KR101798203B1 - Hybrid anticancer prodrug for creating cinnam aldehyde with quinone metide by acidic pH and esterase, and method for preparing the same - Google Patents

Abstract

The present invention relates to a hybrid anticancer pro-drug capable of simultaneously producing cinnamaldehyde and quinone methide. The hybrid anticancer pro-drug of the present invention releases cinnamaldehyde producing reactive oxygen species (ROS) by esterase and acidic pH and quinone methide scavenging glutathione (GSH) respectively, inhibits antioxidation systems, and promotes apoptosis, thereby bringing synergistic anticancer effects and double stimulation responses specific to cancer cells. Thus, the hybrid anticancer pro-drug can be useful as an anticancer agent.

Description

[0001] The present invention relates to a hybrid anticancer prodrug, which simultaneously produces cinnamaldehyde and quinomethide by acidic pH and esterase, and a method for preparing the same,

The present invention relates to a hybrid anticancer prodrug which simultaneously produces cinnamaldehyde and quinomethide by an acidic pH and an esterase, and is a hybrid anticancer prodrug which induces the death of cancer cells while increasing oxidative stress by complementary synergistic action, .

Cancer is defined as "the formation of tumors composed of undifferentiated proliferating undifferentiated cells, ignoring the necessary conditions in the tissues, unlike normal cells that can regularly proliferate and inhibit according to the individual's needs" Cells are transformed into cancer cells by a change in the gene in the cell for a specific reason. In the world, cancer accounts for about 13% of all deaths, and it is possible to develop without any distinction between sexes and ages. It is the second leading cause of death in the world, Research is underway. In this study, it is required to develop an effective anticancer agent that can minimize the side effects due to the diversity of the cancer and the pathogenesis of the cancer, and new anticancer drugs are being marketed continuously.

The stimulus-sensitive system responds to the external environment such as pH, temperature, ionic strength, electric field, magnetic field, light, ultrasonic wave, etc., and changes in the phase transition, swelling, It says. Such a stimulation-sensitive system is mainly used for a release control system that not only protects the drug but also regulates the release rate of the drug or induces the drug to stay in a specific site. In particular, the pH of the cancer site is different from that of the normal body (7.4 ± 0.04), and various functional groups susceptible to pH exist, and thus drug carriers using the drug carriers are actively developed.

On the other hand, cinnamaldehyde (cinnamaldehyde) is the main active ingredient of cinnamomum cassia, a plant of Camelliaceae (Lauraceae), which has been used to treat indigestion, gastritis, blood circulation disorder and inflammation in both the east and the west. Cinnamon bark ). ≪ / RTI > Shin Nam aldehyde contains α, β-carbonyl, also known as micheal receptor pharmacophore, and produces reactive oxygen species (ROS), which reduces the mitochondrial membrane potential The ability to induce apoptosis through the release of cytochrome C as a cytosol in cells has been demonstrated to have anticancer ability through a mechanism dependent on caspases. Despite the excellent anticancer potential of cinnamaldehyde and its derivatives, however, phagocytosis is rapidly phagocytosed in vivo by hepatic macrophages and has a short half-life of less than 1.5 hours (minutes, about 5 minutes) There is a disadvantage that there is no ability. Therefore, clinical application of cinnamaldehyde for chemotherapy requires the development of physicochemical modifications or new drug delivery systems to enhance the anti-cancer effect.

In addition, quinone methide is known to react with GSH (glutathione), an essential antioxidant enzyme in cancer cells, to lower antioxidant levels, thereby increasing antioxidative effects while increasing oxidative stress.

Accordingly, the present inventors have conducted studies to produce a hybrid anticancer prodrug that has synergistic effects to enhance short-term retention time and efficacy, which is a disadvantage of cinnamaldehyde inducing apoptosis, and to enhance its anticancer effect. As a result, ([4 - [[5-methyl-2- (styryl-1,3-dioxan-5-yl) methoxycarbonyloxymethyl] phenyl] benzoate, a prodrug that simultaneously produces quinone Osamp increased the retention time of cinnamaldehyde, further acted selectively on cancer cells, minimized side effects, exhibited the maximum anticancer effect, and confirmed the applicability as a new chemotherapeutic agent, thus completing the present invention.

The present invention aims to provide a hybrid anticancer prodrug which simultaneously produces cinnamaldehyde and quinomethide by acidic pH and esterase, and a method for producing the same.

