" refers to the treatment of diseases caused by or related to tumors. Unless otherwise specified, "→" in formula (I) represents a coordination bond.

[0044] In a first aspect, the present invention provides an amino acid amine-borane compound with the structure shown in formula (I):

[0045]

[0046] Wherein, R is a hydrogen atom, alkyl, aryl, heteroaryl, alkyl containing a substituent, aryl containing a substituent, or heteroaryl containing a substituent; R′ is a hydrogen atom, alkyl, or alkyl containing a substituent; alkyl or alkyl containing a substituent includes, but is not limited to, hydrogen atoms, alkyl, or alkyl containing a substituent.

[0047] Aryl or aryl groups containing substituents include, but are not limited to, aryl groups. Heteroaryl groups or heteroaryl groups containing substituents include, but are not limited to, those containing...

[0048]

[0049] Preferably, the pharmaceutically acceptable salt of the amino acid amine-borane compound is its ammonium salt, lithium salt, sodium salt, potassium salt, rubidium salt, cesium salt, magnesium salt, or calcium salt.

[0050] Preferably, all enantiomers of the amino acid amine-borane compound or a pharmaceutically acceptable salt thereof, and mixtures of enantiomers in any proportion.

[0051] Preferably, the isotopic product of the enantiomer of the amino acid amine-borane compound or its pharmaceutically acceptable salt contains... 10 B 11 B 12 C 13 C 18 O or 2 H atom.

[0052] A second aspect of the present invention provides a method for preparing the above-mentioned amino acid amine-borane compound, comprising the following steps:

[0053]

[0054] (1) Dissolve natural or non-natural amino acids or N-substituted amino acids (1.0 equivalent) and N,O-bis(trimethylsilyl)acetamide (BSA; 1.0-5.0 equivalent) in an organic solvent and react to obtain silicon-protected amino acid derivatives;

[0055] N,O-bis(trimethylsilyl)acetamide is used as a carboxyl group protecting agent.

[0056]

[0057] (2) Add borane complex (1.0 to 5.0 equivalents) to a solution of silicon-protected amino acid derivatives. After the reaction is complete, remove the silicon-protecting group to obtain an amino acid amine-borane compound.

[0058] A third aspect of the present invention provides the use of the above-mentioned amino acid amine-borane compound in the preparation of a pharmaceutical formulation for BNCT, which can be used to treat various types of tumors or to inhibit the progression of tumors.

[0059] Wherein, the boron neutron capture therapy drug is a drug for treating tumors or a drug for inhibiting the development of tumors; the tumor includes, but is not limited to, malignant tumors; the malignant tumors include, but are not limited to, glioblastoma multiforme, malignant meningioma, intramedullary spinal cord glioma, advanced or recurrent head and neck cancer, thyroid cancer, malignant melanoma, recurrent breast cancer, metastatic liver cancer, malignant brain tumor, osteosarcoma, lung cancer, squamous cell carcinoma of the skin or nasopharyngeal carcinoma.

[0060] The amino acid amine-borane compound can be directly dissolved in physiological saline, phosphate buffer, sterile water or glucose solution to prepare boron drug preparations for infusion.

[0061] The amino acid amine-borane compound requires no excipients, and its water solubility is more than 35 times that of commercially available BPA boron drugs, while the boron atom uptake by tumor cells is 5 to 26 times that of commercially available BPA boron drugs.

[0062] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0063] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0064] I. Preparation of Amino Amine-Boronane Compounds

[0065] Example 1: Preparation of phenylalanine amine-borane

[0066]

[0067] Phenylalanine (165.2 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (306.6 mg, 1.5 mmol) were dissolved in 1,4-dioxane (2 mL) and stirred at 10 °C for 24 hours. Borane dimethyl sulfide complex (1 M, 1 mL, 1.0 mmol) was added to the reaction mixture and stirring continued for 24 hours. After the reaction was complete, 2 mL of water was added to quench the reaction, and phenylalanine amine-borane (compound 1) was obtained. The NMR spectrum is shown below. Figures 1-3 As shown.

[0068] 1H NMR (400MHz, D2O) δ7.42–7.23(m,5H),3.38(t,J=6.5Hz,1H),3.05–2.99(m,2H),1.43(s,3H). 13 C NMR (101MHz, D2O) δ177.9,139.0,133.3,133.1,131.6,69.0,40.3. 11 BNMR (128MHz, D2O) δ-21.3.

