KR20140114522A - Novel cyclic peptide compound, a use thereof, and a preparing method thereof - Google Patents

Abstract

Provided are a novel cyclic peptide compound, an antibiotic and anti-cancer use thereof, and a producing method thereof. The compound as an antimicrobial or anti-cancer drug can be used to treat infection or cancer arising from or caused by microorganisms.

Description

[0001] The present invention relates to a novel cyclic peptide compound, a use thereof, and a production method thereof,

Cyclic peptide compounds, their antibacterial and anticancer applications, and their production methods.

Physiologically active substances derived from microorganisms were generally the source of antibacterial, antifungal, and anticancer drugs, and they have been developed as new drugs for treatment of various diseases such as this and have become the basis of new drug development. Antibiotics derived from microorganisms are, for example, amphotericin, erythromycin, streptomycin, tetracycline, and vancomycin. In 2003, daptomycin isolated from actinomycetes Streptomyces was approved by the US Food and Drug Administration (FDA) as a next-generation antibiotic. On the other hand, anticancer drugs derived from bacteria include, for example, doxorubicin, bleomycin, mithramycin, neocarzinostatin, pentostatin, epothilone, have. Thus, research on physiologically active substances derived from bacteria is very important in the development of antibacterial agents, antifungal agents, and anticancer agents.

In order to search physiologically active substances that differ structurally from existing substances, one of the recent natural product researches is to study natural products in a geographical and phylogenetic specific environment. Easily accessible soil microorganisms and terrestrial plants have been extensively studied for natural products over a long period of time, but research on marine-origin bacteria has been relatively inactive. The oceans occupy 70% of the surface of the earth and are presented as a space of opportunity that is largely unexplored. Although marine microorganisms are not precisely known for their diversity, there has been little research in this area so far that only 1% of marine microorganisms are believed to have been cultured or identified.

Therefore, in order to develop new antibiotics and anticancer agents structurally, it is necessary to select marine bacteria producing useful bioactive substances, and to search for and discover new compounds produced by them.

During the selection and study of new marine bacteria, new strains were found. Also, while studying the above strain, it has been found that the strain produces a novel cyclic peptide, and that the novel cyclic peptide has antimicrobial activity and anticancer activity, thus completing the present invention.

A novel cyclic peptide compound, an isomer or derivative thereof, or a pharmaceutically acceptable salt thereof.

Also provided is an antimicrobial composition comprising the compound.

Also provided is a pharmaceutical composition for treating cancer comprising the above compound.

Also provided is a strain of Streptomyces sp. SNJ042 producing the above compound.

According to one aspect, there is provided a novel cyclic peptide compound, an isomer, derivative, or pharmaceutically acceptable salt thereof.

According to one aspect, there is provided an antimicrobial composition comprising the compound.

According to one aspect, there is provided a pharmaceutical composition for treating cancer comprising the above compound.

According to one aspect, there is provided a Streptomyces sp. SNJ042 strain producing the above compound.

Since the compound represented by the formula 1 has an antibacterial activity and an anticancer activity, the compound represented by the formula 1 can be used as an antimicrobial agent or an anticancer agent in the prevention or treatment of an infectious disease or cancer caused or caused by a microorganism.

Fig. 1 is a structural formula of O. nisamycin A. Fig.
Fig. 2 is a structural formula of O. nisamycin B. Fig.
3 is a photograph of a culture medium in which Streptomyces sp. Strain SNJ042 was cultured.
FIGS. 4A and 4B are graphs showing growth inhibition results of the omnosamycin A and B cells against common cell lines, respectively.
FIGS. 5A to 5E are graphs showing the results of inhibition of growth of cancer nerve axon A on cancer cells. FIG.
6A to 6E are graphs showing the results of inhibition of growth of cancer nerve axon B on cancer cell lines.

One aspect provides a compound, an isomer, derivative, or pharmaceutically acceptable salt thereof, comprising a peptide:

In the above formula 1, Val, Thr, Leu, Trp, and Phe mean valine, threonine, leucine, tryptophan, and phenylalanine, respectively.

In the formula 1, R may be H, a C ₁ to C ₂₀ alkyl group, a C ₂ to C ₂₀ alkenyl group, a C ₂ to C ₂₀ alkenyl group, or a C ₃ to C ₂₀ aryl group. The alkyl group may be, for example, an alkyl group of C ₁ to C ₁₅ , C ₁ to C ₁₀ , C ₁ to C ₅ , or C ₁ to C ₃ . The alkyl group may be, for example, a methyl group. The alkenyl group may be, for example, an alkenyl group of C ₂ to C ₁₅ , C ₂ to C ₁₀ , or C ₂ to C ₅ . The alkenyl group may be, for example, a C ₂ to C ₁₅ , C ₂ to C ₁₀ , or C ₂ to C ₅ alkenyl group. The aryl group may be, for example, an aryl group of C ₃ to C ₁₅ , C ₃ to C ₁₀ , or C ₃ to C ₅ .

The term "isomer" refers to a compound that has the same molecular formula but does not have the same linking or spatial arrangement of constituent atoms in the bonsai. Isomers include, for example, structural isomers, and steric isomers.

The term " derivative "refers to a compound obtained by substituting a part of the structure of the above compound with another atom or an atomic group.

The term "pharmaceutically acceptable salts" refers to the inorganic and organic acid addition salts of the compounds.

