KR101120565B1 - Novel cyclic pentadepsipeptide(I) and its use - Google Patents

Abstract

The present invention relates to a novel cyclic pentapsipeptide. Since the cyclic pentapsipeptide of the present invention has excellent drug resistance inhibitory activity and cancer cell proliferation inhibitory activity, it can be used as a medicine for treating antibiotic-resistant bacteria and treating cancer. have.

Cyclic Depsipeptide, Fusarium, Drug Tolerance, Antibiotic Resistant Bacteria, Cancer Treatment

Description

Novel cyclic pentadepsipeptide (I) and its use}

The present invention relates to medicine. The drug of the present invention is a cyclic pentapsipeptide excellent in drug resistance inhibitory activity and cancer cell proliferation inhibitory activity, which is produced in soil-derived Fusarium microorganisms.

Fusarium fungi associated with marine plants have been found to be a source of production of novel cyclic depsipeptides, such as sansalvamid.

Sansalvamid A was first reported in the production of marine microorganisms, Halodule wrightii [Belofsky GN, Jensen PR, Fenical W. (1999) Sansalvamide: A new cytotoxic cyclic depsipeptide produced by a marine fungus of the genus Fusarium . Tetrahedron Lett . 40, 2913-2916. Sansalvamid A consists of four hydrophobic amino acids (phenylalanine, two leucine and valine) and one hydroxy acid ((S) -2-hydroxy-4-methylpentanoic acid (OLeu), The seae centers are all S-shaped. Sansalvamid A has shown significant proliferation inhibitory effect on 60 cell lines of the US National Cancer Center and has been found to be an inhibitor of Topoisomerase I. The anticancer activity of Sansalvamid A may be at least in part through a mechanism associated with the inhibition of topoisomerase I. In addition, the fact that homologues through N-methylation or para-bromination to sansalvamid A shown in FIG. 1 also shows remarkable cytotoxicity to human pancreatic cancer cells similarly to sansalvamid A is used as an excellent anticancer agent. May be [Ujiki MB, Milam B, Ding XZ, Roginsky AB, Salabat MR, Talamonti MS, Bell RH, Gu W, Silverman RB, Adrian TE. (2006) A novel peptide sansalvamide analogue inhibits pancreatic cancer cell growth through G0 / G1 cell-cycle arrest. Biochem. Bioph. Res. Co. 340, 1224-1228.

Recently, N-methyl acid salbamide, the N-methyl homologue of san salbamide, has been produced from Fusarium species isolated from green algae. N-methyl acid salbamide consists of four amino acids (phenylalanine, leucine, N-methylleucine and valine) and one hydroxy acid (OLeu), and in vitro cytotoxicity in the screening of human cancer cell lines in the US National Cancer Center. [Cueto M, Jensen PR, Fenical W. (2000) N-Methylsansalvamide, a cytotoxic cyclic depsipeptide from a marine fungus of the genus Fusarium Phytochemistry. 55, 223-226.

Multidrug resistance (MDR) is one of the major obstacles to successful cancer treatment with chemotherapeutic agents, and various biochemical, medicinal and clinical trials have recently been devised to overcome it [Teodori E, Dei S. , Scapecchi S, Gualtieri F. (2002) The medicinal chemistry of multidrug resistance (MDR) reversing drugs. II Farmaco 57, 385-415. Although multiple mechanisms are involved in multidrug resistance, overexpression of P-glycoprotein and multidrug resistance-related proteins has been shown to be a cause of multidrug resistance in cancer cells [Thomas H, Coley HM. (2003) Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting P-glycoprotein. Cancer Control 10, 159-165; Perez-Tomas R. (2006) Multidrug resistance: retrospect and prospects in anti-cancer drug treatment. Curr. Med . Chem . 13, 1859-1876.

Sansalvamid A is a lipophilic cyclic dipeptipeptide with protease resistance and cell membrane permeability, which is easier to administer orally than other drugs, and the core structure of 4 amino acids and 1 hydroxy acid is more limited due to limited rotation of the bond. Forming a rigid array has the advantage of excellent affinity and long half-life in vivo.

In order to use these structural advantages and cytotoxicity against cancer cells possessed by Sansalvamid A or N-methyl Acid Salvamid in the treatment of cancer, numerous homologues which have been modified so far have been organically synthesized, but are separated from the present invention. There has been no report on pentapsipeptide.

It is an object of the present invention to provide a novel cyclic pentapsipeptide.

Another object of the present invention to provide a pharmaceutical composition for inhibiting drug resistance.

Another object of the present invention to provide a pharmaceutical composition for treating cancer.

The present invention provides a cyclic pentapsipeptide of Formula 1, which is a novel compound.

 The cyclic depsipeptide of Formula 1 is a novel 15-membered cyclic peptapsipeptide which has a difference in the binding order of constituent amino acids and hydroxy acids with acid salbamide A and N-methyl acid salbamide. It turned out to be a compound. In addition, as shown in FIG. 1, various homologs synthesized by using the core structure of Sansalvamid A may have only increased the cytotoxicity through bromination of the benzene ring of phenylalanine or N-methylation of leucine or valine. In addition, no attempt was made to change the binding order of the constituent amino acids and the hydroxy acid. Furthermore, in order to change the binding order of the constituent amino acids and hydroxy acids through artificial organic synthesis, between hydroxy acid ((S) -2-hydroxy-4-methylpentanoic acid (OLeu) and phenylalanine included in the core structure There is a problem that the ester bond of is broken.

