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WO2005003076A1 - Novel heptatrienoic acid substitued bicyclic ketone derivative and pharmaceutical compositions comprising the same - Google Patents

Novel heptatrienoic acid substitued bicyclic ketone derivative and pharmaceutical compositions comprising the same Download PDF

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Publication number
WO2005003076A1
WO2005003076A1 PCT/KR2003/001331 KR0301331W WO2005003076A1 WO 2005003076 A1 WO2005003076 A1 WO 2005003076A1 KR 0301331 W KR0301331 W KR 0301331W WO 2005003076 A1 WO2005003076 A1 WO 2005003076A1
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angiogenesis
ketone derivative
novel
bicyclic ketone
pharmaceutical composition
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French (fr)
Inventor
Ho Jeong Kwon
Hye-Jin Jung
Jong Heon Shin
Chang Jin Kim
Jung-Rae Rho
Hyang-Burm Lee
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Industry Academy Cooperation Foundation of Sejong University
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Industry Academy Cooperation Foundation of Sejong University
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Priority to AU2003304290A priority patent/AU2003304290A1/en
Publication of WO2005003076A1 publication Critical patent/WO2005003076A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/40—Unsaturated compounds
    • C07C59/76—Unsaturated compounds containing keto groups
    • C07C59/80—Unsaturated compounds containing keto groups containing rings other than six-membered aromatic rings
    • C07C59/82—Unsaturated compounds containing keto groups containing rings other than six-membered aromatic rings the keto group being part of a ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/04—One of the condensed rings being a six-membered aromatic ring
    • C07C2602/10—One of the condensed rings being a six-membered aromatic ring the other ring being six-membered, e.g. tetraline

Definitions

  • the present invention relates to novel heptatrienoic acid substituted bicyclic ketone derivative and pharmaceutical composition comprising it, in particular, to novel heptatrienoic acid substituted bicyclic ketone derivative exhibiting anti-angiogenic activity and pharmaceutical composition for preventing or treating diseases associated with unregulated angiogenesis .
  • Angiogenesis the growth of new blood vessels, is essential for a number of physiological processes such as embryonic development, wound healing, and tissue or organ regeneration (Iwaguchi, T. 1993. Angiogenesis and its regulation. Gan To Kagaku Ryoho 20:1-9.; Kuwano, . 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 . Fibrinolysis 1:703-705.).
  • angiogenesis drives angiogenic diseases such as rheumatoid arthritis, diabetic retinopathy, solid tumor, hemangioma and psoriasis (Andre, T., et al . , 1998. Tumoral angiogenesis: physiopathology, prognostic value and therapeutic perspectives. Rev. Med. Interne. 19:904-9134; Battegay, E. J. 1995. Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects. J. Mol . Med. 73: 333-346; Carmeliet, P. and R. K. Jain. 2000. Angiogenesis in cancer and other diseases. Nature 407:249-257; & Fidler, I. J.
  • angiogenesis inhibitors which are natural or synthetic, 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.
  • SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-l/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types.
  • Flk-l/KDR vascular endothelial growth factor receptor
  • antiangiogenic molecules function in multiple ways, including the inhibition of endothelial cell proliferation, migration, protease activity, and tubule formation, as well as the induction of apoptosis (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).
  • marimastat, Neovastat, AG-3340 are synthetic inhibitors of cell invasion (Jia, M. C. 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) , Vitaxin inhibits cell adhesion, TNP-470, thalidomide, 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, J.
  • Angiogenesis inhibitor TNP-470 during bone marrow transplant safety in a preclinical model. Clin . Cancer Res . 7:1026-1032
  • interferon-alpha, suramin and its analogues interfere with angiogenic growth factors, SU6668, SU5416 do their receptors (Ingber, D. et al . , 1990. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature 348:555-557)
  • endostatin, interleukin-12 are endogenous inhibitors of angiogenesis (Boehm, T. et al . , 1997. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.
  • U.S. Pat. No. 5,908,930 discloses the compounds to inhibit tyrosine kinase and antiangiogenic pharmaceutical composition comprising it.
  • U.S. Pat. No. 5,846,562 discloses a pharmaceutical composition comprising fumagillol derivative as active ingredient effective in treating diseases associated with angiogenesis.
  • U.S. Pat. No. 5,994,388 describes an antiangiogenic pharmaceutical composition comprising as active ingredient cytochalasin and isoindolinone derivatives and U.S. Pat. No.
  • Korean patent application laid-open No. 2001-98524 suggests that wondonine A extracted from the Polifera may inhibit angiogenesis.
  • Korean patent application laid-open No. 2000-5903 describes that novel peptide salmosin obtained from toxin of Agkistrodon halys brevicaudus 'may inhibit angiogenesis and thus used as anticancer agent and
  • Korean patent application laid- open No. 2001-53018 discloses a protein capable of inhibiting angiogenesis .
