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    • 专利摘要:
    Bacterial strain of micromonospora matsumotoense producer of paulomycins and paulomycin produced by it. The invention provides a new bacterial strain of micromonospora matsumotoense isolated from its natural environment and deposited in the spanish type culture collection under the access number (cect 9281), which is capable of efficiently producing an antitumor compound of the family of fermentation by fermentation. The paulomycins. Specifically, this strain is a producer of a new paulomycin, designated in the present invention as paulomycin g, which has cytotoxic activity against tumor cells, preferably human, preferably of pancreas, breast and liver. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2613746A1
    申请号:ES201700259
    申请日:2017-03-13
    公开日:2017-05-25
    发明作者:Aida SARMIENTO VIZCAÍNO；Alfredo Javier FERNÁNDEZ BRAÑA；Luis Arsenio GARCÍA DÍAZ；José Luis ACUÑA FERNÁNDEZ；María Gloria BLANCO BLANCO；José Fernando Reyes Benítez；Ignacio PÉREZ-VICTORIA MORENO DE BARREDA；Jesús Melchor MARTÍN SERRANO；María Francisca VICENTE PÉREZ；Nuria DE PEDRO MONTEJO
    申请人:Universidad de Oviedo；
    IPC主号:
    专利说明:

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    BACTERIAL strain of MICROMONOSPORA MATSUMOTOENSE PRODUCER OF PAULOMYCINS AND PAULOMYCIN PRODUCED BY THE SAME
    The present invention falls within the pharmaceutical field, and specifically refers to new natural products with application in oncology, which are obtained by fermentation of a bacterial strain producing antitumor compounds of the paulomycin family. The invention also relates to a new paulomycin, paulomycin G, its method of obtaining and pharmaceutical compositions comprising it, which are useful for the treatment and / or prevention of tumors, preferably of pancreas, breast and liver in humans.
    STATE OF THE TECHNIQUE
    Paulomycins are glycosylated natural products whose structure has a residue of Paulic acid, which includes an isothiocyanate group, with pharmacological interest due to its antibiotic properties. Paulomycins A and B are antibiotics with very potent activities against Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus and other Streptomyces) and have therapeutic use in the treatment of gonococcal infections and Chlamydia (Novak, 1988, Patent PCT / US1987 / 002420). Initially described in S. paulus (Argoudelis et al., 1982, J. Antibiot. 41: 157-169) and later in S. albus J1074 (Majer and Chater, 1987, Mol. Biol. Evol. 4: 406-425 ), a series of paulomycins with various modifications in the two branched carbon chains of the paulomycosa were subsequently isolated from these Streptomyces species (Argoudelis et al., 1988. J. Antibiot. 41: 1316-1330; Majer et al., 1987, J. Gen. Microbiol. 133: 2503-2507); Brana et al., 2014, Arch Microbiol. 196: 345-355). The biosynthetic path of paulomycins is also an active research topic (Li et al. 2015, PLoS One. 10: e0120542; Gonzalez et al., 2016, Microb. Cell. Fact. 15:56) and to date it has not been He has described no chemical synthesis.
    In the oceans, sediments are lately one of the most studied marine sources for the isolation of actinobacteria (Ward and Bora, 2006 Curr. Opin. Microbiol. 9: 279-86; Hame $ -Kocaba§ and Uzel, 2012. J. Microbiol Methods. 88: 342-7). Previous work in the Cantabrian Sea (Bay of Biscay), North East Atlantic, has revealed that actinobacteria with biological activity, mainly species of
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    Streptomyces, are associated with corals and other invertebrates that live up to 4,700 m deep in the underwater canyon of Aviles (Brana et al., 2015, Microb Ecol 69: 512-24, Sarmiento-Vizcaino et al., 2015, Int. J Syst. Evol. Microbiol. 65: 1328-34, Sarmiento-Vizcaino et al., 2016, Microb. Ecol. 71: 375-86). It has also been described that paulomycins A and B are produced by a ubiquitous strain of Streptomyces albidoflavus widely distributed in terrestrial, marine and atmospheric environments on the Cantabrian coast (Sarmiento-Vizcaino etal., 2016, Microb. Ecol. 71: 375-86 ).
    DESCRIPTION OF THE INVENTION
    The present invention provides a new bacterial strain of Micromonospora matsumotoense that has been isolated from its natural environment and deposited in the Espahola Collection of Type Crops under the accession number (CECT 9281), which is capable of efficiently producing by fermentation cytotoxic compounds of the paulomycin family. Specifically, this strain is a producer of a new paulomycin designated in the present invention as paulomycin G, which exhibits cytotoxic activities against different tumor cell lines, preferably human.