It is another object of the present invention to provide a composition for preventing or treating cancer comprising the above-mentioned hybrid anticancer prodrug as an active ingredient.

In order to achieve the above object, the present invention provides a hybrid anticancer prodrug represented by the following formula (1) simultaneously producing cinnamaldehyde and quinone methide, and a process for producing the same.

[Chemical Formula 1]

In Formula 1, R is H- or C ₆ H ₅ COO-, R 'is H- or CH ₃ -, and X is O or NH.

In addition, the present invention provides a pharmaceutical composition and a food composition for preventing or treating cancer comprising the hybrid anticancer prodrug as an active ingredient.

The hybrid anticancer prodrug of the present invention can inhibit the antioxidant system and accelerate apoptosis by releasing cinnamaldehyde and GSH-cleaving quinomethine to produce ROS by acidic pH and Esterase, respectively, It can be used effectively as an anticancer agent because it causes synergy anticancer effect.

1 is a graph showing the ¹ H-NMR spectrum of the cinnamaldehyde derivatives (1) (a) and cinnamaldehyde-releasing compound (2) (b) prepared in Examples 1-1 and 1-2 of the present invention.
Fig. 2 is a graph showing ¹ H-NMR spectrum (a), ¹³ C NMR spectrum (b) and LC-MS (c) of OSamp prepared in Example 1-4 of the present invention.
FIG. 3A shows the sensitivity of the present invention to the acidic pH of OSamp through ¹ H-NMR spectrum. FIG.
FIG. 3B shows the sensitivity of OSamp to the esterase of the present invention through GSH levels.
FIG. 4 is a graph showing the generation of quinone and cinnamaldehyde by OSamp in the SW620 cell line.
FIG. 5A is a graph showing GSH scavenging activity according to OSamp concentration in SW620 cell line. FIG.
FIG. 5B is a graph showing the level of ROS according to the concentration of OSaam in the SW620 cell line.
FIG. 5c is a graph showing the production of intracellular ROS according to the OSamp concentration in a SW620 cell line through a confocal scanning laser microscope.
5d to 5f are cytotoxicities according to OSamp concentration in DU145 (prostate cancer cell line) (d), SW620 (colorectal cancer cell line) (e) and HEK293 (human kidney cells), respectively.
6A is a graph showing changes in the mitochondrial membrane potential of the SW620 cell line treated with OSamp of the present invention.
FIG. 6B shows the release of cytochrome c from the SW620 cell line treated with OSamp of the present invention.
FIG. 7A is a fluorescence microscope photograph of the SW620 cell line treated with OSamp of the present invention. FIG.
FIG. 7B is a graph showing an increase in the amount of SW620 cells treated with Osamp of the present invention through a flow cytometer.
FIG. 7C shows the effect of inducing the degradation of caspase-3 in the SW620 cell line treated with OSamp of the present invention.
FIG. 7d is a view showing the amount of a nucleosome DNA fragment in an OSamp-treated SW620 cell line of the present invention. FIG.
FIG. 7E is a graph showing the expression of p-STAT3 in an OSamp-treated SW620 cell line of the present invention.
FIG. 8 is a graph showing the tumor growth inhibitory effect of tumor size (a), tumor volume (b), and weight change (c) after administration of OSamp in a tumor mouse model in which Osamp was administered according to the present invention.
FIG. 9 is a graph showing tumor growth inhibitory effect through tumor size (a), volume change (b) and weight change (c) according to the OSamp concentration in a tumor mouse model in which Osamp was administered according to the present invention .
10 is a graph showing the results of performing histological examination (a) and immunohistochemistry (b) in a tumor mouse model in which Osamp was administered according to the present invention.
FIG. 11 is a graph showing changes in ALT activity (a) and H & E staining results (b) of liver tissues in a tumor mouse model in which Osamp was administered according to the present invention.

The present invention provides a hybrid anticancer prodrug that simultaneously produces cinnamaldehyde and quinone methide, and provides a hybrid anticancer prodrug represented by Formula 1 below.

[Chemical Formula 1]

In Formula 1, R is H- or C ₆ H ₅ COO-, R 'is H- or CH ₃ -, and X is O or NH.

Hereinafter, the present invention will be described in more detail.