  • Fig. 1 is a photograph to show inhibitory effect of culture extract from Embellisia chlamydospora on the tube formation of BAECs (bovine aortic endothelial cells): (A) Control, (B) bFGF alone and (C) bFGF plus culture extract from E. chlamydospora (20 ⁇ g/ml ) .
  • BAECs bovine aortic endothelial cells
  • Fig. 2 is a photograph to represent cell morphology of E. chlamydospora producing new compound of this invention: (A) Slightly different shapes of conidia (arrow represent a germinated cell) , (B) Scars (arrows) on conidiophores , (C) Hyphal coil on artificial medium (PDA) , (D) Early mycelial growth, ( ⁇ ) Aggregated and thick mycelial mass, and (F) Dematiaceous mycelia and dark brown chlamydospores of Embellisia chlamydospora on an artificial medium, PDA.
  • Fig. 3 is a schematic diagram for the purification of antiangiogenic compound from E. chlamydospora .
  • Fig. 4 is a graph to represent the effect of bicyclic ketone derivative of this invention on the growth of BAECs, normal (CHANG) and cancer (HT29, HepG2, C8161, HT1080, HeLa) cell lines.
  • Fig. 5 is a photograph to show inhibitory effect of bicyclic ketone derivative of this invention on the tube formation of BAECs .
  • Fig. 6 represents microscopic observation to show inhibitory activity of bicyclic ketone derivative of this invention on the invasion of BAECs.
  • Fig. 7 is a graph to show inhibitory activity of bicyclic ketone derivative on the invasion of BAECs.
  • the present compound may be isolated from fungus, Embellisia chlamydospora and chemically synthesized.
  • a wide variety of conventional methods may be applied to isolate and purify the present compound from Embellisia chlamydospora .
  • the isolation and purification of the present derivative is exemplified in Example 3.
  • the present compound represented by the formula (I) has several chiral centers and therefore, it will be appreciated by one skilled in the art that its all optical isomers will be included within the scope of the present compound.
  • the present compound isolated from the natural source is generally a specific optical isomer having optical activity. In contrast, when synthesized chemically, the racemates are generally produced. Therefore, the racemates will be included within the scope of the present compound.
  • the present compound exhibits potently anti-angiogeic activity and therefore, it would be very successful in treating or preventing diseases or disorders associated with unregulated angiogenesis .
  • a pharmaceutical composition for treating or preventing diseases associated with unregulated angiogenesis which comprises: (a) a pharmaceutically effective amount of heptatrienoic acid substituted bicyclic ketone derivative of the formula (I) ; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises the derivative of the formula (I) as active ingredient, and therefore, the common descriptions of them are abbreviated in order to avoid the complexity of this specification leading to undue multiplicity.
  • the pharmaceutical composition is capable of treating or preventing diseases associated with unregulated angiogenesis through effective inhibition of angiogenesis.
  • the pharmaceutical composition inhibits the proliferation and differentiation of endothelial cells in angiogenesis as demonstrated Examples below.
  • the disease associated with unregulated angiogenesis which may be treated or prevented with the present composition, is rheumatoid arthritis, diabetic retinopathy, cancer, hemangioma or psoriasis. More preferably, the disease associated with unregulated angiogenesis is cancer.
  • the pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, macrocrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, stearic acid, magnesium and mineral oil, but not limited to.
  • the pharmaceutical compositions of this invention further may contain wetting agent, sweetening agent, emulsifying agent, suspending agent, preservatives, flavors, perfumes, lubricating agent, or mixtures of these substances.
  • the pharmaceutical composition of this invention may be administered orally or parenterally.
  • the correct dosage of the pharmaceutical compositions of this invention will vary according to the particular formulation, the mode of application, age, body weight and sex of the patient, diet, time of administration, condition of the patient, drug combinations, reaction sensitivities and severity of the disease. It is understood that the ordinary skilled physician will readily be able to determine and prescribe a correct dosage of this pharmaceutical compositions.
  • An exemplary daily dosage unit for human host comprises an amount of from about 0.001 mg/kg to about 100 mg/kg.
  • the pharmaceutical compositions of this invention can be formulated with pharmaceutical acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dosage form.
  • Non-limiting examples of the formulations include, but not limited to, a solution, a suspension or an emulsion, an extract, an elixir, a powder, a granule, a tablet, a capsule, a liniment, a lotion and an ointment .
  • DMEM fetal calf serum
  • RPMI1640 purchased from Life Technology (Grand
  • Matrigel and transwell plate were from Collaborative Biomedical Products (Bedford, MA) and Corning Costar (Cambridge, MA) , respectively.
  • Gelatin type B was purchased from Sigma (St. Louis, MO) .
  • Example 1-1 Preparation of culture extracts from microbial library
  • the microorganisms (fungi) used in this screening were isolated from soil of various regions in the world. 100 ⁇ & of diluted solutions were spread on PDA or L-O medium and incubated at 28°C for 7 days. Then, each colony was transferred on malt extract agar (MEA) medium and cultivated at 28°C for 7 days.