    The inventors of the present invention have isolated said strain, Micromonospora matsumotoense M-412, from the coral reef ecosystems the underwater canon of Aviles. This strain was studied and subsequently identified. Therefore, said strain is described in the present invention, a method for obtaining paulomycins by fermentation therefrom and a new natural product of the paulomycin family with cytotoxic activity against human tumor lines, such as, but not limited to , pancreatic adenocarcinoma, breast adenocarcinoma and liver carcinoma lines.
    Therefore, we report here the discovery of a new natural product, Paulomycin G, obtained from M-412 Micromonospora matsumotoense, isolated from a sediment collected at 2,000 m. deep during an Oceanographic expedition in the underwater canyon of Aviles. All known paulomycins are produced by species of the genus Streptomyces, therefore, paulomycin G is the first member of the family produced by a species of the genus Micromonospora. Paulomycin G is a novel natural product, structurally unique since, according to our knowledge, it is the first member of the paulomycin family that lacks the paulomycosa residue. It is also the smallest known bioactive paulomycin, the minimum paulomycin described. Given its structural characteristics, the
    Paulomycin G is also of interest in biosynthetic studies and could be useful in future
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    investigations of paulomycin biosynthesis, or as a basic structure in the generation of new derivatives by means of combinatorial biosynthesis to generate structural diversity in the paulomycin family.
    Paulomycin G is also the first member of the family to show strong cytotoxic activities against different human tumor cell lines, such as pancreatic adenocarcinoma (MiaPaca_2), breast adenocarcinoma (MCF-7) and liver carcinoma (HepG2). However, these activities do not appear in paulomycin B, which was analyzed in parallel. Based on its cytotoxic activities, Paulomycin G deserves to be considered a candidate in cancer chemotherapy.
    The inventors determined the culture conditions of the strain for the production of the new paulomycin, subsequently purified it, proceeded to its structural elucidation and tested its cytotoxic activity against various human tumor lines.
    The present invention thus represents a solution to the need to have new antitumor compounds with biomedical potential. It also represents a solution to the need to have simple, short and economical procedures for obtaining said compound with antitumor activity, since the procedure described in the present invention allows to produce the said compound by fermentation with an actinobacterium, instead of by chemical synthesis, more complex, long and expensive process. In this sense, in biotechnological processes that involve obtaining structurally complex natural products, such as paulomycins, femnentation production by the producing microorganism is the preferred procedure, since it is simpler, shorter and more economical than the organic synthesis procedure.
    Thus, in a first aspect, the present invention relates to a bacterial strain of Micromonospora matsumotoense M-412 deposited on 02.15.2017 in the Spanish Type Culture Collection under the accession number CECT 9281. From now on, reference will be made to this bacterial strain as "strain of the invention" or "bacterial strain of the invention".
    Another aspect of the invention relates to a supernatant or extract of a culture of the bacterial strain of the invention. This supernatant will be referred to as
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    "supernatant of the invention", and comprises at least one compound of general formula (I) described below.
    The supernatant of the invention comprising at least one compound of formula (I) can be obtained by culturing the strain of the invention in the presence of a suitable culture medium and fermentation conditions. Said fermentation conditions and said culture medium are preferably those described later in the process of the invention. Subsequently, centrifugation of said bacterial culture can be carried out, by any method known to those skilled in the art for this purpose, and to the elimination of sediments deposited as a result of this centrifugation step, thus obtaining the supernatant of the invention which It comprises at least one compound of formula (I) and other metabolites produced and secreted by the strain of the invention in culture.
    Another aspect of the invention relates to the use of the bacterial strain of the invention for the production of compounds of general formula (I):
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    (I)
    or any of its salts or tautomers where,
    R1 is selected from hydrogen, substituted or unsubstituted Ci-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl or substituted or unsubstituted C2-C6 alkynyl.
    R2 is selected from hydrogen, substituted or unsubstituted C! -C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted CrC6 alkylaryl, substituted or unsubstituted benzyl or substituted benzoyl or not replaced
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    R3 and R4 are independently selected from hydrogen, CORa, COORa, substituted or unsubstituted C6 alkyl, substituted or unsubstituted C2-C6 alkenyl or C2-C6 alkynyl
    substituted or unsubstituted, where Ra is selected from hydrogen, Ci-C6 alkyl
    substituted or unsubstituted, C2-C6 alkenyl substituted or unsubstituted, C2-C6 alkynyl
    substituted or unsubstituted, substituted or unsubstituted aryl or a heterocyclic group
    substituted or unsubstituted.
