KR20050044906A - Novel heptatrienoic acid substituted bicyclic ketone derivative and pharmaceutical compositions comprising the same - Google Patents

Abstract

The present invention relates to a bicyclic ketone derivative substituted with a novel heptatrienoic acid and a pharmaceutical composition comprising the same, and more particularly, to a novel heptatrienoic acid showing antiangiogenic activity. Bicyclic ketone derivatives and pharmaceutical compositions for the prevention or treatment of unregulated angiogenesis-related diseases comprising the same.

Description

NOVEL HEPTATRIENOIC ACID SUBSTITUTED BICYCLIC KETONE DERIVATIVE AND PHARMACEUTICAL COMPOSITIONS COMPRISING THE SAME}

The present invention relates to a bicyclic ketone derivative substituted with a novel heptatrienoic acid and a pharmaceutical composition comprising the same, and more particularly to a novel heptatrile showing antiangiogenic activity. It relates to a bicyclic ketone derivative substituted with no acid and a pharmaceutical composition for the prevention or treatment of unregulated angiogenesis-related diseases comprising the same.

Angiogenesis, ie the growth of new blood vessels, is very important for many physiological processes such as embryonic development, healing of wounds and regeneration of tissues or organs (Iwaguchi, T. 1993. Angiogenesis and its regulation.Gan To Kagaku Ryoho 20: 1 Kuwano, M. et al., 2001.Angiogenesis factors.Intern.Med. 40: 565-572 .; & Tobelem G. 1990. Endothelial cell growth: biology and pharmacology in regulation to angiogenesis.Blood Coagul.Fibrinolysis1 : 703-705.). However, persistent unregulated angiogenesis leads to angiogenic diseases such as rheumatoid arthritis, diabetic retinopathy, solid tumors, hemangiomas and psoriasis (Andre, T., et al., 1998. Tumoral angiogenesis: 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; & Fidler, IJ 2000. Angiogenesis and cancer metastasis. Cancer J. Sci. Am. 2: 134-141).

The angiogenesis process consists of multiple stages, stimulating endothelial growth by tumor cytokines, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and extracellular matrix proteins by metalloproteinases. It is composed of the following steps: decomposition step, endothelial cell migration mediated by cell membrane adhesion molecules, endothelial cell proliferation step, and tube formation step (Bussolino, F. et al., 1997. Molecular mechanisms of blood vessel formation. Biochem.Sci. 22: 251-256; Kuwano, M. et al., 2001.Angiogenesis factors.Intern.Med. 40: 565-572; & Risau, W. 1994.Angiogenesis and endothelial cell function.Arzneimittelforschung 44: 416 -417). Thus, inhibition of this process is emerging as a promising new strategy for the treatment of cancer and other human diseases associated with angiogenesis.

A variety of novel angiogenesis inhibitors are being developed for this purpose. Naturally derived or synthesized inhibitors include protease inhibitors, tyrosine kinase inhibitors, chemokines, interleukins and proteolytic fragments of matrix proteins (Abedi, H. and I. Zachary. 1997. Vascular endothelial growth factor stimulates 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, and growth of Multiple tumor types. Cancer Res. 59: 99-106; & 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). These anti-angiogenic molecules act on multiple pathways and also induce apoptosis as well as inhibiting endothelial cell proliferation, migration, protease activity and angiogenesis (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; & Marme, D. 2001. Tumor angiogenesis: new approaches to cancer therapy.Onkologie 1: 1-5). Antiangiogenic function for many of these molecules has been fully reported in vitro and in vivo , and some have been tested in 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. K. 2000. Anti-angiogenesis therapy: concepts and importance of dosing schedules in clinical trials.Drug Resist.Updat. 3: 223-235). For example, marimastat, neovastat and AG-3340 are synthetic inhibitors for cell invasion (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), vitacin inhibits cell adhesion, while TNP-470, thalidomide and combretastatin A-4 inhibit cell proliferation. (Damato, R. et al., 1994. Thalidomide is an inhibitor of angiogenesis.Proc. Natl. Acad. Sci. USA 91: 4082-4085; & 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 inhibit angiogenic growth factors, SU6668 and SU5416 inhibit receptors of these factors (Ingber, D. et al., 1990. Synthetic analogues of fumagillin that inhibit angiogenesis and Suppress tumor growth.Nature 348: 555-557), and endostatin and interleukin-12 are endogenous inhibitors of angiogenesis and tumor growth (Boehm, T. et al., 1997. 88: 277-285) is in the clinical trial stage for various solid tumors.