  • MEA malt extract agar
  • the isolate was inoculated into a 250 ml baffle flask containing 25 ml of the YpSs (glucose (20 g/L), yeast extract (2 g/L), peptone (5 g/L), MgS0 4 • 7H 2 O(0.5 g/L), KH 2 P0 4 (1 g/L, pH 5.6-5.8) culture medium and cultured at 28°C for 7 days on a rotary shaker at 145 rpm. Then, culture broth was extracted with the same volume of acetone.
  • YpSs glucose (20 g/L)
  • yeast extract (2 g/L)
  • peptone 5 g/L
  • KH 2 P0 4 (1 g/L, pH 5.6-5.8
  • Example 1-2 Screening of anti-angiogenic compound-producing microorganism (tube formation assay) Several microbial culture extracts were tested to screen the active compound producer for anti-angiogenesis . Tube formation assay was applied for this purpose. 150 ⁇ i of Matrigel (10 mg/ml) was coated in a 48 well culture plates and polymerized for 2 h at 37°C. The BAECs (bovine aortic endothelial cells, ATCC, 1 x 10 5 cells) were seeded on the surface of the Matrigel and treated with bFGF (basic fibroblast growth factor, 30 ng/ml) . Then, the microbial culture extracts (20 ⁇ g/v ) was added and incubated for 6-18 h.
  • BAECs bovine aortic endothelial cells, ATCC, 1 x 10 5 cells
  • Example 1-3 Taxonomic studies of strain E. chlamydospora Biochemical and physiological characteristics of the strain were examined according to the "Bergey' s Manual of Determinative Bacteriology" and the physiological characteristics including utilization of carbon sources were examined by following the method of Shirling and Gottlieb (Kwon, H. J. 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 E. chlamydospora was observed under light microscope as shown in Fig. 2.
  • Example 2-1 Cultivation of Embellisia chlamydospora
  • MEA medium plate glucose 20 g/L, malt extract 20 g/L, peptone 1 g/L, agar 18 g/L
  • agar pieces of the stock plate were cut under sterile condition and inoculated into a 1 L conical flask containing wheat bran medium (wheat bran 50 g /50 ml) .
  • Seed incubated for 3 days at 28°C was transferred 5-6 spoons per conical flask containing wheat bran medium (wheat bran 100 g /100 ml) for large-scale of cultivation and total 10 flasks were incubated at 28°C for 7 days .
  • Example 2-2 Cell lines and culture conditions BAECs and HT-29 colon cancer cells were grown in MEM, RPMI with 10% fetal bovine serum (FBS) , respectively.
  • FBS fetal bovine serum
  • CHANG-liver normal, HepG2 human hepatoma, HT1080 fibrosarcoma, C8161 melanoma, and HeLa cervical carcinoma cells were grown in DMEM with 10% FBS, 5.5 ml penicillin/streptomycin, and NaHC0 3 at 37°C under 5 % C0 2 , 95% air.
  • EXAMPLE 3 Isolation and Purification of Anti-angiogenic Compound from Embellisia chlamydospora
  • the purification process of active compound from E. chlamydospora is shown in Figure 3.
  • Grown mycelium (1 kg) in wheat bran medium was extracted with 5 volumes of acetone for 24 h and filtered with filter papers (Whatman, 3 mm) .
  • the filtrate was concentrated in vacuo and extracted with the same volume of ethyl acetate.
  • the ethyl acetate extract was concentrated in vacuo and then applied on a silica gel (Merck silica gel 60) column chromatography (6 x 17 cm) which was prepared with CHC1 3 .
  • Active fractions eluted with 9:1 in CHCl 3 :MeOH, were collected, evaporated and applied on preparative thin layer chromatography (silica gel 60 F254) using a solvent system of CHC1 3 : MeOH (8:2) .
  • the active compound was purified on HPLC by C18 reversed-phase column (Shiseido Co., Japan, semi-preparative UG 120 D, 10 mm x 250 mm) eluted with 70% acetonitrile in 0.1% TFA.
  • the active compound was obtained as yellow oil and purity of the compound was confirmed by HPLC.
  • the molecular formula of active compound was C 25 H 32 0 4 on the basis of high-resolution mass data (MW 396.52) .
  • EXAMPLE 4 Determination of Chemical Structure of Antiangiogenic Compound NMR spectra were recorded in CD 3 OD solutions at 500 MHz for 1 H and 125 MHz for 13 C, respectively, on a Varian Unity 500 spectrometer. All of the chemical shifts were recorded in ⁇ (ppm) based on MeOH (3.30/49.50 ppm) using TMS as the internal standard. Mass spectra were obtained by a Jeol JMS-HX 110 high- resolution mass spectrometer. All solvents used were either of spectral grade or redistilled from glass prior to use. The NMR data of purified anti-angiogenic compound are listed in Table 1 : TABLE 1
  • purified anti-angiogenic compound was identified as a novel heptatrienoic acid substituted bicyclic ketone derivative of the formula I and named as "embellistatin” ,
  • Example 5-1 Effect of embellistatin on the growth of various cell lines
  • the present inventors investigated the effect of embellistatin on the growth of various cell lines including bovine aortic endothelial cells (BAECs) using MTT (3- (4,5- dimethylthiazol-2-yl) -2 , 5- diphenyl tetrazolium bromide) colorimetric assay.