In general, Cinnam aldehyde is known to induce apoptosis through the production of reactive oxygen species (ROS), but it has weak cytotoxicity in normal cells. However, its application has been limited by the short half-life in renin-aldehyde in blood and by its low activity compared to general anti-cancer drugs. Therefore, in order to overcome such disadvantages, in the present invention, a novel hybrid anticancer prodrug [4 - [[5-methyl-2- (styryl- dioxan-5-yl) methoxycarbonyloxymethyl] phenyl] benzoate] (OSamp) was prepared.

(2)

Wherein the hybrid anticancer prodrug comprises a portion of an ester compound that produces quinone and a cinnamaldehyde derivative portion, wherein the ester compound portion and the cinnamaldehyde derivative portion are in the form of carbonate (-OCOO-) or carbamate (- NHCOO < - >). At this time, the ester compound portion is linked to -COO-.

The quinomethide and cinnamaldehyde can be produced by esterase and acidic pH, and in particular they can be produced specifically in cancer cells.

At this time, quinone methide is produced through ester bond decomposition by esterase, and the quinone methide produced at this time can alkylate the antioxidant glutathione (GSH) to inhibit the antioxidant system and increase the oxidative stress. In addition, the acidic pH cleaves the acetal bond of the present invention to release cinnamaldehyde, and the released cinnamaldehyde can promote ROS and promote apoptosis. ROS produced by the release of cinnamaldehyde in a state of reduced antioxidant levels is accumulated in large quantities, further promoting apoptosis. Accordingly, the hybrid anti-cancer drug of the present invention exhibits a synergistic anticancer effect through double stimulation reaction and sequential treatment in a cancer cell-specific manner.

In addition,

(a) reacting cinnamaldehyde with an acidic solution to produce a cinnamaldehyde derivative having an acetal bond;

(b) reacting the cinnamaldehyde derivative prepared in the step (a) with carbonyldiimidazole to prepare a cinnamaldehyde releasing compound;

(c) reacting a hydroxyalkylphenyl and a benzoyl compound to produce an ester compound that produces a quinomethide; And

(d) reacting the cinnamaldehyde-releasing compound prepared in the step (b) with the ester compound prepared in the step (c). The present invention also provides a method for producing a hybrid anticancer prodrug.

A representative example of the method for producing the hybrid anticancer prodrug of the present invention can be represented by the following Reaction Scheme 1.

[Reaction Scheme 1]

The process for preparing the hybrid anticancer prodrug of the present invention will be described in detail as follows.

The step (a) is a step of preparing a cinnamaldehyde derivative having an acetal bond by adding cinnamaldehyde to an acidic solution, allowing the reaction to proceed at a high temperature of 70 to 100 ° C, followed by evaporation of the solvent. The acidic solution is preferably p-toluenesulfonic acid or sulfuric acid, but is not limited thereto.

The prepared cinnamaldehyde derivative can decompose in an acid including an acetal bond, thus allowing the release of cinnamaldehyde in cancer cells.

The step (b) is a step of preparing a cinnamaldehyde-releasing compound. The cinnamaldehyde derivative prepared in the step (b) is dissolved in an organic solvent together with carbonyldiimidazole, reacted at 20 to 40 ° C, and the solvent is evaporated .

The step (c) is a step of preparing an ester compound that produces quinone methide. The ester compound is prepared by reacting a hydroxyalkylphenyl and a benzoyl compound in an organic solvent.

The hydroxyalkylphenyl is preferably hydroxymethylphenyl, but is not limited thereto. The benzoyl compound is preferably benzoyl chloride, but is not limited thereto.

(D) is a step of producing a hybrid anticancer prodrug that simultaneously produces cinnamaldehyde and quinomethine, wherein the cinnamaldehyde releasing compound prepared in step (b) and the ester compound prepared in step (c) Reacting in an organic solvent and evaporating the solvent.

The organic solvent used in each step may include, but is not limited to, tetrahydrofuran, dichloromethane, hexane, dioxane, benzene, dimethylsulfoxide, dimethylformamide, and the like.

A representative example of the degradation process of the hybrid anticancer prodrug of the present invention can be represented by the following reaction formula (2).

[Reaction Scheme 2]

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the above-mentioned hybrid anticancer prodrug as an active ingredient.

The hybrid anticancer prodrug of the present invention is produced by sequential release of quinomethide and cinnamaldehyde by esterase and acidic pH, alkylation of antioxidant GSH by quinomethyl release, thereby inhibiting the antioxidant system and increasing oxidative stress . In addition, the active oxygen (ROS) produced by the release of cinnamaldehyde is accumulated in a state of reduced antioxidation level, thereby promoting apoptosis, thereby causing a synergistic anticancer effect through double stimulation reaction and sequential treatment action specific to cancer cells. Can be usefully used.