Cyclic pentapsipeptide of formula (1) is a novel cyclic pentadepsipeptide with different binding order of acid salbamide A and N-methyl acid salbamide with four constituent amino acids and one hydroxy acid. Show cytotoxicity equivalent to Sansalvamid A. In particular, the cyclic pentapsipeptide of formula (1) has inhibitory activity of drug resistance of cells not reported in organosynthesized homologues using acid salbamide A, N-methyl acid salbamide and these as basic ring structures. In the present invention, the inhibitory activity of drug resistance means that cells exposed to a chemotherapeutic agent generate cells that are resistant to a number of structurally unrelated drugs. In the case of maintaining, of course, it is also used as an activity of increasing or restoring the sensitivity of the cells already drug-resistant to the drug.

Accordingly, the cyclic pentapsipeptide of the present invention or a pharmaceutically acceptable salt thereof may be particularly suitable for treating cells with drug resistance, inhibiting the expression of drug resistance, and particularly for the treatment of multi-drug resistant cancer. Can be used.

Preferably, the cyclic pentapsipeptide of Formula 1 or a pharmaceutically acceptable salt thereof of the present invention may be used in combination with conventionally known drug resistance inhibitors, ie, cyclosporine and its analogues, phenothiazine, thioxantheres, verapamil and the like. Can be.

Also preferably the cyclic pentapsipeptides of the formula (1) or pharmaceutically acceptable salts thereof of the present invention may be used in the treatment of tumors in combination with anticancer agents, i.e. standard chemotherapeutic agents, particularly preferably innate or It can be used to treat tumors that are drug resistant.

Cyclic pentadepsipeptide of the formula (1) of the present invention is Fusarium Solani ( Fusarium solani) can be produced in KCCM90040 [Accession No .: KCCM10881P]. In order to produce the cyclic pentapsipeptide of Formula 1, solid culture using cereals is preferable.

Since the cyclic pentapsipeptide of the present invention exhibits cytotoxicity to various cancer cells, it may be used as a therapeutic agent for tumors, and furthermore, as a drug resistance inhibitor of cells.

Cyclic depsipeptide-producing strains were isolated from potatoes in Mungyeong, Korea, and the isolated strains were identified as Fusarium solani. The strain was named Fusarium solani KCCM90040, and was deposited internationally in accordance with the Budapest Treaty under the deposit number KCCM10881P on January 15, 2008 to the Korea Center for Microbiological Conservation.

The strain produced a cyclic pentapsipeptide of Formula 1 and a cyclic pentadepsipeptide of Formula 2 below.

The cyclic pentapsipeptides of Chemical Formulas 1 and 2 are novel as 15-cyclic cyclic peptapsipeptides, which differ in the binding order of N-methyl acid salbamide, constituent amino acids, and hydroxy acids. It turned out to be a compound.

The cyclic pentapsipeptide of the present invention or a pharmaceutically acceptable salt thereof may be used as an active ingredient of a pharmaceutical composition for inhibiting drug resistance of cells. The cells may be tumor cells or antibiotic resistant bacteria.

In addition, the cyclic pentapsipeptide of the present invention or a pharmaceutically acceptable salt thereof may be used as an active ingredient of a pharmaceutical composition for treating cancer having tumor cell proliferation inhibitory activity.

Pharmaceutically acceptable salts refer to non-toxic organic or inorganic acid addition salts of base compounds. Examples of inorganic acids which form suitable salts are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and acid metal salts such as sodium orthophosphate and potassium hydrogen sulfate. Examples of organic acids that form suitable salts are mono-, di- and tricarboxylic acids. Examples of such acids include acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, Cinnamic acid, salicylic acid and 2-phenoxybenzoic acid. In addition, organic acids which form suitable salts include sulfonic acids such as methane sulfonic acid and 2-hydroxyethane sulfonic acid. These salts and base compounds may exist in hydrated or near anhydride form. Acid salts can be isolated by dissolving the free base in an aqueous solution or an aqueous alcohol solution or other suitable solvent containing a suitable acid and evaporating the solution, or by reacting the free base in an organic solvent, in which case the salts can be separated directly or Or by concentrating the solution). In general, acid addition salts of the compounds according to the invention are crystalline substances that are soluble in water and various hydrophilic organic solvents, which appear to have higher melting points and solubility than their free base forms.

As used herein, a patient refers to primates, including humans, and mammals such as goats, horses, cattle, pigs, dogs, cats, mice, and mice.

The dosage of the cyclic pentapsipeptide of Formula 1 or a pharmaceutically acceptable salt thereof can vary widely depending on the particular dosage unit used, the duration of treatment, the age and sex of the patient being treated, the nature of the tumor to be treated and the drug resistance. Can change.

The cyclic pentapsipeptides of formula (1) or pharmaceutically acceptable salts thereof are used in combination with other anticancer agents, particularly chemotherapy agents, which are known to be useful in the treatment of tumors. An effective amount of the cyclic pentapsipeptide of Formula 1 or a pharmaceutically acceptable salt thereof for reversing drug resistance will generally range from about 15 mg / kg to 500 mg / kg. The unit dose may contain 25 to 500 mg of the cyclic pentapsipeptide of Formula 1 or a pharmaceutically acceptable salt thereof and may be administered one or more times daily. The cyclic pentapsipeptide of Formula 1 or a pharmaceutically acceptable salt thereof may be administered orally or parenterally with the pharmaceutical carrier in the form of a conventional dosage unit.

