KR100242240B1 - Process for the preparation of sesquiterpene lacton compounds for angiogenesis inhibitor from carpesium adrotanoides l and composition containing same - Google Patents

Abstract

The present invention relates to a method of preparing a sesquiterpene lacton compound having an activity of inhibiting angiogenesis from Carpesium adrotanoides L, and a composition comprising the same. Since it can suppress vascular induction, it can be usefully used for the prevention and treatment of blindness, cancer metastasis and diabetic retinopathy during corneal transplantation.

Description

FIELD OF THE INVENTION A process for the preparation of a sesquiterpine lactone-based compound that inhibits angiogenesis-induced phenomena from tobacco grass and a composition comprising the same

The present invention provides a method for preparing a sesquiterpene lacton-based compound having angiogenesis-inhibiting activity from the ground and root of tobacco grass (Carpesium adrotanoides L) and a composition for inhibiting angiogenesis-inducing phenomenon comprising the same It is about.

The biggest cause of cancer's life threat is the metastatic capacity of cancer cells. In other words, it is not difficult to remove the cancer by any surgical procedure if it finds a generation site in any cancer. However, the reason why this surgical treatment has a limitation in the treatment of cancer is that the operation can be cured only in the very limited initial stage because the cancer cells are spread to other places than the primary site. Recent investigations have shown that more than 50% of cancer patients whose cancer tissues have been removed by surgery do not see a cure due to metastasis of cancer cells.

Angiogenesis is a process in which new blood vessels are formed from existing blood vessels. Normal development, that is, embryonic development, female reproductive cycle (DM Noden, Am. Rev. Respir. Dis., 140) , 1097-1103 (1989)), as well as essential for growth and wound healing, and are closely related to diseases such as cancer, arthritis, diabetic retinopathy and atherosclerosis (O'Brien, ER et al., J. Pathol. 145, 883-894 (1994). In particular, neovascular induction during metastasis of cancer, one of the processes of developing malignant tumors, is considered an essential process. The neovascular induction shape is double-sided as described above, and the research on this also proceeds with the search for a substance that inhibits it and the search for a substance that promotes the wound (therapeutic accelerator). Some inhibitors are in clinical trials to verify the activity as an anticancer agent. (R. Bicknel et al., TiPS, 16, 57 (1995)).

In the early 1970s, Dr. Folkman of Harvard Medical School proposed the hypothesis that cancer cells secrete specific factors to induce angiogenesis. Later, a team of researchers, including the Polkman Research Group, have been working on this process. Angiogenesis has been shown to play an important role, and the presence of angiogenic factors that induce angiogenesis has also been identified (J. Folkman et al., Science, 235, 442 (1987)). In 1985, Dr. Vallee of Harvard Medical School discovered a protein that induces blood vessels from the secretion of human crystalline cancer cells and named it angiogenin. And solid tumors (soiled tumors) can continue to grow by nourishing and excreting waste products by using new blood vessels induced around them, and these cancer cells are connected to the circulatory system and cancer can spread to other parts of the lungs, liver, etc. It was also revealed (LA Liotta et al., Cell, 64, 327 (1991)). Angiostatin, one of the proteins that inhibit angiogenesis, has been reported to prevent metastasis and growth of lung cancer cells (J. Folkman et al., Cell, 79, 715 (1994)). These facts show that in the early stages of cancer research, cancer cells, while also understood as anaerobic respiratory tissues, provide oxygen and nutrient supply for the growth of cancer cells. In addition, interferon alpha, a factor that inhibits neovascular induction, is used as a therapeutic agent to prevent hemangiomas of the lungs, which is fatal to newborns, and regulates the growth of endothelial cells that grow very rapidly in tumor cells, unlike normal cells. The study showed that antibodies from the growth factor, basic fibroblast growth factor (bFGF), dramatically reduce cancer cell growth. The above results suggest that inducers and inhibitors are in harmony in normal cells, but in neoplastic cells where the coordination is disrupted, new entrance hall inducers are activated to promote cancer metastasis.