  • BAECs bovine aortic endothelial cells
  • MTT 3- (4,5- dimethylthiazol-2-yl) -2 , 5- diphenyl tetrazolium bromide
  • Cells were inoculated at a density of 5 x 10 3 cells per well in 96-well culture plates and incubated for 24 h for stabilization.
  • Various concentrations of embellistatin were added to each well and incubated for 3 days and performed MTT assay. 50 ⁇ i of MTT (2 mg/ml stock solution, Sigma, St.
  • embellistatin inhibited the proliferation of each cell lines with a different growth inhibitory spectrum.
  • embellistatin potently inhibits the growth of BAECs and cancer (HT29, HepG2, C8161, HT1080, HeLa) cell lines than that of normal (CHANG) cell line.
  • Example 5-2 Effect of embellistatin on the tube formation of endothelial cells
  • Vascular endothelial cells undergo rapid in vi tro differentiation into capillary like structures, providing a simple assay (described in Example 1-2) for assessing impact of agents on endothelial differentiation process which requires cell-matrix interaction, intercellular communication as well as cell motility.
  • BAECs cultured on Matrigel layers form normally incomplete and narrow tube-like structures in the absence of angiogenic factors but the capillary network formation is further stimulated by the treatment of angiogenic factor such as bFGF resulting in elongated and robust tube-like structures which are organized by much larger number of cells compared to those of the control (Fig. 5A, B) .
  • angiogenic factor such as bFGF
  • Example 5-3 Effect of embellistatin on the invasion of BAECs
  • migrating endothelial cells must break and traverse their own basement membrane to form new blood vessels and bFGF can stimulate this endothelial cell invasion. Therefore, the present inventors investigated the effect of embellistatin on endothelial cell invasion in vitro using a transwell chamber system with polycarbonate filter inserts that were coated with Matrigel preventing the migration of non-invasive cells.
  • the lower side of the filter was coated with 10 ⁇ i gelatin (1 ⁇ g/ ⁇ A) and the upper side was coated with 10 ⁇ A Matrigel.
  • bFGF (30 ng/ml) , BSA, and embellistatin were added in the lower wells placed in 600 ⁇ A MEM.
  • the BAECs (1 x 10 5 cells) were placed in the upper part of the filter and the chamber was then incubated at 37°C for 18 h.
  • the cells were fixed with 70% methanol and stained with hematoxylin/eosin.
  • the cell invasion was determined by counting the whole cell numbers in a single filter using optical microscope and photographed at a 40x magnification by the ImagePro Plus software . The experiment was repeated twice independently.
  • Figures 6 and 7 show that embellistatin (5 jKg/ml) significantly inhibits bFGF-stimulated invasiveness of BAECs. This data demonstrate that embellistatin inhibits the angiogenesis of endothelial cells in vi tro.

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Abstract

The present invention relates to novel heptatrienoic acid substituted bicyclic ketone derivative and pharmaceutical composition comprising it, in particular, to novel heptatrienoic acid substituted bicyclic ketone derivative exhibiting anti-angiogenic activity and pharmaceutical composition for preventing or treating diseases associated with unregulated angiogenesis. (I)

Description

NOVEL HEPTATRIENOIC ACID SUBSTITUTED BICYCLIC KETONE DERIVATIVE AND PHARMACEUTICAL COMPOSITIONS COMPRISING THE SAME
BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to novel heptatrienoic acid substituted bicyclic ketone derivative and pharmaceutical composition comprising it, in particular, to novel heptatrienoic acid substituted bicyclic ketone derivative exhibiting anti-angiogenic activity and pharmaceutical composition for preventing or treating diseases associated with unregulated angiogenesis .