Said cancer is selected from the group consisting of lung cancer, pancreatic cancer, colon cancer, colorectal cancer, myeloid leukemia, thyroid cancer, MDS, bladder carcinoma, epidermal carcinoma, melanoma, breast cancer, prostate cancer, head and neck cancer, uterine cancer, A cancer selected from the group consisting of gastric cancer, laryngeal cancer, esophageal cancer, bladder cancer, oral cancer, cancer of the mesenchymal origin, sarcoma, teratocarcinoma, neuroblastoma, renal carcinoma, liver cancer, non- Hodgkin's lymphoma, multiple myeloma, Lt; / RTI >

The composition of the present invention may contain one or more known active ingredients having an effect of preventing or treating cancer together with the hybrid anticancer prodrug.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For the desired effect, the hybrid anticancer prodrug of the present invention may be administered in an amount of 1 mg / kg to 10000 mg / kg per day, and may be administered once or several times a day.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of cancer.

The present invention also provides a food composition for preventing or ameliorating cancer comprising the hybrid anticancer prodrug as an active ingredient. When the hybrid anticancer prodrug of the present invention is used as a food additive, the hybrid anticancer prodrug may be added as it is, mixed with another food or food ingredient, and used appropriately according to a conventional method.

In addition, the mixing amount of the hybrid anticancer prodrug may be suitably changed according to the purpose of use (prevention, health or therapeutic treatment), and the hybrid anticancer prodrug may be used in an amount of 0.001 to 50 Weight percent, but is not limited thereto. If the content is less than 0.001% by weight, the effect of improving the cancer may be insignificant. If the content is more than 50% by weight, the rate of increase in the effect of the composition may not be economical.

As a specific example, the hybrid anticancer prodrug of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight, based on the raw material. However, when it is intended for health and hygiene purposes or for the purpose of controlling health, it can be added in an amount below the above range, and there is no problem in terms of safety. Therefore, the active ingredient can be used in an amount exceeding the above range have.

There is no particular limitation on the kind of the food. Examples of foods to which the compound of the present invention can be added include antibiotics such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, , Tea, a drink, an alcoholic beverage, a vitamin complex, and the like.

When the food composition of the present invention is prepared as a beverage, it may contain additional ingredients such as various flavors or natural carbohydrates such as ordinary beverages. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame, and the like. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , Carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may include flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of the above additives is not limited to a great extent, but may be in the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  One. OSamp Manufacturing

1-1. Shin Nam Aldehyde  The derivative ((5-methyl-2- styryl -1,3- dioxan -5- yl ) methanol) Preparation of (1)

Tris (hydroxymethyl) ethane (4.88 g) was dissolved in 70 ml of dry benzene. Then, cinnamaldehyde (5.714 mL) and p-toluenesulfonic acid (40 mg) were added to the reaction solution and reacted at 90 DEG C for 4 hours. After that, the reaction solution was cooled at room temperature, and 1 mL of triethyleneamine was added to terminate the reaction. The benzene in the reaction mixture was evaporated using a rotary evaporator and purified by column chromatography (hexane / ethyl acetate = 7/3) to give the title compound.

The ¹ H NMR spectrum of the title compound is shown in FIG.

1-2. Shin Nam Aldehyde  Preparation of releasing compound (2)

1,1'-Carbonyldiimidazole (4.1 g) and cinnamaldehyde derivative (2) (2) prepared in 1-2 above (3.0 g) were dissolved in 50 mL of dry dichloromethane And reacted at room temperature for 30 minutes. The reaction mixture was evaporated under reduced pressure to remove the dichloromethane and purified by column chromatography using ethyl acetate as eluting solvent to give the title compound (2).

The ¹ H NMR spectrum of the title compound is shown in FIG.

1-3. (4-( Hydroxymethyl ) Phenyl ) Benzoate  synthesis

(4-hydroxymethyl) phenol (2.0 g) was dissolved in 40 mL of dry tetrahydrofuran at room temperature and benzoyl chloride (1.87 mL) was added dropwise to the solution (4 DEG C) in an ice-water bath. Then, the reaction was carried out with stirring for 30 minutes. The solvent in the reaction mixture was evaporated to dryness and the title compound (3) was obtained using silica gel chromatography (hexane / ethyl acetate = 2/1).