In 1993, German scientists released a chemical from soybeans that inhibited the induction of neovascularization (T. Fotsis et al., Proc. Natl. Acad. Sci. USA, 90, 2690 (1993)). It was named Genistein, and it is expected to be able to suppress the large growth of small malignancies. In Japan, soybeans are eaten a lot, so the incidence of prostate cancer is very low compared to westerners. However, it has been reported that Japanese do not eat soy during just a few years after emigrating to the West, and the incidence of prostate cancer increases rapidly. And in 1995, Dr. Faith M. Uckun, of the University of Minnesota, USA, surprisingly received a human BCD leuche when a single antibody was cross-linked with Zenithine in a mouse transplanted with human BCP leukemia cells. Mia cells were reported to die by 99.999% (Faith M. Uckun, Science, 267, 886 (1995)).

Therefore, the discovery of angiogenesis inhibitors is a clue to the cancer's growth mechanism, and can be applied to early diagnosis, prevention and treatment of cancer, as well as rheumatoid, a disease associated with angiogenesis. It will be widely used in the treatment of diseases such as arthritis, chronic inflammation, diabetic retinopathy and hemangioma. For this reason, academia and industry have been continually interested in this and working on research and development (W. Auerbach et al., Pharnac. Ther., 61, 265-311 (1994)).

Inhibitors of angiogenesis-induced angiogenesis have been listed, including platelet factor-4 (PF-4), its synthetic peptides, fumagillin and its derivatives AGM-1470, and ursolic acid (Ursolic). acid), herbimycin A, cartilage-derived inhibitors (CDI), and synthetic inhibitors of obalicin, thalidomide, and microbial secondary metabolite FR-111142. There is this.

Accordingly, the present inventors continued to develop a substance capable of suppressing angiogenesis-inducing phenomena from natural herbal medicines. As a result, the secuterpine lactone-based compound contained in the tobacco grass native to Korea is isolated and thus, It came to completion.

It is an object of the present invention to provide a method for preparing a sescuterpin lactone compound having an activity of inhibiting neovascular induction from the ground or root of tobacco grass.

Another object of the present invention to provide a composition for the prevention of blindness, cancer metastasis and diabetic retinopathy during corneal transplantation comprising a compound having the activity of inhibiting the neovascular induction phenomenon.

1 shows the hydrogen nuclear magnetic resonance (NMR) spectrum of compound I.

2 shows the carbon NMR spectra of compound I.

3 shows the hydrogen NMR spectrum of compound II.

4 shows carbon NMR spectra of compound II.

In accordance with the above object, in the present invention, the ground portion or root of the tobacco paste is ground, the ground portion or root of the ground tobacco extract is extracted with an alcohol solvent, and the extract is concentrated and purified, the following formula 1 compound, Provided are methods for preparing the compound of 2 or a mixture thereof.

According to another object of the present invention, in the present invention, an angiogenesis-inhibiting composition comprising an effective amount of the compound of Formula 1 and a pharmaceutically acceptable carrier, an newborn comprising an effective amount of the compound of Formula 2 and a pharmaceutically acceptable carrier Provided is a composition for inhibiting vascular induction, and a composition for inhibiting angiogenesis, including an effective amount of a mixture of the compounds of Formulas 1 and 2 and a pharmaceutically acceptable carrier.

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

Compounds of formulas (1) and (2), prepared from tobacco grass according to the present invention, were identified as secuterpin lactone crab compounds, telekin and dihydroaromaticin, respectively (Dong, Y. and Ding, Y., Zhiwu Xuedao, 30, 71-75 (1988); Bohlmann, F. and Mahaanta, PK, Phytochemistry, 18, 887-888 (1979)).

The sesquiterpin lactone-based compound is ground or root of the tobacco plant which is a natural herbal medicine, pulverized, the pulverized leaves are extracted with an alcohol solvent, preferably methanol, and then concentrated and coated on a chelate to fill the column with nucleic acid Eluting to remove the non-polar fraction, and then eluting the active substance remaining on the celite with chloroform, and then concentrating the eluate and purifying by silica gel chromatography, thin layer chromatography, and high performance liquid chromatography. It can manufacture.

