Anti-tumor prodrug, and preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of biochemistry; and specifically relates to an anti-tumor prodrug, and a preparation method therefor and the use thereof. The anti-tumor prodrug is composed of the tail of nervonic acid and the head of a hydrophilic chemotherapeutic drug. The hydrophilic chemotherapeutic drug is gemcitabine, and the tumor is glioma. An acid-sensitive amphiphilic prodrug compound is synthesized on the basis of using a nutrient nervonic acid capable of penetrating the blood-brain barrier and a chemotherapeutic drug as raw materials. Once the prodrug compound reaches a tumor site, an acid-sensitive bond is automatically cleaved due to the low pH in the tumor microenvironment, causing the prodrug compound to separate into the nervonic acid and the chemotherapeutic drug to perform respective functions thereof, wherein the chemotherapeutic drug kills glioma cells, and the nervonic acid repairs nerve tissues, thereby fundamentally solving the toxic side effects of the chemotherapeutic drug, and integrating therapeutic and rehabilitative effects.

Description

An antitumor prodrug and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biochemical technology, and particularly relates to an antitumor prodrug and a preparation method and application thereof. BACKGROUND

[0002] Glioma is one of the most common primary brain tumor types, and can cause significant morbidity and mortality. Since most gliomas are characterized by infiltrative growth and malignant transformation, the boundary with the surrounding brain tissue is unclear, and it is easy to relapse, so the treatment is very difficult. The annual incidence of brain glioma in China is 5 / 100,000-8 / 100,000, and the 5-year mortality rate is second only to pancreatic cancer and lung cancer among systemic tumors. There are mainly three methods for treating brain glioma: surgical resection, radiotherapy and chemotherapy. Due to the characteristics of brain glioma, chemotherapy is an essential treatment method. However, due to the existence of the blood-brain barrier, it is difficult for drugs to be effectively delivered to the lesion site to exert efficacy. In addition, studies have shown that damage to the blood-brain barrier is associated with many brain diseases, so how to further develop new drugs and new drug delivery systems on the basis of protecting the blood-brain barrier from damage is still a research focus.

[0003] Based on non-invasive means, systemic administration to brain lesion sites, drug delivery through endogenous transport mechanisms across the blood-brain barrier, limited penetration caused by off-target exposure, etc. are not sufficient to improve brain diseases. In order to improve the efficiency of crossing the blood-brain barrier, physical or chemical methods (exogenous effects) are used to induce the tight junctions of endothelial cells to be disconnected, opening the blood-brain barrier. For example, focused ultrasound combined with microbubbles, arterial injection of hyperosmotic agent mannitol to open the blood-brain barrier. This way of temporarily and locally damaging the blood-brain barrier can promote, increase and improve the entry of drugs into brain tissue. With the continuous development of minimally invasive surgery, a series of techniques can be used to bypass the blood-brain barrier to reach the tumor site through local direct administration. For example, the convection-enhanced delivery method has been applied to clinical trials. Clinically, under the guidance of MRI, by implanting a catheter, the accumulation of drugs in the brain tumor area is increased. Laser interstitial thermotherapy is a method of heating and destroying tumors by directional implantation of laser optical fibers. The above-mentioned non-invasive or invasive methods inevitably cause complications (brain edema, infection, etc.). Therefore, it is urgent to explore a new type of drug that can effectively treat brain glioma and repair damaged brain tissue. SUMMARY

[0004] The present application aims to provide an antitumor prodrug and a preparation method and application thereof. The antitumor prodrug is separated into nervonic acid and a chemotherapy drug in the tumor microenvironment to perform their respective functions. The chemotherapy drug kills glioma cells, and the nervonic acid repairs nerve tissue, fundamentally solving the toxic and side effects of the chemotherapy drug, and integrating treatment and rehabilitation, which has good application prospects.

[0005] The application provides an antitumor prodrug, which is composed of a tail of nervonic acid and a head of a hydrophilic chemotherapeutic drug.

[0006] Further, the hydrophilic chemotherapeutic drug is a water-soluble chemotherapeutic drug such as gemcitabine or hydroxyurea.

[0007] Further, the tumor is brain glioma.

