KR100213897B1 - Cinnam aldehyde derivatives having angiogenesis inhibitory activity - Google Patents

Abstract

The present invention relates to a cinnamic aldehyde derivative derived from Cinnamomum cassia Blume, a preparation method thereof, and a composition containing the same, wherein cinnamon is extracted with a mixture of chloroform and acetone, and the extract is concentrated and extracted with sodium hydroxide solution, After acidification of the base layer and re-extraction with methylene chloride, the methylene chloride extract was concentrated and purified by silica gel chromatography and high performance liquid chromatography to obtain 2-hydroxy cinnamicaldehyde and 2 -O-benzoyl cinnamic synthesized therefrom. Aldehyde inhibits angiogenesis and thus may be useful as a cancer metastasis inhibitor, and as a medicament for preventing and treating blindness due to neovascularization caused by diabetic retinopathy or corneal transplantation.

Description

Cinnamon aldehyde derivative derived from cinnamon having angiogenesis inhibitory activity, a preparation method thereof and a composition containing the same

The present invention relates to a cinnamic aldehyde derivative extracted from Cinnamomum cassia Blume, a method for extracting the same and a composition containing the same, and more particularly to a new cinnamic derivative having an angiogenesis inhibitory activity from cinnamon. Extracting and synthesizing another cinnamic aldehyde derivative therefrom, and pharmaceutical compositions for inhibiting cancer metastasis and blindness containing them.

Cancer is a life-threatening cause of cancer metastasis. In other words, it is not difficult to remove the cancer by any surgical procedure if it finds only the site of generation of cancer. However, the reason why this surgical treatment has a limitation in cancer treatment is that cancer cells spread to many places other than the primary site, and can be cured only by surgery only in a very limited initial period. Recent investigations have shown that more than 50% of cancer patients whose cancerous tissue has been removed by surgery cannot be cured because of cancer cell metastasis.

Angiogenesis is a process in which new blood vessels are formed from existing blood vessels, and are essential for the development (embryonic development, female reproductive cycle), growth and healing of wounds, but various cancers, arthritis, diabetic retinopathy, It is also closely related to diseases such as spontaneous sclerosis. In particular, neovascularization is considered an essential process in metastasis of cancer, one of the processes of developing malignant tumors. Angiogenesis has this double-sided phenomenon, and research into this is also ongoing in both the search for substances that inhibit them and those that promote them (for wound healing accelerators), and some inhibitors are currently being tested for their anticancer activity. Some are also available (TiPS, 16, 57 (1995)).

In the early 1970s, since Dr. Folkman of Harvard Medical School proposed the hypothesis that cancer cells secrete specific factors to induce angiogenesis, neovascular induction plays an important role in the metastasis of cancer cells. The presence of angiogenic factor that inhibits (Science, 235, 442 (1987)). In 1985, angiogenin, an angiogenic factor, was first discovered in the secretion of human adenocarcinoma cells by the team of Dr. Valle of Harvard Medical School. In addition, solid tumors can continue to grow by nourishing and excreting waste products using new blood vessels induced around them, and the fact that these cancer cells are connected to the circulatory system and the cancer has spread to other parts of the lungs, liver, etc. (Cell, 64,327 (1991)). And angiostatin (angiostain), one of the angiogenesis-inhibiting factors, was released in 1994 (Cell, 79,715 (1994)). In addition, interferon-α, a factor that inhibits angiogenesis, is used as a therapeutic agent to prevent hemangioma of the lungs, which is very deadly in newborns. These facts show that, unlike cancer, which was initially understood as an anaerobic respiratory tissue in the early stages of cancer research, oxygen and nutrient supply are essential for the growth of cancer cells. In addition, studies have shown that bFGF, a growth regulator of endothelial cells that grows rapidly in tumor cells but not in normal cells, dramatically reduces the growth of cancer cells. These findings suggest that inducers and inhibitors are in harmony in normal cells, but the neovascular inducer is activated in cancer cells in which the coordination is disrupted during cancerization, thereby promoting cancer metastasis.

In 1993, a chemical that inhibited neovascular induction was isolated from soybeans (Proc. Natl. Acad Sci. USA, 90, 2690 (1993)) by German scientists and named genistein, a small malignant tumor. It is expected that this large growth can be suppressed. In this regard, the incidence of prostate cancer is very low in the case of Japanese ingesting soybeans, but it is reported that the incidence of prostate cancer increases rapidly if the Japanese do not eat soy beans for only a few years after immigrating to the West. . And in 1995, Dr. Yucun of the University of Minnesota, USA, reported that 99.999% of human BCP leukemia cells were abolished when mice injected with human BCP leukemia cells were crosslinked with Zenithine to a single antibody (Faith M. Uckun). et al., Science, 267,886 (1995).

