KR20030075807A - Novel Bicyclic Sesterterpene Derivatives and Pharmaceutical Compositions Comprising the Same - Google Patents

Abstract

PURPOSE: A novel bicyclic sesterterpene-based derivative and a pharmaceutical composition containing the derivative are provided, to obtain a pharmaceutical composition for treating or preventing the uncontrolled angiogenesis related disease. CONSTITUTION: The bicyclic sesterterpene-based derivative is represented by the formula 1, wherein R1, R2 and R3 are independently H, a linear or branched alkyl group, a linear or branched alkenyl group, a linear or branched alkynyl group, a cycloalkyl group, a cycloalkenyl group, an aryl group, an alkaryl group or an aralkyl group. Preferably R1, R2 and R3 are independently H or a methyl group in the formula 1. The pharmaceutical composition comprises the bicyclic sesterterpene-based derivative of the formula 1, and a pharmaceutically acceptable carrier.

Description

Novel Bicyclic Sesterterpene Derivatives and Pharmaceutical Compositions Comprising the Same

The present invention relates to a novel bicyclic cesterterpene derivative and a pharmaceutical composition comprising the same, and more particularly to the treatment of a novel bicyclic cesterterpene derivative and an unregulated angiogenesis-related disease comprising the same. Or to a prophylactic pharmaceutical composition.

Angiogenesis is essential for various physiological processes such as embryo development, wound healing and tissue or organ regeneration (Iwaguchi, T. 1993. Angiogenesis and its regulation.Gan To Kagaku Ryoho 20: 1-9 .; Kuwano , M. et al., 2001. Angiogenesis factors.Intern . Med. 40: 565-572 . ; and Tobelem G. 1990. Endothelial cell growth: biology and pharmacology in regulation to angiogenesis.Blood Coagul.Fibrinolysis 1: 703-705 .). However, persistent unregulated angiogenesis leads to angiogenic diseases such as rheumatoid arthritis, diabetic retinitis, solid tumors, hemangiomas and psoriasis (Andre, T., et al., 1998. Tumoralangiogenesis: physiopathology, prognostic value and therapeutic perspectives.Rev. Med.Interne . 19: 904-9134; Battegay, EJ 1995.Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects.J . Mol.Med . 73: 333-346; Carmeliet, P. and RK . Jain 2000. Angiogenesis in cancer and other diseases Nature 407:. 249-257; and Fidler, IJ 2000. Angiogenesis and cancer metastasis Cancer J. Sci Am 2:... 134-141).

The angiogenesis process promotes endothelial cell growth by tumor cytokines, vascular endothelial growth factor (VEGF) and basal fibroblast growth factor (bFGF), and decomposes extracellular matrix proteins by metalloproteases. It consists of several steps, such as endothelial cell migration mediated by cell membrane adhesion molecules, endothelial cell differentiation and tube formation (Bussolino, F. et al., 1997. Molecular mechanisms of blood vessel formation. Trends Biochem. . Sci 22: 251-256; Kuwano, M. et al, 2001. Angiogenesis factors Intern Med 40: 565-572; and Risau, W. 1994. Angiogenesis and endothelial cell function Arzneimittelforschung 44:..... 416-417 ). Thus, inhibition of these processes has emerged as a new therapeutic strategy for the treatment of cancers and other human diseases associated with angiogenesis.

Various types of angiogenesis inhibitors have been developed for this purpose. Such inhibitors, as natural or synthesized materials, include degradation fragments of protease inhibitors, inhibitors of tyrosine kinase, chemokines, interleukins and matrix proteins (Abedi, H. and I. Zachary. 1997. Vascular endothelial growth factorstimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells.J. Biol. Chem. 272: 15442-15451; Cao, Y. 2001. Endogenous angiogenesis inhibitors and their therapeutic implications.Int . J. Biochem Cell Biol. 33: 357-369; Fong, T. A et al., 1999.SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1 / KDR) that inhibits tyrosine kinase catalysis, tumor vascularization Cancer Res. 59: 99-106; and Kwon, HJ et al., 2001. Anti-angiogenic activity of acalycixenolide E, a novel marine natural product from Acalycigorgia inermis.J. Microbiol.Biotechnol. 11: 656-662).