Measurement of the inhibitory activity of neovascularization of the secuterpin lactone compounds of the present invention, telekines and dihydroaromatics, was verified using a chicken chorioallantoic membrane (CAM). As an analytical method that can be identified as, it is generally called a CAM verification method and is a useful method for measuring the formation and inhibition of neovascularization. When the fertilized egg is hatched at 37 ° C. about 4 days, CAM starts to form. By directly processing the sample and injecting the fat emulsion, the production and distribution of blood vessels can be clearly distinguished with the naked eye. In addition, there is a method of using the eyes of rabbits or mice, but it is very difficult to observe, so it is advantageous to use analytical methods.

Three skew teopin lactone compound tele Keene, dihydro aromatic tisin and mixtures thereof are measured inhibitory activity of angiogenesis resulting IC ₅₀ the purified pool from the tobacco in accordance with the present invention, respectively 6㎍ / ㎖, 8㎍ / ㎖ And 20 μg / ml. These compounds with activity that inhibits neovascularization can be used as agents for the prevention and treatment of blindness, cancer metastasis and diabetic retinopathy that may occur during corneal transplantation.

The secuterpin lactone compounds, telekines, dihydroaromatics, or mixtures thereof, isolated and purified from tobacco plants according to the present invention, may be prepared by tablets, capsules, powders, granules, suspensions, emulsions or parenteral preparations by conventional methods. It may be formulated into a unit dosage form or a multiple dosage form such as, and may be used as a preparation for preventing and treating blindness, cancer metastasis and diabetic retinopathy occurring during corneal transplantation.

The pharmaceutical composition containing telekines, dihydroaromatisine or mixtures thereof isolated in the present invention as an active ingredient may be parenterally or orally administered as desired, and may be 10-20 mg / kg body weight per day. The amount can be administered in one to several portions. Dosage levels for a particular patient may vary depending on the patient's weight, age, gender health status, diet, time of administration, method and excretion, drug combination and severity of the disease.

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

Example 1 Isolation and Purification of Telekin and Dihydroaromaticin from Tobacco Paste

Tobacco grasses used in the present invention were harvested in the fall of 1994 and 1995, which grow wild in Ulleung-gun, Gyeongsangbuk-do and Geumsan-gun, Chungcheongnam-do. The ground portion or root of the 6 kg tobacco pool was finely pulverized, 18 L of methanol was added thereto, and left at room temperature for 48 hours, followed by stirring to separate the liquid and solid portions using a filter paper. The liquid phases were collected, concentrated under reduced pressure, coated on celite, filled in a column, eluted with nucleic acid to remove the nonpolar composition, and then eluted with chloroform to measure the inhibition of neovascularization induction. It was confirmed that there is. The chloroform fraction containing the active ingredient was concentrated under reduced pressure and again dissolved in chloroform to adsorb the active substance onto silica gel (Merck, Art No. 9385), followed by the first change of chloroform and methanol from 10: 0 to 8: 2. Silica gel column chromatography was divided into seven subfractions. Among them, the small fraction showing the activity was subjected to silica gel column chromatography again with a solvent of benzene: ethyl acetate (5: 1), and divided into six small fractions. Each of the partially purified telekines and dihydroaromatics from these subfractions was purified using thin layer chromatography. Finally, pure telekines and dihydroaromatics were purified using HPLC. In this case, an ultracarb 10 ODS (250 × 21.2 mm) manufactured by phenomenex was used as an HPLC column, and the active materials were eluted with 80% methanol as a solvent. The finally isolated pure material yielded 12 mg and 10 mg of telekin and dihydroaromaticin per kg of tobacco grass, respectively.

It was confirmed through the following analytical method that the isolated purified material was telekin and dihydroaromaticin.

UV absorbance analysis was measured using an ultraviolet-visible spectrometer (Shimadzu, UV-265), IR absorbance was measured by a ratio recording infrared spectrometer (Bio-Rad Digilab Division FTS-80), VG70- The molecular weight and molecular formula were determined by high-resolution electron chemical ionization (HRCI) -MS method using a SEQ mass spectrometer. In addition, H, C, Cosy and HMQC spectra were obtained using a nuclear magnetic resonance (Bruker 300 MHz, 600 MHz NMR), and the structures were determined by comprehensive analysis.

The physicochemical properties of telekines and dihydroaromatics analyzed as described above are shown in Table 1 below.

