A boron-containing vincristine compound or a pharmaceutically acceptable salt thereof and a preparation method and application thereof

Abstract

The present invention discloses a boron-containing vincristine compound, a derivative or a pharmaceutically acceptable salt thereof, a preparation method and an application thereof. The boron-containing vincristine compound, a derivative or a pharmaceutically acceptable salt thereof is prepared by mixing vincristine and its derivatives or substitutes with B having neutron absorption ability. 10 The compound obtained by combining and its derivatives and pharmaceutically acceptable salts thereof are applied in BNCT boron neutron therapy to provide high boron content and improve the specificity of the boron carrier to tumor cells.

Description

Technical Field

[0001] The present invention relates to a boron carrier technology, and in particular to a boron-containing vincristine compound or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof. Background Art

[0002] Boron neutron capture therapy (BNCT) is a dual targeted therapy for tumor treatment. 10 The compound (boron carrier, also called boron medicine) is injected into the human body, and local irradiation with neutron beam causes B-10 accumulated in the tumor tissue to undergo a nuclear reaction with thermal neutrons, and the generated 7Li and α particles are used to destroy the tumor tissue. The range of the 7Li particles released by the reaction is about 4-5μm, and the range of the α particles is about 9-10μm, while the diameter of tumor cells is generally less than 10μm, so the radiation killing range of this method is limited to tumor cells, and the damage to surrounding normal tissues is small. Therefore, patients undergoing treatment do not need special protection.

[0003] So far, the boron carriers used in boron neutron capture therapy (BNCT) have gone through three generations of evolution. The first generation of boron carriers used inorganic substances such as boric acid and borax. The high toxicity of boric acid and borax resulted in mediocre tumor treatment effects and more serious side effects. Organic substances 4-dihydroxyboryl-L-phenylalanine (BPA) and sodium 11-hydrogen mercapto-12-boronide (BSH) stood out among the boron carriers due to their selective targeting properties and became the second generation of organic boron carriers. BPA can target and bind to specific proteins and selectively accumulate in proteins related to tumor growth during the rapid proliferation of tumor cells. BSH was developed along with carborane chemistry. The carborane structure generally contains more than 10 boron atoms. As a boron carrier, it will effectively increase the boron concentration in tumors. However, due to the defects at the molecular level, the tumor specificity of BPA and BSH is insufficient. With the development of boron carriers, the third generation of boron carriers are mainly amino acids, nucleotides, boron-containing nanoparticles, etc.; the third generation of amino acid boron carriers have strong metabolic stability and show high tumor specificity, but are limited by their limited specificity of binding to tumor cells. They are currently mostly used to treat brain tumors, but still have limitations for the treatment of tumors in the chest or abdominal organs. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, one of the purposes of the present invention is to provide a boron-containing vincristine compound or a pharmaceutically acceptable salt thereof. 10 The combination is applied in BNCT boron neutron therapy to provide high boron content and improve the specificity of boron carriers to tumor cells.

[0005] A second object of the present invention is to provide a method for preparing the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof as described above.

[0006] The third object of the present invention is to provide a boron-containing vincristine compound or a pharmaceutically acceptable salt thereof for use in the preparation of adjuvant cancer therapeutic drugs.

[0007] One of the purposes of the present invention is achieved by the following technical solution: a boron-containing vincristine compound or a pharmaceutically acceptable salt thereof, which is vincristine and its derivatives or substitutes with B having neutron absorption ability 10 The compound obtained by combining, its derivatives and pharmaceutically acceptable salts thereof have or contain the following chemical structural formula (I) or substitutes and derivatives having structural formula (II):

[0008]

[0009]

[0010] As a further preferred solution, R1 to R6 are C1-C 10 Short chain hydrocarbon groups, C1-C 10 Alkoxy, C1-C 10 A boron-carrying group in a heterochain group containing nitrogen atoms and / or oxygen atoms.

[0011] As a further preferred embodiment, R1 to R6 are the same or different groups.