Given these facts, the discovery of angiogenesis inhibitors is a clue to the mechanism of cancer growth, and can be applied to early diagnosis, prevention and treatment of cancer as well as neovascularization. It is expected to be widely used in the treatment of diseases related to induction, such as rheumatoid arthritis, chronic inflammation, diabetic retinopathy, and hemangioma. For this reason, the academic and industrial sectors have focused their attention on research and development on angiogenesis inhibitors (Pharmac. Ther., 61, 265 (1994)).

The antiangiogenic inhibitors discovered and synthesized in the past include platelet factor-4 (Platelet factor-4, PF-4) and its synthetic peptides, fumagillin and its derivatives AGM-1470, ursolic acid and herb. Mycin A (herbimycinA), cartilage-derived inhibitors (CDI), and synthetic inhibitors of ovalicin.

It is an object of the present invention to provide a novel cinnamic aldehyde derivative and a method for preparing the same as an angiogenesis-inhibiting substance derived from cinnamon.

Another object of the present invention is to effectively inhibit the neovascularization that occurs at the time of las cancer metastasis or corneal transplantation, thereby preventing the metastasis of the cancer and preventing the explanation by diabetic retinopathy or corneal transplantation. To provide.

1 is a hydrogen NMR spectrum of the 2-hydroxy cinnamic aldehyde of the present invention,

2 is an ultraviolet spectrum of 2-hydroxy cinnamic aldehyde of the present invention,

3 is an infrared spectrum of 2-hydroxy cinnamic aldehyde of the present invention,

4 is a hydrogen NMR spectrum of 2-O-benzoyl cinnamic aldehyde of the present invention.

In order to achieve the above object, the present invention provides a cinnamic aldehyde derivative of the general formula (I).

In the above, R is hydrogen or benzoyl group to be.

In the compound of Formula (I), 2-hydroxy cinnamic aldehyde (I-a) wherein R is hydrogen is extracted with a mixture of chloroform and acetone, the extract is concentrated and extracted with sodium hydroxide solution, and a base layer. The acidified product can be obtained from cinnamon by the method comprising the step of re-extracting with methylene chloride, the methylene chloride extract is concentrated and purified by silica gel chromatography and high performance liquid chromatography.

2-O-benzoyl cinnamic aldehyde (I-b), wherein R is a benzoyl group in the compound of formula (I), reacting 2-hydroxy cinnamic aldehyde with triethylamine and benzoyl chloride in CH ₂ Cl ₂ It can manufacture by the method to include.

In order to achieve the above another object, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis and preventing blindness by diabetic retinopathy or corneal transplant containing the compound of Formula (I) as an active ingredient.

The process of isolating and purifying 2-hydroxy cinnamic aldehyde from cinnamon among the new cinnamic aldehyde derivatives of general formula (I) having angiogenesis-inducing inhibitory activity is described in detail as follows: Edible cinnamon of chloroform and acetone Extraction is carried out in the same amount mixture, and the extract is concentrated under reduced pressure. Ethyl acetate and 2% sodium hydroxide solution were added to the concentrate, and the aqueous layer and the organic solvent layer were separated. The aqueous layer was acidified to pH 5-6, preferably pH 5 with 10% acetic acid and re-extracted with methylene chloride. The organic solvent layer containing the active substance was concentrated under reduced pressure, and then purified by silica gel chromatography and HPLC to obtain 2-hydroxy cinnamic aldehyde of the above formula (I) according to the present invention.

Novel Cinnamic Derivatives of the Invention 2-O-Benzoyl Cinnamic aldehyde, which can be obtained by extraction from cinnamon, is reacted with triethylamine and benzoyl chloride in a CH ₂ Cl ₂ solvent at 0 ° C. It can manufacture by making it.

Angiogenesis-inhibiting activity of the cinnamic aldehyde derivatives can be measured by CAM assay that can directly identify angiogenesis using chorioallantoic membrane (CAM) of fertilized eggs. The CAM begins to emerge four days after incubation at 37 ° C. The samples are processed directly on the CAM. Blood vessels processed as a sample and generated in the CAM can be clearly distinguished from other blood vessels by injecting a fat emulsion, and thus the blood vessels can be visually checked for production and distribution. In addition, there is a method of using the eyes of rabbits or mice, but it is advantageous to use CAM analysis because it is very difficult to observe.