1-4. Manufacture of OSamp

Compound (2) (2 g) prepared in 1-2 above and compound (3) (2 g) prepared in 1-3 above were added to a solution of 4- (dimethylamino) pyridine ) In 50 mL of dry dichloromethane and allowed to react at 40 < 0 > C for 24 hours. The reaction mixture was evaporated under reduced pressure to remove the solvent and purified by column chromatography (hexane / ethyl acetate = 2/1) to give the title compound (OSamp).

¹ H NMR, ¹³ C NMR spectrum and LC-MS of the above-prepared title compound (OSamp) were confirmed, and the results are shown in FIGS. 2A to 2C, respectively.

As shown in Figs. 2A to 2C, the presence of acetal cations was confirmed at a peak of 5.2 ppm.

Experimental Example  1. Acidic pH and On Esterase  About OSamp Confirm the sensitivity of

In order to confirm the sensitivity of OSamp to acidity, the reaction was carried out at pH 5.5 for 24 hours, and then ¹ H NMR was performed. The results are shown in FIG.

As shown in Fig. 3A, it was confirmed that the signal of the acetal cation at 5.2 ppm was decreased and the signal of the aldehyde cation at 9.6 ppm appeared at the acidic pH. This indicates that the acetal bond of OSamp is broken at acidic pH and releases cinnamaldehyde.

In order to confirm the sensitivity of OSamp to the esterase, OSamp was added to 350 μM of GSH solution, which was divided into two groups and Esterase was added to only one group. At this time, the amount of GSH reduced by QM emitted from OSamp was quantified by measuring the absorbance at 405 nm using a microplate reader (Biotek Instruments, Winooski, VT). The GSH amount of the treated cells was compared with the basal GSH content measured in the untreated cells, which is shown in FIG. 3B.

As shown in FIG. 3B, GSH levels were not changed when OSamp was added in the absence of esterase, but OSamp was found to erase GSH in a concentration-dependent manner in the presence of esterase. This indicates that the esterase cleaves the ester bond of OSaam and cleaves GSH by generating QM.

Experimental Example  2. In the tumor environment OSamp ≪ / RTI > Shin Nam of Aldehyde  Confirm creation

To OSamp is to determine whether the generation of the quinone nonme lactide and cinnamaldehyde in the tumor environment, colorectal cancer cell line (SW620) to 80% confluency during a 6-well plate to reach the ^{(confluency) (5 × 10 5} / well ). After the SW620 was treated with OSamp for 24 hours, the GSH and renal aldehyde levels in the cell lysate were analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) and the results are shown in FIG. 4 .

As shown in Fig. 4, OSamp treatment significantly reduced intracellular GSH levels and increased cinnamaldehyde levels compared to untreated cells. Indicating that quinone and glutamic aldehyde, which cleave GSH in the cells, are produced.

Experimental Example  3. In the tumor environment Concentration of OSamp In accordance Oxidative stress  Confirmation of increase

To determine the effect of OSamp concentration on the tumor environment, Shin - Nam aldehyde (CA) or OSamp was treated by concentration in the SW620 cell line group. The controls did not do anything. First, the GSH level of OSamp was measured in SW620 cells to confirm the GSH scavenging ability. The results are shown in FIG. 5A.

As shown in Fig. 5 (a), it was confirmed that OSamp significantly decreased intracellular levels of GSH in a concentration-dependent manner. Half of intracellular GSH was cleared by 50 μM OSamp.

In order to confirm the ability of OSamp to increase oxidative stress, Shin Nam aldehyde or OSamp was treated for 12 hours by concentration, and SW620 cells were stained with DCDF-DA (dichlorodihydrofluorescein-diacetate) as an intracellular ROS probe. The cells were then observed using a confocal scanning laser microscope and flow cytometry. The results are shown in FIG. 5B.

As shown in FIG. 5B, OSamp increased the intracellular ROS level in a concentration-dependent manner, and DCFH-DA fluorescence was significantly shifted to the right. At the same concentration of 50 μM, OSamp produced more ROS than cinnamaldehyde. To further confirm the production of ROS due to OSamp, the cells were pretreated with H ₂ O ₂ - scavenged catalase. Catalase inhibited OSamp-induced accumulation of ROS, confirming that DCFH-DA fluorescence was shifted to the left.