[0012] As a further preferred embodiment, the substitution position of the substituent of the structural formula (I) occurs on the benzene ring or on the hydrocarbon carbon chain.

[0013] As a further preferred embodiment, the derivative is a derivative containing one or more alicyclic structures, benzene ring structures, piperazine ring structures, amino functional groups (-NH2) and oxygen heterocyclic rings in the vincristine molecule, and the oxygen heterocyclic rings, such as furan rings, thiophene rings, etc., give it more chemical reactivity; including derivatives formed by short chains of -CH2-bonds in the middle of the carbonyl group; the pharmaceutically acceptable salt is a salt obtained by adding an acid to the boron-containing vincristine and its derivatives or a salt obtained by adding a base, and the acid is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, tartaric acid, methanesulfonic acid or isethionic acid; the base is sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, ammonia, triethylamine or triethanolamine.

[0014] The second object of the present invention is achieved by the following technical solution: a method for preparing the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof as described above, wherein B having a very strong neutron absorption ability 10 Introduced into the structure (I), a new type of boron carrier / boron drug is obtained. The preparation method described in the embodiment of the present application comprises the following steps:

[0015] (1) Nitration reaction: Using vincristine having the chemical formula (I) as a raw material, in the presence of nitrous acid and concentrated sulfuric acid at 40-50° C., the ortho- and para-positions of the alkyl and amino groups on vincristine are substituted to generate a nitro-substituted product having the chemical formula (a);

[0016]

[0017] (2) Redox reaction: Under the conditions of iron and hydrochloric acid, the nitrogen on the benzene ring is +3 valent, and the nitrogen on the amino group is -3 valent. The reaction is carried out in an iron, hydrochloric acid, and H2O system. After the redox reaction, the nitro group on the vincristine nitro substituent (a) is reduced to an amine group to obtain a vincristine amine substituent of the chemical formula (b);

[0018]

[0019] (3) Diazotization reaction: The vincristine amine substituent (b) is subjected to a diazotization reaction with hydrochloric acid and sodium nitrite at 0-5° C. to obtain a diazonium salt compound having a chemical formula (c);

[0020]

[0021]

[0022] (4) Substitution reaction: an iodine atom is introduced into the diazonium salt compound having the chemical formula (c), and the diazonium salt compound (c) is heated with KI, whereby -N2Cl on the diazonium salt compound (c) is replaced by -I, thereby obtaining a vincristine halogen substituted product (d);

[0023]

[0024] (5) Grignard reagent reaction: Add vincristine halogen substitute (d) into a dry apparatus, react with metal magnesium in anhydrous ether, heat under reflux, and generate a compound having a chemical formula (e);

[0025]

[0026] (6) Synthesis reaction: The above-mentioned Grignard reagent (e) and B 10 (OCH2CH3)3 reacts at room temperature, wherein the Grignard reagent (e) and B 10The mass ratio of (OCH2CH3)3 is greater than 1:6 to obtain a boron-containing vincristine compound having a chemical structural formula (II), a derivative thereof or a pharmaceutically acceptable salt thereof:

[0027]

[0028] The third objective of the present invention is achieved by the following technical solution: use of a boron-containing vincristine compound or a pharmaceutically acceptable salt thereof in the preparation of an adjuvant cancer treatment drug.

[0029] Specifically, the cancer adjuvant therapeutic drugs include adjuvant therapeutic drugs for hematopoietic malignancies, thymic carcinoma and other related diseases.

[0030] The present invention also provides a boron carrier, which comprises the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof as described above. The boron carrier is used for boron neutron therapy to treat hematopoietic malignancies, thymic carcinoma and other related diseases.

[0031] The present invention also provides a pharmaceutical composition, which comprises the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof as described above, and a medically and pharmaceutically acceptable carrier.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The boron-containing vincristine or its pharmaceutically acceptable salt described in the present application is a product synthesized by using vincristine and boron-10 with strong neutron absorption capacity, which can provide a high-content boron carrier, has a high affinity for tumor cells, good stability and low biological toxicity.