The amino acid of the compound may be substituted with another amino acid having similar physicochemical properties. Valine, leucine, tryptophan, or phenylalanine may be substituted with another amino acid having a non-polar R group or a derivative thereof. For example, valine may be substituted with alanine (Ala), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp) or methionine (Met). For example, leucine may be substituted with alanine (Ala), valine (Val), isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp) or methionine (Met). For example, tryptophan may be substituted with alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), or methionine (Met). For example, phenylalanine can be substituted with alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), tryptophan (Trp) or methionine (Met). The threonine may be substituted with another amino acid having a polar R group or a derivative thereof. For example, threonine can be substituted by substitution with glycine (Gly), serine (Ser), cysteine (Cys), tyrosine (Tyr), asparagine (Asn) or glutamine (Gln).

The Trp may be 4-OCH ₃ -Trp in which a methoxy group is bonded to the 4th carbon of the R group.

Phe may be? -OH-Phe, which is a bond of? Carbon with a hydroxy group.

The compound may be a compound wherein the amino group of the amino acid is bonded to a C ₁ to C ₂₀ alkyl group. For example, the amino acid of Trp, Leu, Val ¹ , Val ⁵ , Thr ² , or a combination thereof may be a compound wherein the amino group is bonded to a C ₁ to C ₂₀ alkyl group. For example, the alkyl group may be methyl.

The compound may be a compound represented by the general formula (1).

[Chemical Formula 1]

In Formula 2, R may be H, a C ₁ to C ₂₀ alkyl group, a C ₂ to C ₂₀ alkenyl group, a C ₂ to C ₂₀ alkenyl group, or a C ₃ to C ₂₀ aryl group. The alkyl group may be, for example, an alkyl group of C ₁ to C ₁₅ , C ₁ to C ₁₀ , C ₁ to C ₅ , or C ₁ to C ₃ . The alkyl group may be, for example, a methyl group. The alkenyl group may be, for example, an alkenyl group of C ₂ to C ₁₅ , C ₂ to C ₁₀ , or C ₂ to C ₅ . The alkenyl group may be, for example, a C ₂ to C ₁₅ , C ₂ to C ₁₀ , or C ₂ to C ₅ alkenyl group. The aryl group may be, for example, an aryl group of C ₃ to C ₁₅ , C ₃ to C ₁₀ , or C ₃ to C ₅ .

The compound may be a compound represented by the general formula (2).

(2)

The compound may be a compound represented by the general formula (3).

(3)

An aspect of the present invention provides an antimicrobial composition comprising the novel cyclic peptide compound.

The composition may have an antibacterial activity against microorganisms. The composition may have an antimicrobial activity against, for example, a pathogenic microorganism or a non-pathogenic microorganism. The composition may have antibacterial activity against, for example, Gram negative microorganisms or Gram positive microorganisms. The composition may be, for example, Staphylococcus aureus, Bacillus subtilis, Kocuria rhizophila, Proteus hauseri, Salmonella enterica, enterica), Escherichia coli, or a combination thereof.

The composition may further comprise known active ingredients having antimicrobial activity against microorganisms.

The pharmaceutical composition may further comprise a carrier, an excipient or a diluent. Carriers, excipients and diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Vaginal cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories or sterilized injection solutions according to conventional methods. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

In the above pharmaceutical composition, the solid preparation for oral administration may be a tablet, a pill, a powder, a granule, or a capsule. The solid preparation may further comprise an excipient. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid preparation may further comprise a lubricant such as magnesium stearate or talc. In the above pharmaceutical composition, the liquid preparation for oral administration may be a suspension, a solution, an emulsion, or a syrup. The liquid formulation may comprise water, or liquid paraffin. The liquid preparations may contain excipients such as wetting agents, sweetening agents, perfumes, or preservatives. In the above pharmaceutical composition, the preparation for parenteral administration may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an oil, a lyophilized or a left-over preparation. Non-aqueous solvents or suspensions may include vegetable oils or esters. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The suppository base may be witepsol, macrogol, tween 61, cacao paper, laurin, or glycerogelatin.

The non-pharmaceutical composition may be used in combination with other non-pharmaceutical compositions or ingredients contained therein. The non-pharmaceutical composition may be, for example, a disinfectant cleaner, a shower foam, an oral cleanser, a wet tissue, a hand cleanser, a humidifier filler, a mask, an ointment or a filter filler.

One aspect provides a pharmaceutical composition for preventing or treating cancer comprising the novel cyclic peptide compound.

The cancer may be selected from the group consisting of, for example, hepatocellular carcinoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, bacterial sarcoma, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, Cancer, breast cancer, cholangiocarcinoma, cholangiocarcinoma, retinoblastoma, choroidal melanoma, bladder cancer, bladder cancer, peritoneal cancer, pituitary adenocarcinoma, pituitary adenocarcinoma, papillary adenoma, pituitary adenoma, multiple myeloma, Neoplasms, neuroblastoma, neuroblastoma, pyelonephritis, renal cancer, malignant soft tissue tumor, malignant bone tumor, malignant melanoma, benign ovarian cancer, Malignant lymphoma, malignant mesothelioma, malignant melanoma, ovarian tumor, vulvar cancer, urethral cancer, unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoma, gastrointestinal stromal tumor, Wilm's tumor , Breast cancer, sarcoma, penile cancer, pharyngectomy, pregnancy choroidal disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic brain tumor, rectal cancer, rectal carcinoid tumor, vaginal cancer, spinal cord tumor, neuroendocrine tumor, pancreatic cancer, Cancer, squamous cell carcinoma, lung cancer, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, laryngeal cancer, or a combination thereof.

