阅读说明：本技术 基于阿霉素药物的诊疗一体化荧光药物分子及其制备和应用 (Diagnosis and treatment integrated fluorescent drug molecule based on adriamycin drug and preparation and application thereof ) 是由 张晓琳 乔威威 李建军 马思阳 于 2020-11-17 设计创作，主要内容包括：基于阿霉素药物的诊疗一体化荧光药物分子及其制备和应用,属于有机化学和药物化学技术领域。本发明诊疗一体化药物分子简称CRDB是由香豆素酰肼衍生物通过酰腙键与阿霉素结合实现荧光团与药物分子的组装。具体制备方法为：将化合物溶于甲醇溶液中,向溶液中加入阿霉素后滴入三氟乙酸,常温搅拌6-8小时后,向溶液中慢速滴加正已烷,过滤取得溶液中析出的黄色固体为CRDB。本发明基于阿霉素药物的诊疗一体化荧光药物分子靶向性好、副作用小、治疗效率高,还具备纳米药物所欠缺的成本低、合成易于调控、荧光检测无辐射,药物易定量。(Diagnosis and treatment integrated fluorescent drug molecules based on an adriamycin drug, and preparation and application thereof, and belongs to the technical field of organic chemistry and pharmaceutical chemistry. The diagnosis and treatment integrated drug molecule CRDB is formed by combining coumarin hydrazide derivatives with adriamycin through acylhydrazone bonds to assemble fluorophores and drug molecules. The preparation method comprises the following steps: dissolving the compound in methanol solution, adding adriamycin into the solution, then dripping trifluoroacetic acid into the solution, stirring the solution at normal temperature for 6 to 8 hours, then slowly dripping n-hexane into the solution, and filtering the solution to obtain a yellow solid which is CRDB and is separated out from the solution. The diagnosis and treatment integrated fluorescent drug based on the adriamycin drug has the advantages of good molecular targeting property, small side effect, high treatment efficiency, low cost, easy synthesis regulation and control, no radiation in fluorescent detection and easy quantification of the drug, which are all defects of nano drugs.)

1. Diagnosis and treatment integrated fluorescent drug molecules based on an adriamycin drug are characterized in that the structural formula of the drug molecules is as follows:

2. the method of claim 1, wherein the synthetic route is as follows:

the preparation method comprises the following steps: dissolving the compound in a methanol solvent, adding adriamycin into the solution, then dripping trifluoroacetic acid into the solution, stirring the solution at normal temperature for 6 to 8 hours, then slowly dripping n-hexane into the solution until a solid product is precipitated, and filtering the solution to obtain a yellow solid precipitated in the solution, namely CRDB.

3. The method for preparing an adriamycin drug-based diagnosis and treatment integrated fluorescent drug molecule according to claim 2, wherein 0.2-0.24mmol coumarin hydrazide is added in every 30ml methanol solvent.

4. The method for preparing an adriamycin drug-based diagnosis and treatment integrated fluorescent drug molecule according to claim 2, wherein the addition amount of adriamycin is the same as the molar amount of coumarin hydrazide.

5. The method of claim 2, wherein the amount of trifluoroacetic acid added is 0.04-0.06ml per 30ml of methanol solvent.

6. The use of the fluorescent drug molecule of claim 1, wherein the acylhydrazone bond is more easily cleaved under acidic conditions to release the drug doxorubicin, which is a unimolecular therapeutic drug molecule constructed based on the fluorescent coumarin and doxorubicin drugs, and can be observed through a fluorescence signal at 450 nm.

Technical Field

The invention belongs to the technical field of organic chemistry and pharmaceutical chemistry, and particularly relates to an integrative diagnosis and treatment fluorescent drug molecule based on an adriamycin drug, and preparation and application thereof.

Background

Cancer is one of the diseases with the highest global mortality at present, the number of cancer deaths worldwide is increasing with the continuous rising of the incidence rate, and how to realize cancer diagnosis and treatment has a key role in improving the quality of life and prolonging the life of cancer patients.

The tumor drugs are various, and in order to improve the curative effect of the drugs in the chemotherapy process, the tumor drugs need to be capable of gathering the tumor sites to be treated in a targeted manner, and the tumor drugs are kept nontoxic and harmless in the in-vivo transportation process so as to avoid damaging normal tissues. This requires that the drug be designed to be encapsulated and released only within the specific environment within the tumor tissue. How to release can utilize the acidity or the high concentration of a certain substance in the tumor microenvironment to reasonably derive chemical bonds to trigger the drug release.