Further, in order to further confirm the formation of intracellular ROS in the cells stained with DCFH-DA, the cells were observed through a confocal scanning laser microscope, and the results are shown in FIG. 5C.

As shown in FIG. 5c, OSamp induces ROS production in a dose-dependent manner, and catalase significantly inhibited OSamp-induced ROS accumulation. This indicates that QAM-mediated GSH cleavage induces the accumulation of intracellular ROS in OSamp, as it sensitizes cells to renal aldehyde producing ROS.

In order to confirm the toxicity of OSamp to various cells, Shin Nam aldehyde or Osamp was treated at different concentrations in DU145 (prostate cancer cell line), SW620 (colorectal cancer cell line) and HEK293 (human kidney cell) , 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) analysis was conducted, and the results are shown in FIGS. 5D to 5F, respectively.

As shown in Figures 5d-5f, 50 [mu] M cinnamaldehyde induced about 20% apoptosis, but OSamp showed dose-dependent toxicity significantly higher than cinnamaldehyde. OSamp at the same dose of 50 [mu] M showed more than 80% apoptosis induction in both DU145 and SW620 cells. Specifically, OSamp was found to have an IC ₅₀ of ~ 21 μM for DU145 and ~16 μM for SW620 cells. On the other hand, OSamp showed low toxicity (IC ₅₀ of ~ 40 μM) for normal cells (HEK 293).

Experimental Example  4. OSamp Of mitochondrial cell death pathway

A unique characteristic of early apoptosis is the interruption of active mitochondria, including changes in mitochondrial membrane potential. To determine the effect of OSamp on the mitochondrial membrane potential, the SW620 cell line was divided into two groups, Shin Nam aldehyde or OSamp. The controls did not do anything. First, a mitochondrial membrane potential probe, JC-1, was used. The results are shown in FIG. 6A.

As shown in Fig. 6a, Shinnam aldehyde did not significantly affect the mitochondrial permeability transition, but OSamp treatment caused a significant loss of mitochondrial membrane potential in a concentration-dependent manner. In the presence of catalase, the loss of mitochondrial membrane by OSamp was significantly inhibited.

In addition, since one of the representative features of the mitochondrial apoptotic pathway is cytosolic mitochondrial cytochrome c, it was observed whether or not OSamp induces release of cytochrome c with cytochrome c, and the result is shown in FIG. 6B.

As shown in Figure 6b, cinnamaldehyde induced the effect on cytoplasmic cytochrome c potential in mitochondria. On the other hand, OSamp significantly increased the level of cytosolic cytochrome c in a concentration dependent manner. Catalase significantly inhibited the levels of cytosolic cytochrome c. These results indicate that oxidative stress, which increases the apoptotic chain induced by OSamp, is associated with mitochondrial perturbation.

Experimental Example  5. OSamp  Assessment of cell killing ability

To further confirm OSamp-mediated apoptotic cell killing capacity, groups of SW620 cell lines were treated with Shin Nam aldehyde or OSamp at different concentrations. The controls did not do anything. Catalase (CAT) was added 10 minutes before OSamp treatment in the catalase-added group. Then, the cells were stained with Annexin V (FITC) labeled with fluorescein isothiocyanate (FITC) and propidium iodide (PI), which is a cell survival marker, and fluorescence microscopic photographs of the cells were observed. Respectively.

As shown in Fig. 7A, apoptotic cells display both strong red (PI) and green (FITC) fluorescence and inhibit co-internationalization of catalase. This indicates that OSamp promotes the production of ROS inducing apoptosis.

Additional analysis of apoptosis was performed by flow cytometry, and the results are shown in Figure 7b.

As shown in Figure 7b, after 8 hours of treatment, cinnamaldehyde (50 [mu] M) did not show any significant apoptosis, but OSamp showed a significant increase in apoptosis in a concentration-dependent manner, The increase in cell mass was confirmed. However, ROS-mediated apoptosis was markedly inhibited by catalase.

The effect of OSamp on the expression of caspase-3, an important enzyme participating in apoptosis, was also observed, which is shown in FIG. 7c.

As shown in Figure 7c, cinnamaldehyde (50 [mu] M) had no effect on the degradation of caspase-3, but OSamp induced a large amount of caspase-3 degradation in a concentration dependent manner, -3 as a strong band.

Also, the effect of OSamp on the DNA fragment characteristic of apoptosis was observed, which is shown in Fig. 7d.