The term "prophylactic " refers to any act that inhibits disease or delays onset by administration of the composition. The term "treatment" refers to any action that improves or alleviates a symptom of a disease by the administration of the composition.

One aspect provides Streptomyces sp. SNJ042 strain (accession number: KCTC18240P).

The strain may produce the novel peptide compound.

The strain includes its variants. The mutant may be, for example, a mutant caused by a natural mutation or an anthropogenic mutation. Anthropogenic mutations can be caused by chemical mutagens such as ultraviolet light, physical mutagens, or base compounds.

The strain includes a spore, a bacterium, or a culture thereof.

One aspect of the present invention is a method of isolating a strain of Streptomyces sp. SNJ042 (Accession No .: KCTC18240P); And separating the novel peptide compound from the culture broth. The present invention also provides a method for producing the novel peptide compound.

The method comprises culturing Streptomyces sp. SNJ042 strain (Accession No. KCTC18240P).

The step of culturing may be to culture the strain in a liquid medium or a solid medium. The medium may include as a carbon source, for example, glucose, starch syrup, dextrin, starch, molasses, animal oil, or vegetable oil. The medium may include, for example, wheat bran, soybean meal, wheat, malt, cottonseed, fish, cones titer, juice, yeast extract, ammonium sulfate, sodium nitrate or urea as a source of nitrogen. The culture medium may contain, if necessary, inorganic salts which promote salt formation, potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid or other ion production.

The culture may be culturing under aerobic conditions with shaking or standing. The incubation temperature may be, for example, about 20 째 C to about 37 째 C, or about 26 째 C to about 30 째 C.

The method comprises separating the novel peptide compound from the culture broth.

The step of separating may comprise the step of concentrating, centrifuging, filtering, or performing chromatography on the culture. The chromatography can be, for example, column chromatography, planar chromatography, paper chromatography or thin film chromatography depending on the form of stationary phase. Chromatography can be, for example, gas chromatography, liquid chromatography, or affinity chromatography, depending on the physical properties of the mobile phase. The liquid chromatography may be, for example, high performance liquid chromatography (HPLC). Chromatography can be, for example, according to a separation method, ion exchange chromatography, size-exclusion chromatography. The chromatography may be, for example, normal chromatography or reverse phase chromatography.

One aspect provides a method of sterilizing or bacteriumifying a microorganism, comprising applying, spraying, coating, dipping, or a combination thereof in an in vitro, an antimicrobial composition comprising the novel cyclic peptide compound.

In the above method, the microorganism may be, for example, a pathogenic microorganism or a non-pathogenic microorganism. The microorganism may be, for example, a gram negative microorganism or a gram positive microorganism. Microorganisms include, for example, Staphylococcus aureus, Bacillus subtilis, Kocuria rhizophila, Proteus hauseri, Salmonella enterica ), Escherichia coli, or a combination thereof.

The term " applying "refers to applying the composition to the surface of an object. The term " spray "refers to spraying the composition. The term "coating" refers to coating the surface of an object with the composition. The term "immersion" refers to immersing an object in the composition.

The term "sterilization" refers to the action of killing microorganisms. The term "bacterium" refers to an action that inhibits the growth or proliferation of microorganisms.

An aspect of the present invention provides a method for preventing or treating an infectious disease caused by or caused by a microorganism, comprising the step of administering an antimicrobial pharmaceutical composition containing the novel cyclic peptide compound to a subject other than a human. do.

The term "individual" includes all mammals, including humans.

The preferred dosage of the compound varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, the compound represented by the formula 1 can be administered in an amount of, for example, about 0.0001 mg / kg to about 100 mg / kg, or about 0.001 mg / kg to about 100 mg / have. The compound represented by Formula 1 in the composition may be contained in an amount of about 0.0001% by weight to about 10% by weight, or about 0.001% by weight to about 1% by weight based on the total weight of the entire composition.

The pharmaceutical dosage forms of the compounds may be in the form of their pharmaceutically acceptable salts. Such compounds may be used alone or in combination with other pharmaceutically active compounds.

The pharmaceutical composition may be administered to a mammal, including a mouse, a mouse, a livestock, and a human, in a variety of routes. The method of administration may be, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injection.

Examples of infectious diseases caused by or caused by microorganisms include Staphylococcus aureus, Bacillus subtilis, Kocuria rhizophila, Proteus heresieri Proteus hauseri, Salmonella enterica, Escherichia coli, or a combination thereof. The disease may be, for example, food poisoning, skin inflammation, otitis media, cystitis, sepsis, pneumonia, diarrhea, or a combination thereof.

One aspect provides a method of preventing or treating cancer, comprising administering to a subject a pharmaceutical composition for the prevention or treatment of cancer comprising the novel cyclic peptide compound.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are for illustrative purposes only, and the scope of the present invention is not limited to these embodiments.

Example  One. Streptomyces  genus( Streptomyces sp .) SNJ042  Isolation of strain

In order to screen a strain producing a substance having an antibacterial or anticancer activity, the strain SNJ042 derived from the ocean was isolated. The whole genome of SNJ042 strain was isolated and 16S ribosomal DNA was sequenced by PCR (SEQ ID NO: 1).