With the development of science and technology, the traditional medicine with single curative effect cannot meet the requirements of the medical industry. Doctors want to know the delivery and action of the medicine, and patients often want to know what happens in their bodies intuitively, so that the doctors and the patients have urgent needs for the medicine which integrates observation and treatment. Molecules for realizing diagnosis and treatment integration by combining fluorescence diagnosis and tumor treatment are also reported successively, but the existing tumor treatment method for realizing diagnosis and treatment integration is mainly realized based on nano ions, and on the whole, the nano medicines have the advantages of high cost, poor repeatability, difficult quantification, strong heavy metal toxicity and the like. Compared with the prior art, the small-molecule fluorescent drug integrating diagnosis and treatment has more advantages, and the molecules have the advantages of good targeting property, small side effect, high treatment efficiency, low cost, easiness in synthesis regulation and control, no radiation in fluorescence detection, easiness in drug quantification and the like which are lacking in nano-drugs, so that the development of diagnosis and treatment integrated single-molecule drugs capable of synchronously monitoring curative effects is an urgent need.

Disclosure of Invention

Aiming at the defects, the invention provides an integrative diagnosis and treatment fluorescent drug molecule based on an adriamycin drug, which has good targeting property, small side effect and high treatment efficiency.

The diagnosis and treatment integrated medicine molecular structural formula adopted for solving the technical problems is shown as the formula I:

the diagnosis and treatment integrated drug molecule CRDB is formed by combining coumarin hydrazide derivatives with adriamycin through acylhydrazone bonds to assemble fluorophores and drug molecules.

The invention also discloses a preparation method of the diagnosis and treatment integrated drug molecule, and the synthetic route of the method is as follows:

the preparation method comprises the following steps: dissolving the compound in a methanol solvent, adding adriamycin into the solution, then dripping trifluoroacetic acid into the solution, stirring the solution at normal temperature for 6 to 8 hours, then slowly dripping n-hexane into the solution until a solid product is precipitated, and filtering the solution to obtain a yellow solid precipitated in the solution, namely CRDB.

Further, 0.2 to 0.24mmol of coumarin hydrazide per 30ml of methanol solvent is added.

Further, the addition amount of the adriamycin is the same as the molar amount of the coumarin hydrazide.

Further, trifluoroacetic acid is added in an amount of 0.04-0.06ml per 30ml of methanol solvent.

The invention simultaneously protects the application of drug molecules CRDB, and the acylhydrazone bond of the single-molecule diagnosis and treatment drug molecules constructed based on the fluorescent coumarin and the adriamycin drugs is easier to crack and release the drug adriamycin under the acidic condition, and the single-molecule diagnosis and treatment drug molecules can be observed through a fluorescent signal at 450 nanometers.

The principle is as follows: the coumarin organic fluorophore with blue light emission and the broad-spectrum drug adriamycin are combined through the acylhydrazone bond by reasonable and ingenious design, so that the fluorescent tumor treatment prodrug integrating diagnosis and treatment is constructed. The acylhydrazone of the molecule is not easy to decompose under the neutral condition of normal tissues, but is easy to decompose under the slightly acidic environment condition in the tumor tissues, and the drug molecule adriamycin can be released aiming at the tumor tissues. And before the drug is released, because the absorption spectrum of the drug adriamycin is well overlapped with the emission spectrum of the fluorophore coumarin, the coumarin transfers energy to the adriamycin through efficient fluorescence resonance energy transfer to cause weak fluorescence of the coumarin, when the coumarin reaches the interior micro-acid environment of a tumor, the adriamycin is released due to the breakage of an acylhydrazone bond, the distance between the coumarin serving as an energy transfer donor and the adriamycin serving as an energy transfer acceptor is increased, the fluorescence resonance energy transfer is incomplete, and the fluorescence of the coumarin is enhanced, so that the fluorescent signal transmission is realized while the adriamycin drug release treatment is realized.

Has the advantages that: the diagnosis and treatment integrated fluorescent drug based on the adriamycin drug has the advantages of good molecular targeting property, small side effect, high treatment efficiency, low cost, easy synthesis regulation and control, no radiation in fluorescent detection and easy quantification of the drug, which are all defects of nano drugs.

Drawings

FIG. 1 is a molecular structure diagram of compound adriamycin (1), coumarin hydrazine hydrate (2) and drug molecule CRDB (3).

Fig. 2 is a ultraviolet absorption spectrum diagram of compound adriamycin (1), coumarin hydrazine hydrate (2) and drug molecule CRDB (3) in ethanol.

Fig. 3 is a graph showing the uv absorption spectra of compound doxorubicin (1), coumarin hydrazine hydrate (2), and drug molecule CRDB (3) in PBS buffer (pH 7.4).

Fig. 4 is a graph showing the ultraviolet absorption spectra of compound doxorubicin (1), coumarin hydrazine hydrate (2), and drug molecule CRDB (3) in an acetic acid buffer solution (pH 5.0).