[0069] Example 2: Preparation of tryptophanamine-borane

[0070]

[0071] Tryptophan (204.2 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (613.2 mg, 3.0 mmol) were dissolved in chloroform (3 mL) and stirred at 20 °C for 36 hours. Boranetetrahydrofuran complex (1 M, 2 mL, 2.0 mmol) was added to the reaction mixture and stirring continued for 24 hours. After the reaction was complete, 2 mL of methanol was added to quench the reaction, and tryptophan amine-borane (compound 2) was isolated. The NMR spectrum is shown below. Figures 4-6 As shown.

[0072] 1 H NMR(400MHz,D2O)δ7.71(d,J=8.0Hz,1H),7.49(d,J=8.2Hz,1H),7.31–7.20(m,2H),7.16(q,J=7 .5Hz,1H),3.46(t,J=6.2Hz,1H),3.36–3.28(m,1H),3.17(dd,J=14.9,7.0Hz,1H),1.26(s,3H). 13 C NMR (101MHz, D2O) δ178.5,140.3,130.6,129.0,126.1,123.4,122.4,115.9,111.55,59.0,30.35. 11 B NMR (128MHz, D2O) δ-22.2.

[0073] Example 3: Preparation of methionine amine-borane

[0074]

[0075] Methionine (149.2 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (306.6 mg, 1.5 mmol) were dissolved in tetrahydrofuran (3 mL) and stirred at 50 °C for 36 hours. Borane dimethyl sulfide complex (1 M, 1 mL, 1.5 mmol) was added to the reaction mixture and stirring continued for 24 hours. After the reaction was complete, 2 mL of methanol was added to quench the reaction, and methionine amine-borane (compound 3) was isolated. The NMR spectrum is shown below. Figures 7-9 As shown.

[0076] 1 H NMR (400MHz, D2O) δ3.15 (t, J = 6.2 Hz, 1H), 2.44 (td, J = 7.5, 4.1 Hz, 2H), 1.99–1.83 (m, 5H), 1.60–0.73 (m, 3H). 13 C NMR (101MHz, D2O) δ177.4, 61.7, 30.6, 28.9, 14.0. 11 BNMR (128MHz, D2O) δ-21.3.

[0077] Example 4: Preparation of threonine amine-borane

[0078]

[0079] Threonine (119.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (1.02 g, 5.0 mmol) were dissolved in diethyl ether (2 mL) and stirred at 60 °C for 48 hours. Boranetetrahydrofuran complex (1 M, 5 mL, 5 mmol) was added to the reaction mixture and stirring continued for 36 hours. After the reaction was complete, 2 mL of water was added to quench the reaction, and threonine amine-borane (compound 4) was isolated. The NMR spectrum is shown below. Figures 10-12 As shown.

[0080] 1 H NMR (400MHz, D2O) δ3.80 (dq, J = 8.1, 6.4Hz, 1H), 2.92 (d, J = 7.9Hz, 1H), 1.77–1.34 (m, 3H), 1.20 (d, J = 6.5Hz, 3H). 13 C NMR (101MHz, D2O) δ172.8, 65.9, 60.4, 19.4. 11 B NMR (128MHz, D2O) δ-21.2.

[0081] Example 5: Preparation of isoleucine amine-borane

[0082]

[0083] Isoleucine (131.2 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (408.8 mg, 2.0 mmol) were dissolved in acetonitrile (3 mL), and the mixture was stirred at 25 °C for 24 hours. Borane dimethyl sulfide complex (1 M, 1.5 mL, 1.5 mmol) was added to the reaction mixture, and stirring continued for 36 hours. After the reaction was complete, 2 mL of ethanol was added to quench the reaction, and isoleucine amine-borane (compound 5) was isolated. The NMR spectrum is shown below. Figures 13-15 As shown.

[0084] 1 H NMR(400MHz,D2O)δ3.01–2.90(m,1H),1.67(ddd,J=11.9,9.4,5.0Hz,1H),1.57–1.02(m,5H),0.90–0.74(m,6H). 13 C NMR (101MHz, D2O) δ174.1,59.5,35.8,24.4,14.6,11.0. 11 B NMR (128MHz, D2O) δ-21.9.