In the treatment of tumors using the cyclic pentapsipeptide of Formula 1 or a pharmaceutically acceptable salt thereof, an anticancer agent, particularly a chemotherapeutic agent, is effective at inhibiting the tumor. It should be administered with an acceptable salt.

Tumors that can be treated by the methods of the invention include melanoma, lymphoma, leukemia and sarcomas, as well as benign and malignant tumors or neoplasms. Examples of such tumors include skin tumors (eg malignant melanoma and mycosis), blood tumors such as leukemia (eg acute lymphocytic leukemia, acute or chronic myeloid leukemia), lymphomas (eg Hodgkin's disease or malignant lymphoma) , Gynecological tumors (eg ovarian tumors and uterine tumors), urinary tumors (eg prostate tumors, bladder or testicular tumors), soft tissue sarcomas, osteogenic or non-osteosarcoma, breast tumors, pituitary tumors, thyroid tumors, adrenal cortical tumors, Gastrointestinal tumors (eg, esophageal tumors, gastric tumors, intestinal tumors and colon tumors), pancreatic tumors, liver tumors, laryngeal tumors, papillomas and lung tumors. Of course, tumors that are typically multidrug resistant or multidrug resistant are most effectively treated by the methods of the present invention. Such tumors include colon tumors, lung tumors, gastric tumors and liver tumors.

Chemotherapeutic agents used with the cyclic pentapsipeptide of Formula 1 or a pharmaceutically acceptable salt thereof include cytotoxins commonly used in the treatment of tumors. Examples of chemotherapeutic agents include cyclophosphamide, methotrexate, prednisone, 6-mercaptopurine, procarbazine, daunorubicin, vincristine, vinblastine, chlorambucil, cytosine arabinoside, 6-thio Guanine, thio TEPA, 5-fluorouracil, 5-fluoro-2-deoxywoodyrind, 5-azacytidine, nitrogen mustard, 1,3-bis (2-chloroethyl) -1-nitrosouurea (BCNU), (1- (2-Chloroethyl) -3-cyclohexyl-1-nitrosouurea) (CCNU), Busulfan, Adremycin, Bleomycin, Bindesin, Cychloroycin or Methylglyoxal bix ( Guanyl hydrazone) (ie MGBG). The effective amount of chemotherapeutic agent used in the methods of the present invention will vary and vary widely depending on such factors as the patient, the shape and size of the tumor tissue and the specific chemotherapeutic agent used. The amount is any amount that is effective, and one skilled in the art can easily determine the amount. In general, chemotherapeutic agents will be required in small amounts when administered with a cyclic pentapsipeptide of Formula 1 or a pharmaceutically acceptable salt thereof, as compared to when chemotherapeutic agents are administered alone, the main reason being This is because a large amount does not need to cause a problem of drug resistance. Of course, mixtures of chemotherapeutic agents may be used, and assistive measures such as surgical resection or radiation therapy may also be useful for the treatment of tumors. Although it has been described above that the cyclic depsipeptide of Formula 1 or a salt thereof and a chemotherapeutic agent are administered together, it does not necessarily mean that they are formulated or co-administered in the same dosage form. Here, the expression together means that the cyclic pentapsipeptide of Formula 1 or a pharmaceutically acceptable salt thereof and a chemotherapeutic agent are administered in a mixed dosage form or dividedly administered during the course of treatment.

Preferred route of administration is oral administration. The cyclic pentapsipeptides of formula (1) or pharmaceutically acceptable salts thereof for oral administration are solid or liquid preparations, for example capsules, pills, tablets, troches, rosin, melts, powders, solutions, suspensions. It may be formulated as an agent or an emulsion. Solid unit dosage forms include capsules, which may be in the form of conventional hard or soft gelatin sheaths, which are for example surfactants, lubricants and inerts such as lactose, sucrose, calcium phosphate and corn starch. Contains fillers. In another embodiment, the compounds of the present invention comprise a conventional tablet base (e.g. lactose, sucrose and corn starch) as a binder (e.g. acacia, corn starch or gelatin), a disintegrant to aid in the degradation and dissolution of the tablet after administration. (E.g. potato starch, alginic acid, corn starch and guar gum), lubricants (e.g. talc, stearic acid or stearic acid) to improve the outflow of tablet granules and to prevent the tablet's drug from adhering to tableting dies and punch surfaces Magnesium acid, calcium stearate or zinc stearate), and may be formulated into tablets by mixing with dyes, colorants and flavoring agents to enhance the aesthetic properties of the tablets and to make them more suitable for patients. Suitable excipients useful for oral liquid dosage forms include diluents such as water and alcohols (eg, ethanol, benzyl alcohol and polyethylene alcohol) with or without pharmaceutically acceptable surfactants, suspending agents or emulsions.