Fig. 5 is a graph of the fluorescence spectra of drug molecule CRDB (3) in acetic acid buffer (pH 5.0) and PBS buffer (7.4).

Detailed Description

The invention is described in more detail below with reference to specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be purchased from chemical companies.

Synthesis of the Compound of example 1

The compound coumarin hydrazide (80mg,0.22mmol) was dissolved in 30ml of methanol, equimolar amount of doxorubicin was added to the solution, one drop of trifluoroacetic acid was added, stirring was carried out at room temperature for six hours, n-hexane was slowly dropped into the solution until a solid precipitated in the solution was filtered to obtain 155 mg of a yellow solid, and the yield was 77%.

Example 2 ultraviolet absorption Spectroscopy of Compounds in ethanol

Adriamycin, coumarin hydrazine hydrate and drug molecule CRDB are weighed and respectively prepared into 10-5 mu m ethanol solution, and the ultraviolet absorption spectrum is measured, and the test result is shown in figure 2. As can be seen from fig. 2, the uv absorption of the drug molecule CRDB is almost identical to the sum of the uv absorptions of the two fluorescent compounds that make up it, indicating that there is no interaction between the two compounds in the ground state.

Fluorescence spectrometry of the compound of example 3 by uv absorption spectroscopy in PBS buffer (pH 7.4)

Doxorubicin, coumarin hydrazine hydrate and the drug molecule CRDB were weighed to prepare 10-5 μm PBS buffer solution (pH 7.4), and the uv absorption spectrum was measured, and the test results are shown in fig. 3. As can be seen from fig. 3, the uv absorption of the drug molecule CRDB is almost identical to the sum of the uv absorption of the two fluorescent compounds that make up it, indicating that there is no interaction between these two compounds in the ground state.

Fluorescence spectroscopy for ultraviolet absorption of the compound of example 4 in acetic acid buffer (pH 5.0)

Doxorubicin, coumarin hydrazine hydrate and drug molecule CRDB were weighed to prepare 10-5 μm acetic acid buffer solutions (pH 5.0), and the uv absorption spectra were determined, and the test results are shown in fig. 4. As can be seen from fig. 4, the ultraviolet absorption of the drug molecule CRDB is almost identical to the sum of the ultraviolet absorption of the two fluorescent compounds that make up it, indicating that there is no interaction between these two compounds in the ground state.

Absorption spectra data of the compound of example 5 in solvent

TABLE 1 absorption spectra data of compounds in solvents

As can be seen from Table 1, in the ethanol solution, drug molecule CRDB has a maximum absorption peak at a wavelength of 428nm, corresponding to a molar absorption coefficient of 36488M-1 cm-1. Doxorubicin had a maximum absorption peak at 489nm, corresponding to a molar absorption coefficient of 8282M-1 cm-1. And the coumarin hydrazine hydrate has a maximum absorption peak at the wavelength of 417nm, and the corresponding molar absorption coefficient is 54574M-1 cm-1.

In PBS solution at pH 7.0, drug molecule CRDB has a maximum absorption peak at a wavelength of 438nm, corresponding to a molar absorption coefficient of 21350M-1 cm-1. The doxorubicin has a maximum absorption peak at a wavelength of 480nm, and the corresponding molar absorption coefficient is 3739M-1 cm-1. And the coumarin hydrazine hydrate has a maximum absorption peak at a wavelength of 428nm, and the corresponding molar absorption coefficient is 42432M-1 cm-1.

In an acetic acid buffer solution with pH 5.0, CRDB as a drug molecule has a maximum absorption peak at 439nm, and the corresponding molar absorption coefficient is 23161M-1 cm-1. The doxorubicin has a maximum absorption peak at a wavelength of 495nm, and the corresponding molar absorption coefficient is 3653M-1 cm-1. While the compound 1 has a maximum absorption peak at a wavelength of 428nm, and the corresponding molar absorption coefficient is 29031M-1 cm-1.

EXAMPLE 6 fluorescence Spectroscopy profiles of drug molecules CRDB at different pH

The drug molecule CRDB was weighed and prepared into 10-5 μm acetic acid buffer solution (pH 5.0) and 10-5 μm PBS buffer solution (pH 7.4), respectively, and the fluorescence spectrum was measured, and the test results are shown in fig. 5. As can be seen from the attached FIG. 5, the drug molecule CRDB has weak fluorescence under neutral conditions, but the coumarin has obviously enhanced fluorescence under acidic conditions, and the analysis is that doxorubicin and coumarin hydrazine hydrate are broken under acidic conditions. Under neutral conditions, fluorescence resonance energy transfer occurs between coumarin and adriamycin, so that coumarin is weak in fluorescence, but the fluorescence of coumarin can be released after the coumarin is broken.

The above description is only for the purpose of creating a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution and the inventive concept of the present invention within the technical scope of the present invention.