Among the compounds and peptides, the substances to which the clinical approach has been carried out are mainly synthesized compounds, and US Pat. No. 5,908,930 discloses a compound which inhibits tyrosine kinase and a pharmaceutical composition comprising the same. U. S. Patent No. 5,846, 562 discloses a pharmaceutical composition comprising as an active ingredient a fumagillol derivative effective for the treatment of anti-angiogenic diseases. U. S. Patent No. 5,994, 388 discloses cytochalasin and iso An antiangiogenic agent comprising derivatives of indolinone (isoindolinone) as an active ingredient, US Patent No. 6,228,879 discloses EM-138, a novel compound that can suppress the onset of angiogenesis and angiogenic diseases It is starting. Korean Patent Publication No. 2001-98524 suggests that wondonin A extracted from sponge animals inhibits angiogenesis. 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 several compounds and peptides have been developed as anti-angiogenic agents as described above, novel anti-angiogenic compounds with structures not associated with known inhibitors are not only for the development of new anti-angiogenic agents, but also for anti-angiogenesis. It will be valuable as a useful tool for the chemical genetic approach of research.

Based on this, the present inventors searched for a low molecular weight compound having antiangiogenic activity from the metabolite of the microorganism, and as a result, the present inventors found that the novel compound obtained from the culture extract of Embelsia chlamidospora was potent antiangiogenic It was confirmed that it is a product compound.

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.

1 is a photograph showing the M Valley cyano Cloud Midousuji Fora tube formation inhibition of BAECs (bovine aortic endothelial cell) according to culture extracts:

(A) Control (B) bFGF alone and (C) bFGF and Embelsia chlamidospora culture extract (20 μg / ml).

Figure 2 is a photograph showing cell morphology of M. Valley cyano Cloud Midousuji Fora generating new compounds of the present invention (denotes the arrow is a germination cell) some other form of minute living person (conidia), (B), minute living person bottle ( Marks (arrows) on conidiophores, (C) Hypal coils on artificial media (PDA), (D) initial mycelial growth, (E) aggregated mycelial mass and (F) artificial media , PDA, M. Valley cyano climb to matiahgwa mycelia Midousuji Fora (dematiaceous mycelia) and spores yuhyeop dark brown on.

Figure 3 is a schematic diagram showing the purification process of the anti-angiogenic compound from Embelia chlamidospora .

4 is a graph showing the effect of the bicyclic ketone derivatives of the present invention on the growth of BAEC, normal (CHANG) and cancer (HT29, HepG2, C8161, HT1080, HeLa) cell lines.

5 is a graph showing the inhibitory effect of the bicyclic ketone derivatives of the present invention on tube formation of BAECs.

6 shows microscopic observations showing the inhibitory activity of the bicyclic ketone derivatives of the invention against invasion of BAECs.

7 shows microscopic observations showing the inhibitory activity of the bicyclic ketone derivatives of the invention on invasion of BAECs.

Accordingly, it is an object of the present invention to provide a novel heptatrienoic acid substituted bicyclic ketone 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.

Other objects and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the appended claims and drawings.

According to one embodiment of the present invention, the present invention provides a novel heptatrienoic acid substituted bicyclic ketone derivative represented by Formula I:

Formula I

The compounds of the present invention are isolated or chemically synthesized from the fungus M. Valley cyano Cloud Midousuji Fora (Embellisia chlamydospora). The method for separating and purifying the compound of the present invention from Embelia chlamidospora can be through various known purification methods. Isolation and purification of the derivatives of the present invention are illustrated in Example 3.

There are several asymmetric carbon centers in the nucleus of the compound structure of the present invention represented by the above formula (I), and thus, it is clear to those skilled in the art that all stereoisomers of the compounds of the present invention are included in the scope of the present invention. Compounds of the invention that are isolated in nature are generally optically active as one particular stereoisomer, but racemates are typically produced when chemically synthesizing the compounds of the invention. Accordingly, racemates will also be included within 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 are excellent for the treatment or prevention of diseases or disorders associated with unregulated angiogenesis.