[0085] Example 6: Preparation of leucine amine-borane

[0086]

[0087] Leucine (131.2 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (408.8 mg, 2.0 mmol) were dissolved in dichloromethane (3 mL) and stirred at 0 °C for 48 hours. Boranetetrahydrofuran complex (1 M, 1 mL, 1.0 mmol) was added to the reaction mixture, and stirring continued for 48 hours. After the reaction was complete, 2 mL of methanol was added to quench the reaction, and leucine amine-borane (compound 6) was obtained.

[0088] 1 H NMR (400MHz, D2O) δ3.08 (t, J = 7.1 Hz, 1H), 1.68–1.48 (m, 2H), 1.47 (t, J = 7.0 Hz, 4H), 0.83 (dd, J = 6.5, 5.4 Hz, 6H). 13 C NMR (101MHz, D2O) δ178.7,61.7,41.6,24.4,21.8,21.7. 11 B NMR (128MHz, D2O)δ-20.4.

[0089] Example 7: Preparation of valine amine-borane

[0090]

[0091] Valine (117.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (817.6 mg, 4.0 mmol) were dissolved in acetone (3 mL) and stirred at 45 °C for 36 hours. Borane dimethyl sulfide complex (1 M, 2 mL, 2.0 mmol) was added to the reaction mixture and stirring was continued for 18 hours. After the reaction was complete, 2 mL of tert-butanol was added to quench the reaction, and valine amine-borane (compound 7) was isolated.

[0092] 1 H NMR (400MHz, D2O) δ2.96 (d, J = 5.3Hz, 1H), 2.07–1.93 (m, 2H), 1.79–1.09 (m, 1H), 0.94 (s, 4H), 0.93 (s, 3H). 13 C NMR (101MHz, D2O) δ176.1, 59.5, 31.1, 19.2, 17.8.

[0093] Example 8: Preparation of lysine amine-borane

[0094]

[0095] Lysine (146.2 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (204.4 mg, 1.0 mmol) were dissolved in tetrahydrofuran (3 mL) and stirred at 20 °C for 24 hours. Borane tetrahydrofuran complex (1 M, 1 mL, 1.5 mmol) was added to the reaction mixture and stirring continued for 36 hours. After the reaction was complete, 2 mL of water was added to quench the reaction, and lysine amine-borane (compound 8) was obtained.

[0096] 1 H NMR (400MHz, D2O) δ3.67(t,J=6.1Hz,1H),2.65(t,J=7.5Hz,2H),1.83(dq,J=10.1,6.2Hz,2H),1.66–1.55(m,2H),1.47–0.88(m,5H). 13 C NMR (101MHz, D2O) δ175.4, 58.5, 40.1, 30.8, 27.2, 25.3.

[0097] Example 9: Preparation of glutamate-borane

[0098]

[0099] Glutamic acid (147.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (408.8 mg, 2.0 mmol) were dissolved in 1,4-dioxane (3 mL) and stirred at 30 °C for 36 hours. Boranetetrahydrofuran complex (1 M, 1.5 mL, 1.5 mmol) was added to the reaction mixture and stirring was continued for 48 hours. After the reaction was complete, 2 mL of ethanol was added to quench the reaction, and glutamate-borane (compound 9) was obtained.

[0100] 1 H NMR (400MHz, D2O) δ4.19–4.05(m,1H),2.31(ddd,J=8.9,7.3,4.4Hz,2H),2.15–2.02(m,2H),1.69–0.88(m,3H). 13 C NMR (101MHz, D2O) δ181.9, 175.9, 56.7, 34.0, 25.2.

[0101] Example 10: Preparation of tyrosine amine-borane

[0102]

[0103] Tyrosine (181.2 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (204.4 mg, 1.0 mmol) were dissolved in acetonitrile (3 mL), and the mixture was stirred at 25 °C for 40 hours. Borane dimethyl sulfide complex (1 M, 3.5 mL, 3.5 mmol) was added to the reaction mixture, and stirring was continued for 36 hours. After the reaction was complete, 2 mL of isopropanol was added to quench the reaction, and tyrosine amine-borane (compound 10) was isolated.

[0104] 1 H NMR(400MHz,D2O)δ7.07(d,J=8.6Hz,2H),6.75(d,J=8.6Hz,2H),3.23(d,J=13.1Hz, 1H), 2.89 (dd, J=14.8, 4.8Hz, 1H), 2.81 (dd, J=14.2, 8.6Hz, 1H), 1.41–0.65 (m, 3H). 13 C NMR (101MHz, D2O) δ169.3,156.6,130.2,123.4,115.4,54.5,36.2.