    Another aspect of the invention relates to a process for obtaining a compound of general formula (I) described above, hereafter "process of the invention", comprising:
    to. cultivate the bacterial strain of the invention in a culture medium in
    fermentation conditions, and
    b. purify the compound of general formula (I) produced by the strain in culture of
    the stage (a).
    The cultivation of step (a) of the process of the invention can be carried out, for example, but not limited to, inoculating the strain or spores of the strain in an appropriate culture medium. Those skilled in the art will recognize the bacterial culture media that can be employed at this stage of the process of the invention. The "culture medium" is a suitable nutritive medium, that is, comprising the nutrients necessary for the maintenance and in vitro growth of the strain of the invention, for the development of its fermentation activity and, therefore, for the production of the compounds of formula (I) described above. Said culture medium may be liquid, solid or semi-solid. In order for the strain of the invention to grow properly in the culture medium, it must meet a number of conditions such as temperature, agitation, humidity, lightness and adequate oxygen pressure, as well! as a correct degree of acidity or alkalinity (pH). Likewise, the culture medium must be free of all contaminating microorganisms.
    Thus, the cultivation of step (a) takes place in a suitable nutrient medium comprising, for example, but not limited to, agar or gelatin or albumin, carbon sources (eg glucose, sucrose or mannitol), nitrogen sources ( for example, peptones), sulfur, phosphorus, sources of vitamins, amino acids and hormones and / or growth factors (for example, came extract or yeast extract), MOPS, inorganic salts (for example, calcium in the form of CaCI2, magnesium, manganese, sodium or potassium), hydrogen ions, etc. In a preferred embodiment, the culture medium
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    used in the inversion procedure is a culture medium comprising nutrients and physicochemical properties similar to those of the marine environment, more preferably the medium is marine R5A.
    The strain of the invention can be cultivated, for example, but not limited to, by flask culture with agitation, and small or large-scale fermentation (which includes continuous, batch or batch fermentation, batch or feed feed, or in solid state) carried out in a laboratory or industrial bioreactor in a suitable medium and under conditions that allow to express and / or isolate the compounds of formula (I) described above. The compounds of the invention are secreted, together with other metabolites or compounds, in the nutrient medium, and these can be recovered directly from the medium.
    The person skilled in the art will recognize the appropriate fermentation conditions to be applied in step (a) of the process of the invention. Preferably, said conditions comprise incubation under agitation, more preferably in orbital shaker, of the culture of the invention for 5 to 8 days, more preferably for 7 days; at an essentially constant temperature between 25 and 30 ° C, preferably between 27 and 29 ° C, more preferably at 28 ° C; at between 200 and 300 rpm, preferably between 250 and 270 rpm, more preferably at 250 rpm and at a constant pH between 6.0 and 7.5, preferably between 6.0 and 7.0, more preferably at 6.7.
    After cultivation of step (a) of the process of the invention, an additional centrifugation step can take place after which the sediments are discarded and the supernatants are selected, thus obtaining the supernatant of the invention. Said supernatants can then be filtered in step (b) of the process of the invention and can then be subjected to an extraction procedure, for example, but not limited to solid phase extraction, in order to elute the compound of the invention present therein.
    The compound of the invention secreted to the culture medium by the strain of the invention can be recovered from the medium using methods known in the art, for example by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation and / or precipitation.
    The compound of the invention produced by the strain in culture can be purified by a variety of methods known in the art including, but not
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    limitation, chromatography (for example, HPLC, ion exchange, affinity, hydrophobic, chromato-focus, and molecular exclusion), electrophoretic procedures (for example, preparatory isoelectric focusing), differential solubility (for example, precipitation with ammonium sulfate), SDS-PAGE, precipitation or extraction, in order to obtain the substantially pure compound of the invention.
    The compound of the invention produced by the strain in culture can be detected using methods known in the art. These detection procedures may include, for example, but not limited to, UPLC, UV, NMR, mass spectrometry, or the like.
    Another aspect of the invention relates to a compound of general formula (I) or any of its salts or tautomers (hereinafter "compound of formula (I) of the invention", "compound of the invention" or "paulomycin G "):
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    (I)
    or any of its salts or tautomers where,
    R1 is selected from hydrogen, C-alkyl, substituted or unsubstituted CG, substituted or unsubstituted C2-C6 alkenyl or substituted or unsubstituted C2-C6 alkynyl.