DESCRIPTION OF THE RELATED ART
Angiogenesis, the growth of new blood vessels, is essential for a number of physiological processes such as embryonic development, wound healing, and tissue or organ regeneration (Iwaguchi, T. 1993. Angiogenesis and its regulation. Gan To Kagaku Ryoho 20:1-9.; Kuwano, . 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 . Fibrinolysis 1:703-705.). However, persistent unregulated angiogenesis drives angiogenic diseases such as rheumatoid arthritis, diabetic retinopathy, solid tumor, hemangioma and psoriasis (Andre, T., et al . , 1998. Tumoral angiogenesis: physiopathology, prognostic value and therapeutic perspectives. Rev. Med. Interne. 19:904-9134; Battegay, E. J. 1995. Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects. J. Mol . Med. 73: 333-346; Carmeliet, P. and R. K. Jain. 2000. Angiogenesis in cancer and other diseases. Nature 407:249-257; & Fidler, I. J. 2000. Angiogenesis and cancer metastasis. Cancer J. Sci . Am. 2:134-141) . The process is consisted of multi-steps such as stimulation of endothelial growth by tumor cytokines, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) , degradation of extracellular matrix proteins by metalloproteinases, migration of endothelial cells mediated by cell membrane adhesion molecules, endothelial cell proliferation, and tube formation (Bussolino, F. et al . , 1997. Molecular mechanisms of blood vessel formation. Trends Biochem. Sci . 22:251-256; Kuwano, . et al . , 2001. Angiogenesis factors. Intern. Med. 40:565-572; & Risau, . 1994. Angiogenesis and endothelial cell function. Arzneimi ttelforschung 44:416-417). Therefore, inhibition of these processes is emerging as a promising new strategy for the treatment of cancer and other human diseases related with angiogenesis. A new diverse class of angiogenesis inhibitors has been developed for this purpose. The inhibitors, which are natural or synthetic, 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-l/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res . 59:99-106; & Kwon, H. J. et al . , 2001. Antiangiogenic activity of acalycixenolide E, a novel marine natural product from Acalycigorgia inermis . J. Microbiol . Biotechnol . 11:656-662) . These antiangiogenic molecules function in multiple ways, including the inhibition of endothelial cell proliferation, migration, protease activity, and tubule formation, as well as the induction of apoptosis (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). The antiangiogenic function of many of these molecules is well documented in vitro and in vivo, and some are currently being tested in clinical trials (Deplanque, G. and A. L. Harris. 2000. Anti-angiogenic agents: clinical trial design and therapies in development. Bur. J". Cancer 36:1713-1724; Liekens, S. E. D. Clercq, and J. Neyts . 2001. Angiogenesis: regulators and clinical applications. Biochem. Pharmacol . 61:253-270; & Mross, K. 2000. Anti-angiogenesis therapy: concepts and importance of dosing schedules in clinical trials. Drug Resist. Updat . 3 : 223-235) . For example, marimastat, Neovastat, AG-3340 are synthetic inhibitors of cell invasion (Jia, M. C. 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) , Vitaxin inhibits cell adhesion, TNP-470, thalidomide, 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, J. . 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 its analogues interfere with angiogenic growth factors, SU6668, SU5416 do their receptors (Ingber, D. et al . , 1990. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature 348:555-557), and endostatin, interleukin-12 are endogenous inhibitors of angiogenesis (Boehm, T. et al . , 1997. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277-285) that are in clinical trials against a variety of solid tumors . Among the compounds and peptides described previously, the substances that have been researched clinically are predominantly synthetic ones. U.S. Pat. No. 5,908,930 discloses the compounds to inhibit tyrosine kinase and antiangiogenic pharmaceutical composition comprising it. U.S. Pat. No. 5,846,562 discloses a pharmaceutical composition comprising fumagillol derivative as active ingredient effective in treating diseases associated with angiogenesis. U.S. Pat. No. 5,994,388 describes an antiangiogenic pharmaceutical composition comprising as active ingredient cytochalasin and isoindolinone derivatives and U.S. Pat. No. 6,228,879 discloses novel compound EM-138 prevent angiogenesis and the occurrence of angiogenic diseases . Korean patent application laid-open No. 2001-98524 suggests that wondonine A extracted from the Polifera may inhibit angiogenesis. Besides synthetic compounds, several peptides have been investigated clinically. Korean patent application laid-open No. 2000-5903 describes that novel peptide salmosin obtained from toxin of Agkistrodon halys brevicaudus 'may inhibit angiogenesis and thus used as anticancer agent and Korean patent application laid- open No. 2001-53018 discloses a protein capable of inhibiting angiogenesis . Although several chemicals and peptides have been developed as antiangiogenic agents as described above, novel antiangiogenic chemicals with unrelated structure from known inhibitors are valuable tools for chemical genetics approach of angiogenesis study as well as development of new antiangiogenic therapeutic drugs . Based on this idea, the present inventors have screened low molecular weight compounds with anti-angiogenic activity from microbial metabolites. As a result, the present inventors have found a new compound from the culture extract of Embellisia chlamydospora, as a potent anti-angiogenic compound. Throughout this application, various publications and patents are referenced and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains .
SUMMARY OF THE INVENTION Accordingly, it is an object of this invention to provide novel heptatrienoic acid substituted bicyclic ketone derivatives . It is another object of this invention to provide a pharmaceutical composition for treating or preventing diseases associated with unregulated angiogenesis.
Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjugation with the appended claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a photograph to show inhibitory effect of culture extract from Embellisia chlamydospora on the tube formation of BAECs (bovine aortic endothelial cells): (A) Control, (B) bFGF alone and (C) bFGF plus culture extract from E. chlamydospora (20 μg/ml ) .