[0008] The application further provides a preparation method of the antitumor prodrug, which comprises the following steps: (1) nervonic acid is dissolved in a DMF solution containing HATU and DIPEA to perform reaction, so as to obtain a post-reaction solution; (2) a DMF solution containing gemcitabine is added into the post-reaction solution to perform stirring reaction, so as to obtain a mixture solution; and (3) the mixture solution is concentrated under reduced pressure, and then subjected to column chromatography separation and purification, so as to obtain the antitumor prodrug.

[0009] Further, the ratio of the gemcitabine to the nervonic acid is 0.5-2:1.

[0010] Further, in step (1), the reaction is performed under the condition of ice bath for 1-2 h.

[0011] Further, in step (2), the stirring reaction is performed at room temperature for 24-36 h.

[0012] Further, in step (3), the eluent with a volume ratio of dichloromethane to methanol of 10 / 1 is used in the column chromatography separation and purification process, and 200-300 mesh silica gel is used as a filling carrier.

[0013] The application further provides application of the antitumor prodrug in preparation of a drug for treating brain glioma.

[0014] The application adopts nervonic acid and a chemotherapeutic drug gemcitabine to construct a prodrug. Nervonic acid is also named shark acid, and is named because it is firstly found in the brain of a shark. Nervonic acid is a recognized core natural component of brain nerve fibers and nerve cells, and is also a unique double-effect miraculous material capable of repairing and dredging damaged brain nerve fibers and promoting regeneration of nerve cells. Deficiency of nervonic acid will cause sequelae of cerebral apoplexy, senile dementia, cerebral palsy, brain atrophy, memory decline, insomnia and amnesia and other brain diseases. Nervonic acid is difficult to be generated in human body, and can only be supplemented by external intake. Nervonic acid can completely penetrate the blood-brain barrier, directly act on nerve fibers to repair and dredge, regenerate damaged and shed protective sheaths, dissolve necrotic tissues blocking the channel, and induce self-growth and division of nerve fibers.

[0015] Nervonic acid has a free carboxyl group, so it can be used to form acid-sensitive ester bonds, hydrazone bonds, imine bonds, etc. with chemotherapeutic drugs to make amphiphilic prodrug compounds. When the nervonic acid carrying the chemotherapeutic drug passes through the blood-brain barrier into the tumor, due to the acidic tumor microenvironment, the acid-sensitive bond breaks, forming nervonic acid and the chemotherapeutic drug, respectively, and performing their respective functions.

[0016] In combination with the chemotherapeutic drug gemcitabine against multiple solid tumors, acylation at the 4-(N)-position of gemcitabine does not impair the efficacy of gemcitabine. Due to the acidic tumor microenvironment, the amide bond between nervonic acid and gemcitabine is cleaved; the amide bond can also be hydrolyzed by intracellular amidases such as cathepsin B or cathepsin D after entering the cell, thereby regenerating gemcitabine and nervonic acid with drug activity. The chemotherapeutic drug gemcitabine kills tumor cells; nervonic acid protects nerve tissue.

[0017] It is known that gemcitabine is rapidly deaminated to the inactive metabolite 2,2-difluorodeoxyuridine in the blood and is rapidly excreted by the urine; in the liver, it is extensively degraded by cytidine deaminase to inactive metabolites. The conjugated polymer of gemcitabine increases the half-life of the drug in the blood plasma by reducing its clearance in the kidneys, and this half-life depends on the molecular weight of the polymer. Therefore, the conjugation of gemcitabine with nervonic acid increases the residence time in the blood circulation.

[0018] The present application has the beneficial effect that: based on the nutrient substance nervonic acid that can penetrate the blood-brain barrier and the chemotherapeutic drug, an acid-sensitive amphiphilic prodrug compound is synthesized, which automatically breaks down into nervonic acid and the chemotherapeutic drug to perform their respective functions when it reaches the tumor site due to the lower pH in the tumor microenvironment, the chemotherapeutic drug kills glioma cells, and nervonic acid protects nerve tissue, fundamentally solving the toxic side effects of chemotherapeutic drugs, and integrating treatment and rehabilitation, which has good prospects for use. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0020] Figure 1 is a synthetic route of the brain glioma prodrug in the present application; Figure 2 is a mass spectrum analysis result of the nervonic acid-gemcitabine prodrug compound; Figure 3 is a nuclear magnetic spectrum of the nervonic acid-gemcitabine prodrug compound; Figure 4 is an infrared spectrum of the nervonic acid-gemcitabine prodrug compound; Figure 5 is an acid sensitivity verification of the nervonic acid-gemcitabine prodrug compound; Figure 6 is a high performance liquid chromatogram of the nervonic acid-gemcitabine prodrug compound; Figure 7 is an ion chromatogram analysis of the nervonic acid-gemcitabine prodrug compound; Figure 8 is a nuclear magnetic spectrum of the coupling of nervonic acid and the chemotherapeutic drug hydroxyurea. DETAILED DESCRIPTION