The antiangiogenic molecules affect a number of processes including induction of cell apoptosis as well as inhibition of endothelial cell differentiation, migration, proteolytic activity and tube formation (Folkman, J. and D. Ingber. 1992. .. Inhibition of angiogenesis Semin cancer Biol 3:. 89-96; Kishi, K. et al, 2000. Recent studies on anti-angiogenesis in cancer therapy Nippon Rinsho 58:.. 1747-1762; and Marme, D. 2001. Tumor angiogenesis: new approaches to cancer therapy.Onkologie 1: 1-5) .The anti-angiogenic function of these molecules has been well studied in vitro and in vivo , some of which are currently undergoing clinical trials (Deplanque, G and AL Harris.2000.Anti-angiogenic agents: clinical trial design and therapies in development.Eur . J. Cancer 36: 1713-1724; Liekens, SED Clercq, and J. Neyts. 2001. Angiogenesis: regulators and clinical applications. Biochem.Pharmacol . 61: 253-270; and Mross, K. 2000. Anti-angiogenesis therapy: concepts an d importance of dosing schedules in clinical trials.Drug Resist.Updat. 3: 223-235).

For example, marimastat, Novastat and AG-3340 are synthesized cell invasion inhibitors (Jia, MC et al., 2000. Suppression of human microvascular endothelial cell invasion and morphogenesis ..... with synthetic matrixin inhibitors Targeting angiogenesis with MMP inhibitors Adv Exp Med Biol 476:. 181-194), TNP-470, thalidomide (thalidomide), and the other comb breather statin A-4 (combretastatin A-4 ) Inhibits cell differentiation (Damato, R. et al., 1994. Thalidomide is an inhibitor of angiogenesis. Proc. Natl. Acad. Sci. USA 91: 4082-4085; and Stern, JW et al., 2001. Angiogenesis inhibitor TNP-470 during bone marrow transplant: safety in a preclinical model.Clin.Cancer Res. 7: 1026-1032), interferon-alpha, suramin and derivatives thereof interfere with angiogenic growth factors, SU6668 and SU5416 inhibit the receptor enzymatic activity of angiogenic growth factors (Ingber, D. et al., 1990. Synthetic anal oguesof fumagillin that inhibit angiogenesis and suppress tumor growth Nature 348:. 555-557), endostatin and interleukin -12 is as inhibitors of intrinsic angiogenesis and is the current clinical trials for various solid tumors (Boehm, T. et al ., 1997. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88: 277-285).

Among the compounds and peptides, the clinically performed materials are mainly synthesized compounds. US Patent No. 5,908,930 discloses a compound that inhibits a new tyrosine kinase and discloses it as an angiogenic agent. 5,846,562 discloses an oral dose therapeutic agent comprising a fumagillol derivative effective as an active ingredient in the treatment of anti-angiogenic diseases. US Pat. It discloses an antiangiogenic agent comprising derivatives of isoindolinone as an active ingredient, US Patent No. 6,228,879 discloses EM-138, a novel compound that can inhibit angiogenesis and angiogenic diseases , Korean Patent Laid-Open No. 2001-98524, Wondonin A extracted from sponge animals inhibits angiogenesis. It discloses. In addition to the synthesized compounds, several peptides have been used in clinical approaches, and Korean Patent Publication No. 2000-5903 can be used as an anticancer agent because salmocin, a novel peptide extracted from the venom of salsa, can inhibit angiogenesis. Korean Patent Publication No. 2001-53018 discloses a protein for inhibiting angiogenesis.

Although various compounds and peptides as described above have been developed as antiangiogenic agents, the development of antiangiogenic compounds having structures that are not related to conventionally known inhibitors is not only a developmental aspect of new antiangiogenic agents. It is of great value as a reverse genetic approach to angiogenesis.

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

Accordingly, it is an object of the present invention to provide a novel bicyclic sesterterpene derivative.

Another object of the present invention is to provide a pharmaceutical composition for the treatment or prevention of unregulated angiogenesis-related diseases comprising the novel compound as an active ingredient.

Figure 1 is a photograph showing the inhibition of tube formation of BAECs (bovine aortic endothelial cells) by the extract of Embelia chlamidospora;

Figure 2 is a photograph showing the cell morphology of Embelia chlamidospora producing novel compounds of the present invention;

3 is a photograph of a compound of the present invention showing inhibition of tube formation of BAECs;

4 is a graph showing the effect of the compounds of the present invention on the viability of BAECs;

5A-5D are graphs showing the effect of the compounds of the present invention on the proliferation of BAECs, CHANG-Liver normal cell lines, HS-578T breast cancer cell lines and HT1080 cell lines, respectively;

6 is a photograph showing the effect of the compounds of the present invention on cell invasion of BAECs;

7 is a graph showing the effect of the compounds of the present invention on cell invasion of BAECs;

8 is a photograph of methylated derivatives of the present invention showing inhibition of tube formation of BAECs; And

Figure 9 is a photograph showing the effect of methylated derivatives of the present invention on cell invasion of BAECs.