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    More preferably, the group R1 is selected from hydrogen, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted iso-propyl, substituted or unsubstituted n-butyl, tert-butyl substituted or unsubstituted, substituted or unsubstituted vinyl, substituted or unsubstituted allyl or substituted or unsubstituted ethynyl.
    In another preferred embodiment, the R1 group is selected from hydrogen and methyl.
    In another preferred embodiment, the R1 group is hydrogen.
    R2 is selected from hydrogen, substituted or unsubstituted CrC6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ci-C6 alkylaryl, substituted or unsubstituted benzyl and substituted or unsubstituted benzoyl 5 replaced.
    More preferably, the group R2 is selected from hydrogen, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted iso-propyl, substituted or unsubstituted n-butyl, tert-butyl substituted or unsubstituted, substituted or unsubstituted vinyl, substituted or unsubstituted allyl, substituted or unsubstituted ethynyl, substituted or unsubstituted acetyl, substituted or unsubstituted benzyl and substituted or unsubstituted benzoyl.
    Even more preferably, the R2 group is selected from hydrogen, acetyl, benzyl and benzoyl.
    In another more preferred embodiment, the R2 group is hydrogen.
    R3 and R4 are independently selected from hydrogen, CORa, COORa, substituted or unsubstituted (V C6 alkyl, substituted or unsubstituted C2-C6 alkenyl and substituted or unsubstituted C2-C6 alkynyl.
    Ra is selected from hydrogen, substituted or unsubstituted Ci-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group.
    More preferably R3 and R4 are independently selected from hydrogen, CORa, COORa and substituted or unsubstituted C ^ Ce alkyl, where Ra is selected from hydrogen and C or substituted or unsubstituted C6 alkyl. Particularly preferred Ra substituents are methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, tert-butyl, sec-butyl and iso-butyl.
    Even more preferred is the compound in which R3 and R4 are hydrogen.
    In particular, the present invention provides, among others, the compound with the following formula (II):
    image3
    Another aspect of the invention relates to a composition, hereinafter "composition of the invention", comprising the strain of the invention, the supernatant 5 of the invention or the compound of formula (I) of the invention. preferred embodiment, said composition further comprises another antitumor agent.
    Said composition may be a pharmaceutical or cosmetic composition, comprising the strain of the invention, the supernatant of the invention or the compound of formula (I) of the invention, and a pharmaceutically or cosmetically acceptable excipient or vehicle. Preferably, the composition of the invention comprises the strain of the invention, the supernatant of the invention or the compound of formula (I) of the invention in a therapeutically effective amount.
    By "therapeutically effective amount" is meant the amount of strain of the invention, supernatant of the invention or compound of the invention, which when administered to the subject to treat a tumor process produces the desired effect. Such desired effect may be, by example but without limiting ourselves, eliminating or reducing the number of tumor cells.The therapeutically effective amount may vary depending on a variety of factors, for example but not limited to, the type of tumor and its severity, as well as age, weight, sex. , physical condition, capacity of response or tolerance, etc. of the individual to whom the composition of the invention will be administered.
    The pharmaceutically or cosmetically acceptable excipients and vehicles that can be used in the composition of the invention are those known to those skilled in the art.
    The term "excipient" refers to a substance that aids in the absorption of the elements of the composition of the invention, stabilizes said elements, activates or aids in the preparation of the composition in the sense of giving it consistency. So that,
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    the excipients could have the function of keeping the ingredients together, such as for example starches, sugars or cellulose, the sweetening function, the coloring function, the protection function of the composition, for example, to isolate it from air and / or humidity, the filling function of a pill, capsule or any other form of presentation, such as for example the case of dibasic calcium phosphate, the disintegrating function to facilitate the dissolution of the components and their absorption, without exclude other types of excipients not mentioned in this paragraph.
    The "pharmacologically acceptable vehicle", like the excipient, is a substance or combination of substances that is used in the composition to dilute any of the components included therein to a certain volume or weight. The term "vehicle" refers to a diluent, adjuvant, excipient or carrier with which the composition of the invention should be administered; obviously, said vehicle must be compatible with said composition.Pharmaceutically acceptable vehicles can be, but not limited to, solids, liquids, solvents or surfactants. Examples of vehicles are, but are not limited to, water, oils or surfactants, including those of petroleum, animal, vegetable or synthetic origin, for example, and without limitation, peanut oil, soybean oil, mineral oil, sesame oil , castor oils, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxyols, poloxamers, polyoxyethylene, polyethylene glycols, dextrose, glycerol, digitonin and the like. The pharmacologically acceptable vehicle is an inert substance or action analogous to any of the elements included in the composition of the present invention. The function of the vehicle is to facilitate the incorporation of other elements, allow a better dosage and administration or give consistency and form to the composition. When the form of presentation is liquid, the pharmacologically acceptable vehicle is the diluent.