In addition, the cyclic pentapsipeptide of the present invention or a pharmaceutically acceptable salt thereof is an injection that is dissolved in a physiologically acceptable diluent together with a pharmaceutical carrier, for example, in a parenteral route, ie, subcutaneous, intravenous, muscle. It may be administered intraperitoneally or intraperitoneally, and the carrier may be a pharmaceutically acceptable surfactant (e.g. soap or detergent), suspending agent (e.g. pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcell Water, saline, dextrose and its pseudosugars, alcohols (e.g., ethanol, isopropane or hexadecyl alcohol), glycols (e.g., propylene glycol, or with or without emulsifiers and other pharmaceutical auxiliaries) Polyethylene glycol), glycerol ketal (e.g. 2,2-dimethyl-1,3-dioxolane-4-methanol), ether (e.g. poly (ethylene-glycol) 400), oils, fatty acids, fats Sterile or mixed liquors such as acid esters or glycerides or acetylated fatty acid glycerides. Examples of oils that can be used in the parenteral formulations of the present invention include petroleum, animal oils, vegetable oils or synthetic oils such as peanut oil, soybean oil, sesame oil, cotton oil, corn oil, olive oil, mineral oil and mineral oil. Suitable fatty acids include oleic acid, stearic acid and isostearic acid. Suitable fatty acid esters are ethyl oleate and myristic acid isopropyl. Suitable surfactants include fatty acid alkali metal salts, ammonium salts and triethanolamine salts, and suitable detergents include cationic detergents (e.g. dimethyl dialkyl ammonium halides, alkyl pyridinium halides and alkylamine acetates), anionic detergents (e.g. alkyls, aryls). And olefin sulfonates, alkyls, olefins, ethers and monoglyceride sulfates and sulfosuccinates, nonionic detergents (eg fatty amine oxides, fatty acid alkanolamides and polyoxyethylenepolypropylene copolymers), and amphoteric detergents (Eg, alkyl-β-amino propionate and 2-alkylimidazoline quaternary ammonium salts) and mixtures thereof. Typically, the parenteral compositions of the present invention contain from about 0.5% to about 25% by weight of the cyclic pentapsipeptide of Formula 1 or a pharmaceutically acceptable salt thereof in solution. It may also be advantageous to use preservatives and buffers. The composition may contain a nonionic surfactant having a dimension-lipophilic balance (HLB) value of about 12 to about 17 to reduce or eliminate irritation at the injection site. The amount of surfactant in such compositions is in the range of about 5 to about 15 weight percent. The surfactant can be a single component with the HLB or a mixture of two or more components with the desired HLB. Examples of surfactants used in parenteral compositions are polyethylene sorbitan fatty acid esters such as sorbitan monooleate and high molecular weight adducts of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol.

In order to produce the cyclic pentapsipeptide of Formula 1 of the present invention, a Fusarium strain, for example, Fusarium solani ( Fusarium) solani ) KCCM90040 [Accession No .: KCCM10881P] Any method of culturing, synthetic method using biosynthesis or organic synthesis can be used.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Example  1: Isolation and Identification of Strains

(1) Separation and Morphological Identification

Fusarium strain producing cyclic depsipeptide was isolated from potato of Mungyeong, Korea. The isolated strains were identified by Samson et al. And Nelson et al. [Samson RA, Hoekstra ES, Oorschot V, Connie AN. (1981) Introduction to food-borne fungi. Published and distributed by Centraalbureau voor Schimmelcultures ; Nelson PE, Toussoun TA, Marasas WF. (1983) Fusarium species: An illustrated manual for identification. The Pennsylvania State University. Press ].

The isolated Fusarium strains were examined for morphological characteristics in carnation lip agar (CLA) and real potato dextrose agar (RPDA).

Small conidia are present in large quantities, generally single cells, from ovoid to kidney (Figure 2a). Conidia disease was branching as shown in Figure 2b. Fusarium Solini's microconidia and conidia are similar to those found at the Fusarium Oxy Forum. However, the small conidia of the Fusarium Oxy Forum have larger and thicker cell walls than Solani's, and the conidia form shorter short cells (Nelson et al., 1983). Growth of the isolated Fusarium strain was fast, the surface of the slant culture was mostly covered with white mycelium, the back of the dark cream form (Fig. 2c).

Fusarium strain isolated through this morphological characteristics was identified as Fusarium Solani.

(2) Molecular Biology Identification

end. DNA  extraction

Whole genomic DNA of the isolated Fusarium Solani strain of the present invention was extracted from the mycelium grown in potato dextrose agar medium by Corel et al. [Correll JC, Klittich CJR, Leslie JF. (1987) Nitrate nonutilizing mutants of Fusarium oxysporum and their use in vegetative compatibility tests. Phytopathology , 77, 16401646].

After culturing the Fusarium Solani strain of the present invention to fill the liquid nitrogen in the medium covered with mycelia, the liquid nitrogen is evaporated at room temperature. After the liquid nitrogen has evaporated, the process is repeated once more. After all of the liquid nitrogen has been evaporated, add a 65 ° C Lysis solution [50 mM Tris pH 8.0, 50 mM ethylenediaminetetraacetic acid (EDTA), 3% sodium dodecylsulfate (SDS), 1% 2-mercaptoethanol and 0.1 m / ml proteinase K] The reaction is carried out at 65 ° C. for one hour. After the reaction, add 0.5 ml phenol solution, shake carefully, and centrifuge for 5 minutes at a speed of 8,000 rpm to separate the phenol layer and the aqueous layer. After carefully transferring the aqueous layer to another tube, in order to remove the remaining phenol layer in the aqueous layer, add 0.4 ml of a solution containing chloroform and asoamyl alcohol in a ratio of 24: 1, shake, and centrifuge to shake the aqueous layer. Move to. Add 0.05 ml of 7.5M ammonium acetate solution and mix carefully. Thereafter, 0.88 ml of 95% ethanol at -20 ° C. was added, followed by centrifugation at 13,000 rpm for 20 minutes to precipitate DNA. Discard the supernatant, wash the DNA with 70% ethanol, and centrifuge again to precipitate the DNA. Discard 70% ethanol and store TE solution (10 mM Tris, 1 mM EDTA, pH8.0).