The SNJ042 strain was identified as Streptomyces sp. This strain was named Streptomyces sp. SNJ042 strain, and the strain was deposited with the depository institution on Feb. 6, 2012 (accession number: KCTC18240P).

Example  2. Streptomyces sp. SNJ042  Culture of strain

Streptomyces sp. SNJ042 strain was inoculated into a sterilized YEME solid medium (10 g of malt extract, 4 g of yeast, 2 g of glucose, 34 g of artificial sea salt and 18 g of agar per 1 L of water) Respectively.

The SNJ042 strain cultivated in the solid medium was inoculated into a sterilized 50 ml of A1 + C liquid medium (10 g of starch per liter of water, 4 g of yeast, 2 g of peptone, 1 g of calcium carbonate and 34 g of artificial sea salt) , And secondary cultured at 30 DEG C for 2 days while shaking at 200 rpm.

10 ml of the secondary cultured medium was inoculated into 1 L of the A1 + C liquid medium and cultured at 30 DEG C for 6 days with shaking at 200 rpm.

Example  3. Oh, my God  Separation and purification of A and B

12 L of SNJ042 culture broth cultivated in Example 2 was extracted with 24 L of ethyl acetate and depressurized to obtain 3.5 g of crude extract. The crude extract was divided into six fractions using two reverse phase chromatographies (Sepak® C ₁₈ 2 g, reversed-phase). Six fractions of the eluent were used, with 80% (v / v) water / methanol decreasing by 20% water, and the last fraction was fractionated with 50% (v / v) methanol / dichloromethane to give a total of six fractions (40 ml each).

Fraction 4 of 20% (v / v) water / methanol fraction was analyzed by liquid chromatography-mass spectrometry and hydrogen nuclear magnetic resonance spectroscopy. The composition of the fractions was analyzed using LC / MS coupled with Agilent Technologies' 1200 Series LC and 6130 Series Mass Spectrometer. The mass spectrum was measured using a Thermo Scientific LTQ-Orbitrap ESI Mass Spectrometer Respectively. It was confirmed by liquid chromatography-mass spectrometry and hydrogen nuclear magnetic resonance spectroscopy analysis that the culture medium of the strain contained new secondary metabolites.

The fraction 4 concentrated by vacuum drying was dissolved in 20 ml of methanol and subjected to reversed-phase semi-preparative HPLC column (C) using a concentration gradient system (30% (v / v) → 10% (v / v) water / methanol, ₁₈ reversed-phase semi-preparative HPLC column, particle diameter 5 mm, 250 x 10 mm (length x inner diameter), elution rate 2 ml / min, UV detector) (Phenomenex). After separation, 7.5 mg and 4.0 mg of offosamycin A and B, respectively, were obtained.

Example  4. Oh, my God  Physicochemical characterization of A and B

O. nisamines A and B are white solid, stable at room temperature, and soluble in methanol, acetone, etc., which are medium organic solvents. The structures of O. nisamines A and B were determined by nuclear magnetic resonance spectrum, infrared and ultraviolet spectroscopic data and high resolution mass spectrometry data. The nuclear magnetic resonance spectrum ( ¹ H NMR, ¹³ C NMR) was 900 MHz NMR of Bruker and pyridine-d ₅ was used as the solvent. The mass spectrum was measured using a Thermo Scientific LTQ-Orbitrap ESI mass spectrometer in mass / charge (m / z) form. The infrared spectrum was obtained using a FT-IR-4200 spectrometer from Jasco. Infrared spectroscopy was performed using a Lamda 35 UV / VIS spectrometer from Perkin Elmer.

The structural localization of these compounds by nuclear magnetic resonance spectra is shown in Tables 1 and 2.

[Ohmyungsamycin A]