Fig. 2 is a photograph to represent cell morphology of E. chlamydospora producing new compound of this invention: (A) Slightly different shapes of conidia (arrow represent a germinated cell) , (B) Scars (arrows) on conidiophores , (C) Hyphal coil on artificial medium (PDA) , (D) Early mycelial growth, (Ξ) Aggregated and thick mycelial mass, and (F) Dematiaceous mycelia and dark brown chlamydospores of Embellisia chlamydospora on an artificial medium, PDA. Fig. 3 is a schematic diagram for the purification of antiangiogenic compound from E. chlamydospora . Fig. 4 is a graph to represent the effect of bicyclic ketone derivative of this invention on the growth of BAECs, normal (CHANG) and cancer (HT29, HepG2, C8161, HT1080, HeLa) cell lines.
Fig. 5 is a photograph to show inhibitory effect of bicyclic ketone derivative of this invention on the tube formation of BAECs . Fig. 6 represents microscopic observation to show inhibitory activity of bicyclic ketone derivative of this invention on the invasion of BAECs. Fig. 7 is a graph to show inhibitory activity of bicyclic ketone derivative on the invasion of BAECs.
DETAILED DESCRIPTION OF THIS INVETNION
In one aspect of this invention, there is provided a heptatrienoic acid substituted bicyclic ketone derivative represented by the following formula (I) :
Figure imgf000009_0001
The present compound may be isolated from fungus, Embellisia chlamydospora and chemically synthesized. A wide variety of conventional methods may be applied to isolate and purify the present compound from Embellisia chlamydospora . The isolation and purification of the present derivative is exemplified in Example 3. The present compound represented by the formula (I) has several chiral centers and therefore, it will be appreciated by one skilled in the art that its all optical isomers will be included within the scope of the present compound. The present compound isolated from the natural source is generally a specific optical isomer having optical activity. In contrast, when synthesized chemically, the racemates are generally produced. Therefore, the racemates will be included within the scope of the present compound. The present compound exhibits potently anti-angiogeic activity and therefore, it would be very successful in treating or preventing diseases or disorders associated with unregulated angiogenesis .
In another aspect of this invention, there is provided a pharmaceutical composition for treating or preventing diseases associated with unregulated angiogenesis, which comprises: (a) a pharmaceutically effective amount of heptatrienoic acid substituted bicyclic ketone derivative of the formula (I) ; and (b) a pharmaceutically acceptable carrier.
Figure imgf000010_0001
The pharmaceutical composition comprises the derivative of the formula (I) as active ingredient, and therefore, the common descriptions of them are abbreviated in order to avoid the complexity of this specification leading to undue multiplicity. The pharmaceutical composition is capable of treating or preventing diseases associated with unregulated angiogenesis through effective inhibition of angiogenesis. In particular, the pharmaceutical composition inhibits the proliferation and differentiation of endothelial cells in angiogenesis as demonstrated Examples below. According to a preferred embodiment, the disease associated with unregulated angiogenesis, which may be treated or prevented with the present composition, is rheumatoid arthritis, diabetic retinopathy, cancer, hemangioma or psoriasis. More preferably, the disease associated with unregulated angiogenesis is cancer. In the pharmaceutical compositions of this invention, the pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, macrocrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, stearic acid, magnesium and mineral oil, but not limited to. The pharmaceutical compositions of this invention, further may contain wetting agent, sweetening agent, emulsifying agent, suspending agent, preservatives, flavors, perfumes, lubricating agent, or mixtures of these substances. The pharmaceutical composition of this invention may be administered orally or parenterally. The correct dosage of the pharmaceutical compositions of this invention will vary according to the particular formulation, the mode of application, age, body weight and sex of the patient, diet, time of administration, condition of the patient, drug combinations, reaction sensitivities and severity of the disease. It is understood that the ordinary skilled physician will readily be able to determine and prescribe a correct dosage of this pharmaceutical compositions. An exemplary daily dosage unit for human host comprises an amount of from about 0.001 mg/kg to about 100 mg/kg. According to the conventional techniques known to those skilled in the art, the pharmaceutical compositions of this invention can be formulated with pharmaceutical acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dosage form. Non-limiting examples of the formulations include, but not limited to, a solution, a suspension or an emulsion, an extract, an elixir, a powder, a granule, a tablet, a capsule, a liniment, a lotion and an ointment .
The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims .
EXAMPLES Materials The materials used in Examples hereunder were commercially available : Silica gel was from Merck Co. (Germany) and all solvents were analytical or first grade. Basic fibroblast growth factor (bFGF) was from Upstate Biotechnology (Lake Placid, NY) . MEM,
DMEM, and RPMI1640 were purchased from Life Technology (Grand
Island, NY) . Matrigel and transwell plate were from Collaborative Biomedical Products (Bedford, MA) and Corning Costar (Cambridge, MA) , respectively. Gelatin type B was purchased from Sigma (St. Louis, MO) .