[0034] 2. The boron-containing vincristine compound or its pharmaceutically acceptable salt described in the present application is absorbed into the body by passive transport, can bind to the ABCB1 protein of cancer cells, and highly expressed ABCB1 prevents the β-tubulin of cancer cells from polymerizing, thus completing its transmembrane transport, and has good application prospects in boron neutron therapy.

[0035] 3. The boron-containing vincristine compound or a pharmaceutically acceptable salt thereof described in the present application is used as a boron carrier (boron drug) to expand the selection range of boron drugs used in BNCT treatment.

[0036] 4. The boron-containing vincristine compound or its pharmaceutically acceptable salt described in the present application is mainly effective as a boron carrier for mesodermal cancers such as hematopoietic malignancies, thymic carcinomas, etc. It can be used as a boron drug for treating hematopoietic malignancies, thymic carcinomas, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The CNMR diagram of the boron-containing vincristine product (II) of Example 1 of the present invention;

[0038] Figure 2 The boron-10 reagent B of Example 1 of the present invention 10 CNMR pattern of (OCH2CH3)3. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0040] The term "comprising" and other equivalent descriptions involved in the specification and claims of this application are intended to cover non-exclusive inclusions, including both the contents clearly described in the specification and claims, and the steps or units that are not described in the specification and claims but are inherent in the product, method or structure.

[0041] The present invention provides a boron-containing vincristine compound or a pharmaceutically acceptable salt thereof, characterized in that vincristine having a chemical structural formula (I) is used as a raw material, and boron-10 is introduced into its structure to obtain a compound having or containing a chemical structural formula (II):

[0042]

[0043] B 10 It is a stable isotope of boron. In BNCT therapy, we need to find atoms with strong neutron absorption ability, and B 10 The absorption capacity of thermal neutrons emitted by BNCT devices is very strong, especially the boron drugs (such as BPA, BSH, etc.) in BNCT must contain high-enriched B 10 , generally the enrichment degree is required to be more than 99%. In the present application, the boron-containing vincristine is a new boron carrier obtained by introducing boron-10 into the structure of vincristine.

[0044] The preparation method comprises the following steps:

[0045] (1) Nitration reaction: The benzene ring is affected by the amino group and the alkyl group. It is easy to undergo substitution reaction with nitric acid under concentrated sulfuric acid as a catalyst and heating conditions to generate trinitro substitution products (ortho-para positions), that is, a total of 6 positions of the two benzene rings are substituted. The specific operation is to add 20ml of concentrated sulfuric acid to a 250ml round-bottom flask, place it in an ice water bath, and then measure 18ml of concentrated nitric acid and carefully add it to the concentrated sulfuric acid while shaking. After adding, let the mixed acid cool to room temperature. Weigh 3g of vincristine and add it to the mixed acid in batches (4-5 times), and shake the flask. Let vincristine fully contact with the mixed acid. When the temperature of the reactant no longer rises but tends to decrease, continue to add the mixed acid. The temperature of the reactant should be kept between 40-50℃. If it exceeds 50℃, cool the flask in a cold water bath. After the addition is completed, place the flask on a water bath and heat it to 60°C (the temperature of the reaction mixture is 50-60°C) within about 10 minutes and maintain it for 30 minutes, shaking the flask intermittently. After cooling, pour the reaction mixture into a separatory funnel. Let it stand to separate the layers, separate the acid layer, and pour it into a designated recovery bottle. The crude vincristine nitro-substituent is first washed with an equal volume of cold water, and then washed with 10% sodium carbonate solution until the washing liquid is no longer acidic. Finally, wash with water until neutral. Separate the crude vincristine nitro-substituent (a), place it in a dry small conical flask, add anhydrous calcium chloride to dry, and shake the conical flask intermittently. Pour the clear and transparent vincristine nitro-substituent into a 30ml distillation flask and connect an air condenser. Heat and distill on an asbestos net to collect the fraction at 204-210°C.