 According to another aspect of the present invention, the present invention provides an unmodulated comprising (a) a pharmaceutically effective amount of a novel heptatrienoic acid substituted bicyclic ketone derivative represented by the following formula (I) and (b) a pharmaceutically acceptable carrier: Provided are pharmaceutical compositions for the treatment or prevention of diseases associated with neovascularization:

Formula I

Since the pharmaceutical composition of the present invention includes the derivative of Formula (I) as an active ingredient, duplicate descriptions are omitted to avoid excessive complexity of the present specification.

The pharmaceutical composition of the present invention has the use of the treatment or prevention of angiogenesis-related diseases by effectively inhibiting vestibular angiogenesis. More specifically, the pharmaceutical compositions of the present invention inhibit the proliferation and differentiation of endothelial cells in the angiogenic stage, which is illustrated in the examples below.

According to a preferred embodiment of the present invention, the disease which can be treated or prevented using the composition of the present invention as an uncontrolled angiogenesis-related disease is rheumatoid arthritis, diabetic retinitis, cancer, angioma or psoriasis. More preferably, the unregulated angiogenesis related disease is cancer.

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 humectant, sweetener, flavoring agent, emulsifier, suspending agent, preservative, flavoring agent, perfume, lubricant, and the like. The pharmaceutical composition of the present invention can be administered orally or parenterally.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, food, time of administration, condition of the patient, drug combination, response sensitivity, and degree of illness of the patient. Do. 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 composition of the present invention may be formulated using a pharmaceutically acceptable carrier and / or excipient in a unit dosage form or the like according to a method which can be easily carried out by those skilled in the art. Can be prepared. The formulations may then be in the form of solutions, suspensions or emulsions or in the form of extracts, elixirs, powders, granules, tablets, capsules, linens, lotions, and ointments.

The following examples are only intended to illustrate the invention, and are not intended to limit the scope of the invention defined by the appended claims by these examples.

sample

Samples used in the following examples were purchased commercially:

Silica gel was purchased from Merck (Germany) and all solvents were of analytical grade or grade 1. Basic fibroblast growth factor (bFGF) was purchased from Upstate Biotech Corporation (Lake Placid, NY). MEM, DMEM and RPMI1640 were purchased from Life Technology, Inc. (Grand Island, NY). Matrigel and transwell plates were purchased from Collaborative Biomedical Products, Inc. (Bedford, Mass.) And Corning Costa (Cambridge, Mass.), Respectively. Gelatin Type B was purchased from Sigma (St. Louis, Mo.).

Example 1 Screening of Microorganisms Producing Antiangiogenic Compounds

Example 1-1 Preparation of Culture Extracts from a Microbial Library

The microorganisms (fungi) used in this search were separated into soil from various regions of the world. 100 μl of diluted solution was spread on PDA or L · O medium and incubated at 28 ° C. for 7 days. Each colony was then transferred to MEA (malt extract agar) medium and incubated at 28 ° C. for 7 days. The isolated strain was 25 ml of YpSs (glucose (20 g / L), yeast extract (2 g / L), peptone (5 g / L), MgSO ₄ 7H ₂ O (0.5 g / L), KH ₂ PO ₄ (1 g / L, pH 5.6-5.8) inoculated in a 250 ml corrugated flask containing culture medium and incubated for 7 days at 145 rpm rotary stirrer at 28 ° C. The culture broth was then subjected to the same Extract with volume of acetone.

Example 1-2 Screening of Microorganisms Producing Antiangiogenic Compounds (Tube Formation Analysis)

Several microbial culture extracts were tested to screen for microbes that produce antiangiogenic compounds. To this end, tube formation analysis was performed. 150 μl of Matrigel (10 mg / ml) was applied to a 48 well culture plate and polymerized at 37 ° C. for 2 hours. BAECs (bovine aortic endothelial cells, ATCC, 1 × 10 ⁵ cells) were inoculated on the matrigel surface and treated with bFGF (basic fibroblast growth factor, 30 ng / ml). Then, microbial culture extract (20 μg / ml) was added and incubated for 6-18 hours. Morphological changes in the cells were observed under a microscope and photographed at 40 × magnification using ImagePro Plus software (Media Cybernetics, Inc.). Cytotoxicity of tubular-forming endothelial cells was measured by trypan blue staining. The experiment was repeated twice independently. As a result, fungi (fungus) M. Valley cyano Cloud Midousuji Fora the culture extract obtained from (Embellisia chlamydospora) was strongly inhibited the tube formation of endothelial cells induced by bFGF (Fig. 1).