I. DNA  Electrophoresis and Homology  Sympathy

Hugh et al. Proposed P28SL (5'-ACA AAT TAC AAC TCG GGC CCG AGA-3 ') of the Fusarium specific primer and P58SL (5'-AGT ATT CTG GCG GGC ATG CCT GT-3) of SEQ ID NO: 2 Control PCR analysis using ') was used [Hue, FX, M. Huerre, MA Rouffault, and C.D. Bievre. Specified detection of Fusarium species in blood and tissues by a PCR technique. Journal of Clinical Microbiology, 37: 2434-2438. 1999]. The Fusarium specific primer pairs amplify fragments of genes encoding rDNA of the genus Fusarium strain by PCR. The binding sites of the P28SL and P58SL primer pairs are the ITS2 and 5.8S and 28S portions of rDNA, which are conserved in Fusarium strains.

1 ng of DNA extracted from A., P28SL and P58SL primer pairs and PCR pre-mixture purchased from Promega were used, and PCR conditions were initially denatured at 94 ° C for 10 minutes. Reacted. Thereafter, DNA fragments were amplified by repeating denaturation at 94 ° C for 1 minute, annealing at 60 ° C for 1 minute, and extension reaction at 72 ° C for 1 minute. The last extension after amplification was allowed to react at 72 ° C. for 10 minutes.

The PCR reaction product was prepared by 2% agarose gel and electrophoresed in Tris-acetate-EDTA buffer. After electrophoresis, the dye was added to an ethidium bromide solution and stained, and the amplified band was confirmed by exposing the stained gel to ultraviolet rays.

PCR amplification products (F) of the Fusarium strain of the present invention is 300 to 400 in the same manner as Fusarium monoliforme NRRL 13569 (S-2) and Fusarium Oxysis Forum KTCC 16909 (S-1) used as a control DNA fragment size between bp is shown (FIG. 3).

After purifying the PCR amplification product of the Fusarium strain of the present invention, the sequence was analyzed in Macrogen (Korea), and homology was examined through BLAST search of GenBank database. The ITS-5.8 rDNA sequence of the Fusarium strain of the present invention is shown in SEQ ID NO: 3. The ITS-5.8 rDNA sequence of the Fusarium strain of the present invention showed at least 98% homology with that of Fusarium solani (FIG. 4).

Therefore, the strain of the present invention is Fusarium solani ( Fusarium solani ) was named KCCM90040, and was deposited with the Korea Microorganism Conservation Center on January 15, 2008 under the deposit number KCCM10881P under the Budapest Treaty.

Example  2: annular Depsipeptide  Production and separation

(One) Fusarium  Restricted badge In bros  culture

1 x 10 ⁵ spore / mL of Fusarium solani solani ) KCCM90040 was used as FSA broth; 25 g of sucrose, 4.25 g of NaNO 3, 5 g of NaCl, 2.5 g of MgSO 47 H 2 O, 1.36 g of KH 2 PO 4, 0.01 g of FeSO 47 H 2 O, and 0.0029 g of ZnSO 47 H 2 O per liter ) Was inoculated in 100 mL and incubated at 25 ° C. for 7 days.

(2) Culture in Grain Medium

1 x 10 ⁵ spore / mL of Fusarium solani solani ) KCCM90040 was inoculated into a rice medium adjusted to 40% by weight water content of 50 g of rice in distilled water and incubated for 7 days at 25 ℃.

(3) cyclic in culture Depsipeptide  extraction

To the culture solution containing the mycelium of (1), chloroform corresponding to twice as much as the culture solution was shaken vigorously, and after taking the chloroform layer as a lower layer, it was dried and dissolved again with methanol.

The grain medium cultured with the Fusarium of (2) is dried at room temperature for 12 hours. The dried mycelium was homogeneous, extracted overnight with 75 mL of acetonitrile: methanol: water in a volume ratio of 16: 3: 1dml, and then filtered through sterile filter paper. The filtrate was removed twice fat with 25 mL n-heptane, and the bottom layer was dried. Then, the filtrate was dissolved in 50 mL of a solvent in which methanol: water was mixed in a 55: 45 volume ratio, and twice with 45 mL of methane dichloride. Extracted. The methane dichloride layer was dried and then dissolved again with methanol.

(4) cyclic in the extract Depsipeptide  detach

Each of the extracts of (3) prepared from the cultures of (1) and (2) was prepared using a Shiseito Pack C18 column (0.46 × 25 cm) [Shiseido, Japan] in an acetonitrile: water 70:30 volume ratio. The mixed solution was eluted at a flow rate of 1 mL per minute for 40 minutes and a peak was detected at 210 nm.

Secondary metabolites could not be identified in the extract extracted from the Fusarium restriction medium broth of (1) (FIG. 5). However, in the extract extracted from the grain medium of (2), the peaks were confirmed at 9.7 minutes and 13.4 minutes, respectively (Fig. 6). In FIG. 6, the compound eluted at 9.7 minutes was classified as Compound A, and the compound eluted at 13.4 minutes was designated as Compound B.

Compounds A and B were separated using a Grom-sil pack ODS preparative column (1.0 × 25 cm) with acetonitrile: water at a 65:35 volume ratio at a flow rate of 3 mL per minute, and each of these compounds Using a Shiseido Pack C18 column again, a solution of acetonitrile: water in a 70:30 volume ratio was purified at a flow rate of 1 mL per minute.

(5) Molecular weight confirmation

Compounds (A) and (B) of (4) measured molecular weight using an electrospray ionization mass spectrometer (ESI-MS). As a result, it was confirmed that the molecular weights were 586.36 and 600.36 m / z, respectively (FIG. 7 and 8).