[0046]

[0047] (2) Redox reaction: In a 250 ml round-bottom flask, place 5 g of iron powder (40-100 mesh), 30 ml of water and 5 ml of hydrochloric acid, and shake vigorously to mix thoroughly. Install a reflux condenser and slowly boil over low heat for 5 min. After cooling slightly, add 10 ml of vincristine (a) from the top of the condenser in batches, and shake the reaction mixture after each addition. If the reaction is too intense and the mixture in the flask boils, immerse the round-bottom flask in cold water for a moment to slow down the reaction. When all the vincristine (a) is added, heat the flask in a boiling hot water bath for 30 min to complete the reduction, then cool the reactants to room temperature and slowly add 50% NaOH solution while shaking to make the reactants alkaline. Then, the reaction flask is replaced with a steam distillation apparatus, and steam distillation is performed until a clear liquid is distilled out. The distillate is placed in a separatory funnel to separate the crude vincristine amine substitution product (b). After adding 5 g of sodium chloride to the water layer to saturate it, it is extracted twice with 10 ml of ether. The crude vincristine amine substitution product and the ether extract are combined and dried with granular sodium hydroxide. The dried mixed solution is carefully poured into a dry 50 ml distillation flask, and the ether is evaporated on a hot water bath. Then, an air condenser is used instead, and it is heated on an asbestos net to collect the fraction at 180-185°C.

[0048]

[0049] (3) Diazotization reaction: In an apparatus containing the vincristine amine substituent of chemical formula (b), add hydrochloric acid and sodium nitrate at 0-5°C to react fully to generate a diazonium salt compound of chemical formula (c). Control the amount of NaNO2 added until the reaction solution turns starch-potassium iodide test paper blue. When NaNO2 is excessive, remove it with urea or sulfuric acid. The purpose of the excess acid is to prevent the diazonium salt from coupling with the unreacted primary amine.

[0050]

[0051] (4) Substitution reaction: The method for testing excess nitrite during the reaction is to use potassium iodide starch paper test. The presence of a drop of excess nitrite solution can turn the potassium iodide starch paper blue. Since air can also oxidize and discolor the potassium iodide starch paper under acidic conditions, the test time is based on the color development within 0.5-2 seconds. Excess nitrite is not conducive to the next coupling reaction, so excess nitrite is often added with urea or aminosulfonic acid to consume excess nitrite. When nitrite is excessive, a small amount of raw material aromatic primary amine can also be added to react with excess nitrite to remove it. Vincristine diazo compound (c) and KI with a mass concentration of 20% are heated at 90°C for 10 minutes. The ratio of vincristine diazo compound (c) to KI is 1:30. Iodine atoms are introduced to obtain vincristine halogen substitution (d), but this method cannot be used to introduce chlorine atoms or bromine atoms.

[0052]

[0053] (5) Grignard reagent reaction: Add a vincristine halogen substitute having the chemical formula (d) into a dry apparatus, react with metallic magnesium in anhydrous ether, and heat under reflux to generate a compound having the chemical formula (e).

[0054]

[0055] (6) Synthesis reaction of introducing boron-10: the above-mentioned Grignard reagent (e) and B 10 (OCH2CH3)3 reacts at room temperature to introduce B-10 into the compound of the above chemical formula (e), wherein the Grignard reagent (e) and B 10 The mass ratio of (OCH2CH3)3 is greater than 1:6, and a compound having the chemical structural formula (II) is obtained.

[0056]

[0057] The following are specific examples of the present application. In the following examples, except for those specifically specified in the present application, the raw materials, reagents, instruments and equipment used can all be purchased.