Since the cinnamic aldehyde derivatives of the present invention have angiogenesis-inhibiting activity, it can inhibit cancer metastasis and prevent blindness due to renal angiogenesis that occurs during diabetic retinopathy or corneal transplantation, thereby preventing cancer metastasis inhibitors, and blindness and It can be used as a therapeutic agent.

As described above, cinnamon-derived cinnamic aldehyde derivatives may be formulated into a unit dosage form or a hydration dosage form such as tablets, capsules, powders, granules, suspensions, emulsions or parenteral preparations by conventional methods.

The pharmaceutical composition containing cinnamic aldehyde derivatives of the present invention as an active ingredient can be administered orally or orally as desired, and it is 10 to 2 mg of 2-hydroxy cinnamicaldehyde per kilogram of body weight per day, and 2-O-. An amount of 5 to 10 mg of benzoyl cinnamicaldehyde may be administered in one to several times. Dosage levels for a particular patient may vary depending on the particular compound to be used, body weight, patient's age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of disease, and the like.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these Examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1

2-Isolation and Purification of Hydroxy Cinnamicaldehyde

200 g of cinnamon purchased from Geumsan, Chungcheongnam-do, Korea was pulverized, and a mixture of 2 L of chloroform and acetone (1: 1) was added thereto, and the mixture was left at room temperature for 24 hours, followed by stirring and filtering. The filtrate was collected, concentrated under reduced pressure, and then separated into an aqueous layer and an organic solvent layer by adding 1 L ethyl acetate and 1 L 2% sodium hydroxide solution. The separated water layer was extracted three times with 1 L of ethyl acetate to remove the portion dissolved in the organic solvent, and then made acidic (pH 5) using a 10% acetic acid solution. The acidified water layer was extracted three times with 1 L of methylene chloride, and the organic solvent layer and the water layer were separated using a separatory funnel. Subsequently, the angiogenesis-inhibiting activity of the organic solvent layer and the water layer was measured, and the organic solvent layers identified as containing the active substance were collected and concentrated to dryness under reduced pressure to obtain a yellowish brown solid substance. The obtained solid was dissolved in methylene chloride, adsorbed onto silica gel (Art No. 9835, Merck), and then subjected to two silica gel column chromatography by changing the ratio of hexane and ethyl acetate from 9: 1 to 1: 1. After the fractions were separated, 2-hydroxy cinnamic aldehyde was purified again using HPLC. At this time, Phenomenex's Ultracarb 10 ODS (250 × 21.2 mm) was used as the HPLC column, and 80% methanol and 20% water were used as the elution solvent. Finally, 11 mg of 2-hydroxy cinnamic aldehyde was obtained per 100 g of cinnamon.

¹³ C-NMR (acetone-d ₆ ): 1940, 157.1, 148.4, 132.7, 129.4, 128.9, 121.6, 120.2, 116.6 MS (CI): m / z 149, 131, 120, 91, 65.

Example 2

2-O-Benzoyl Cinnamicaldehyde Synthesis

As in Example 1, 20 mg of 2-hydroxy cinnamic aldehyde separated and purified from cinnamon was placed in a 50 ml round bottom flask and dissolved by adding 20 ml of dichloromethane solvent. The flask was charged to 0 ° C. in an ice bath with nitrogen, and then 1 ml of triethylamine and 0.5 ml of benzoyl chloride were added and stirred for 3 hours. After the reaction was completed, the mixture was concentrated under reduced pressure, 50 ml of ethyl acetate was added to the concentrated filtrate, which was transferred to a separatory funnel and washed twice with 20 ml of water. The organic solvent layer was dried using anhydrous sulfate and then concentrated under reduced pressure. The concentrated solution was dissolved in methylene chloride and adsorbed onto silica gel (Merck, Art No. 9385), and then purified by silica gel column chromatography while changing the ratio of hexane and ethyl acetate from 9: 1 to 6: 4 to 2-O-. Benzoyl cinnamic aldehyde was obtained in a yield of 72%. IR (KBr): 2763, 1725, 1677, 1627, 1602, 1263, 1209, 1124 cm ^-1 . MS (CI): m / z 253, 149, 132, 122, 105, 94, 78.

Example 3

Structure Analysis of Active Substances

UV absorbance was measured using a UV-265 spectrophotometer of 2-Hydroxy cinnamic aldehyde and 2-O-benzoyl cinnamic aldehyde Shimadzu obtained as described above, FTS of Bio-Rad Digilab Division The IR absorbance was measured using an -80 spectrophotometer and the high resolution MS was measured by VG70-SEQ mass spectrometry to determine molecular weight and molecular formula. In addition, ¹ H, ¹³ C, Cozy and HMQC spectra were obtained using a nuclear magnetic resonance (Bruker 300 MHz, 500 MHz NMR), and the structures were determined by comprehensive analysis of these spectra.