EXAMPLE 1: Screening of Anti-angiogenic Compound-Producing Microorganism
Example 1-1: Preparation of culture extracts from microbial library The microorganisms (fungi) used in this screening were isolated from soil of various regions in the world. 100 μ& of diluted solutions were spread on PDA or L-O medium and incubated at 28°C for 7 days. Then, each colony was transferred on malt extract agar (MEA) medium and cultivated at 28°C for 7 days. The isolate was inoculated into a 250 ml baffle flask containing 25 ml of the YpSs (glucose (20 g/L), yeast extract (2 g/L), peptone (5 g/L), MgS04 • 7H2O(0.5 g/L), KH2P04(1 g/L, pH 5.6-5.8) culture medium and cultured at 28°C for 7 days on a rotary shaker at 145 rpm. Then, culture broth was extracted with the same volume of acetone.
Example 1-2: Screening of anti-angiogenic compound-producing microorganism (tube formation assay) Several microbial culture extracts were tested to screen the active compound producer for anti-angiogenesis . Tube formation assay was applied for this purpose. 150 μi of Matrigel (10 mg/ml) was coated in a 48 well culture plates and polymerized for 2 h at 37°C. The BAECs (bovine aortic endothelial cells, ATCC, 1 x 105 cells) were seeded on the surface of the Matrigel and treated with bFGF (basic fibroblast growth factor, 30 ng/ml) . Then, the microbial culture extracts (20 βg/v ) was added and incubated for 6-18 h. The morphological changes of the cells were observed under microscope and photographed at a 40x magnification by the ImagePro Plus software (Media Cybernetics, Inc.). Cytotoxicity of tube-forming endothelial cells was evaluated by trypan blue staining. The experiment was repeated twice independently. As a result, culture extract from fungus Embellisia chlamydospora potently inhibited the tube formation of endothelial cells induced by bFGF (Fig. 1) .
Example 1-3: Taxonomic studies of strain E. chlamydospora Biochemical and physiological characteristics of the strain were examined according to the "Bergey' s Manual of Determinative Bacteriology" and the physiological characteristics including utilization of carbon sources were examined by following the method of Shirling and Gottlieb (Kwon, H. J. 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 E. chlamydospora was observed under light microscope as shown in Fig. 2.
EXAMPLE 2 : Organism and Culture Condition
Example 2-1: Cultivation of Embellisia chlamydospora The stock culture of Embellisia chlamydospora was maintained on MEA medium plate (glucose 20 g/L, malt extract 20 g/L, peptone 1 g/L, agar 18 g/L) at 28°C. For seed cultivation, agar pieces of the stock plate were cut under sterile condition and inoculated into a 1 L conical flask containing wheat bran medium (wheat bran 50 g /50 ml) . Seed incubated for 3 days at 28°C was transferred 5-6 spoons per conical flask containing wheat bran medium (wheat bran 100 g /100 ml) for large-scale of cultivation and total 10 flasks were incubated at 28°C for 7 days .
Example 2-2: Cell lines and culture conditions BAECs and HT-29 colon cancer cells were grown in MEM, RPMI with 10% fetal bovine serum (FBS) , respectively. CHANG-liver normal, HepG2 human hepatoma, HT1080 fibrosarcoma, C8161 melanoma, and HeLa cervical carcinoma cells were grown in DMEM with 10% FBS, 5.5 ml penicillin/streptomycin, and NaHC03 at 37°C under 5 % C02, 95% air.
EXAMPLE 3: Isolation and Purification of Anti-angiogenic Compound from Embellisia chlamydospora The purification process of active compound from E. chlamydospora is shown in Figure 3. Grown mycelium (1 kg) in wheat bran medium was extracted with 5 volumes of acetone for 24 h and filtered with filter papers (Whatman, 3 mm) . The filtrate was concentrated in vacuo and extracted with the same volume of ethyl acetate. The ethyl acetate extract was concentrated in vacuo and then applied on a silica gel (Merck silica gel 60) column chromatography (6 x 17 cm) which was prepared with CHC13. Active fractions, eluted with 9:1 in CHCl3:MeOH, were collected, evaporated and applied on preparative thin layer chromatography (silica gel 60 F254) using a solvent system of CHC13 : MeOH (8:2) . The active compound was purified on HPLC by C18 reversed-phase column (Shiseido Co., Japan, semi-preparative UG 120 D, 10 mm x 250 mm) eluted with 70% acetonitrile in 0.1% TFA. The active compound was obtained as yellow oil and purity of the compound was confirmed by HPLC. The molecular formula of active compound was C25H3204 on the basis of high-resolution mass data (MW 396.52) .