According to one embodiment of the present invention, the present invention provides a novel bicyclic sesterterpene derivative represented by Formula 1 below:

In the above formula, R ₁ , R ₂ and R ₃ are each independently H, a straight or crushed alkyl group, a straight or crushed alkenyl group, a straight or crushed alkynyl group, a cyclo alkyl group, a cyclo alkenyl group, an aryl group, an alkaryl group Or an aralkyl group.

The compounds of the present invention are obtained by isolation and purification from the fungal microorganism Embellisia chlamydospora .

Methods for isolating and purifying the compounds of the present invention from Embelia chlamidospora can be through various purification methods known to those skilled in the art. Specific examples of methods for isolating and purifying compounds of the present invention are specified in the Examples below.

According to a preferred embodiment of the invention, R ₁ , R ₂ and R ₃ of the compounds of the invention are each independently H, C ₁ -C ₉ straight or crushed alkyl group, C ₂ -C ₉ straight or crushed alkenyl group, straight chain Or a ground alkynyl group, a C ₃ -C ₈ cycloalkyl group, a C ₅ -C ₇ cyclo alkenyl group, an aryl group, an alkanyl group or an aralkyl group.

According to a more preferred embodiment of the invention, R ₁ , R ₂ and R ₃ of the compounds of the invention are each independently H, a C ₁ -C ₉ straight chain or a ground alkyl group, and according to a more preferred embodiment of the invention , R ₁ , R ₂ and R ₃ of the compound of the present invention are each independently H or a methyl group, and according to the most preferred embodiment of the present invention, R ₁ , R ₂ and R ₃ of the compound of the present invention are H.

It is apparent to those skilled in the art that there are several asymmetric carbon centers in the nucleus of the compound structure of the present invention represented by Formula 1, and that there may be stereoisomers of the compound of the present invention, and as a result, all of the compounds represented by Formula 1 Stereoisomers are included within the scope of the present invention. On the other hand, the compound of formula (I) separated from nature is generally optically active as one stereoisomer, but when synthesizing the compound of the present invention chemically, racemates are usually prepared. Therefore, it is apparent to those skilled in the art that racemates are also included in the scope of the present invention.

The compounds of the present invention have antiangiogenic activity as biological features. Accordingly, the compounds of the present invention have the use of treating or preventing various diseases or disorders caused by unregulated angiogenesis.

Accordingly, according to another aspect of the present invention, the present invention provides (a) a pharmaceutically effective amount of a novel bicyclic sesterterpene derivative represented by the following formula:

Formula 1

R ₁ , R ₂ and R ₃ in the above formulas are each independently H, a straight chain or pulverized alkyl group, a straight or pulverized alkenyl group, a straight or pulverized alkynyl group, a cycloalkyl group, a cyclo alkenyl group, an aryl group, an alkaryl group or an aralkyl group ; And

(b) provides a pharmaceutical composition for the treatment or prophylaxis of an unregulated angiogenesis-related disease comprising a pharmaceutically acceptable carrier.

Since the content of the preferred embodiment of the compound represented by Formula 1 in the pharmaceutical composition of the present invention is the same as that of the compound of the present invention described above, the description thereof is omitted in order to avoid excessive complexity of the present specification.

The pharmaceutical composition of the present invention effectively inhibits angiogenesis and thus has the use of treatment or prevention of angiogenesis-related diseases. The pharmaceutical composition of the present invention does not act on the endothelial cell proliferation step, which is an important step in the angiogenesis step, but inhibits angiogenesis by acting on the differentiation step of endothelial cells, which is clearly illustrated in the following examples. It is.