Compound 1 Compound 2 Appearance Colorless needle structure Colorless prism Molecular formula C ₁₅ H ₂₀ O ₃ C ₁₅ H ₂₀ O ₃ Molecular Weight 248 248 UV absorbance (MeOH) 211 nm 210 nm, 223 nm (shoulder) Infrared Spectrum (KBr) 3410, 2920, 1730, 1260cm ^-1 2952, 2908, 1756, 1722, 1658, 1447, 1383, 972 cm ^-1 mp 156-158 ℃ 147-148 ℃ Availability Most organic solvents Most organic solvents Insoluble water water

Example 2 Screening for Inhibitory Activity of Angiogenesis

The measurement of neovascular induction inhibitory activity was performed by CAM analysis using a chicken chorioallantoic membrane (CAM).

(Step 1) Day 1 (0 times)

The fertilized egg (Pulmuone Co., Ltd.) was hatched in an incubator maintained at a temperature of 37 to 38 ° C. and a humidity of 90% or more (here, 0 days ovulation refers to within 3 to 4 days after the fertilized egg was laid and stored at 18 ° C.).

(Step 2) 3rd day (2 times)

The pointed end of the fertilized egg was cut with a knife, laid horizontally, punched with a 5 ml syringe, and 2 ml of albumin was extracted. The pores were sealed with glass tape so that the fertilized eggs were not dried or infected.

(Step 3) 4th day (three times)

On the side with the air sac of the fertilized egg (the opposite side of the syringe hole), use a sharp knife to make a circular window with a diameter of 2 to 3 cm and tear it off (be careful not to let the shell fall inside). (The fertilized egg here means the cross-shaped thin blood vessels visible when the window is opened.) Only the identified ones were covered with a wide glass tape and re-cultured.

(Step 4) 5th day (4,5 times)

At this time, CAM is produced and its diameter is about 2 to 5 mm. The sample was dissolved in a suitable solvent such as distilled water or ethanol, and then divided into quarters with scissors, and then dropped in 10 µl onto a thermosox coverslip (Nunc), which was allowed to stand overnight under UV in a clean bench. It was dried in the bench. After tearing off the glass tape of the fertilized egg, find CAM and check it, and then turn the thermox treated with the tweezers over carefully and place it again with a glass tape. Scissors, knives, tweezers, etc. used at this time are sterilized with 70% ethanol, and tweezers are sterilized every time a sample is administered. Other laboratory instruments should be used with caution to avoid infecting the fertilized egg.

(Step 5) 7th day (6,5 times)

The glass tape was peeled off with a knife, 1 ml of 10% Intralipose (fat emulsion) was taken with a syringe, and bubbles were removed and injected directly under 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. And the degree of inhibition of neovascularization (%) was calculated as in Equation 1 below.

A = 1

At this time, retinoic acid purchased from Sigma was used as a comparative sample.

According to the above method, the concentration of the telekines compound, the dihydroaromaticin compound, and a mixture of these compounds (IC ₅₀ ) which inhibited neovascular induction by 50% was measured. As a result, 6 µg / ml, 8 µg / ml and 20 It was confirmed to be μg / ml.

According to the present invention, purified telekines and dihydroaromatics and mixtures thereof isolated from the ground or roots of tobacco plants, natural herbals, inhibit neovascularization and thus develop as cancer metastasis inhibitors and upon diabetic retinopathy or corneal transplantation. It can be usefully used as an agent for the prevention and treatment of blindness.

Claims (6)

A composition for inhibiting angiogenesis, comprising an effective amount of a compound of Formula 1 and a pharmaceutically acceptable carrier. Formula 1

The method of claim 1, The composition is a blindness, cancer metastasis and diabetic retinopathy preventing and treating at the time of corneal transplant. A composition for inhibiting angiogenesis, comprising an effective amount of a compound of Formula 2 and a pharmaceutically acceptable carrier. Formula 2

The method of claim 3, wherein The composition is a blindness, cancer metastasis and diabetic retinopathy preventing and treating at the time of corneal transplant. A composition for inhibiting angiogenesis, comprising an effective amount of a mixture of a compound of Formula 1 and a compound of Formula 2 and a pharmaceutically acceptable carrier. The method of claim 5, The composition is a blindness, cancer metastasis and diabetic retinopathy preventing and treating at the time of corneal transplant.

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