[0058] Example 1

[0059] This embodiment provides a synthetic boronated vincristine compound, and the synthesis method of the compound is as follows:

[0060] (1) Nitration reaction: Add 20 ml of concentrated sulfuric acid to a 250 ml round-bottom flask and place it in an ice-water bath. Then, weigh 18 ml of concentrated nitric acid and carefully add it to the concentrated sulfuric acid, shaking while adding. After adding, let the mixed acid cool to room temperature. Weigh 3 g of vincristine and add the mixed acid to vincristine in 4-5 portions. After each addition of the mixed acid, shake the flask to allow vincristine to fully contact the mixed acid. When the temperature of the reactant no longer rises but tends to decrease, continue to add the mixed acid. The temperature of the reactant should be maintained between 40-50°C. If it exceeds 50°C, cool the flask in a cold water bath. After the addition is completed, place the flask in a water bath and heat it. Heat the water bath to 60°C in about 10 minutes and keep it for 30 minutes. Shake the flask intermittently. After cooling, pour the reaction mixture into a separatory funnel. Let it stand to separate the layers, separate the acid layer, and pour it into a designated recovery bottle. The crude vincristine nitro-substituent is first washed with an equal volume of cold water, and then with a 10% sodium carbonate solution until the washing liquid is no longer acidic. Finally, wash with water until neutral. Separate the crude vincristine nitro-substituent, place it in a dry small conical flask, add anhydrous calcium chloride to dry, and shake the conical flask intermittently. Pour the clear and transparent vincristine nitro-substituent into a 30ml distillation flask and connect an air condenser. Heat and distill on an asbestos net, collect the fraction at 204-210°C, and obtain vincristine nitro-substituent (a), the structural formula of which is as follows.

[0061]

[0062] (2) Redox reaction: In a 250 ml round-bottom flask, add 5 g of iron powder (40-100 mesh), 30 ml of water and 5 ml of hydrochloric acid, and mix thoroughly; install a reflux condenser and slowly boil over low heat for 5 min; after cooling slightly, add 10 ml of the above-mentioned vincristine nitrosubstituent (a) in batches from the top of the condenser, and shake well each time. After all the vincristine nitrosubstituent (a) has been added, heat the flask in a boiling hot water bath for 30 min to complete the reduction, cool to room temperature, and slowly add 50% NaOH solution to make the reactants alkaline. Then change the reaction flask into a steam distillation apparatus and perform steam distillation until a clear liquid is evaporated. Put the distillate into a separatory funnel to separate the crude vincristine amine substituent. After adding 5 g of sodium chloride to saturate the water layer, extract it twice with 10 ml of ether, combine the crude vincristine amine substituent and the ether extract, and dry it with granular sodium hydroxide. The dried mixed solution was poured into a dry 50 ml distillation flask, and the ether was evaporated on a hot water bath. Then, an air condenser was used to heat on an asbestos net, and the fractions at 180-185°C were collected to obtain vincristine amine substituted product (b), the structure of which is as follows:

[0063]

[0064] (3) Diazotization reaction: add excess hydrochloric acid and an equimolar amount of sodium nitrite at 0-5°C (wherein, the equimolar amount refers to the equimolar amount of sodium nitrite at the site on vincristine that can undergo diazotization reaction). Specifically, hydrochloric acid and sodium nitrite are slowly added to the solution of vincristine amine substituent (b), and the reaction pH is adjusted according to the specific situation to maintain a strong acid environment. The nitro ion reacts with the vincristine amine substituent (b) to form the corresponding vincristine diazo compound (c). Because most diazonium salts are more stable at low temperatures, the decomposition rate of diazonium salts is accelerated at higher temperatures. In addition, nitrous acid is also easy to decompose at higher temperatures. The role of the inorganic acid in the reaction is to first dissolve the aromatic amine, then react with sodium nitrite to form nitrous acid, and finally form a diazonium salt compound. Diazonium salt compounds are generally easy to decompose. They are relatively stable only in excess acid solution, and they can prevent the diazonium salt compounds from coupling with unreacted primary amines. Therefore, a large excess of acid is actually used during diazotization, and excess NaNO2 should be removed with urea or sulfuric acid.

