Norcantharidin derivative, synthetic method and application thereof

Abstract

The application discloses a norcantharidin derivative, a synthetic method and application thereof, and belongs to the technical field of medicine preparation. Norcantharidin and aminobenzene boronic acid are dissolved in an ice acetic acid solution, after reaction, the obtained white filter residue is dried to obtain a crude product; then the crude product is dissolved in methanol and repeatedly recrystallized to obtain transparent needle-like crystalline norcantharidin derivative. The amino group of aminobenzene boronic acid is used to connect norcantharidin and aminobenzene boronic acid to obtain the norcantharidin derivative, which can antagonize leukopenia caused by chemotherapy drugs on the basis of retaining the anticancer activity. The synthetic method is simple, the yield is high, the cost is low, the toxic and side effects of norcantharidin in treating liver cancer can be effectively reduced, the oral bioavailability is high, and the curative effect is improved.

Description

TECHNICAL FIELD

[0001] The application relates to a hybrid compound, a synthesis method and application, in particular to a norcantharidin derivative with the function of increasing the number of white blood cells and synthesis and application thereof, and belongs to the technical field of medicine preparation. BACKGROUND

[0002] At present, the treatment of tumors has become a global problem to be solved, and in clinical treatment, anti-tumor drugs are often used for treatment. Anti-tumor drugs are generally cytotoxic drugs, which have strong killing effect on tumor cells, but also have strong inhibitory effect on normal tissues, especially bone marrow stem cells which are active in metabolism, so bone marrow suppression is a common adverse reaction of chemotherapy drugs. The half-life of white blood cells is 6-8h, so the bone marrow suppression caused by chemotherapy drugs is first manifested as a decrease in the number of white blood cells, which makes patients interrupt or delay chemotherapy, resulting in a great impact on the treatment effect.

[0003] Norcantharidin (NCTD) is a derivative of cantharidin, which is extracted from cantharides and artificially synthesized into an anticancer drug. The configuration is similar to that of cantharidin, but there is no methyl at 1 and 2. It has the effects of anti-cancer and increasing the number of white blood cells. NCTD can stimulate the cell cycle progression of human bone marrow granulocyte-monocyte cells in vitro within 24 hours, and form a dose-dependent manner. It is the first anticancer drug with the effect of increasing white blood cells today. At present, it is mainly used for the treatment of primary liver cancer in clinic. However, adverse reactions such as irritation of the urinary system and gastrointestinal reactions occur when the clinical application dose is slightly large. Animal experiments also show that pathological changes occur in kidney and liver tissues when the drug dose increases to a certain extent. The limitation of the drug dose of NCTD hinders the application of the drug. Therefore, how to obtain a low-toxicity targeted drug of norcantharidin becomes the key to solving the above problems.

[0004] Sialic acid is a kind of sugar residue including nine kinds of carbonic acid sugars, which usually exists in the form of glycoprotein or cell surface terminal sugar. The role of high sialylation in tumor growth and metastasis has been widely reported. It has been proved that phenylboric acid molecules have specific binding capacity with sialic acid. In fact, phenylboric acid can form complexes with various sugar groups, but these complexes are unstable under physiological conditions. Only at low pH value, this binding can be maintained. In the weakly acidic microenvironment of tumors, non-free phenylboric acid cannot form stable structures with other sugar groups, but can specifically bind with sialic acid to form stable complexes. Under physiological conditions, phenylboric acid cannot form stable binding with other sugar groups. SUMMARY

[0005] This invention addresses the shortcomings of existing technologies by providing a norepinephrine derivative with a simple synthesis process, low production cost, high yield, non-toxicity, and whitening effect, as well as its synthesis method and applications.

[0006] The technical solution to achieve the objective of this invention is to provide a demethylcantharidin derivative, the structural formula of which is:

[0007] .

[0008] The technical solution of this invention also includes a method for synthesizing a cantharidin derivative, the steps of which are as follows:

[0009] (1) Dissolve norcantharidin and aminophenylboronic acid in glacial acetic acid solution at a molar ratio of 1:1 to 3 at a temperature of 40 to 60°C. Then stir the reaction for 2 to 8 hours at an oil bath temperature of 40 to 80°C. Let the solution stand until it cools to room temperature, filter it, and dry the white filter residue to obtain the crude product.

[0010] (2) Dissolve the product obtained in step (1) in methanol and recrystallize it repeatedly 2 to 3 times to obtain transparent needle-shaped crystals of cantharidin derivative.

[0011] 3. The method for synthesizing a cantharidin derivative according to claim 2, characterized in that: the molar ratio of cantharidin to aminophenylboronic acid is 1:1.5; the oil bath temperature is 50℃; and the stirring reaction is carried out for 6 hours.

