KR0156227B1 - Vascularization-inhibitor containing udca derivatives - Google Patents

Abstract

The present invention provides an angiogenesis inhibitor containing one of ursodeoxycholic acid derivatives represented by the following general formulas as an active ingredient in an amount effective to inhibit angiogenesis.

(Wherein Me is a methyl group, Et is an ethyl group and TBSO is t-butyldimethylsilylioside.)

Description

Angiogenesis inhibitors containing derivatives of ursodeoxycholic acid

The present invention relates to an angiogenesis inhibitor containing a derivative of ursodeoxycholic acid (weakly referred to as UDCA).

Normally, cell growth and DNA synthesis are tightly regulated by a variety of regulatory factors that act in positive and also negative dimensions. As normal cells develop into solid tumors, they undergo several changes. At the physiological level, growth is stimulated, immunity is reduced, and new angiogenesis is induced. This ability to induce new blood vessels, namely angioginesis and neovascularization, is a hallmark of most malignancies and is a necessary requirement for the growth of solid tumors. Moreover, new blood vessels that penetrate the tumor form the site where most tumor cells enter the circulation. Angiogenesis is also required for expansion of metastatic colonies. In addition to the growth of malignant cells, other diseases are also characterized by abnormal angiogenesis including neovascular glaucoma, diabetic retinopathy and rheumatoid arthritis.

Therefore, a substance that inhibits angiogenesis may be usefully used for effective prevention and / or treatment of various diseases associated with angiogenesis.

The inventors of the present inventors, who have studied the physiological activities of choline acid and UDCA and confirmed their effective cancer cell differentiation induction, continued to study the synthesis of various derivatives of UDCA, among which compounds having the ability to inhibit angiogenesis (described below) TK-090, TK-230, TK-180, TK-092, and TK-170B) were screened to complete the present invention.

UDCA is a degradation product of cholesterol and consists of 24 carbon atoms, and the A and B rings are combined in the cis form. It has a beta methyl group at 18, 19 position, an alpha alcohol group at position 3, and a beta alcohol group at position 7.

It is an object of the present invention to provide an angiogenesis inhibitor containing at least one of the following ursodeoxycholic acid derivatives in an amount effective to inhibit angiogenesis.

(Wherein Me is a methyl group, Et is an ethyl group and TBSO is t-butyldimethylsilylioside.)

Other objects and applications of the present invention will become apparent to those skilled in the art from the following detailed description.

Hereinafter, the present invention will be described in detail.

Since the compounds according to the present invention exhibit an effective antiangiogenic action, they can be used for the purpose of preventing and / or treating a disease associated with angiogenesis.

Angiogenesis inhibitor of the present invention can be administered in an amount effective to inhibit angiogenesis in order to inhibit angiogenesis through various routes of administration, the formulation, dosage and the like of the purpose of administration, the route of administration and the state of the subject and It can be easily determined by those skilled in the art in consideration of the weight and the like.

Preferably, the angiogenesis inhibitor composition contains together one of the aforementioned various kinds of ursodeoxycholic acid derivatives and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include any standard pharmaceutical carrier such as sterile solutions, tablets, coated tablets and capsules.

Typically such carriers include excipients such as starch, milk, sugar, certain kinds of clays, gelatin, stenic acid, talc, vegetable oils or oils, gums, glycols, or other known excipients. Such carriers may also include flavoring and coloring additives and other ingredients.

Compositions containing such carriers can be formulated by known methods. However, compositions containing ursodeoxycholic acid derivatives in an amount effective to inhibit the formation of blood vessels have never been known.

In the method of the present invention, the ursodeoxycholic acid derivative-containing composition can be administered by well-known methods, for example, by oral, intravenous, intramuscular, transdermal administration, etc. no.

In practicing the present invention, the amount of ursodeoxycholic acid derivative contained in the composition is very wide. An effective amount for inhibiting angiogenesis is 0.01 mg-1 g ursodeoxycholic acid derivative. The exact amount and frequency of administration and the nature of the particular derivative to be used can be readily determined by one skilled in the art. Preparation of the compounds of the present invention can be carried out according to the following reaction route starting from ursodeoxycholic acid (1) below, and specific reaction conditions are shown in the examples.

The English abbreviation used in the present invention has the following meaning.

Identification of the material according to the invention was carried out using NMR (200 MHz product from Verion) according to Lambert et al. (Lambert et al., Organic Structural Analysis, MacMillan Publ. Co., NY 1993).

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited only to these examples.

Example 1

In the present invention, the ability to inhibit angiogenesis of ursodeoxycholic acid derivatives was measured using the CAM assay (Crum et al., Science, 230, 1375, 1985). The fertilized eggs of the Abeica-based Okyong system were purchased and maintained at 18 ° C. for 45 hours and transferred to a 37 ° C. incubator maintained at 60% humidity. We used this as a zero-fold. At 2 days of age, the end of the egg was punctured and a syringe was used to extract 2 ml of albumin. On the third day, the air bag side of the egg was disinfected with iodine tincture, a circular window 3 cm in diameter was made with a scalpel, and sealed with a general adhesive tape (Scotch). Each test compound was applied to 1 mg of Termanox 13 mm disks, and left to dry for 40 minutes. The glass tapes of the 4 and 5 day embryos were removed, topped with a dry thermanox disk, and the windows were sealed again with glass tape.

After 2 more days of incubation in the incubator, 10% emulsion (Intralipid, Green Cross, Inc.) was injected into the CAM membrane to photograph the emulsion-filled CAM, and the amount of inhibition of angiogenesis was observed.

Whether angiogenesis was inhibited was considered to have angiogenesis inhibitory ability when the part without angiogenesis was less than 3 mm in diameter, and a relative angiogenesis ratio was measured based on the size of the blood vessel formed when no treatment was performed. It was evaluated by the amount of inhibition of angiogenesis.