(1) Molecular formula: C ₇₅ H ₁₁₉ N ₁₃ O ₁₆

(2) Molecular weight: 1458.8249

(3) Color: White

(4) Infrared absorption band (KBr): 3297, 2963, 1640, 1535, 1509, 1201 cm ^-1

^{(5) 1 H-NMR (} pyridine- d 5, 900 MHZ): see Table 1

^{(6) 13 C-NMR (} pyridine- d 5, 225 MHZ): see Table 1

Nuclear magnetic resonance spectrum of O location ^One H ¹³ C location ^One H ¹³ C One 174.3 C 39 173.2 C 2 4.70 58.3 CH 40 4.96 54.3 CH 2-NH 9.28 40-NH 9.39 3 2.23 32.8 CH 41 2.40 31.9 CH 4 1.12 19.3 CH ₃ 42 1.14 19.0 CH ₃ 5 0.97 18.6 CH ₃ 43 1.03 19.7 CH ₃ 6 172.7 C 44 171.7 C 7 5.49 60.0 CH 45 5.65 54.8 CH 7-NH 9.66 46a 1.74 38.9 CH ₂ 8 5.94 73.2 CH 46b 1.58 9 143.0 C 47 1.43 25.1 CH 10 7.68 127.0 CH 48 0.67 23.1 CH ₃ 11 7.41 128.6 CH 49 0.67 21.9 CH ₃ 12 7.27 127.5 CH 50 3.52 31.3 CH ₃ 13 7.41 128.6 CH 51 173.5 C 14 7.68 127.0 CH 52 5.29 55.6 CH 15 174.5 C 52-NH 8.06 16 5.41 58.3 CH 53 2.57 31.0 CH 16-NH 8.02 54 1.20 19.0 CH ₃ 17 2.66 33.1 CH 55 1.16 19.7 CH ₃ 18 1.36 19.8 CH ₃ 56 170.6 C 19 1.20 20.0 CH ₃ 57 5.61 62.4 CH 20 169.9 C 58 5.05 66.5 CH 21 4.59 70.6 CH 59 1.34 20.5 CH ₃ 22a 4.45 17.1 CH ₂ 60 3.68 34.5 CH ₃ 22b 4.32 61 171.4 C 23 112.9 C 62 5.86 52.5 CH 24 7.19 124.3 CH 62-NH 10.02 24-NH 11.80 63 6.04 69.4 CH 25 139.5 C 64 1.52 16.8 CH ₃ 26 7.30 105.8 CH 65 173.4 C 27 7.27 122.7 CH 66 5.24 57.9 CH 28 6.67 99.5 CH 66-NH 8.65 29 154.9 C 67 2.26 31.8 CH 30 3.83 55.4 CH ₃ 68 0.96 19.8 CH ₃ 31 118.4 C 69 0.96 18.8 CH ₃ 32 2.51 40.8 CH ₃ 70 174.2 C 33 169.4 C 71 3.11 71.4 CH 34 3.19 70.9 CH 72 2.18 32.2 CH 35 3.03 29.0 CH 73 1.09 19.1 CH ₃ 36 1.22 21.9 CH ₃ 74 1.09 19.1 CH ₃ 37 1.02 19.7 CH ₃ 75 2.54 35.9 CH ₃ 38 3.18 39.9 CH ₃ 75 2.54 35.9 CH ₃

[Ohmyungsamycin B]

(1) Molecular formula: C ₇₆ H ₁₂₁ N ₁₃ O ₁₆

(2) Molecular weight: 1472.8514

(3) Color: White

(4) Infrared absorption band (KBr): 3297, 2963, 1640, 1531, 1509, 1199 cm ^-1

^{(5) 1 H-NMR (} pyridine- d 5, 900 MHZ): see Table 2

^{(6) 13 C-NMR (} pyridine- d 5, 225 MHZ): see Table 2

Nuclear Magnetic Resonance Spectra of O location ^One H ¹³ C location ^One H ¹³ C One 174.3 C 39 173.2 C 2 4.70 58.3 CH 40 4.96 54.3 CH 2-NH 9.26 40-NH 9.39 3 2.23 32.8 CH 41 2.40 31.9 CH 4 1.12 19.3 CH ₃ 42 1.14 19.0 CH ₃ 5 0.97 18.6 CH ₃ 43 1.03 19.7 CH ₃ 6 172.7 C 44 171.7 C 7 5.49 60.0 CH 45 5.65 54.8 CH 7-NH 9.66 46a 1.74 38.9 CH ₂ 8 5.94 73.2 CH 46b 1.58 9 143.0 C 47 1.43 25.1 CH 10 7.68 127.0 CH 48 0.67 23.1 CH ₃ 11 7.41 128.6 CH 49 0.67 21.9 CH ₃ 12 7.27 127.5 CH 50 3.52 31.3 CH ₃ 13 7.41 128.6 CH 51 173.5 C 14 7.68 127.0 CH 52 5.29 55.6 CH 15 174.5 C 52-NH 8.06 16 5.41 58.3 CH 53 2.57 31.0 CH 16-NH 8.02 54 1.20 19.0 CH ₃ 17 2.66 33.1 CH 55 1.16 19.7 CH ₃ 18 1.36 19.8 CH ₃ 56 170.6 C 19 1.20 20.0 CH ₃ 57 5.61 62.4 CH 20 169.9 C 58 5.07 66.5 CH 21 4.59 70.6 CH 59 1.34 20.5 CH ₃ 22a 4.45 17.1 CH ₂ 60 3.68 34.5 CH ₃ 22b 4.32 61 171.4 C 23 112.9 C 62 5.86 52.5 CH 24 7.19 124.3 CH 62-NH 10.17 24-NH 11.80 63 6.04 69.4 CH 25 139.5 C 64 1.52 16.8 CH ₃ 26 7.30 105.8 CH 65 173.4 C 27 7.27 122.7 CH 66 5.24 57.9 CH 28 6.67 99.5 CH 66-NH 8.65 29 154.9 C 67 2.05 27.6 CH 30 3.83 55.4 CH ₃ 68 0.95 19.8 CH ₃ 31 118.4 C 69 0.95 18.8 CH ₃ 32 2.51 40.8 CH ₃ 70 174.2 C 33 169.4 C 71 3.08 71.5 CH 34 3.19 70.9 CH 72 2.16 32.2 CH 35 3.03 29.0 CH 73 1.11 19.1 CH ₃ 36 1.22 21.9 CH ₃ 74 1.11 19.1 CH ₃ 37 1.02 19.7 CH ₃ 75 2.54 35.9 CH ₃ 38 3.18 39.9 CH ₃ 75 2.54 35.9 CH ₃

The chemical structure of O. nisamin A analyzed from nuclear magnetic resonance spectral data is shown below.

The chemical structure of O. nisamycin B analyzed from nuclear magnetic resonance spectrum data is shown below.