[0021] The detailed description set forth below is intended as a description of various example embodiments of the application and is not intended to represent the only embodiments in which the application can be practiced. The term "exemplary" used throughout this description means "serving as an example, instance, or illustration," and should not necessarily be construed as preferred or advantageous over other examples.

[0022] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, any numerical range recited herein is intended to include all sub-ranges of the same numerical limits. In

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present application, the preferred methods and materials are described herein. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference in their entirety for the disclosure and

[0024] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. For example, the specification can be used to design equivalent methods and materials for carrying out the same functions as described herein. Therefore, the foregoing description is to be considered only as illustrative of the principles of the application and not in limitation of the scope of the application. The scope of the application is to be defined by the appended claims.

[0025] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0026] The brain glioma prodrug in the present application is composed of two parts: the tail of nervonic acid and the head of hydrophilic chemotherapeutic drug.

[0027] Nervonate acid (NA) is a major fatty acid found in various sphingolipid species in the central nervous system, can easily cross the blood-brain barrier to reach brain tissue, and has important biological functions in brain development and injury repair healthcare. In addition, nervonate acid also has a modifiable carboxyl group, so it can form an acid-sensitive linkage with a water-soluble chemotherapeutic drug to prepare an amphiphilic prodrug compound that can penetrate the blood-brain barrier.

[0028] In the following, HATU: 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIPEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide; wherein HATU is purchased from Shanghai Aladdin Biochem Technology Co., Ltd.; DIPEA is purchased from Tianjin Kemio Chemical Reagent Co., Ltd.; DMF is purchased from Tianjin Kemio Chemical Reagent Co., Ltd.; gemcitabine is purchased from Shanghai Aladdin Biochem Technology Co., Ltd.; nervonate acid is purchased from Shanghai Ronghe Medicine Science and Technology Development Co., Ltd.

[0029] The specific preparation method of the brain glioma prodrug is as follows: nervonate acid (1 mmol) is dissolved in a DMF solution containing HATU and DIPEA (the ratio of nervonate acid to activating agent (HATU and DIPEA) is controlled at 1:1.2, and the reaction effect is the best), and the reaction is carried out under ice bath conditions for 1-2 h. Then, a DMF solution containing gemcitabine (1 mmol) is added. The mixture is stirred at room temperature for 24-36 h. After the reaction is completed, the reaction solution is concentrated under reduced pressure, column chromatography separation and purification are carried out using dichloromethane and methanol with a volume ratio of 10 / 1 as the eluent and 200-300 mesh silica gel as the filling carrier to obtain the nervonate acid-gemcitabine prodrug compound. The specific synthesis route is shown in Figure 1.

[0030] The present application is specifically described below by way of examples.

[0031] The specific preparation method of the brain glioma prodrug in Example 1 includes the following steps: nervonate acid (1 mmol, 366 mg) is dissolved in a 5 mL DMF solution containing HATU (1.2 mmol, 456 mg) and DIPEA (1.2 mmol, 155 mg) (the condensing agent HATU and DIPEA are 1.2-1.5 times the reaction amount for the best effect), and the reaction is carried out under ice bath conditions for 2 h. Then, a DMF (5 mL) solution containing gemcitabine (1 mmol, 263 mg) is added. The mixture is stirred at 25°C for 24 h. After the reaction is completed, the reaction solution is concentrated under reduced pressure, column chromatography separation and purification are carried out using dichloromethane and methanol with a volume ratio of 10 / 1 as the eluent and 200-300 mesh silica gel as the filling carrier to obtain the nervonate acid-gemcitabine prodrug compound.