Example 1-3: Taxonomic Study of the Embelia Chlamidospora Variants

The biochemical and physiological properties of the strains were tested according to "Bergey's Manual of Determinative Bacteriology", and the physiological properties including the utilization of carbon sources were tested according to 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). The cell morphology of Embelia chlamidospora was observed under an optical microscope and is shown in FIG. 2.

Example 2: Organisms and Culture Conditions

Example 2-1: Cultivation of Embelia chlamidospora

Em Valley cyano Cloud stock culture (stock culture) of Midousuji Fora MEA medium plates (glucose 20 g / L, malt extract 20 g / L, peptone 1 g / L, agar 18 g / L) was kept at 28 ℃ in. For seed cultivation, agar pieces of stockplates were cut out under sterile conditions and seeded in 1 L conical flasks containing wheat bran medium (bran 50 g / 50 mL). Seeds incubated at 28 ° C. for 3 days were added by 5-6 tablespoons per conical flask containing bran medium (bran 100 g / 100 ml) for large-scale cultivation and a total of 10 flasks were incubated at 28 ° C. for 7 days. It was.

Example 2-2 Cell Lines and Culture Conditions

HT-29 colon cancer cells and BAECs were incubated in RPMI and MEM, respectively, containing 10% FBS (fetal bovine serum). CHANG-Liver normal, HepG2 human liver cancer, HT1080 fibrosarcoma, C8161 melanoma and HeLa cervical cancer cells at 37 ° C. under 5% CO 2 and 95% air in DMEM containing 10% FBS, 5.5 ml penicillin / streptomycin and NaHCO ₃ Incubated at.

Example 3: Embelia Chlamidospora And purification of antiangiogenic compounds from

The purification process of the active compound from Embelia chlamidospora is shown in FIG. 3. The grown mycelium (1 kg) in bran medium was extracted with 5 volumes of acetone for 24 hours and filtered with filter paper (Whatman, 3 mm). The filtrate was concentrated in vacuo and extracted with the same volume of ethyl acetate. Ethyl acetate was concentrated in vacuo and then subjected to silica gel (Merck silica gel 60) column chromatography (6 × 17 cm) and prepared with CHCl ₃ . The active fractions eluted with CHCl ₃ : MeOH (9: 1) were collected, evaporated and subjected to preparative thin layer chromatography (silica gel 60 F254), and CHCl ₃ : MeOH (8: 2) was used as the solvent system. The active compound was purified by HPLC using a C18 reversed phase column (Shiseido Co., Japan, semi-preparative UG 120 mm, 10 mm x 250 mm) and eluted with 70% acetonitrile in 0.1% TFA. The active compound was obtained as a yellow oil, and the purity of the compound was confirmed by HPLC. The molecular formula of the active compound was C ₂₅ H ₃₂ O ₄ based on the high resolution mass data (MW 396.52).

Example 4 Determination of Chemical Structure of Antiangiogenic Compounds

NMR spectra were recorded using a Varian Unity 500 spectrometer at 500 MHz for ¹ H and 125 MHz for ¹³ C in CD ₃ OD solvent. All chemical shifts were reported in δ (ppm) based on MeOH (3.30 / 49.50 ppm) and TMS was used as internal standard. Mass spectra were collected on a Jeol JMS-HX 110 high resolution mass spectrometer. All solvents used were either spectral or re-distilled in glass prior to use.

NMR data of purified anti-angiogenic compounds are shown in Table 1:

The physicochemical properties of the purified antiangiogenic compounds are shown in Table 2:

Thus, purified anti-angiogenic compounds have been identified as novel heptatrienoic acid substituted bicyclic ketone derivatives of formula I and named "embellistatin".