The pharmaceutical compositions of the present invention may be used for the treatment or prevention of unregulated angiogenesis-related diseases or disorders, and preferably for the treatment or prevention of rheumatoid arthritis, diabetic retinopathy, malignant tumors, hemangiomas or psoriasis. And most preferably used for the treatment or prevention of malignant tumors.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, or the like.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-100 mg / kg.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example

Experimental material

Where to buy the experimental materials used in the examples below is as follows:

Silica gel was purchased from Merck Co., Germany and all solvents used analytical grade or first class reagents. bFGF was purchased from Upstate Biotechnology, Lake Placid, NY, MEM and heparin were purchased from Life Technology, Grand Island, NY, and Matrigel was a collaborative biomedical product. (Collaborative Biomedical Products, Bedford, MA), transwell plates were purchased from Corning Costar, Cambridge, MA, gelatin type B and Triton X-100 were obtained from Sigma Chem. Co., St. Louis. , MO).

Example 1 Screening of Microorganisms Producing Antiangiogenic Substances

Example 1-1: Obtaining Various Microbial Culture Extracts

The microorganisms (fungus) used for screening were isolated strains by collecting soils from all over the world, which were separated by dilution plate method. First, 0.1 ml of each dilution of the sample was smeared on PDA, L · O medium, incubated at 28 ° C. for 7 days, and then transplanted into single malt extract agar medium and cultured at 28 ° C. for 7 days, followed by different fungal strains. Were screened. The isolated strains were extracted by mixing 25 ml of YpSs medium in a 250 ml baffled Erlenmeyer flask and incubating for 7 days at 28 ° C. and 145 rpm to mix the culture solution and acetone in a 1: 1 ratio (50% acetone). Extraction status).

Example 1-2 Screening of Microorganisms Producing Antiangiogenic Materials (Tube Formation Assay)

The presence of antiangiogenic agents in extracts of various microbial cultures was analyzed as follows:

First, 150 μl of Matrigel (10 mg / ml) was coated on a 48 well plate and polymerized at 37 ° C. for 2 hours. Subsequently, BAECs (bovine aortic endothelial cells, ATCC, 1 × 10 ⁵ cells) were inoculated on Matrigel and treated with bFGF (basic fibroblast growth factor, 30 ng / ml). Then, the culture extract (20 μg / ml) of each microorganism was added and incubated for 18 hours. Morphological changes of the cells were observed under a microscope and photographed 40 times with ImagePro Plus software (Media Cybernetics, Inc.). Cytotoxicity of tubular endothelial cells was assessed by trypan blue staining. All experiments were repeated twice independently.

As a result, it was confirmed that one experimental group strongly inhibits the tube formation of endothelial cells induced by bFGF (see FIG. 1). In FIG. 1, panel A is BAECs without addition of bFGF and microbial culture extract, panel B is BAECs stimulated with bFGF only, and panel C is a photograph of BAECs with addition of bFGF and microbial culture extract. As can be seen in Figure 1, it can be seen that the tube formation of endothelial cells is strongly inhibited by the extract of the microorganism.

Example 1-3 Identification of Microorganisms

Identification of microorganisms in this experiment was performed by analyzing the biochemical and physiological characteristics according to Bergey's Manual of Determinative Bacteriology , and by Shirling and Gottlieb's method (Kwon, HJ et al., 2001. Anti-angiogenic activity of acalycixenolide E, a novel marine natural product from Acalycigorgia inermis. J. Microbiol. Biotechnol. 11: 656-662). In addition, analysis of cell morphology was performed under an optical microscope.

As a result of the identification of the microorganism, the culture extract showing antiangiogenic activity in Example 1-2 was found to be derived from Embellisia chlamydospora , a kind of fungus.

Figure 2 shows the cell morphology of Embelia chlamidospora isolated and identified by the inventors. In FIG. 2, the panel A shows the shape of the conidia, and the arrow shows the germinated cells and the conidia in the B panel. Panel C shows mycelial coils in artificial media (PDA), panel D shows initial mycelium, panel E shows aggregated mycelium, panel F shows dematiaceous mycelium and dark brown on PDA It represents the consort of spoilers. As can be seen in FIG. 2, the isolated and identified Embellia chlamidospores of the present invention show a typical cell morphological aspect.

Example 2: Culture and Maintenance of Cells

Example 2-1: Cultivation of M. Valley cyano Cloud Midousuji Fora (Embellisia chlamydospora)

Stocks of Embelia chlamidospora were maintained at 28 ° C. on MEA media plates (20 g / l glucose, 20 g / l malt extract, 1 g / l peptone and 18 g / l agar). For seed culture, the agar pieces of the stock plate were cut under sterile conditions and seeded in conical flasks (1 L) containing bran medium (bran 50 g / 50 mL). Seeds incubated at 28 ° C. for 3 days transfer 5-6 tablespoons per conical flask containing bran medium (bran 100 g / 100 ml) for large-scale cultivation, and a total of 10 flasks are transferred at 28 ° C. for 7 days. Incubated.