[0032] The mass spectrum analysis result of the nervonic acid-gemcitabine prodrug compound is shown in Figure 2. As shown in Figure 2, m / z 610.44 is the molecular ion peak of the combination of nervonic acid and gemcitabine, proving that the NA-Gem prodrug molecule is synthesized in this embodiment.

[0033] The nuclear magnetic spectrum of the nervonic acid-gemcitabine prodrug compound is shown in Figure 3. As shown in Figure 3, the signal peak at a chemical shift of 10.98 ppm is the signal peak of the hydrogen of the amide bond, and the signal peaks at 4.15 ppm and 3.87 ppm are the signal peaks of the hydrogens of the hydroxyl groups of gemcitabine; the signal peaks at 0.84-1.53 ppm belong to the signal peaks of the hydrogens of nervonic acid.

[0034] The chemical structure of NA-Gem is further characterized by FTIR, and the result is shown in Figure 4. As shown in Figure 4, the characteristic signal peaks of the amide bond (-CO-NH-) are at 1660 cm -1 and 1556 cm -1 . This indicates that the combination of nervonic acid and gemcitabine is successful. The acylation at the 4-(N)-position of gemcitabine does not damage the efficacy of gemcitabine. Gemcitabine is a pyrimidine antitumor drug, which belongs to cell cycle-specific antimetabolites. Its main metabolite is mixed with DNA in cells, and mainly plays a role in the G1 / S phase of the cell cycle. The acid sensitivity verification of the nervonic acid-gemcitabine prodrug compound is shown in Figure 5. As shown in Figure 5, when the pH is low, the acid-sensitive bond automatically breaks, and the prodrug compound separates into nervonic acid and chemotherapeutic drugs to perform their respective functions, having good acid sensitivity. As can be seen from the above, the nervonic acid-gemcitabine prodrug compound is successfully obtained by using the specific preparation method in this embodiment 1.

[0035] At the same time, the purity of the prodrug is detected by using a high-performance liquid chromatograph, as shown in Figure 6. Combined with the maximum absorption value of the ultraviolet spectrum of the prodrug, the mobile phase is methanol: water = 90%: 10%. Finally, it is found that the purity of the prodrug prepared in this embodiment is 98.66%.

[0036] The in vitro blood-brain barrier model is constructed by using a Transwell (permeable cell culture chamber). The prodrug compound is dissolved in cell culture medium and incubated (cultured) with endothelial cells in the upper chamber. The ion chromatograph is used to quantitatively analyze the fluorine element in the cell culture medium in the lower chamber of the Transwell. The results show that the fluorine element changes in a gradient with time, proving that the prodrug compound can pass through the blood-brain barrier. The specific results are shown in Figure 7. The nuclear magnetic spectrum of the coupling of nervonic acid and the chemotherapeutic drug hydroxyurea is shown in Figure 8.

[0037] Finally, it is to be explained that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. An antitumor prodrug, characterized in that, The antitumor prodrug is composed of a tail of nervonic acid and a head of a hydrophilic chemotherapeutic drug.

2. The antitumor prodrug according to claim 1, wherein The hydrophilic chemotherapeutic drug is gemcitabine.

3. The antitumor prodrug according to claim 1, wherein The tumor is brain glioma.

4. The method for preparing the antitumor prodrug according to any one of claims 1-3, characterized in that, The method comprises the following steps: (1) reacting nervonic acid in a DMF solution containing HATU and DIPEA to obtain a post-reaction solution; (2) adding a DMF solution containing gemcitabine to the post-reaction solution and stirring to obtain a mixture solution; (3) concentrating the mixture solution under reduced pressure and then performing column chromatography separation and purification to obtain the antitumor prodrug.

5. The production method according to claim 4, wherein The ratio of gemcitabine to nervonic acid is 0.5-2:

1.

6. The production method according to claim 4, wherein In step (1), the reaction is performed under ice bath conditions for 1-2 h.

7. The production method according to claim 4, wherein In step (2), the stirring is performed at room temperature for 24-36 h.

8. The production method according to claim 4, wherein In step (3), the eluent used in the column chromatography separation and purification process is dichloromethane and methanol with a volume ratio of 10 / 1, and the filling carrier is 200-300 mesh silica gel.

9. Use of the antitumor prodrug according to any one of claims 1-3 in the preparation of a drug for treating brain glioma.

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