Compounds A and B were very similar in molecular weight to previously reported cyclic depsipeptides. The origin, molecular weight and literatures of the reported compounds are summarized in Table 1.

Song et al. (2006): Song HH, Ahn JH, Lim YH, Lee C, (2006) Analysis of beauvericin and unusual enniatins co-produced by Fusarium oxysporum FB1501 (KFCC 11363P). J. Microbiol . Biotechnol . 16, 11111119

Belovskisky et al. (1999): Belofsky GN, Jensen PR, Fenical W. (1999) Sansalvamide: A new cytotoxic cyclic depsipeptide produced by a marine fungus of the genus Fusarium. Tetrahedron Lett . 40, 2913-2916

Cueto et al. (2000): Cueto M, Jensen PR, Fenical W. (2000) N-Methylsansalvamide, a cytotoxic cyclic depsipeptide from a marine fungus of the genus Fusarium Phytochemistry. 55, 223-226

Oh et al. (2006): Oh DC, Jensen PR, Fenical W. (2006) Zygosporamide, a cytotoxic cyclic depsipeptide from the marine-derived fungus Zygosporium masonii . Tetrahedron Lett . 47, 8625-8628

Example  3: annular Depsipeptide  Structural analysis

(1) Compound A

The FT IR-8400S infrared spectrometer (Shimatsu, Japan) was used to confirm the functional group of Compound A. Compound A had amide bond (1654.42 cm ⁻¹ ) and ester bond (1745.52 cm ⁻¹ ) as a result of IR analysis (FIG. 9). The maximum UV spectrum was 287 nm in methanol and the melting point was 82 ° C. as measured by a melting point meter (Thermo fisher scientific Inc. Waltham, USA).

^{1D-NMR (1 H NMR,} 13 C NMR, and DEPT) measurement was performed using a Bruker DMX 600 spectrometer system, 2D- NMR (COSY, HMQC, and HMBC) methanol using a Bruker AVANCE 800 spectrometer system (CD ₃ OD).

¹ H NMR and ¹³ C NMR spectral results showed that Compound A is a typical cyclic depsipeptide (Table 2).

DEPT and 2D-NMR spectral results (COSY, HMQC, and HMBC) show that Compound A is composed of five units: lysonic acid (OLeu), leucine (Leu), valine (Val), phenylalanine (Phe), and leucine (Leu) I can confirm that there is. The order of the 5 units in Compound A was determined by HMBC correlation data analysis (FIG. 10).

This confirmed that Compound A is a cyclic pentapsipeptide of Formula 2.

[Formula 2]

The compound of Formula 2 is a novel cyclic pentadepsipeptide showing a difference in the binding sequence of the acid salbamide, four amino acids, and one hydroxy acid, and was named neo-acid salbamide.

(2) Compound B

Compound B had amide bonds (1653.24 cm ⁻¹ ) and ester bonds (1742.65 cm ⁻¹ ) from IR analysis (FIG. 11). The maximum UV spectrum was 213 nm in methanol and the melting point was 82 ° C.

1D-NMR ( ¹ H NMR, ¹³ C NMR, and DEPT) measurements were made using a Bruker DMX 600 spectrometer system, while COSY, HMQC, and HMBC in 2D-NMR were measured on CDCl ₃ using a Bruker DMX 600 spectrometer system. , NOESY (nuclear overhauser effect spectroscopy) was measured in CDCl ₃ using a Varian VNS 600 spectrometer system (Palo Alto, CA, USA).

¹ H NMR and ¹³ C NMR spectral results showed that Compound B is a typical cyclic depsipeptide (Table 3).

DEPT and 2D-NMR spectral results (COSY, HMQC, and HMBC) indicate that Compound A is a leucoic acid (OLeu), N-methylleucine (N-MeLeu), valine (Val), phenylalanine (Phe), and leucine (Leu). It was confirmed that it consists of 5 units. The order of these five units in Compound B was determined by HMBC correlation data analysis (FIG. 12).

This confirmed that the compound B is a cyclic penta depsi peptide of formula (1).

[Formula 1]

The compound of Formula 1 is a novel cyclic pentapsipeptide showing a difference in the binding sequence of N-methyl acid salbamide, four amino acids, and one hydroxy acid, and was named neo-N-methyl acid salvamide.

In addition, NOESY correlation analysis was performed to confirm the arrangement of hydroxy acids (OLeu), indicating that CH ₃ protons (3.180 ppm) of NMeLeu were H-2 (4.970 ppm) of OLeu and Phe, Leu and Val residues, respectively. NOESY correlation with H-8 (4.803 ppm), H-16 (4.694 ppm) and H-23 (4.590 ppm). Therefore, these protons were all located on the same side, and it was confirmed that the stereochemical structure of the hydroxy acid (OLeu) was S-array (FIG. 12).

(3) Determination of stereochemical structure

After the acid hydrolysis to confirm the stereochemical structure of the amino acids contained in Compounds A and B, each amino acid was derivatized with Marfey's reagent, and then analyzed by HPLC with a control group. It is shown to 14 (compound B), respectively. All amino acids contained in Compound A was confirmed to be L-type. In addition, all of the amino acids of Compound B are L-type, and since the stereochemical structure of hydroxy acid (OLeu) is S-array, it was confirmed that the compound of Compound B is a compound having the stereochemical structure of FIG. 15.