As shown in Figure 7d, cinnamaldehyde hardly induced DNA fragmentation, but OSamp increased the amount of nucleosomal DNA fragment in a concentration-dependent manner.

STAT3 (signal transduction and activator of transcription 3) is a potential transcription factor for tumor and anti-apoptotic activity and is structurally activated through phosphorylation mainly on Tyr-705 in many human tumors. Structurally activated STAT3 is considered a therapeutic target for the treatment of a variety of human cancers. Thus, the effect of OSamp on the expression of phosphorylated (activated) STAT3 (p-STAT3) in SW620 cells was observed and is shown in Figure 7e

As shown in Fig. 7E, strong expression of p-STAT3 appeared in untreated SW620 cells (control group). Shinnam aldehyde had little effect on the expression of p-STAT3, but OSamp significantly inhibited STAT-3 phosphorylation in a concentration-dependent manner in tyr-705. Inhibition of OSAMP-induced STAT-3 phosphorylation was catalase-mediated, and it was confirmed that OSamp, which produces ROS, exhibits anticancer activity as an inhibitor of STAT3 activation.

Experimental Example  6. OSamp of Therapeutic  Confirm antitumor activity

In order to confirm the in vivo anticancer activity of OSamp, SW620 cells were observed using a xenograft mouse model. First, administration of OSamp was started when SW620 cells were subcutaneously inoculated under the back of a nude mouse (male, 6 weeks old, Orient Bio, Korea) and developed into a small tumor (10 mm ³ ). OSamp was intravenously administered to the tail at intervals of 3 days, and tumor size and body weight were observed for 23 days, and the results are shown in Figs. 8a to 8c.

As shown in Figures 8a to 8c, prominent tumor growth was observed in untreated mice. The administration of 2 mg / kg Shin Nam aldehyde showed a slight inhibitory effect on tumor growth and the same amount of OSamp significantly inhibited tumor growth without changing body weight.

In addition, the therapeutic effect of OSamp was compared with the same amount of the common chemotherapeutic drug, camptothecin (CPT). To confirm the dose-dependent efficiency of OSamp, various amounts of OSamp were intravenously administered to the tail at intervals of 3 days, and tumor size and body weight were observed for 23 days, and the results are shown in FIGS. 9a to 9c.

As shown in Figs. 9A to 9C, OSamp showed weak anticancer activity when administered at 1 mg / kg, and showed remarkable anticancer activity without change in body weight when administered at 2 mg / kg or more. The tumor size did not show any significant difference between 2 mg / kg and 4 mg / kg.

Histologic examination and immunohistochemistry were performed to further confirm the anticancer activity of OSamp. The results are shown in FIG.

As shown in Figure 10a, tumors of untreated mice consisted of many tumor cells that retained normal membrane and nuclear structure, whereas tumors of OSamp-treated mice and CPT-treated mice had many non-nucleated apoptotic cells Was observed.

Also, as shown in Fig. 10B, tumors of the OSamp-treated mice also showed large regions of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end) positive cells, indicating that OSamp induces apoptotic cell death in tumors.

Experimental Example  7. OSamp Toxicity of

In order to confirm the toxicity of OSamp, administration of OSamp was started in nude mice (male, 6 weeks old, Orient Bio, Korea) at intervals of 3 days for 2 weeks, and ALT activity was measured. The results are shown in FIG. 11 .

As shown in Fig. 11A, OSamp did not cause a change in ALT activity as compared to untreated mice.

Also, as shown in Fig. 11B, the liver tissue of the OSamp-treated mice exhibited similar changes to the tissues of the control mice, indicating that OSAMP in an amount showing a potential anticancer activity does not cause chronic damage to the liver.

Through the above experimental results, the OSamp according to the present invention inhibits the antioxidant system and promotes apoptosis by releasing cinnamaldehyde and GSH which are activated by acidic pH and esterase to produce ROS, respectively, And it was confirmed that it could cause an anticancer effect.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation Example 1. Preparation of pharmaceutical preparations

1. Manufacturing of powder

20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

2. Preparation of tablets

Compound of formula 1 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

3. Preparation of capsules

Compound of formula 1 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

4. Preparation of injections

Compound of formula 1 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

5. Manufacture of liquids

20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  2. Manufacture of food preparation

1. Health food manufacturing

A compound of formula 1, a vitamin mixture, a vitamin A acetate 70g, a vitamin E 1.0, a vitamin B1 0.13, a vitamin B2 0.15, a vitamin B6 0.5, a vitamin B12 0.2g, a vitamin C10, a biotin 10g, a nicotinamide 1.7, Calcium carbonate 0.5, an inorganic mixture suitable amount, ferrous sulfate 1.75, zinc oxide 0.82, magnesium carbonate 25.3, potassium phosphate monobasic 15, calcium phosphate dibasic 55, potassium citrate 90, calcium carbonate 100 and magnesium chloride 24.8, And a health food was prepared according to a conventional method. At this time, although the composition ratio of the vitamin and mineral mixture is relatively mixed with the ingredient suitable for health food, it may be arbitrarily modified.