[0105] Example 11: Preparation of serine amine-borane

[0106]

[0107] Serine (105.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (306.6 mg, 1.5 mmol) were dissolved in 1,4-dioxane (2 mL), and the mixture was stirred at 20 °C for 36 hours. Boranetetrahydrofuran complex (1 M, 1.5 mL, 1.5 mmol) was added to the reaction mixture, and stirring was continued for 24 hours. After the reaction was complete, 2 mL of water was added to quench the reaction, and serine amine-borane (compound 11) was isolated.

[0108] 1 H NMR (400MHz, D2O) δ3.95 (d, J = 3.7Hz, 1H), 3.81 (d, J = 5.0Hz, 1H), 3.20 (t, J = 4.4Hz, 1H), 1.73–0.86 (m, 3H). 13 C NMR (101MHz, D2O) δ172.8, 62.9, 58.4.

[0109] Example 12: Preparation of 4-dihydroxyboryl-L-phenylalanine (BPA)amine-borane

[0110]

[0111] 4-Dihydroxyboryl-L-phenylalanine (208.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (204.4 mg, 1.0 mmol) were dissolved in tetrahydrofuran (3 mL) and stirred at 25 °C for 24 hours. Borane dimethyl sulfide complex (1 M, 3 mL, 3.0 mmol) was added to the reaction mixture and stirring continued for 36 hours. After the reaction was complete, 2 mL of methyl water was added to quench the reaction, and 4-dihydroxyboryl-L-phenylalanine amine-borane (compound 12) was isolated.

[0112] 1 H NMR (400MHz, D2O) δ7.50 (d, J = 7.4Hz, 2H), 7.13 (d, J = 7.5Hz, 2H), 3.48 (t, J = 6.2Hz, 1H), 2.71–2.51 (m, 2H), 1.26–0.97 (m, 3H). 13 C NMR (101MHz, D2O) δ172.1,138.3,136.2,133.4,130.5,57.6,36.3.

[0113] Example 13: Preparation of proline amine-borane

[0114]

[0115] Proline (115.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (306.6 mg, 1.5 mmol) were dissolved in 1,4-dioxane (3 mL) and stirred at 40 °C for 24 hours. Boranetetrahydrofuran complex (1 M, 2 mL, 2.0 mmol) was added to the reaction mixture and stirring was continued for 12 hours. After the reaction was complete, 2 mL of methanol was added to quench the reaction, and proline amine-borane (compound 13) was isolated.

[0116] 1 H NMR(400MHz,D2O)δ3.47-3.34(m,2H),2.86-2.77(m,1H),2.23-2.11(m,1H),1.95-1.82(m,2H),1.78-1.63(m,1H),1.16–0.94(m,3H). 13 C NMR (101MHz, D2O) δ173.4, 59.2, 46.7, 29.8, 24.6.

[0117] Example 14: Preparation of alanine amine-borane

[0118]

[0119] Alanine (89.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (306.6 mg, 1.5 mmol) were dissolved in N,N-dimethylformamide (3 mL), and the mixture was stirred at 10 °C for 48 hours. Boranetetrahydrofuran complex (1 M, 2 mL, 2.0 mmol) was added to the reaction mixture, and stirring was continued for 36 hours. After the reaction was complete, 2 mL of ethanol was added to quench the reaction, and alanine amine-borane (compound 14) was obtained.

[0120] 1 H NMR (400MHz, D2O) δ3.09 (q, J = 7.1Hz, 1H), 1.37 (d, J = 7.2Hz, 3H), 1.18–0.93 (m, 3H). 13 C NMR (101MHz, D2O) δ176.4, 53.2, 17.6.

[0121] Example 15: Preparation of cysteine ​​amine-borane

[0122]

[0123] Cysteine ​​(105.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (204.4 mg, 1.0 mmol) were dissolved in 1,2-dichloroethane (3 mL) and stirred at 60 °C for 24 hours. Boranetetrahydrofuran complex (1 M, 1 mL, 1.0 mmol) was added to the reaction mixture, and stirring continued for 36 hours. After the reaction was complete, 2 mL of water was added to quench the reaction, and cysteine ​​amine-borane (compound 15) was isolated.