Example 4: Cytotoxicity Measurement

In the SRB method, a multidrug-resistant cancer cell line and a multidrug-resistant cancer cell line of known cyclic hexadepsipeptides (buricine, Enniatin H, I and MK1688) and the compounds of Formulas 1 and 2 were used. Cytotoxicity was measured.

Lung cancer cell line (A549), ovarian cancer cell line (SK-OV-3) and skin cancer cell line (SK-MEL-2), uterine sarcoma cell line (MEA-SA) and its multidrug resistant cancer cell line (MES-SA / DX5) Purchased from the American Type Culture Collection (USA), colon cancer cell line (HCT15) was supplied by the US National Cancer Center, and HCT15's multidrug resistant cancer cell line (HCT15 / CL02) was continuously and cascaded from HCT15 to Doxorubicin at the Korea Research Institute of Chemical Technology. Was supplied with the cell line prepared by exposure. EC ₅₀ was determined at a concentration at which 50% cell growth was inhibited under assay conditions. Doxorubicin was used together as a control.

Crude extracts of Sansalvamid show in vitro cytotoxicity to most cancer cells and have an IC ₅₀ value of 9.8 μg / ml for colorectal cancer cell line HCT116 reported by Belofsky et al. (Belofsky et al., 1999). In addition, N- methyl acid imide of Salvador in vitro cytotoxicity test of the cancer cell lines in the US National Cancer Institute reported by 8.3 μM GI ₅₀ to be a Ceuto [Ceuto et al., 2000 ]. According to the results of Tables 4 and 5, the cyclic pentapsipeptides of formulas (1) and (2) are equivalent to acid salbamide A or N-methyl acid salbamide for most cancer cell lines, regardless of whether they possess multidrug resistance. Levels of cytotoxicity were shown. In addition, the cyclic pentapsipeptide of Formula 1 was superior to the proliferative inhibitory activity against multi-drug resistant cancer cell lines compared to the cyclic pentapsipeptide of Formula 2 (FIGS. 16 and 17).

Example  5: Multi-drug resistance  Inhibitory activity measurement

The multidrug resistance inhibitory activity of the cyclic pentapsipeptides of Formulas 1 and 2 was compared with multidrug-resistant cancer cell lines (MES-SA and HCT15), ie, MES-SA / DX5 and HCT15. It was confirmed in / CL02. The inverse effect of the multidrug resistance of the cyclic pentapsipeptides of Formulas 1 and 2 on the cytotoxicity of paclitaxel (TAX) against multidrug resistant cancer cell lines was measured (Table 6). Verapamil (VER), which has P-glycoprotein inhibitory activity and is used as a multi-drug resistance inhibitor, was used as a control.

Cyclic pentapsipeptide of formula (2) slightly enhanced the cytotoxicity of paclitaxel to cancer cell lines with multidrug resistance, but its width was not large. Significantly enhanced the cytotoxicity of Taxel (FIGS. 18 and 19). Therefore, the N-methyl group in the cyclic pentapsipeptide of Formula 1 seems to be an important factor in the expression of multi-drug resistant activity. Cyclic pentapsipeptide of Formula 1 exhibited multi-drug resistance inhibitory activity similar to verapamil used as a positive control.

Example  6: Antibacterial  And Antifungal  Active measurement

(One) Antibacterial  activation

Antibacterial against three Gram-positive bacteria ( L. monocytogenes ATCC 14028; S. aureus ATCC 35556; B. cereus ATCC 13061), three Gram-negative bacteria ( E. coli ATCC 8739 P. aeruginosa ATCC 9026; S. typhimurium ATCC 14028) Activity was measured.

Compounds of formulas (1) and (2) were dissolved in DMSO at concentrations of 0.1, 0.5, 1, and 2 mM, respectively, and then dropped onto sterile paper discs (5 mm in diameter) and the DMSO was evaporated. Tryptic soy aga was inoculated with bacteria at a concentration of 1 × 10 ⁷ CFU / mL, and the paper disc was placed and incubated at 37 ° C. for 24 hours, and then the clear zone and its diameter were observed.

Both compounds of Formulas 1 and 2 showed no antibacterial activity against all of the bacteria tested.

(2) Antifungal  activation

Four fungal strains ( Mucor rouxii , Penicillium citrinum , Fusarium oxysporum, and Aspergillus oryzae ) antifungal activity was measured.

Compounds of formulas (1) and (2) were dissolved in methanol at concentrations of 1 and 10 mM, respectively, and then dropped onto sterile paper discs (8 mm in diameter) and the methanol was evaporated. The paper disc was placed on a fungus mycelium cultured in potato dextrose agar and incubated at 25 ° C. for 48 hours, and then the clear zone and its diameter were observed. As a control, a paper disc in which only methanol was dropped was used.

Compounds of formulas (1) and (2) did not show inhibitory activity at 4 mM concentrations against the four fungal strains. However, both Mucor compounds of Formulas 1 and 2 Although no clear zone was observed at 10 mM concentration for rouxii , inhibition of mycelial growth was confirmed (FIG. 20A). Compound of Formula 2 is Fusarium Although no clearzone was shown for oxysporum , the compound of Formula 1 showed weak inhibitory activity (FIG. 20B).

Formulation example  1: preparation of tablets

Component content

100 mg of cyclic pentapsipeptide of Formula 1

Corn Starch 68mg

Lactose 90mg

Microcrystalline Cellulose 40mg

Magnesium Stearate 2mg

According to the conventional method of preparing tablets, the above ingredients were added to the indicated contents, mixed uniformly, stirred and granulated. Then, using a tableting machine after drying to prepare a tablet containing 100mg of the cyclic pentapsipeptide of the active ingredient formula (1) per tablet.