EXAMPLE 4: Determination of Chemical Structure of Antiangiogenic Compound NMR spectra were recorded in CD3OD solutions at 500 MHz for 1H and 125 MHz for 13C, respectively, on a Varian Unity 500 spectrometer. All of the chemical shifts were recorded in δ (ppm) based on MeOH (3.30/49.50 ppm) using TMS as the internal standard. Mass spectra were obtained by a Jeol JMS-HX 110 high- resolution mass spectrometer. All solvents used were either of spectral grade or redistilled from glass prior to use. The NMR data of purified anti-angiogenic compound are listed in Table 1 : TABLE 1
Figure imgf000017_0001
Physicochemical properties of purified anti-angiogenic compound are shown in Table 2 : TABLE 2
Figure imgf000018_0001
Therefore, purified anti-angiogenic compound was identified as a novel heptatrienoic acid substituted bicyclic ketone derivative of the formula I and named as "embellistatin" ,
EXAMPLE 5: Anti-Angiogenic Activity of Embellistatin
Example 5-1 : Effect of embellistatin on the growth of various cell lines The present inventors investigated the effect of embellistatin on the growth of various cell lines including bovine aortic endothelial cells (BAECs) using MTT (3- (4,5- dimethylthiazol-2-yl) -2 , 5- diphenyl tetrazolium bromide) colorimetric assay. Cells were inoculated at a density of 5 x 103 cells per well in 96-well culture plates and incubated for 24 h for stabilization. Various concentrations of embellistatin were added to each well and incubated for 3 days and performed MTT assay. 50 μi of MTT (2 mg/ml stock solution, Sigma, St. Louis, MO) was added and the plate was incubated for an additional 4 h. After removal of medium, 100 μA of DMSO was added. The plate was read at 540 nm by universal microplate reader (Bio-Tek Instruments, Inc., Winooski, VT) . As shown in Fig. 4, embellistatin inhibited the proliferation of each cell lines with a different growth inhibitory spectrum. Notably, embellistatin potently inhibits the growth of BAECs and cancer (HT29, HepG2, C8161, HT1080, HeLa) cell lines than that of normal (CHANG) cell line. These data suggest that embellistatin could exhibit the anti-angiogenic activity by inhibition of endothelial cell growth. The viability of endothelial cells was not affected up to 5 g/ml of embellistatin treatment, implying that the growth inhibitory activity of embellistatin shown in Fig. 4 is not due to mere cytotoxicity of the compound.
Example 5-2: Effect of embellistatin on the tube formation of endothelial cells Vascular endothelial cells undergo rapid in vi tro differentiation into capillary like structures, providing a simple assay (described in Example 1-2) for assessing impact of agents on endothelial differentiation process which requires cell-matrix interaction, intercellular communication as well as cell motility. BAECs cultured on Matrigel layers form normally incomplete and narrow tube-like structures in the absence of angiogenic factors but the capillary network formation is further stimulated by the treatment of angiogenic factor such as bFGF resulting in elongated and robust tube-like structures which are organized by much larger number of cells compared to those of the control (Fig. 5A, B) . To examine the effect of embellistatin on this process, BAECs stimulated by bFGF were treated with embellistatin. Figure 5C shows that embellistatin efficiently inhibits the tube formation induced by bFGF. These cells are not stained by trypan blue, implying that the inhibition of tube formation by embellistatin is not due to merely cytotoxicity on the cells.
Example 5-3: Effect of embellistatin on the invasion of BAECs As another important property of angiogenesis, migrating endothelial cells must break and traverse their own basement membrane to form new blood vessels and bFGF can stimulate this endothelial cell invasion. Therefore, the present inventors investigated the effect of embellistatin on endothelial cell invasion in vitro using a transwell chamber system with polycarbonate filter inserts that were coated with Matrigel preventing the migration of non-invasive cells. The lower side of the filter was coated with 10 μi gelatin (1 μg/ μA) and the upper side was coated with 10 μA Matrigel. bFGF (30 ng/ml) , BSA, and embellistatin were added in the lower wells placed in 600 μA MEM. The BAECs (1 x 105 cells) were placed in the upper part of the filter and the chamber was then incubated at 37°C for 18 h. The cells were fixed with 70% methanol and stained with hematoxylin/eosin. The cell invasion was determined by counting the whole cell numbers in a single filter using optical microscope and photographed at a 40x magnification by the ImagePro Plus software . The experiment was repeated twice independently. Figures 6 and 7 show that embellistatin (5 jKg/ml) significantly inhibits bFGF-stimulated invasiveness of BAECs. This data demonstrate that embellistatin inhibits the angiogenesis of endothelial cells in vi tro.

Claims

What is claimed is:
1. A heptatrienoic acid substituted bicyclic ketone derivative represented by the following formula (I) :
Figure imgf000022_0001
2. A pharmaceutical composition for treating or preventing diseases associated with unregulated angiogenesis, which comprises: (a) a pharmaceutically effective amount of heptatrienoic acid substituted bicyclic ketone derivative of claim 1; and (b) a pharmaceutically acceptable carrier.
3. The pharmaceutical composition according to claim 2, wherein the disease associated with unregulated angiogenesis is selected from the group consisting of rheumatoid arthritis, diabetic retinopathy, cancer, hemangioma and psoriasis.
4. The pharmaceutical composition according to claim 3 , wherein the disease associated with unregulated angiogenesis is cancer.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
JPH09110780A (en) * 1995-10-17 1997-04-28 Takeda Chem Ind Ltd Tan-2342-related compound, its production and use thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09110780A (en) * 1995-10-17 1997-04-28 Takeda Chem Ind Ltd Tan-2342-related compound, its production and use thereof

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