Example  5. Oh, my God  Identification of antimicrobial activity of A and B

Four species of Aspergillus fumigatus (HIC6094), Trichophyton rubrum (IFO9185), Trichophyton mentagrophytes (IFO40996), and Candida albicans (ATCC 10231) , Staphylococcus aureus (ATCC 6538p), Bacillus substilis (ATCC 6633), Kocuria rhizophila (NBRC 12708), Salmonella enteric (TCC14028), Proteus herb The antimicrobial activity of O.nosamycin A and B against six species of Bacillus, Proteus hauseri (NBRC3851), and Escherichia coli (ATCC 35270) was confirmed.

Each strain required for the test was a 15% glycerol stock stored at -80 ° C. Six bacteria were streaked on Luria Bertani (LB) agar medium, C. albicans were streaked on Yeast Peptone Dextrose (YPD) agar medium and the other three fungi were cultured on Potato Dextrose agar (PDA) medium. Bacteria were incubated in a 37 ° C incubator for 12 to 16 hours, in the case of C. albicans in a 28 ° C incubator for 48 hours, and for the other three fungi in a 28 ° C incubator for 2 weeks. After the pre-culture, bacteria were inoculated into LB (5 ml) and C. albicans in YPD (5 ml) liquid medium by inoculation with a single colony isolated from the solid medium. Culture conditions were shake cultured at 37 ° C and 180 rpm for 12-16 hours, and C. albicans was cultured at 28 ° C and 250 rpm for 12 hours and 16 hours with shaking. The other three fungi were obtained by spore culture using 0.1% tween 80 solution without liquid culture. The medium used for the antifungal activity test was Potato Dextrose Broth (PDB, Difco), and the medium used for the antibacterial activity test was m Plate Count Broth (mPCB, Difco).

Each test substance was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / ml. The test substance was diluted to 100 μg / ml in a 96-well plate with two-fold dilution To a concentration of 0.39 [mu] g / ml. The bacteria used in the test were 1 × 10 ⁵ cells / ml for bacteria, 1 × 10 ³ cells / ml for C. albicans, and 1 × 10 ⁵ spores / ml for three fungi except C. albicans. Growth inhibition activity was confirmed after culturing for 12 to 16 hours at 37? And for 48? At 28? For fungi. As a positive control, 10 mg / ml of ampicillin was used as the antimicrobial agent and 10 mg / ml of amphotericin B was used as the antifungal agent. All the media used in the test were subjected to high pressure steam sterilization.

As a result, Ohnosamycin A and B significantly inhibited the growth of Bacillus subtilis, Cocuria lipidophilus, and Proteus hastilleri. The minimum inhibitory concentration (MIC, mg / ml) of the compound completely inhibiting the growth of the bacteria was determined and the results are shown in Table 3.

Test strain The MIC (mg / ml) The MIC (mg / ml) Staphylococcus aureus - - Bacillus Subtilis 6.25 50 Cocuria rihizopila 1.56 12.5 Proteus Hauser 3.12 25 Salmonella entericica - - Escherichia coli - -

("-": does not inhibit growth)

As shown in Table 3, it was confirmed that O. nisamycin A and B had a strong growth inhibitory activity against pathogenic bacteria.

Example 6. Toxicity test of omnosamycin A and B against a common cell line

The cells were cultured in RPMI 1640 medium (A549, HCT116, SNU638) containing 10% fetal bovine serum, 1% penicillin-K-streptomycin, Dulbecco's modified Eagle's medium (K-HEP- -231) and minimal essential medium (MRC-5) were incubated at 37 ℃ in a constant temperature and humidity 5% CO ₂ incubator. The cell passage was performed once every 3 days using 0.25% trypsin-1 mM EDTA.

MRC5 cells were cultured in RPMI 1640 medium (A549, HCT116, SNU638) containing 10% fetal bovine serum, 1% penicillin-K-streptomycin, Dulbecco's modified Eagle's medium (K- MB-231) and minimal essential medium (MRC-5) were incubated at 37 ℃ in a constant temperature and humidity 5% CO ₂ incubator. The cell passage was performed once every 3 days using 0.25% trypsin-1 mM EDTA.

190 μl of 7 × 10 ³ cells / ml (MRC-5) per sample and 10 μl of sample were added to each well of a 96-well plate in a 96-well microplate for 72 hours at 37 ° C. in a 5% CO ₂ incubator Lt; / RTI > 50 μl of 50% TCA (trichloroacetic acid) was added to each well, fixed at 4 ° C for 30 minutes, washed 5 times with distilled water, and dried at room temperature. 100 μl of a 0.4% SBR solution dissolved in 1% acetic acid was added to each well, and the mixture was stained for 1 hour, washed 5 times with 1% acetic acid, and then dried. The stained SRB was then dissolved in 200 μl of 10 mM Tris buffer (pH 10.0) and the absorbance was measured at 515 nm using an ELISA reader, and the toxicity of Ohnosamycin A and B was measured.

As a result, the 50% growth inhibitory concentration (IC ₅₀ ) value for the common cell line of C. nisamycin A was determined, and the results are shown in Table 4 and FIG. 4A.

Concentration (μM) Standard Deviation Cell survival rate (%) IC ₅₀ ([mu] M) 2.5 1.56 81 > 40 5 7.77 87 10 4.71 83 20 4.02 85 40 2.02 92

In addition, the 50% growth inhibitory concentration (IC ₅₀ ) value for the common cancer cell line of C. nisamycin B was determined, and the results are shown in Tables 5 and 4B.