[0124] 1 H NMR (400MHz, D2O) δ4.63 (dt, J = 6.8, 5.2Hz, 1H), 3.68–3.51 (m, 2H), 1.38–0.97 (m, 3H). 13 C NMR (101MHz, D2O) δ171.8, 56.9, 54.4.

[0125] Example 16: Preparation of Aspartic Amine-Boronane

[0126]

[0127] Aspartic acid (105.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (408.8 mg, 2.0 mmol) were dissolved in N-methylpyrrolidone (3 mL), and the mixture was stirred at 25 °C for 36 hours. Borane dimethyl sulfide complex (1 M, 1 mL, 1.0 mmol) was added to the reaction mixture, and stirring was continued for 24 hours. After the reaction was complete, 2 mL of methanol was added to quench the reaction, and aspartic acid amine-borane (compound 16) was isolated.

[0128] 1 H NMR (400MHz, D2O) δ3.89 (dd, J = 3.75, 8.69Hz, 1H), 2.59-2.88 (m, 2H), 1.63-0.96 (m, 3H). 13 C NMR (101MHz, D2O) δ175.1, 173.4, 54.4, 35.4.

[0129] Example 17: Preparation of N-methylphenylalanine amine-borane

[0130]

[0131] N-methylphenylalanine (179.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (306.6 mg, 1.5 mmol) were dissolved in tetrahydrofuran (2 mL) and stirred at 30 °C for 36 hours. Borane tetrahydrofuran complex (1 M, 1 mL, 1.0 mmol) was added to the reaction mixture and stirring continued for 24 hours. After the reaction was complete, 2 mL of methanol was added to quench the reaction, and N-methylphenylalanine amine-borane (compound 17) was isolated.

[0132] 1 H NMR (400MHz, D2O) δ7.42–7.23(m,5H),3.38(t,J=6.5Hz,1H),3.05–2.99(m,2H),2.52(s,3H),1.52–1.17(m,3H). 13 C NMR (101MHz, D2O) δ172.2,138.0,129.3,128.1,125.6,65.0,37.3,33.5.

[0133] Example 18: Preparation of N,N-dimethylphenylalanine amine-borane

[0134]

[0135] N-methylphenylalanine (179.1 mg, 1.0 mmol) and N,O-bis(trimethylsilyl)acetamide (306.6 mg, 1.5 mmol) were dissolved in tetrahydrofuran (2 mL), and the mixture was stirred at 30 °C for 36 hours. Borane tetrahydrofuran complex (1 M, 1 mL, 1.0 mmol) was added to the reaction mixture, and stirring continued for 36 hours. After the reaction was complete, 2 mL of methanol was added to quench the reaction, and N,N-dimethylphenylalanine amine-borane (compound 18) was isolated.

[0136] 1 H NMR (400MHz, D2O) δ7.42–7.23(m,5H),3.38(t,J=6.5Hz,1H),3.05–2.99(m,2H),2.50(s,6H),1.72–1.25(m,3H). 13 C NMR (101MHz, D2O) δ173.1,137.6,129.0,127.9,126.0,64.6,37.3,35.9,33.5.

[0137] II. Verification of the water solubility, safety, and efficacy of amino acid amine-borane compounds

[0138] 1. Determination of water solubility of amino acid amine-borane prepared in Examples 1-3

[0139] 30.0 mg of compounds 1–3 were each added to 200.0 μL of distilled water and mixed thoroughly to obtain suspensions. Each suspension was sonicated for 2 h, followed by centrifugation at 5000 rpm for 10 min. 100 μL of the supernatant was collected and diluted to 5 mL. The boron content in the supernatant was determined by inductively coupled plasma mass spectrometry (ICP-MS), repeated 6 times, and the average value was taken. Commercially available drug BPA was used as a control.

[0140] The test results showed that the water solubility of compound 1 was 52.36 g / L, the water solubility of compound 2 was 100.93 g / L, the water solubility of compound 3 was 88.25 g / L, while the water solubility of the control group, commercially available boron drug BPA, was 1.46 g / L.

[0141] The results showed that amino acid amine-borane compounds 1–3 had good water solubility, reaching up to 69.1 times that of commercially available BPA boron drugs.

[0142] 2. Cytotoxicity test of the amino acid amine-borane prepared in Examples 1-8

[0143] This experiment used mouse fibroblasts (L929 cells, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) for cytotoxicity testing. L929 cells were cultured in DMEM medium containing 10% (v / v) fetal bovine serum, 1% (v / v) penicillin, and streptomycin. The cells were placed in a cell culture incubator at 37°C and 5% CO2, and passaged 3–5 times before use. The viability of L929 cells was evaluated using the Almar Blue assay kit; generally, a cell viability >75% was considered to indicate that the compound was not cytotoxic.