[0012] The present invention also provides an application of the norepinephrine derivative, wherein the norepinephrine derivative is used in combination with antitumor chemotherapy drugs cisplatin, fluorouracil, camptothecin, and doxorubicin, or in combination with antitumor natural active ingredients and traditional Chinese medicines such as evodia rutaecarpa, astragalus, angelica, and epimedium, for targeted therapy of liver cancer, breast cancer, and lung cancer; or the norepinephrine derivative is used as a preoperative medication for cancer.

[0013] This invention utilizes the condensation reaction between the amino group on aminophenylboronic acid and norcantharidin to modify norcantharidin with phenylboronic acid. By controlling the reaction conditions, the reactive oxygen species bridge structure of norcantharidin is preserved, resulting in norcantharidin-phenylboronic acid. The obtained compound retains its antitumor activity while exhibiting significantly reduced toxicity, especially with almost no damage to vital organs such as the kidneys, and retains the white blood cell-boosting effect of norcantharidin, actively targeting the liver. This invention also uses sialic acid as a target site to design targeted drugs modified with phenylboronic acid as a ligand. By recognizing the highly distributed sialic acid on the tumor surface, targeted selection of tumor cells is achieved, improving drug delivery and efficacy efficiency.

[0014] The beneficial effects of the technical solution of this invention are as follows:

[0015] (1) The norcantharidin is targeted to the liver after being acidified by phenylboronic acid, so that the drug is enriched in the target area and the treatment effect is improved. It is found through screening of various tumor cells that the norcantharidin phenylboronic acid derivative retains the activity on tumor cells. Through acute toxicity experiments on mice, it is shown that the toxicity reaction is greatly reduced after modification of norcantharidin, and the maximum tolerance of mice is more than 10 times relative to norcantharidin.

[0016] (2) The synthesized norcantharidin derivative retains the white blood cell increasing effect of norcantharidin and can antagonize the bone marrow suppression effect caused by chemotherapy drugs.

[0017] (3) The synthesized norcantharidin derivative has an oral bioavailability of about 50% in rats, and has high oral bioavailability.

[0018] (4) The production cost is low, the synthesis process is simple, and high-purity products can be obtained through simple reaction and recrystallization, so that industrial production is easy to realize. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a hydrogen spectrum (deuterated DMSO dissolution) diagram of the norcantharidin derivative provided by the embodiment of the present application;

[0020] Figure 2 is a carbon spectrum (deuterated DMSO dissolution) diagram of the norcantharidin derivative provided by the embodiment of the present application;

[0021] Figure 3 is a mass spectrum diagram of the norcantharidin derivative provided by the embodiment of the present application. DETAILED DESCRIPTION

[0022] The technical solutions of the present application will be further described below in combination with the drawings and embodiments. EMBODIMENT

[0023] Step 1

[0024] 840 mg (5 mmol) of norcantharidin and 1027.5 mg of aminophenylboronic acid (7.5 mmol) were placed in a two-neck flask, and glacial acetic acid was added. Stirring was performed at 50°C until dissolution. The reaction was heated and stirred at 50°C in an oil bath for 6 hours, and a large amount of white precipitate was generated. The reaction solution was cooled to room temperature, and the obtained white filter residue was dried to obtain a crude product. The reaction process is as follows:

[0025] .

[0026] Step 2

[0027] The crude product was dissolved in methanol, evaporated overnight, concentrated to get a large amount of crystallization, filtered, and the filter residue was dried at 60°C to get the final product. The filtrate was evaporated and recrystallized twice to get 1220 mg of white needle-like crystals (denoted as NCTD-PBA), with a yield of 85%.

[0028] The compounds in the present application are characterized and the performance of the products is measured as follows:

[0029] 1. Nuclear magnetic resonance characterization

[0030] 10 mg of the product was dissolved in 0.6 mL of deuterated DMSO and tested on a 400 MHz nuclear magnetic resonance instrument, 1 The H-NMR spectrum is shown in Figure 1 : 1 H NMR (400 MHz, DMSO- d 6) δ 8.18 (s, 2H), 7.83 (dt, J = 7.4, 1.2 Hz, 1H), 7.57 (dd, J = 2.3, 1.2 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.21 (ddd, J = 7.9, 2.2, 1.2 Hz, 1H), 4.81 (q, J = 1.9, 1.5 Hz, 2H), 3.19 (s, 2H), 1.70 (d, J = 1.6 Hz, 4H).

[0031] 15 mg of the product was dissolved in 0.8 mL of deuterated DMSO and tested on a 400 MHz nuclear magnetic resonance instrument, 13 The C-NMR spectrum is shown in Figure 2 : 13 C NMR (101 MHz, DMSO- d 6) δ 177.34, 134.53, 132.97, 132.20,129.05, 128.53, 79.30, 50.25, 28.51.