Concentration (μM) Standard Deviation Cell survival rate (%) IC ₅₀ ([mu] M) 2.5 79 4.789 > 40 5 74 5.319 10 71 2.398 20 77 5.590 40 78 1.804

Example  7. Oh, my God  Confirmation of antitumor activity of A

The SRB test (Sulforhodamine B assay) was performed using the method of Doll R and Peto R (1981). (Human lung carcinoma), HCT116 (Human colon carcinoma), SNU638 (Human stomach carcinoma), SK-HEP-1 (Human liver carcinoma), MDA- 5 (Human lung fibroblast) cells were used.

The cells were cultured in RPMI 1640 medium (A549, HCT116, SNU638) containing 10% fetal bovine serum, 1% penicillin-K-streptomycin, Dulbecco's modified Eagle's medium (K-HEP- -231) and minimal essential medium (MRC-5) were incubated at 37 ℃ in a constant temperature and humidity 5% CO ₂ incubator. The cell passage was performed once every 3 days using 0.25% trypsin-1 mM EDTA.

The cultured cells were seeded in 96-well microplates at 7 × 10 ³ cells / ml (MRC-5), 5 × 10 ³ cells / ml (SK-HEP-1, SNU638) per well. 4 × 10 ³ cells / ml (A549, MDA-MB-231), 3.5 × 10 ³ cells / ml (HCT116) Were added to each well of a 96-well plate and cultured in a 5% CO ₂ incubator at 37 ° C for 72 hours. 50 [mu] l of 50% TCA was added to each well, fixed at 4 [deg.] C for 30 minutes, washed 5 times with distilled water and dried at room temperature. To each well 1, 100 μl of a 0.4% SBR solution dissolved in 1% acetic acid was added and stained for 1 hour, washed 5 times with 1% acetic acid, and then dried. After that, the stained SRB was dissolved in 200 μl of 10 mM Tris buffer (pH 10.0), absorbance was measured using an ELISA reader at 515 nm, and the inhibitory effect of cancer cells was measured.

As a result, it was found that 50% inhibition of hepatocellular carcinoma cells (SK-HEP-1), breast cancer cells (MDA-MB-231), stomach cancer cells (SNU638), colon cancer cells (HCT116), and lung cancer cells (A549) The inhibitory concentration (IC ₅₀ ) values were determined and the results are shown in Table 6 and Figures 5A-5E.

cell Concentration (nM) Cell survival rate (%) IC ₅₀ (nM) SK-HEP-1 1600 -15.424 816 800 51.940 400 94.979 200 97.802 100 97.384 50 95.006 25 96.578 12.5 100.734 MDA-MB-231 1600 -3.986 688 800 31.815 400 97.362 200 101.628 100 77.287 50 92.590 25 84.737 12.5 101.376 SNU638 1600 17.199 532 800 17.550 400 69.907 200 71.046 100 66.297 50 64.414 25 58.874 12.5 66.510 HCT116 1600 7.388 359 800 7.526 400 47.829 200 48.037 100 59.701 50 48.640 25 47.568 12.5 64.485 A549 1600 -2.640 551 800 7.348 400 103.037 200 148.248 100 173.705 50 65.552 25 78.322 12.5 100.960

In addition, 50% growth was observed for the hepatocarcinoma cells SK-HEP-1, breast cancer cells (MDA-MB-231), stomach cancer cells (SNU638), colon cancer cells (HCT116), and lung cancer cells (A549) The inhibitory concentration (IC ₅₀ ) values were determined and the results are shown in Table 7 and Figures 6a to 6e.

cell Concentration (μM) Cell survival rate (%) IC ₅₀ ([mu] M) SK-HEP-1 2.5 101 16.8 5 94 10 74 20 41 40 35 MDA-MB-231 2.5 92 12.7 5 83 10 59 20 35 40 26 SNU638 2.5 88 13.5 5 81 10 66 20 34 40 18 HCT116 2.5 99 12.4 5 90 10 77 20 27 40 20 A549 2.5 92.78 15.6 5 82 10 63 20 43 40 35

As shown in Tables 6 and 7, it was confirmed that ocisamycin A and B have strong cytotoxicity against cancer cell lines.