[0144] The compounds obtained in Examples 1-8 were dissolved in sterile fetal bovine serum (FBS) buffer at a concentration of 10 mM, filtered through a microporous membrane for sterilization, and then further sterilized at 60°C for 24 h before being stored under sterile conditions. The 10 mM FBS solutions of the compounds from Examples 1-8 were diluted with DMEM culture medium to obtain 0.5 mM, 1 mM, 2.5 mM, and 5 mM DMEM culture media of the compounds from Examples 1-8. L929 cells were seeded at a density of 1500 cells / well in 96-well plates and cultured under the above conditions for 24 h. After washing off the DMEM culture medium, DMEM culture medium containing different concentrations (0.5 mM, 1 mM, 2.5 mM, and 5 mM) of the compounds obtained in Examples 1-8 was added. Cells cultured in DMEM culture medium without additional compounds were used as a blank control. The cells were co-cultured in an incubator for 24 h. Subsequently, the cell viability was detected using the Almar Blue assay kit after 24 hours of co-culture, and the fluorescence intensity at 600 nm emission wavelength under an excitation wavelength of 530 nm was measured using a microplate reader to evaluate cell viability. The experimental results are as follows: Figures 16-23 As shown.

[0145] Depend on Figure 16 It can be seen that at lower culture concentrations of compound 1 (1 mM and 0.5 mM), the survival rate of L929 cells reached over 80%, indicating that compound 1 has essentially no cytotoxicity. Figure 17 It can be seen that at all culture concentrations of compound 2, the survival rate of L929 cells reached over 75%, indicating that compound 2 did not exhibit cytotoxicity at any concentration. Figure 18 It can be seen that at all culture concentrations of compound 3, the survival rate of L929 cells reached over 75%, indicating that compound 3 did not exhibit cytotoxicity at any concentration. Figure 19 It can be seen that at culture concentrations of 2.5 mM, 1 mM, and 0.5 mM of compound 4, the survival rate of L929 cells reached over 80%, indicating that compound 4 has essentially no cytotoxicity. Figure 20 It can be seen that at culture concentrations of 2.5 mM, 1 mM, and 0.5 mM of compound 5, the survival rate of L929 cells was not significantly different from that of the control group, and was even higher than that of the control group, indicating that compound 5 has essentially no cytotoxicity. Figure 21 It can be seen that at lower culture concentrations of compound 6 (1 mM and 0.5 mM), the survival rate of L929 cells was not significantly different from that of the control group, and was even higher than that of the control group, indicating that compound 6 has essentially no cytotoxicity. Figure 22 It can be seen that at a low culture concentration of compound 7 (0.5 mM), the survival rate of L929 cells was greater than 75%, indicating that compound 7 has essentially no cytotoxicity. Figure 23It can be seen that at culture concentrations of 2.5 mM, 1 mM and 0.5 mM of compound 8, the viability of L929 cells reached more than 80%, indicating that compound 8 has basically no cytotoxicity.

[0146] In summary, the various essential amino acid amine-boranes prepared by this method are not cytotoxic.

[0147] 3. Experiment on boron atom uptake in 4T1 cells of amino acid amine-borane prepared in Examples 1-7

[0148] This experiment used mouse breast cancer cells (4T1 cells, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) as a tumor cell line to perform a boron uptake assay. 4T1 cells were cultured in 1640 medium containing 10% (v / v) fetal bovine serum and placed in a cell culture incubator at 37°C and 5% CO2. The cells were cultured and passaged 3–5 times before use.