[0065]

[0066] (4) Substitution reaction: The method for testing excess nitrite during the reaction is to use potassium iodide starch paper test. The presence of a drop of excess nitrite solution can turn the potassium iodide starch paper blue. Since air can also cause potassium iodide starch paper to oxidize and change color under acidic conditions, the test time is based on the color development within 0.5-2 seconds. Excess nitrite is not conducive to the next coupling reaction, so excess nitrite is often added with urea or aminosulfonic acid to consume excess nitrite. When nitrite is excessive, a small amount of raw material aromatic primary amine can also be added to react with excess nitrite to remove it. Vincristine diazo compound (c) and KI with a mass concentration of 20% are heated at 90°C for 10 minutes. The ratio of vincristine diazo compound (c) to KI is 1:30. Iodine atoms are introduced to obtain vincristine halogen substitution (d), the structural formula of which is as follows:

[0067]

[0068] (5) Grignard reagent reaction: Add 2g of magnesium metal, 20mL of anhydrous ether and a small amount of vincristine halogen substitute (d) to a dry apparatus. After the reflux of anhydrous ether disappears, add the remaining vincristine halogen substitute (d) and 50mL of anhydrous ether. After the addition is complete, continue the reaction for 2h, increase the reaction temperature and recover the anhydrous ether. When the reaction temperature reaches 80°C, stop the reaction to obtain vincristine Grignard reagent (e).

[0069]

[0070] (6) Synthesis reaction of introducing boron-10: the above-mentioned Grignard reagent (e) and B 10(OCH2CH3)3 undergoes nucleophilic substitution reaction at room temperature. The Grignard reagent (e) reacts with B 10 The mass ratio of (OCH2CH3)3 is greater than 1:6, and B-10 is introduced to finally obtain the boron-containing vincristine product (II), the structure of which is as follows:

[0071]

[0072] like Figure 1 As shown, it is the CNMR data of the boron-containing vincristine product (II) of the present invention, Figure 2 Boron-10 reagent B 10 CNMR diagram of (OCH2CH3)3. Figure 1-2 , it can be seen that if the boron-containing reagent is not thoroughly distilled and impurities are removed, a peak will appear near 57. Figure 1 The peak at position 57 is not obvious, which proves that the boron-containing reagent B 10 (OCH2CH3)3 is removed completely.

[0073] Note: During the preparation of boron-containing vincristine, some by-products may be generated. The generated by-products are other monosubstituted or polysubstituted products. The generated by-products and their pharmaceutically acceptable salts have the same efficacy as the main product. The by-products include but are not limited to the following substances:

[0074]

[0075]

[0076] Confirmation of the borylated vincristine product is shown in the figure above.

[0077] Note: The substituents on the benzene ring of vincristine in this patent application and the boride having the above structural formula all belong to the content of this patent application.

[0078] Test Examples

[0079] Experimental Example 1 Biological Toxicity Test of the Compound Prepared in Example 1 of the Present Invention

[0080] Prepare 5 Kunming mice (mice source Guangdong Biomedical Animal Experiment Center), weighed mass of 21.6g, 22.4g, 21g, 22.1g, 22.5g, respectively, give 5 Kunming mice, respectively, 50mg / kg, 100mg / kg, 150mg / kg, 200g / kg, 210mg / kg of the product of Example 1 above, vincristine containing boron, to mice tail injection. According to the above dosage and Kunming mice weight, the amount of vincristine added was calculated to be 0.00108g, 0.00224g, 0.00315g, 0.00442g, 0.004725g. Dissolve the above mass vincristine in 2mL dimethyl sulfoxide to prepare vincristine containing boron solution. After injection, mark them as 1-5 groups and observe for 3 days.

[0081] Results and observations: No abnormalities were observed in the Kunming mice in groups 1 and 2, so it is speculated that the toxicity caused less harm to the Kunming mice. In group 3, the Kunming mice became sluggish 15 minutes after gavage, specifically slow limb movements. After 15 minutes, they returned to normal. The vitality of the Kunming mice decreased on the first and second days, and returned to normal on the third day. The vitality of the Kunming mice in group 4 decreased 5 minutes after gavage, and they refused to eat and did not move for the next 3 days. They started eating on the third day, but their vitality was still very low. The vitality of the Kunming mice in group 5 decreased 3 minutes after gavage, and they refused to eat and did not move for the next 3 days until they died.