Example 2-2: Culture of Various Experimental Cell Lines

BAECs and HCT-116 colon cancer cells (Korea Cell Line Bank) were cultured in MEM and RPMI medium containing 10% fetal bovine serum. CHANG-Liver Normal Cells (ATCC) and HS-578T Breast Cancer Cells (Korea Cell Line Bank) Cultured at 37 ° C. under 5% CO ₂ and 95% air in DMEM containing% fetal bovine serum, 5.5 mL penicillin / streptomycin and NaHCO ₃ .

Example 3: Embelisia Klimidospora ( Embellisia chlamydospora And purification of active compounds from

The grown mycelium was extracted with 10-fold volume of acetone for 24 hours and filtered with chromatography paper. The extract was concentrated in vacuo and fractionated using methanol and DMSO. The methanol layer was then dried under reduced pressure and then subjected to silica gel (70-230 mesh) column chromatography (ψ6 × 17 cm) using chloroform / methanol as solvent to obtain each fraction. Solvent conditions increased the polarity by increasing methanol from 0% to 100% step by step. Among the obtained fractions, an angiogenic active fraction was collected in chloroform: methanol (9: 1), which was purified thin layer chromatography (TLC, silica gel 60 F ₂₅₄ ) using chloroform / methanol (8: 2) as a developing solvent. ) To obtain a fraction with antiangiogenic activity. Finally, the obtained antiangiogenic activity fraction was subjected to C ₁₈ reverse phase HPLC (semi-preparative UG 120 Å 5 μm, 10 mmφ ×) using acetonitrile / 0.1% trifluoroacetic acid (70:30) as a solvent. 250 mm, flow rate 3 ml / min) to purify the compound having antiangiogenic activity, the purity was confirmed by TLC and HPLC.

Example 4 Structure Determination and Properties of Antiangiogenic Active Compounds

To determine the structure of the active compound purified in Example 3, ¹ H-NMR (500 MHz) and ¹³ C-NMR (125 MHz) were CDed using a Varian Unity 500 spectrophotometer. _It was carried out in ₃ OD solution. All chemical shifts were reported in δ (ppm) based on MeOH (3.30 / 49.50 ppm) using TMS as internal standard. IR spectra were measured using a Matterson Galaxy spectrophotometer. Mass spectra were also measured using a Jeol JMS-HX 110 high resolution mass spectrophotometer. All solvents used were either spectral grade or redistilled prior to use.

The NMR measurement results are shown in Table 1 below:

# ¹ H ¹³ C HMBC 12345678910111213141516171819202122232425 2.75, 1H, dd (11.7, 2.0) 2.46, 1H, br d (16.6) 1.94, 1H, ddd (16.6, 11.7, 6.8) 5.41, 1H, m3.99, 1H, dd (9.8, 3.9) 1.76, 1H , m; 1.64, 1 H, m 2.08, 1 H, m; 1.79, 1 H, m 5.19, 1 H, br dd (9.3, 3.9) 2.31, 1 H, m; 2.12, 1 H, m 2.29, 1 H, m; 2.04, 1H, m5.32, 1H, dd (10.7, 4.9) 2.35, 1H, dd (13.2, 10.8) 1.73, 1H, m2.61, 1H, hex (6.8) 3.84, 1H, dd (10.7, 6.8) 3.70, 1H, dd (10.7, 6.8) 1.27, 3H, d (6.8) 1.57, 3H, br s1.65, 3H, br s1.65, 3H, br s0.97, 3H, s 209.41 s148.35 s151.53 s50.19 d29.38 t129.62 d136.58 s76.36 d30.17 t35.21 t133.06 s124.70 d24.12 t40.68 t137.91 s122.53 d39.66 t49 .47 s38.14 d65.13 t14.00 q9.72 q14.93 q14.74 q16.29 q C-1, C-2, C-3, C-5, C-17, C-18C-4, C-6, C-7C-6, C-7, C-9, C-10, C- 22C-7, C-8C-11, C-12C-15, C-16C-17, C-24C-1, C-4, C-15, C-16, C-18C-2, C-3, C-4, C-20, C-21C-3, C-19, C-21C-3, C-19, C-20C-6, C-7, C-8C-10, C-11, C- 12C-14, C-15, C-16C-1, C-4, C-17, C-18