Formulation example  2: preparation of injectables

Component content

50 mg of cyclic pentapsipeptide of Formula 1

Sodium Metabisulfite 1.5mg

Methyl Paraben 1.0mg

Propyl Paraben 0.1mg

Appropriate amount of purified water for injection

According to a conventional method for preparing an injection, the above components are dissolved in boiling water with a given content, stirred, and then cooled and filled into a 2 ml sterile vial, and supplemented with an appropriate amount of purified water for injection to 2 ml. Injections containing 50 mg of the cyclic pentapsipeptide of Formula 1 were prepared.

Formulation example  3: Preparation of Syrup

Component content

Cyclic pentadepsipeptide of Formula 1 200mg

2 g of concentrated juice

5g sucrose

100 mg sodium citrate

Fragrance 70mg

Water

According to a conventional method of preparing syrup, the above components are mixed in an appropriate amount of water in a given content, heated to dissolve, cooled, and filled into a container to give 200 mg of a cyclic penta of formula 1 per bottle of a 200 ml volume of beverage. A syrup comprising the depsi peptide was prepared.

Figure 1 shows the chemical structures of the acid salbamide A homologues.

Figure 2 is a morphological characteristic of the strain of the present invention is a photograph showing the appearance and appearance of the small conidia (a), conidia disease (b) and slant culture (c).

Figure 3 is agarose gel electrophoresis picture of the PCR amplification product using the Fusarium-specific primer for fusarium strain identification. M is a DNA marker in units of 100 bp, F is a PCR amplification product of the strain of the present invention, S-1 and S-2 are the amplification products of the Fusarium monoliforme NRRL 13569 and the Fusarium Oxis Forum KTCC 16909, respectively. Indicates.

Figure 4 compares the homology of the ITS-5.8 rDNA sequence of the Fusarium solani of the sample (sample) of the present invention.

Figure 5 is an HPLC chromatogram of the extract extracted from the culture medium after immersing the strain of the present invention in Fusarium restriction medium.

Figure 6 is an HPLC chromatogram of the extract extracted from the culture medium after the solid culture of the strain of the present invention in grain medium.

7 is a result of measuring the molecular weight by using an electron injection ionization mass spectrometer of Compound A produced in the strain of the present invention.

8 is a result of measuring the molecular weight by using an electron injection ionization mass spectrometer of Compound B produced in the strain of the present invention.

9 is an IR spectrum measured by the FT IR-8400S infrared spectrometer of Compound A.

10 analyzes the HMBC correlation data of Compound A.

FIG. 11 is an IR spectrum measured by the FT IR-8400S infrared spectrometer of Compound B.

FIG. 12 analyzes HMBC and NOESY correlation data for Compound B.

FIG. 13 is an HPLC chromatogram for identifying stereochemical structures of amino acids included in Compound A. FIG.

FIG. 14 is an HPLC chromatogram for identifying stereochemical structures of amino acids included in Compound B. FIG.

15 shows the stereochemical structure of Compound B.

Figure 16 shows the cytotoxicity of the compound of formula (1) (a) is for cancer cell lines without multi-drug resistance, (b) is for cancer cell lines with multi-drug resistance.

Figure 17 shows the cytotoxicity of the compound of formula (2) (a) is for cancer cell line without multi-drug resistance, (b) is for cancer cell line with multi-drug resistance.

Figure 18 shows the multi-drug resistance inhibitory activity of the compounds of Formula 1 and Formula 2, (a) is for HCT15 cell line, (b) is for HCT15 / CL02.

Figure 19 shows the multi-drug resistance inhibitory activity of the compounds of formula 1 and formula (a) is for the MEA-SA cell line, (b) is for MEA-SA / DX5.

Figure 20 shows the antifungal activity at a concentration of 10 mM of the compounds of Formulas 1 and 2, (a) is Mucor for rouxii , (b) Fusarium It's about oxysporum .

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Novel cyclic pentadepsipeptide (II) and its use <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Fusarium specific primer P28SL <400> 1 acaaattaca actcgggccc gaga 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Fusarium specific primer P58SL <400> 2 agtattctgg cgggcatgcc tgt 23 <210> 3 <211> 305 <212> DNA <213> Fusarium solani <400> 3 gggcctggcg ttggggatcg gcggagcccc ctgtgggcac acgccgtccc tcaaatacag 60 tggcggtccc gccgcagctt ccattgcgta gtagctaaca cctcgcaact ggagagcggc 120 gcggccatgc cgtaaaacac ccaacttctg aatgttgacc tcgaatcagg taggaatacc 180 cgctgaactt aagcatatca ataagcggag gaaaagaaac caacagggat tgccccagta 240 acggcgagtg aagcggcaac agctcaaatt tgaaatctgg ctctcgggcc cgagttgtaa 300 tttgt 305  

Claims (4)

Cyclic pentapsipeptide of Formula 1 below.

[Formula 1] A pharmaceutical composition for inhibiting pharmaceutical resistance containing any one or more selected from the group consisting of a cyclic pentapsipeptide of Formula 1 and a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1] The pharmaceutical composition for inhibiting drug resistance according to claim 2, which is for inhibiting drug resistance of tumor cells. A pharmaceutical composition for treating cancer having tumor cell proliferation inhibitory activity containing any one or more selected from the group consisting of a cyclic pentapsipeptide of Formula 1 and a pharmaceutically acceptable salt thereof.

[Formula 1]

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