[0082] Conclusion: The mice can survive at a low dose of 200 mg / kg of the boron-containing vincristine of the present invention, but the mice cannot survive at a dose higher than 200 mg / kg.

[0083] Experimental Example 2 In vitro antitumor test of the compound prepared in Example 1 of the present invention

[0084] 200mg / kg boronized vincristine was injected into the tail vein of thymic cancer mice. Three days later, the tail was cut and 20μl of blood was collected and mixed with 1ml of normal saline (containing 4mgEDTA.2Na / 100ml or heparin, 1mg heparin anticoagulated blood 2-5ml). After centrifugation at 4000r / min for 3min, the CH3OH extract was combined, and then the CH3OH was evaporated to dryness. The residue was taken, 0.8mL CH3OH was added to dissolve, and then vortex mixed and centrifuged. The supernatant obtained was used as a sample. EDTA solution was added to a polytetrafluoroethylene beaker, and the absorbance was measured under the same conditions as the determination of the boron standard solution. Subsequently, the mouse was dissected, 0.2g of the mouse thymus tissue was taken, cut into pieces, 1.8ml of normal saline was added, and homogenized with a glass homogenizer in an ice bath to make a 10% tissue homogenate. The tissue homogenate was centrifuged at 4000r / min for 10min at 4°C, and the supernatant was used for determination.

[0085] The concentration of boronated vincristine in Kunming mice was determined by spectrophotometry. The specific operation was as follows: a certain gradient concentration of boron standard solution (20 μg / mL) was respectively aspirated into 7 polyethylene beakers, EDTA solution was added to each beaker to mask interfering ions such as iron, aluminum, and copper, the pH was adjusted to 5.0 with sodium hydroxide or hydrochloric acid standard solution, ammonium acetate buffer solution of pH 5.2 and azomethine H acid colorimetric solution were added, the solution was transferred to a volumetric flask, diluted to the scale with water, mixed, and placed in the dark at room temperature for 3 h, and the absorbance was measured in sequence at a wavelength of 415 nm with water as a reference using a 1 cm absorption cell, and a working curve was drawn.

[0086] Take 1.0 mL of the blood and thymus tissue supernatant of the Kunming mice and measure the absorbance. Find the corresponding boron amount according to the drawn standard curve, and then calculate the boron content in the sample. Perform a blank test at the same time. The azomethine H acid colorimetric reagent must be prepared and used immediately.

[0087] The results showed that the plasma levels of boron-containing vincristine in Kunming mice were all between 9 and 15 mg / kg, which was almost non-toxic to Kunming mice.

[0088] Comparative Example

[0089] Comparative Example 1 In vitro anti-tumor experiment of existing organic boron carrier BPA

[0090] The organic boron carrier BPA was synthesized by China Science High Energy (Guangzhou) Medical Technology Development Co., Ltd.

[0091] 450mg / kg of organic boron carrier BPA was injected into the tail vein of thymic cancer mice. Three days later, the tail was cut and 20μl of blood was taken and mixed with 1ml of physiological saline (containing 4mgEDTA.2Na / 100ml or heparin, 1mg heparin anticoagulated blood 2-5ml). After centrifugation at 4000r / min for 3min, the CH3OH extract was combined, and then the CH3OH was evaporated to dryness. The residue was taken, 0.8mL of CH3OH was added to dissolve, and then vortex mixed and centrifuged. The supernatant obtained was used as a sample. EDTA solution was added to a polytetrafluoroethylene beaker, and the absorbance was measured under the same conditions as the determination of the boron standard solution. Subsequently, the mouse was dissected, 0.2g of the mouse thymus tissue was taken, cut into pieces, 1.8ml of physiological saline was added, and homogenized with a glass homogenizer in an ice bath to make a 10% tissue homogenate. The tissue homogenate was centrifuged at 4000r / min for 10min at 4℃, and the supernatant was used for determination.