Mass spectra, IR and other measurement results are shown in Table 2 below:

shape White crystals (or amorphous solid, colorless oil) Solubility Soluble in methanol and chloroform, insoluble in water UV _λmax (nm) 262 FAB-MS (m / z) 403 (M + H) ⁺ IR _νmax (cm ^-1 ) 1700 TLC (R _f ) ^* 0.6 ^* Hexane: using a (4: 1) as a developing solvent EtoAc

From the results of Table 1 and Table 2, the compound purified in Example 3 of the present invention was found to have a molecular formula of C ₂₅ H ₃₈ O ₄ , the structural formula of the novel double ring sester as shown in the formula (2) It was found that the compound is a ruterpene compound, and this compound was named "embellistatin".

Example 5 Analysis of Various Activities of Compounds of the Invention (Emberstatin)

Example 5-1: Analysis of the impact on the tube formation of endothelial cells

It was carried out according to the tube formation analysis described in Example 1-2, each experiment was carried out twice independently, the results are shown in FIG.

In FIG. 3, panel A is BAECs not treated with bFGF and emberstatin, panel B is BAECs stimulated with bFGF only, and panel C is a photograph of BAFs treated with bFGF and emberstatin.

Vascular endothelial cells rapidly differentiate into capillary-like structures in vitro, and these properties simplify the analysis of the effects of certain substances on endothelial cell differentiation processes that require cell-substrate interactions, intercellular communication, and cell mobility. Provide a way to do it. BAECs cultured in the Matrigel layer form incomplete but narrow tubular structures even without angiogenic factors, and when angiogenic factors such as bFGF are added, the capillary net structure forms a strong tubular structure that is elongated. This structure is formed by a greater number of cells compared to the control. Looking at the effect of the compound of the present invention on this mechanism, it can be seen that it effectively inhibits the tube formation by bFGF (see: C panel of Figure 3). Cells whose tube formation was inhibited by the compounds of the present invention are not stained by trypan blue, which shows that the inhibitory effect of tube formation by the compounds of the present invention does not result from cytotoxicity.

Example 5-2 Analysis of Impact on the Viability of BAECs

The optimal amount that does not exhibit the cytotoxicity of emberstatin was determined as follows. First, BAECs cultured in MEM medium containing 10% fetal bovine serum were dispensed by 5 × 10 ³ cells in 96-well plates, and cultured in the same medium for 3 hours to fix. Cells were then washed using PBS, exchanged again with fresh identical medium, and then emberstatin was added to each well at concentrations of 0, 1, 5, 10 and 20 μg / ml and incubated for 72 hours. . After incubation, 50 μl of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide, 2 mg / ml stock solution, Sigma) was added to each well and for 4 hours After standing, the medium was removed and 100 μl DMSO was added to each well. Subsequently, absorbance was measured at 540 nm using a universal microplate reader (BIO-TEK INSTRUMENTS, INC.) To measure cell viability. The experiment was conducted twice independently, and the results are shown in FIG. 4.

As can be seen in Figure 4, the addition of emberstatin at a concentration of 20 ㎍ / ㎖ was found to exhibit cell viability of more than 95%. Therefore, it can be seen that the inhibitory effect on tube formation from Example 5-1 was not due to cytotoxicity to endothelial cells.

Example 5-3 Analysis of Effect on Growth of BAECs

Proliferation of endothelial cells is an important step in the process of angiogenesis, which consists of multiple stages. Therefore, whether the compound of the present invention exhibits antiangiogenic activity by inhibiting proliferation of BAECs was analyzed as follows: First, cells (BAECs, CHANG- liver normal cell line, HS-578T breast cancer cell line and HT1080 cell line) were analyzed as follows. 5 × 10 ³ cells were dispensed into 96 well plates and cultured in medium containing 10% fetal bovine serum. Subsequently, the cells were washed with PBS, and then exchanged with fresh fresh medium. Emberstatin was then added to each well at a concentration of 5 μg / ml, incubated for 4 days, and subjected to MTT analysis (same as in Example 5-2) at 24 hour intervals. Experimental results are shown in FIGS. 5A-5D. Figure 5a is a BAECs, Figure 5b is CHANG- liver normal cell line, Figure 5c is a graph for HS-578T breast cancer cell line and Figure 5d is a HT1080 cell line.