Korea Biotechnology Research Institute KCTC18240P 20130206

<110> SEOUL NATIONAL UNIVERSITY R & DB Foundation <120> Novel cyclic peptide compound, a use thereof, and preparing          method thereof <130> PN099692 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1469 <212> DNA <213> 16S rDNA of Streptomyces sp. SNJ042 strain <400> 1 tcaggacgaa cgctggcggc gtgcttaaca catgcaagtc gaacgatgaa ccggtttcgg 60 ccggggatta gtggcgaacg ggtgagtaac acgtgggcaa tctgccctgc actctgggat 120 aagcccggga aactgggtct aataccggat acgacactcc gaggcatctc ggggtgtgga 180 aagttccggc ggtgcaggat gagcccgcgg cctatcagct tgttggtggg gtaatggcct 240 accaaggcga cgacgggtag ccggcctgag agggtgaccg gccacactgg gactgagaca 300 cggcccagac tcctacggga ggcagcagtg gggaatattg cacaatgggc gaaagcctga 360 tgcagcgacg ccgcgtgagg gatgacggcc ttcgggttgt aaacctcttt cagcagggaa 420 gaagcgaaag tgacggtacc tgcagaagaa gcaccggcta actacgtgcc agcagccgcg 480 gt; ttgtcgcgtc gattgtgaaa gcccggggct taaccctggg tctgcagtcg atacgggcag 600 gctagagttc ggcaggggag actggaattc ctggtgtagc ggtgaaatgc gcagatatca 660 ggaggaacac cggtggcgaa ggcgggtctc tgggccgata ctgacgctga ggagcgaaag 720 cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacgg tgggcactag 780 gtgtgggcaa cattccacgt tgtccgtgcc gcagctaacg cattaagtgc cccgcctggg 840 gagtacggcc gcaaggctaa aactcaaagg aattgacggg ggcccgcaca agcggcggag 900 catgtggctt aattcgacgc aacgcgaaga accttaccaa ggcttgacat acatcggaaa 960 cggccagaga tggtcgcccc cttgtggtcg gtgtacaggt ggtgcatggc tgtcgtcagc 1020 tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgtc ctgtgttgcc 1080 agcaacctct tcggaggttg gggactcacg ggagactgcc ggggtcaact cggaggaagg 1140 tggggacgac gtcaagtcat catgcccctt atgtcttggg ctgcacacgt gctacaatgg 1200 ccggtacaat gagctgcgat gccgtgaggt ggagcgaatc tcaaaaagcc ggtctcagtt 1260 cggattgggg tctgcaactc gaccccatga agtcggagtc gctagtaatc gcagatcagc 1320 attgctgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacgt catgaaagtc 1380 ggtaacaccc gaagccggtg gcctaacccc ttgtgggagg gagctgtcga aggtgggact 1440 ggcgattggg aagaagtcgt aacaaggta 1469

Claims (20)

A compound comprising the peptide, an isomer, derivative, or pharmaceutically acceptable salt thereof.
[Formula 1]

Wherein R is H, a C ₁ to C ₂₀ alkyl group, a C ₂ to C ₂₀ alkenyl group, a C ₂ to C ₂₀ alkenyl group, or a C ₃ to C ₂₀ aryl group. 2. The compound of claim 1, wherein R is H or methyl, an isomer, derivative, or pharmaceutically acceptable salt thereof. The compound according to claim 1, wherein the compound represented by formula
Val is substituted with Ala, Leu, Ile, Phe, Trp or Met;
Thr is substituted with Gly, Ser, Cys, Tyr, Asn or Gln;
Leu is substituted with Ala, Val, Ile, Phe, Trp or Met;
Trp is substituted with Ala, Val, Leu, Ile, Phe or Met; or
Phe is substituted with Ala, Val, Leu, Ile, Trp or Met, an isomer, derivative, or pharmaceutically acceptable salt thereof. The method according to claim 1, wherein the Trp is 4-OCH ₃ -Trp of the compound, acceptable isomers, derivatives, or a pharmaceutical salt thereof, the applicable one. 2. The compound of claim 1, wherein Phe is HO-Phe, an isomer, derivative, or pharmaceutically acceptable salt thereof. The compound according to claim 1, wherein N in the peptide bond in the amino acid of Trp, Leu, Val ¹ , Val ⁵ , Thr ² , or a combination thereof in Formula 1 is a C ₁ to C ₂₀ alkyl group is bonded, Or a pharmaceutically acceptable salt thereof. 7. The compound of claim 6, wherein the alkyl group is methyl, an isomer, derivative, or pharmaceutically acceptable salt thereof. The compound of claim 1, wherein the compound is represented by formula (1), an isomer, derivative, or pharmaceutically acceptable salt thereof.
[Formula 2]

Wherein R is H, a C ₁ to C ₂₀ alkyl group, a C ₂ to C ₂₀ alkenyl group, a C ₂ to C ₂₀ alkenyl group, or a C ₃ to C ₂₀ aryl group. 2. The compound according to claim 1, wherein the compound is represented by Formula 3, an isomer thereof, a derivative, or a pharmaceutically acceptable salt thereof.
[Formula 3]

The compound according to claim 1, wherein the compound is represented by Formula 4, an isomer thereof, a derivative, or a pharmaceutically acceptable salt thereof.
[Formula 4]

An antimicrobial composition comprising the compound of claim 1. 12. The composition of claim 11, wherein the composition is selected from the group consisting of Staphylococcus aureus, Bacillus subtilis, Kocuria rhizophila, Proteus hauseri, Salmonella enterica Salmonella enterica), Escherichia coli, or a combination thereof. The antimicrobial composition according to claim 11, which is a pharmaceutical composition or a composition other than medicines. A pharmaceutical composition for preventing or treating cancer comprising the compound of claim 1. 15. The pharmaceutical composition according to claim 14, wherein the cancer is colon cancer, stomach cancer, breast cancer or liver cancer. Streptomyces sp. SNJ042 strain (accession number: KCTC18240P) producing the compound of claim 1. Culturing Streptomyces sp. SNJ042 strain (Accession No .: KCTC18240P); And
And separating the compound of claim 1 from the culture. A method for sterilizing or bacterium of microorganisms, comprising applying, spraying, coating, dipping or a combination thereof in an in vitro, an antimicrobial composition comprising the compound of claim 1. A method for preventing or treating an infectious disease caused or caused by a microorganism, comprising the step of administering to a subject an antimicrobial pharmaceutical composition comprising the compound of claim 1. A method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition for the prevention or treatment of cancer comprising the compound of claim 1.

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