[0092] The concentration of organic boron carrier BPA in Kunming mice was determined by spectrophotometry. The specific operation was as follows: a certain gradient concentration of boron standard solution (20 μg / mL) was respectively absorbed into 7 polyethylene beakers, EDTA solution was added to each beaker to mask interfering ions such as iron, aluminum, and copper, and the pH was adjusted to 5.0 with sodium hydroxide or hydrochloric acid standard solution. Ammonium acetate buffer solution of pH 5.2 and azomethine H acid colorimetric solution were added, and the solution was transferred to a volumetric flask, diluted to the scale with water, mixed, and placed in the dark at room temperature for 3 h. The absorbance was measured in sequence at a wavelength of 415 nm with water as a reference using a 1 cm absorption cell, and a working curve was drawn.

[0093] Take 1.0 mL of the blood and thymus tissue supernatant of the Kunming mice and measure the absorbance. Find the corresponding boron amount according to the drawn standard curve, and then calculate the boron content in the sample. Perform a blank test at the same time. The azomethine H acid colorimetric reagent must be prepared and used immediately.

[0094] The results showed that the plasma content of the organic boron carrier BPA in Kunming mice was 8.94 μg / g. This shows that compared with the existing organic boron carrier BPA, the boron-containing vincristine of the present invention has a broader application prospect in drug development.

[0095] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A boron-containing vincristine compound or a pharmaceutically acceptable salt thereof, characterized in that: The boron-containing vincristine compound has the following structural formula: or or or or or 2. The boron-containing vincristine compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt obtained by adding an acid to the boron-containing vincristine, and the acid is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, tartaric acid, methanesulfonic acid or isethionic acid.

3. A method for preparing a boron-containing vincristine compound or a pharmaceutically acceptable salt thereof, characterized in that: The steps include: (1) Nitration reaction: Using vincristine as a raw material, a nitro-substituted vincristine (a) is obtained through a nitration reaction; （a） (2) Redox reaction: After a redox reaction, the nitro group on the vincristine nitro substituent (a) is reduced to an amine group to obtain the vincristine amine substituent (b); (3) Diazotization reaction: After the vincristine amine substituent (b) undergoes diazotization reaction, a diazonium salt compound (c) is obtained; (4) Substitution reaction: an iodine atom is introduced into the diazonium salt compound (c), and the -N2Cl on the diazonium salt compound (c) is replaced by -I to obtain a vincristine halogen substituted product (d); (5) Grignard reagent reaction: Add vincristine halogen substitute (d) into a dry apparatus, react with magnesium in anhydrous ether, heat under reflux to generate compound (e); (6) Synthesis reaction: Compound (e) is introduced into boron-10 to obtain a compound having the chemical formula (II): （Ⅱ）。 4. The method for preparing the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: In the nitration reaction of step (1), the reaction reagents are nitrous acid and concentrated sulfuric acid, and the reaction temperature is 40-50°C.

5. The method for preparing the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: In the redox reaction of step (2), the reaction reagents are iron, hydrochloric acid and H2O.

6. The method for preparing the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: In the diazotization reaction of step (3), the reaction reagents are hydrochloric acid and sodium nitrite, and the reaction temperature is 0-5°C.

7. The method for preparing the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: In the substitution reaction of step (4), the reaction reagent is KI.

8. Use of the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 in the preparation of an adjuvant cancer therapy drug.

9. The use according to claim 8, wherein the cancer adjuvant therapeutic drug comprises an adjuvant therapeutic drug for hematopoietic malignancies and thymic carcinoma-related diseases.

10. A boron carrier, characterized in that: The invention comprises the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7.

11. A pharmaceutical composition, characterized in that The invention comprises the boron-containing vincristine compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 7, and a medically and pharmaceutically acceptable carrier.

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