1 is a hydrogen NMR spectrum of the 2-hydroxy cinnamic aldehyde of the present invention, FIG. 2 is an ultraviolet spectrum, FIG. 3 is an infrared spectrum, and FIG. 4 is a hydrogen NMR spectrum of the 2 -O-benzoyl cinnamic aldehyde of the present invention. .

The physicochemical characteristics of 2-hydroxy cinnamic aldehyde and 2-O-benzoyl cinnamic aldehyde which have been structurally analyzed as described above are as follows.

[Experimental stage]

Day 1 (day 0): The fertilized eggs were hatched in the incubator. At this time, the temperature of the incubator is 37 to 38 ℃, the humidity was checked from time to time to maintain more than 90%. Here, the 0-day ovulation means that within 3-4 days after the fertilized egg is laid and stored at 18 ℃.

Day 3 (Day 2): Cut the pointed end of the fertilized egg with a knife, lay it horizontally, puncture it with a 5 ml syringe, and extract 2 ml of albumin. The pores were sealed with glass tape so that the fertilized eggs were not dried or infected, and the pores were placed downward and incubated again.

Day 4 (3 times): A round window measuring 2-3 cm in diameter to the side with the air sac of the fertilized egg (the opposite side of the hole with a syringe), closed with only wide glass tapes identified as fertilized egg, and then incubated again. . A circular spear can be made by using a sharp knife to circularly flap the shell of the fertilized egg and then tear it off with tweezers, taking care not to let the shell powder fall inside. A fertilized egg is a cross-shaped thin blood vessel that is visible when a window is opened.

Day 5 (Day 4.5): At this time, CAMs are formed, with diameters of 2 to 5 mm.

Example 4

Screening for Angiogenesis Inhibitory Activity

Angiogenesis-inhibiting activity was assessed according to the following procedure using fertilized eggs of chickens according to the CAM assay. Fertilized eggs were purchased from Pulmuone Co., Ltd. The Thermanox coverslip for sample processing was obtained from Nunc, and the 10% Intralipose (fat emulsion) and 1ml and 5ml syringes required for observation were obtained from Green Cross. Each purchased. Laboratory equipment requires an incubator that can maintain humidity above 90% to hatch the fertilized egg, a clean bench to prevent infection of the fertilized egg, and a dissecting microscope equipped with a camera to observe and photograph the results. Do. Retinoic acid purchased from Sigma was used as a comparative sample.

The sample was dissolved in a suitable solvent such as distilled water or ethanol and then dropped 10 μl onto a quartered Thermonox coverslip and dried in a clean bench. Here, the Therma Knox coverslip was cut with scissors and cut into quarters and left overnight under the UV of the clean bench. The glass tape of the fertilized egg was torn off with a knife to find and check the CAM, and then trumpet coverslips treated with the tweezers were turned upside down, carefully placed, and then again covered with the glass tape. The scissors, knife, and tweezers used at this time were sterilized with 70% ethanol, and the tweezers were sterilized every time a sample was loaded. Other experimental instruments were also used with care not to infect fertilized eggs.

Day 7 (6.5-day fold): The glass tape was ripped off with a knife, 1 ml of intralipose (fat emulsion) was removed with a syringe to remove bubbles, and then injected into the lower portion of the CAM. At this time, a clear blood vessel can be observed on a white background. Be careful not to injure your blood vessels when injecting intralipose with a syringe. Observed fertilized eggs are photographed with a camera mounted on an anatomical microscope. Observation of the formation and inhibition of neovascularization by the above method, the inhibition degree was calculated by the following formula and the results are shown in Table 1.

As described above, 2-hydroxy cinnamic aldehyde isolated and purified from cinnamon according to the present invention and 2-O-benzoyl cinnamic aldehyde synthesized therefrom inhibits neovascular induction, and as a cancer metastasis inhibitor, and diabetic retinopathy or It can be usefully used as a medicament for preventing and treating blindness due to neovascularization generated during corneal transplantation.

Claims (3)

2-O-benzoyl cinnamic aldehyde of the following general formula (I-b): 2-O-benzoyl cinnamic aldehyde of formula (I-b) comprising reacting ₂ -hydroxy cinnamic aldehyde of formula (I-a) with triethylamine and benzoyl chloride in CH ₂ Cl ₂ Manufacturing method. A pharmaceutical composition for inhibiting cancer metastasis and preventing blindness by diabetic retinopathy or corneal transplant, comprising the compound of formula (I) as an active ingredient: In the above, R is hydrogen or benzoyl group ( )to be.