Example 2

(Wherein Me is a methyl group)

2.00 g (0.0051 M) of UDCA and 1.4 mL (0.01 M) of DBU were dissolved in 10 mL, 1.5 mL (0.02 M) of bromoethyl was added and stirred for 1.5 hours at room temperature. Washed with water and 100 mL of ethyl acetate. The organic layer obtained by two-sided extraction was dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure.

Column chromatography was performed using ethyl acetate: n-hexane (5: 4) as the eluent to obtain 2.0 g of the target substance. At this time, Rf was 0.42 and the final yield was 97%. Angiogenesis inhibition by CAM analysis was 0%.

Example 3

(Wherein Me is a methyl group)

2.0 g (0.0049 M) of TK-088 and 0.52 g (0.011 M) of sodium hydride were stirred at 0 ° C. for 30 minutes, and 1.42 mL (0.02 M) of iodine methane was added slowly, followed by stirring at room temperature for 3 hours. The organic layer was washed with water, extracted five times with 100 ml of ethyl acetate, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure.

Column chromatography was performed using ethyl acetate: n-hexane (5: 3) as the eluent to obtain 2.0 g of the target substance. The maximum Rf was 0.5 and the final yield was 33%.

Dissolved in CDC ¹³ and TMS and measured by C ¹³ NMR are as follows;

The empirical formula for this material is C ₂₆ H ₄₄ O ₄ , named 7-hydroxy-3-methyloxy cholan-24-ionic acid (3α, 5β, 7β) methyl ester. Angiogenesis inhibition by CAM analysis was 75%.

Example 4

(Wherein Me is a methyl group)

Phenyl acetate 0.465 (0.0034M) was dissolved in 10 ml, DCC 0.88 g (0.0043 M) was added below 0 ° C, and stirred for 30 minutes. 0.7 g (0.0017 M) of TK-088 were added, and it stirred at room temperature for 7 hours. The organic layer was washed with water, extracted three times with 20 ml of ethyl acetate, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure.

Column chromatography was performed using ethyl acetate: n-hexane (1: 5) as the eluent to obtain 2.0 g of the target substance. At this time, Rf was 0.28 and the final yield was 22%.

The empirical formula for this material is C ₃₃ H ₄₈ O ₅ , designated 7-hydroxy-3-panylacetyloxycholane-24-ionic acid (3α, 5β, 7β) methyl ester. Angiogenesis inhibition by CAM analysis was 50%.

Example 5

(Wherein TBSO is t-butyldimethylsilyloxide)

0.5 g (0.0012 M) of ursodeoxycholic acid was dissolved in 15 ml, and 0.18 g (0.0012 M) of TBSCI and 0.168 g (0.0025 M) of imidazole were added and stirred at room temperature for 3 hours. The organic layer was washed with water, extracted five times with 100 ml of ethyl acetate, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure.

Column chromatography was performed using ethyl acetate: n-hexane (1: 3) as the eluent to obtain 0.51 g of the target substance. At this time, Rf was 0.15 and the final yield was 86%.

Angiogenesis inhibition by CAM analysis was 0%.

Example 6

(Wherein Me is a methyl group)

1.0 g (0.0026 M) of ursodeoxycholic acid was dissolved in 30 ml of DMF, adjusted to 4 ° C. or less, DCC 0.6 g (0.0029 M) and HOBT 0.4 g (0.0029 M) were added and stirred for 40 minutes. 1.26 g (0.01 M) of glycine methyl ester was added to 5 ml and 5 ml of triethanolamine, and then stirred at room temperature overnight. The mixture was neutralized with a 1N aqueous hydrochloric acid solution, washed with water, and then extracted three times with 50 ml of ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Column chromatography was performed using ethyl acetate: n-hexane (3: 1) as an eluent to obtain 0.63 g of the target substance. At this time, Rf was 0.37 and the final yield was 51%.

The empirical formula for this material was C ₂₇ H ₄₅ O ₅ N, named 3,7-dihydroxycholane-24-ionic acid (3α, 5β, 7β) -N-methyl glycinate. Angiogenesis inhibition by CAM analysis was 100%.

Example 7

(Wherein Et is an ethyl group)

Ursodeoxycholic acid 2.0 (0.0051M) was dissolved in 1.4 mL (0.01 M) of DBU, and 1.5 mL (0.02 M) of bromoethane was added thereto, followed by stirring at room temperature for 3 hours. The organic layer was washed with water and extracted twice with 100 ml of ethyl acetate, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. Column chromatography was performed using ethyl acetate: n-hexane (5: 4) as the eluent to obtain 2.01 g of the target substance. At this time, Rf was 0.38 and the final yield was 97%.

Example 8

[Acute Toxicity Test]

The acute dynamics test of the derivative of ursodeoxycholic acid and ursodeoxycholic acid obtained in Examples 3 to 7 was carried out, and the results are shown in Table 1.

In the test, 60 ICR male rats weighing 18 to 25 g were divided into 1 to 6 groups with 10 animals as 1 group, and the test substance suspended in 0.5% carboxymethyl cellulose solution was administered at 5000 mg / kg. Afterwards, the number of dead animals and the half lethality during 14LF were measured.

As can be seen from Table 1, no dead animals were observed when the derivative of ursodeoxycholic acid of the present invention was administered, and the half lethality (LD: Lerhal Dose) was 5000 mg / kg or more, indicating that the material was highly stable. have.

Claims (1)

An angiogenesis inhibitor containing one type of ursodeoxycholic acid derivative represented by the following general formula as an active ingredient in an amount of 0.01 mg to 1 g. (Wherein Me is a methyl group, Et is an ethyl group and TBSO is t-butyldimethylsilylioside.)