[0032] See the attached Figure 1 , 2 , which are the hydrogen spectrum (deuterated DMSO dissolution) and carbon spectrum (deuterated DMSO dissolution) of the norcantharidin derivative provided in the present embodiment, respectively; see the attached Figure 3The mass spectrum of the norcantharidin derivative provided in this embodiment is shown in the following figure. The results prove that the product NCTD-PBA compound obtained in step 2 of this embodiment has been successfully prepared.

[0033] 2. Investigation of the effect of the compound on the bone marrow suppression of chemotherapeutic drugs

[0034] Most anticancer drugs have the side effect of bone marrow suppression, which greatly limits their clinical application. Norcantharidin can raise white blood cells by shortening the bone marrow maturation and release time of white blood cells and promoting the differentiation of bone marrow hematopoietic stem cells into granulocytes and monocytes, and its advantage is that it can inhibit tumor cells without reducing the number of white blood cells in the peripheral blood and has no obvious immunosuppression on the body. In order to investigate whether the present application still has the effect of raising white blood cells, a chemotherapeutic drug, cyclophosphamide, was used to evaluate the effect of the present application on the white blood cells of mice after modeling.

[0035] The test scheme is as follows:

[0036] Modeling: Leukopenia mice were modeled by cyclophosphamide, and except for the blank group, the experimental mice were intraperitoneally injected with cyclophosphamide 100 mg / kg once a day for three consecutive days, and the number of white blood cells was measured by cutting the tail to take blood on the fourth day to prepare the leukopenia model mice.

[0037] Experimental grouping: Except for the blank group, the mice were divided into a model group, a norcantharidin group B, and a norcantharidin-PBA group C. Each group had 10 mice, and the drug administration was performed by intraperitoneal injection once a day.

[0038] Drug administration: The blank group and the model group were normally fed, the drug administration groups were intraperitoneally injected with drugs, and the drug administration was continuously performed for 4-7 days. The number of white blood cells was counted by cutting the tail of the mice at 4h after drug administration on the 4th and 7th days.

[0039] White blood cell counting: 190 μL of white blood cell diluent (2% glacial acetic acid) was added to 10 μL of blood, and then the mixture was fully mixed and filled into a counting cell. Then, the mixture was left to stand for 2-3 min, and the total number of white blood cells in four large squares in the four corners was counted under a low-power microscope. Finally, the number of white blood cells per liter of blood was calculated.

[0040] Table 1. Peripheral white blood cell count of mice

[0041]

[0042] The counting results show that the present application retains the white blood cell raising effect of norcantharidin, and from the perspective of raising white blood cells to the normal level, the present application has a good repairing effect on the bone marrow suppression caused by anticancer chemotherapeutic drugs such as cyclophosphamide, and can provide a new product for the application of chemotherapeutic drugs.

[0043] The norcantharidin derivative provided by the application is a new compound obtained by connecting the amino group of aminobenzene boronic acid with norcantharidin, and has excellent liver targeting property. The norcantharidin derivative provided by the application is combined with antitumor chemotherapy drugs such as cisplatin, fluorouracil, camptothecin and adriamycin, or combined with antitumor natural active ingredients and traditional Chinese medicines such as evodiamine, astragalus, angelica, and yohimbine, and can be used for targeted treatment of liver cancer, breast cancer and lung cancer. The norcantharidin derivative can also be used as a preoperative drug for cancer.

[0044] The synthesis method is simple, one-step synthesis, high yield, and can be synthesized on a large scale in industry. The norcantharidin derivative retains the antitumor effect, increases the number of white blood cells, increases the concentration of the drug in the liver, improves the therapeutic effect, and reduces the side effects of norcantharidin in treatment.

Claims

1. A cantharidin derivative, characterized in that... Its structural formula is: 。 2. A method for synthesizing the norcantharidin derivative of claim 1, characterized in that It comprises the following steps: (1) Norcantharidin and aminobenzoic acid are dissolved in glacial acetic acid solution at a molar ratio of 1:1-3 at a temperature of 40-60°C, and then stirred and reacted at an oil bath temperature of 40-80°C for 2-8 hours. After the solution is cooled to room temperature, filtration is performed, the obtained white filter residue is dried, and a crude product is obtained; (2) The product obtained in step (1) is dissolved in methanol and repeatedly recrystallized 2-3 times to obtain transparent needle-like crystalline norcantharidin derivative.

3. The method for synthesizing norcantharidin derivatives according to claim 2, wherein: The molar ratio of norcantharidin to aminobenzoic acid is 1:1.5, the oil bath temperature is 50°C, and the stirring reaction is 6 hours.

4. Use of the norcantharidin derivative of claim 1 in the preparation of a targeted therapeutic drug for liver cancer, breast cancer, lung cancer, or a preoperative drug, wherein the norcantharidin derivative is used in combination with an antitumor chemotherapy drug, such as cisplatin, fluorouracil, camptothecin, or doxorubicin, or in combination with a traditional Chinese medicine, such as evodiamine, astragalus, angelica, or yohimbine.

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