[0149] The 4T1 tumor cell line, which is in the logarithmic growth phase and has a cell content of 80%–85%, was seeded into 6-well plates (1 × 10⁶ cells per well). 6 (cells). After cell adhesion, cells were treated with 1 mM 1,4-dihydroxyboronylphenylalanine (BPA) and the compounds prepared in Examples 1-7 above for 24 h, respectively, with cells cultured in DMEM medium without the added compounds used as a blank control. The culture medium was discarded, and the cells were washed three times with PBS. After washing with PBS, 0.25% trypsin was added and digested at 37°C for 3 min. Digestion was stopped by adding regular culture medium, and the cell suspension was transferred to centrifuge tubes. After repeated pipetting to form a single-cell suspension, the cells were counted, centrifuged at 1500 rpm for 5 min, and the supernatant was aspirated. The cells were then centrifuged with concentrated nitric acid (per 10 mM). 6 Cells were digested with 0.5 mL of solution for 2 hours, then diluted to 10 mL with ultrapure water and filtered through a microporous membrane. The zero point was adjusted using a sample that had not been incubated with boron-containing culture medium, and the boron concentration in the solution was determined by ICP-MS to calculate the intracellular boron concentration. Three samples were taken, and the average value was calculated.

[0150] Experimental results are as follows Figure 24 As shown, in 4T1 cells, the intracellular boron concentration obtained by the control group BPA was 5.00 μg / 10⁻¹⁰. 6 The intracellular boron concentration obtained with compound 2 (tryptophanamine-borane) was 133.85 μg / 10 cells. 6 The number of cells was 26.8 times that of the control group BPA. The intracellular boron concentration obtained with compound 1 (phenylalanine amine-borane) was 91.65 μg / 10⁻⁶ cells, which was 26.8 times that of the control group BPA. 6 The number of cells was 18.3 times that of the control group BPA. The intracellular boron concentration obtained with compound 7 (valineamine-borane) was 35.67 μg / 1010. 6The intracellular boron concentration obtained with compound 6 (leucine amine-borane) was 22.33 μg / 10 cells, which was 7.1 times that of the control group BPA. 6 The intracellular boron concentration obtained with compound 3 (methionine amine-borane) was 20.76 μg / 10 cells, which was 4.5 times that of the control group BPA. 6 The intracellular boron concentration obtained with compound 4 (threonine amine-borane) was 13.00 μg / 10 cells, which was 4.2 times that of the control group BPA. 6 The intracellular boron concentration obtained with compound 5 (isoleucine amine-borane) was 1.67 μg / 10 cells, which was 2.6 times that of the control group BPA. 6 The number of cells was 3.0 times that of the control group (BPA).

[0151] In summary, except for isoleucine and lysine, the boron uptake results of the essential amino acid amine-borane in 4T1 cells were all superior to those of BPA, reaching up to 26.8 times that of BPA.

[0152] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. An amino acid amine-borane compound, characterized in that, Having a compound of formula (I) or a pharmaceutically acceptable salt thereof: Where R is , , , , , , , , , , , , , , , , , , or ; R′ represents a hydrogen atom; → indicates a coordinate bond.

2. The amino acid amine-borane compound according to claim 1, characterized in that, The pharmaceutically acceptable salts of the compounds of formula (I) are their ammonium salts, lithium salts, sodium salts, potassium salts, rubidium salts, cesium salts, magnesium salts, or calcium salts.

3. The method for preparing the amino acid amine-borane compound according to claim 1 or 2, characterized in that, Includes the following steps: 1) Combine amino acids and N , O - Bis(trimethylsilyl)acetamide dissolves in an organic solvent and reacts to yield a silicon-protected amino acid; 2) Add the borane complex to the reaction mixture of step 1) to obtain the amino acid amine-borane compound.

4. The method for preparing the amino acid amine-borane compound according to claim 3, characterized in that, amino acids, N , O The equivalent ratio of the bis(trimethylsilyl)acetamide and borane complex is 1:(1~5):(1~5).

5. The use of the amino acid amine-borane compound according to claim 1 or 2 in the preparation of a drug or preparation for treating tumors, or in the preparation of a drug or preparation for inhibiting the progression of tumors.

6. The use of the amino acid amine-borane compound according to claim 1 or 2 in the preparation of a pharmaceutical formulation for the treatment of tumors by boron neutron capture.

7. The application according to claim 5 or 6, characterized in that, The tumors mentioned are glioblastoma multiforme, malignant meningioma, intramedullary spinal cord glioma, advanced or recurrent head and neck cancer, thyroid cancer, malignant melanoma, recurrent breast cancer, metastatic liver cancer, malignant brain tumor, osteosarcoma, lung cancer, squamous cell carcinoma of the skin, or nasopharyngeal carcinoma.

8. The application according to claim 5 or 6, characterized in that, The pharmaceutical preparation is an injectable formulation made by mixing an amino acid amine-borane compound with physiological saline, phosphate buffer solution, sterile water or glucose solution.

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