5A to 5D, the proliferation of BAECs was slightly reduced by emberstatin, and the proliferation of the remaining three cell lines was hardly affected by emberstatin. The results show that the antiangiogenic activity of emberstatin does not appear by inhibiting endothelial cell proliferation, but by controlling endothelial cell differentiation.

Example 5-4 Analysis of Effect on Cell Invasion of BAECs

One of the main characteristics of angiogenesis is the movement of endothelial cells, the destruction of their basement membrane (basement membrane) must be made through the formation of neovascularization, bFGF can promote this endothelial cell invasion. Therefore, it was investigated whether the compound of the present invention emberstatin affects this endothelial cell invasion.

Invasion of BAECs was performed in vitro using a transwell chamber system with a polycarbonate filter. First, the bottom of the filter was coated with 10 μl gelatin (1 μg / μl), and the top of the filter was coated with 10 μl of Matrigel. BFGF (30 ng / ml), BAS and emberstatin (1, 5 or 10 μg / ml) were added to the lower wells placed in 600 μl MEM. Subsequently, after placing BAECs (1 × 10 ⁵ cells) on top of the filter, the chamber was incubated at 37 ° C. for 18 hours. Then, the cells were fixed with 70% methanol, stained with hematoxylin / eosin, and the degree of cell invasion was determined by counting the whole cells in one filter under the microscope, and photographed 40 times with ImagePro Plus software. It was. The experiment was conducted twice independently. The experimental results are shown in FIGS. 6 and 7.

6 and 7, the cell invasion rate of the control group treated only with bFGF was significantly increased compared to the control group, and when treated with 5 or 10 μg / ml emberstatin, the cell induction induced by bFGF was significantly reduced. It can be seen that. Therefore, it can be seen that the compound of the present invention emberstatin inhibits angiogenesis of endothelial cells by controlling the differentiation of endothelial cells.

Example 6 Preparation of Methylated Emberstatin Derivatives

1.5 mg of emberstatin (3.73 μmol) isolated in Example 3 was dissolved in 0.5 mL of acetone, and methyl iodide (24 μl, 74.6 μmol) and K ₂ CO ₃ (5.2 mg, 37.3 μmol) were added. After the addition, the reaction was stirred for 3 hours. After the reaction, 0.8 mg of methylated emberstatin was isolated by analysis of TLC and HPLC. The molecular weight and molecular structure of the methylated emberstatin were confirmed by ESI MS and NMR. In the chemical formula 1, the methylated emberstatin was identified. The other molecular weight and physicochemical properties were as follows: Color: white, Molecular weight: 418, Formula: C ₂₆ H ₄₀ O ₄ , Maximum absorbance: 200 nm, 260 nm, ¹ H NMR (300 MHz, CD ₃ OD) δ5.41 5.39 (1 H, m), 5.32 (1 H, dd , J = 10.2, 5.0 Hz), 5.20 5.18 (1 H, m), 3.99 (1 H, dd, J = 10.2, 4.1 Hz), 3.85 (3 H, s), 3.78 (1 H, dd, J = 10.3, 7.6 Hz), 3.67 (1 H, dd, J = 10.3, 7.6 Hz), 2.78 (1 H, dd, J = 11.1, 2.3 Hz), 2.69 2.61 (1 H, m), 2.49 2.43 (1 H , m), 2.37 2.29 (2 H, m), 2.21 1.96 (3 H, m), 1.83 1.77 (2 H, m), 1.76 (3 H, s), 1.76 (3 H, s), 1.58 (3 H, s), 1.24 (3H, d, J = 7.0 Hz), 0.98 (3H, s).

Example 7 Activity Analysis of Methylated Emberstatin

Example 7-1: Analysis of the impact on the tube formation of endothelial cells

It was carried out according to the tube formation analysis described in Example 1-2, each experiment was carried out twice independently, the results are shown in FIG.

In Figure 8, A panel is BAECs untreated with bFGF and methylated emberstatin, B panel is BAECs stimulated with bFGF only, C and D panels with bFGF and methylated emberstatin (5, 10 μg / ml respectively) This is a photograph of processed BAECs. The effect of methylated emberstatin is shown to inhibit tube formation by bFGF, but at the same treatment concentration (5 μg / ml), the degree of inhibition of tube formation was weakened compared to unmethylated emberstatin, but twice the treatment concentration ( 10 μg / ml) showed distinct activity.

Example 7-2 Analysis of Effect on Cell Invasion of BAECs

The experiment was performed according to the cell invasion assay described in Example 5-4, and each experiment was performed twice independently, and the results are shown in FIG. 8.