    The composition of the present invention can be formulated for administration to an animal, preferably a mammal, including man, in a variety of ways known in the state of the art. Examples of preparations include any solid composition (tablets, pills, capsules, powders, granules, bars, pencils, vaporizers, aerosols, etc.), semi-solid (ointment, cream, ointment, gel, hydrogel, foam, lotion, soap, jelly , gelatin, etc.) or liquid (aqueous or non-aqueous solutions, hydroalcoholic or hydro-glycol solutions, suspensions, emulsions, syrups, anhydrous compositions, aqueous dispersions, oils, milks, balms,
    liniments, serums, etc.) for oral, topical or parenteral administration. The composition of
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    The present invention may also be in the form of sustained release formulations or any other conventional release system. The term "sustained release" is used in the conventional sense by referring to a system of vehiculization of a compound that provides for the gradual release of said compound over a period of time and preferably, but not necessarily, with relatively constant levels of compound release. over a period of time. Illustrative examples of sustained release vehicles or systems include, but are not limited to, liposomes, mixed liposomes, oleosomes, niosomes, ethosomes, milicapsules, microcapsules, nanocapsules, sponges, cyclodextrins, vesicles, micelles, mixed surfactant micelles, phospholipid mixed micelles - surfactant, microspheres, microspheres, nanospheres, lipospheres, microemulsions, nanoemulsions, miniparticles, miliparticles, microparticles, nanoparticles, solid lipid nanoparticles, nanostructured lipidic supports, polymeric materials, biodegradable or non-biodegradable microparticles, such as bioparticles, microparticles biodegradable microspheres.
    Such compositions and / or their formulations may be administered in a variety of ways, including, but not limited to, parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrastromal, intraarticular, intrasynovial, intrathecal, intralesional, intraarterial, intracardiac, intramuscular, intranasal, intracranial, cutaneous or subcutaneous, intraorbital, intracapsular, topical, ophthalmological or ocular, through transdermal or rectal or vaginal patches, by administering a suppository or encapsulated, percutaneous, nasal spray, surgical implant, internal surgical pump, pump infusion or via catheter.
    The compositions of the present invention are suitable for application by medical devices that allow the release of the active ingredient in concentrations suitable for the treatment of tumor processes or bacterial infections. These devices should preferably be suitable for administration of the active ingredient locally, allowing the treatment to act in the affected area and not be dispersed. The devices may, for example, but not be limited, carry the active ingredient inside or be coated therewith.
    The use of the strain of the invention, of the supernatant of the invention, of the compound of formula (I) of the invention or of the composition of the invention can be used in the treatment of cancer.
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    Therefore, another aspect of the invention relates to the use of the supernatant of the invention, of the compound of formula (I) of the invention or of the composition, preferably pharmaceutical, of the invention, for the preparation of a medicament. Alternatively, this aspect of the invention relates to the supernatant of the invention, the compound of formula (I) of the invention or the composition of the invention for use as a medicament.
    Another aspect of the invention relates to the use of the supernatant of the invention, the compound of formula (I) of the invention or the composition of the invention for the preparation of a medicament for the treatment of cancer.
    The compound of formula (I) of the invention is tumor growth inhibitor and is therefore useful in the treatment of cancer.
    Thus, pharmaceutical compositions comprising an effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof together with a pharmaceutically acceptable excipient are included in the present description.
    The use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament is also included in the present description.
    Also included in the present description is the use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof to inhibit the growth of a tumor.
    As used herein, "inhibit" means decrease, slow down or stop. Therefore, a compound of this invention may decrease, slow down or stop the growth of a tumor cell. As used herein, "growth" means increase in tarnaho, or proliferation or both. Therefore, a compound of this invention can inhibit the increase in tarnaho of a tumor cell and / or can prevent the tumor cell from dividing and increase the number of tumor cells. A "tumor cell" is a cell that constitutes a neoplasm (new growth), which can be cancerous (malignant) or non-cancerous (benign). A cancerous tumor cell can invade normal surrounding tissues and blood / lymphatic vessels and form metastases in tissues away from the original tumor. On the contrary, a non-cancerous tumor cell can grow and compress adjacent normal tissues but cannot
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    invade normal tissues and blood / lymphatic vessels, and also can not metastasize in tissues away from the original tumor.