Example 5: Antiangiogenic Activity of Embelistatin

Example 5-1 Effect of Embelistatin on Growth of Various Cell Lines

We describe the effects of embellistatin on the growth of various cell lines including right aortic endothelial cells (BAECs) in MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetra Jolly bromide) colorimetric assay. Cells were seeded in 96-well culture plates at a density of 5 × 10 ³ per well and incubated for 24 hours for stabilization. Various concentrations of embellistatin were added to each well and incubated for 3 days, followed by MTT assay. 50 μl of MTT (2 mg / ml stock solution; Sigma, St. Louis, Mo.) was added and the plate was incubated for an additional 4 hours. After removing the medium, 100 μl of DMSO was added. The plate was read at 540 nm with a universal microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). As shown in FIG. 4, embelistatin inhibited the proliferation of each cell line with different growth inhibition spectra. Notably, embelistatin strongly inhibits the growth of BAECs and cancer (HT29, HepG2, C8161, HT1080, HeLa) cell line lines rather than the growth of normal (CHANG) cell line lines. These data suggest that embellistatin can exhibit antiangiogenic activity by inhibiting endothelial cell growth. The viability of endothelial cells was not affected by embellistatin treatment up to 5 μg / ml, which means that the growth inhibitory activity of embellistatin shown in FIG. 4 is not due to the simple cytotoxicity of the compound. .

Example 5-2 Effect of Embellistatin on Tube Formation of Endothelial Cells

Vascular endothelial cells undergo rapid in vitro differentiation to form capillary-like structures that allow a simple assay to evaluate the effect of a sample on endothelial differentiation processes that require cell-matrix interactions, intercellular contact, and cell mobility. In Example 1-2). BAECs cultured on Matrigel layers usually form incomplete and narrow tube-like structures in the absence of angiogenic factors. However, capillary network formation is more stimulated by treatment of angiogenic factors such as bFGF, resulting in a prolonged and robust tubular-like structure, which is organized by a greater number of cells than the control group (Fig. 5A and B). To test the effects of embellistatin on this process, BAECs stimulated with bFGF were treated with embellistatin. 5C shows that embelistatin effectively inhibits bFGF induced vascular formation. These cells are not stained with trypan blue, meaning that inhibition of tube formation by embellistatin is not due to simple cytotoxicity to the cells.

Example 5-3 Effect of Embellistatin on BAECs Invasion

As another important feature of angiogenesis, moving endothelial cells must break through their basement membranes to form new blood vessels and bFGF stimulates this endothelial invasion. Therefore, the inventors investigated the effects of embellistatin on endothelial cell invasion in vitro using a transwell chamber system. The system was equipped with a polycarbonate filter insert, which was applied with a Matrigel to prevent migration of non-invasive cells. The bottom of the filter was applied with 10 μl of gelatin (1 μg / μl) and the top with 10 μl of Matrigel. bFGF (30 ng / ml), BSA and Embelistatin were added to the lower wells placed in 600 μl of MEM. After placing the BAECs (1 × 10 ⁵ cells) on top of the filter, the chamber was incubated at 37 ° C. for 18 hours. Cells were fixed with 70% methanol and stained with hematoxylin / eosin. Cell invasion was determined by counting total cell numbers in a single filter, using an optical microscope and photographed at 40x magnification with ImagePro Plus software. The experiment was repeated twice independently. 6 and 7 show that embellistatin (5 μg / ml) significantly inhibits bFGF-stimulated invasion of BAECS. This data indicates that embellistatin inhibits angiogenesis of endothelial cells in vitro .

Claims (4)

Heptatrienoic acid substituted bicyclic ketone derivatives represented by Formula I: Formula I A pharmaceutical composition for the treatment or prophylaxis of an unregulated angiogenesis-related disease comprising (a) a pharmaceutically effective amount of a heptatrienoic acid substituted bicyclic ketone derivative of claim 1 and (b) a pharmaceutically acceptable carrier. The method of claim 2, wherein the unregulated angiogenesis-related disease is selected from the group consisting of rheumatoid arthritis, diabetic retinitis, cancer, hemangioma, and psoriasis. Pharmaceutical composition for. 4. The pharmaceutical composition of claim 3, wherein the uncontrolled angiogenesis-related disease is cancer.