2. Health drink  Produce

According to a conventional health drink manufacturing method, the compound of Formula 1, 15 g of vitamin E, 100 g of vitamin E (powder), 19.75 g of iron lactate, 3.5 g of zinc oxide, 3.5 g of nicotinic acid amide, 0.2 g of vitamin A, 0.25 g of vitamin B1, B2 was mixed with a predetermined amount of water, and the mixture was stirred and heated at 85 for about 1 hour. The resulting solution was filtered, sterilized in a sterilized 2 L vessel, sterilized by sealing, and stored in a refrigerator to prepare a health drink. At this time, although the composition ratio of the ingredients suitable for the beverage is comparatively mixed, the mixture ratio may be arbitrarily varied according to the demand, the demanded country, the intended use, and the regional or national preference.

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

Claims (10)

A mixed anticancer prodrug drug represented by the following formula (1), which simultaneously produces cinnamaldehyde and quinone methide.
[Chemical Formula 1]

In Formula 1, R is H- or C ₆ H ₅ COO-, R 'is H- or CH ₃ -, and X is O or NH. The hybrid chemotherapeutic prodrug according to claim 1, wherein the compound of formula (1) is represented by the following formula (2).
(2)

The hybrid anticancer prodrug according to claim 1, wherein the cinnamaldehyde and quinone methide are produced by an esterase and an acidic pH. The hybrid anticancer prodrug drug of claim 1, wherein the hybrid anticancer prodrug drug promotes apoptosis. The hybrid anticancer prodrug according to claim 1, wherein the hybrid anticancer prodrug inhibits the production of glutathione. a) reacting cinnamaldehyde with an acidic solution to prepare a cinnamaldehyde derivative represented by the following formula:
[Chemical Formula]

Wherein R is H- or C ₆ H ₅ COO-, R 'is H- or CH ₃ - and X' is -OH or NH ₂ ;
(b) reacting the cinnamaldehyde derivative prepared in the step (a) with carbonyldiimidazole to prepare a cinnamaldehyde releasing compound;
(c) reacting a hydroxyalkylphenyl and a benzoyl compound to produce an ester compound that produces a quinomethide; And
(d) reacting the cinnamaldehyde-releasing compound prepared in step (b) with the ester compound prepared in step (c). Manufacturing method:
[Chemical Formula 1]

In Formula 1, R is H- or C ₆ H ₅ COO-, R 'is H- or CH ₃ -, and X is O or NH. 1. A pharmaceutical composition for preventing or treating cancer comprising an anticancer prodrug drug represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

In Formula 1, R is H- or C ₆ H ₅ COO-, R 'is H- or CH ₃ -, and X is O or NH. The pharmaceutical composition for preventing or treating cancer according to claim 7, wherein the compound of formula (1) is a hybrid anticancer prodrug represented by the following formula (2).
(2)

The method of claim 7, wherein the cancer is selected from the group consisting of lung cancer, pancreatic cancer, colorectal cancer, colorectal cancer, myeloid leukemia, thyroid cancer, MDS, bladder carcinoma, epidermal carcinoma, melanoma, breast cancer, prostate cancer, head and neck cancer, Which is composed of uterine cancer, ovarian cancer, brain cancer, stomach cancer, laryngeal cancer, esophageal cancer, bladder cancer, oral cancer, cancer of the mesenchymal origin, sarcoma, teratocarcinoma, neuroblastoma, renal carcinoma, liver cancer, non-Hodgkin's lymphoma, multiple myeloma, Or a pharmaceutically acceptable salt, solvate or prodrug thereof. 1. A food composition for preventing or ameliorating cancer, comprising a hybrid anticancer prodrug represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

In Formula 1, R is H- or C ₆ H ₅ COO-, R 'is H- or CH ₃ -, and X is O or NH.

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