In Figure 9, A panel is BAECs untreated with bFGF and methylated emberstatin, B panel is BAECs stimulated with bFGF only, C and D panels with bFGF and methylated emberstatin (5, 10 μg / ml, respectively). This is a photograph of processed BAECs. The effect of methylated emberstatin is shown to inhibit cell invasion by bFGF, but at the same treatment concentration (5 μg / ml), the degree of inhibition of cell invasion is weakened compared to unmethylated emberstatin, but doubled. At concentrations (10 μg / ml), there was a marked inhibitory effect.

As described in detail above, the present invention provides a novel anti-angiogenic compound and a pharmaceutical composition comprising the same. The anti-angiogenic compounds of the present invention do not inhibit endothelial cell proliferation and inhibit endothelial cell migration and tubulation, which are important processes for angiogenesis, and thus can be used as safe angiogenesis inhibitors and therapeutic agents for angiogenic diseases in vivo. Can be.

Claims (11)

A novel bicyclic sesterterpene derivative represented by the following formula (1): Formula 1 R ₁ , R ₂ and R ₃ in the above formulas are each independently H, a straight chain or pulverized alkyl group, a straight or pulverized alkenyl group, a straight or pulverized alkynyl group, a cycloalkyl group, a cyclo alkenyl group, an aryl group, an alkaryl group or an aralkyl group . A compound according to claim 1, wherein R ₁ , R ₂ and R ₃ of the compound of Formula 1 are each independently H, C ₁ -C ₉ straight or crushed alkyl group, C ₂ -C ₉ straight or crushed alkenyl group, straight or pulverized A bicyclic sesterterpene derivative characterized by being an alkynyl group, a C ₃ -C ₈ cycloalkyl group, a C ₅ -C ₇ cyclo alkenyl group, an aryl group, an alkali group or an aralkyl group. The bicyclic sesterterpene derivative according to claim 2, wherein R ₁ , R ₂ and R ₃ of the compound of Formula 1 are each independently H, C ₁ -C ₉ straight chain or crushed alkyl group. The bicyclic sesterterpene derivative according to claim 3, wherein R ₁ , R ₂ and R ₃ of the compound of Formula 1 are each independently H or a methyl group. The bicyclic sesterterpene derivative according to any one of claims 1 to 4, wherein the bicyclic sesterterpene derivative has antiangiogenic activity. (A) A novel bicyclic sesterterpene derivative represented by the following formula (1) in a pharmaceutically effective amount: Formula 1 R ₁ , R ₂ and R ₃ in the above formulas are each independently H, a straight chain or pulverized alkyl group, a straight or pulverized alkenyl group, a straight or pulverized alkynyl group, a cycloalkyl group, a cyclo alkenyl group, an aryl group, an alkaryl group or an aralkyl group ; And (b) A pharmaceutical composition for the treatment or prophylaxis of uncontrolled angiogenesis-related diseases comprising a pharmaceutically acceptable carrier. A compound according to claim 6, wherein R ₁ , R ₂ and R ₃ of the compound of Formula 1 are each independently H, C ₁ -C ₉ straight or pulverized alkyl group, C ₂ -C ₉ straight or pulverized alkenyl group, straight or pulverized A pharmaceutical composition for the treatment or prophylaxis of an unregulated angiogenesis-related disease, characterized by being an alkynyl group, a C ₃ -C ₈ cycloalkyl group, a C ₅ -C ₇ cyclo alkenyl group, an aryl group, an alkaryl group or an aralkyl group. 8. The treatment of uncontrolled angiogenesis-related diseases according to claim 7, wherein R ₁ , R ₂ and R ₃ of the compound of Formula 1 are each independently H, C ₁ -C ₉ straight chain or ground alkyl group. Or prophylactic pharmaceutical compositions. The method of claim 8, wherein R ₁ , R ₂ and R ₃ of the compound of Formula 1 are each independently H or a methyl group, characterized in that the pharmaceutical composition for the treatment or prevention of unregulated angiogenesis-related diseases. 10. The unregulated angiogenesis-related disease according to any one of claims 6 to 9, wherein the unregulated angiogenesis-related disease is rheumatoid arthritis, diabetic retinitis, malignant tumor, hemangioma or psoriasis. Pharmaceutical compositions for the treatment or prophylaxis. The pharmaceutical composition of claim 10, wherein the unregulated angiogenesis-related disease is a malignant tumor.