    Also included in the present description is the use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof to treat cancer.
    The use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament with antitumor activity is also included in the present description.
    The use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for the treatment of cancer is also included in the present description.
    Also included in the present description is a method of treating a subject, including a human being, diagnosed with cancer, which consists in treating said subject with a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate.
    In another preferred embodiment, the medicament referred to in the present invention is for the treatment of cancer by inhibiting the growth of the cells constituting the tumor, thereby preventing the invasion of normal tissues and blood / lymphatic vessels by tumor cells and, therefore, prevents metastasis. Examples of cancers that can be treated include pancreas and breast, but are not limited to ovary, prostate, testicle, melanoma, kidney, central nervous system and leukemia. The term "acceptable pharmaceutical composition" consists of a biologically suitable material, that is, that the material can be administered to the subject without causing substantially harmful biological effects.
    The term "medicament", as used in this invention, refers to any substance used for the treatment of tumors in man, or any other animal, and plants. In the context of the present invention, this term refers to a preparation comprising the supernatant of the invention, the compound of formula (I) of the invention or the composition of the invention.
    The medicament referred to in the present invention can be for human or veterinary use. The "medicine for human use" is any substance or combination of substances that is presented as having properties for the treatment or prevention of diseases in humans or that can be used in humans
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    or be administered to human beings in order to restore, correct or modify physiological functions by exercising a pharmacological, immunological or metabdlican action, or to establish a medical diagnosis. The "veterinary medicinal product" is any substance or combination of substances that is presented as having curative or preventive properties with respect to animal diseases or that can be administered to the animal in order to restore, correct or modify its physiological functions by exercising a pharmacological, immunological or metabolic action, or to establish a veterinary diagnosis, including, but not limited to, drug premixes. "Medication premix" or "premix for medicated foods" means any veterinary medicinal product prepared in advance with a view to the subsequent manufacture of medicated foods. "Medicated food" means any mixture of veterinary medicinal product (s) and food (s) prepared previously for sale and intended to be administered to animals without transformation, due to curative or preventive properties or Other properties of the medicine.
    The medicament of the invention can be used both alone and in combination with other medicaments or compositions for the treatment of tumor processes. Thus, the medicaments of the present invention can be used together with other active ingredients or therapies as a combination therapy. The other active ingredients may be part of the same composition or they may be provided by a different composition, being administered at the same time or at different times.
    The composition of the present invention, preferably pharmaceutical or cosmetic, may alternatively or additionally include another antitumor compound.
    The term "treatment", as understood in the present invention, refers to combating the effects caused as a consequence of the disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) that includes:
    (i) inhibit the disease or pathological condition, that is, stop its development;
    (ii) alleviate the disease or the pathological condition, that is, cause the regression of the infection, disease or the pathological condition or its symptomatology;
    (iii) stabilize the disease or the pathological condition.
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    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    DESCRIPTION OF THE FIGURE
    FIG. 1. Phylogenetic tree obtained by the “neighbor-joining” method obtained by matrix analysis of distances of the 16S rRNA gene sequences, showing the position of Mtsumotoense M-412 Micromonospora and its closest phylogenetic relatives
    The numbers of the nodes are bootstrap values (1000 samples; only values> 70% are presented). The bar indicates 0.2% sequence divergence.
    EXAMPLES
    The invention will now be illustrated by tests carried out by the inventors, which show the effectiveness of the strain of the invention in the production of a new compound of the paulomycin family (designated herein as paulomycin G), with antitumor activity against human tumor lines.
    EXAMPLE 1. EXPERIMENTAL SECTION
    General Experimental Procedures
    Semi-preparative HPLC analyzes and separations were carried out using an Alliance chromatographic system with a SunFire C18 column (10 pm, 10 x 250 mm, Waters). For UPLC analyzes, an Acquity UPLC equipped with a BEH C18 column (1.7 pm, 2.1 x 100 mm, Waters) was used. The optical rotation was determined with a JASCO P-2000 polarimeter. IR spectra were measured with a JASCO FT / IR-4100 spectrometer equipped with a PIKE MIRacle ™ ATR accessory (simple reflection). NMR spectra were recorded on a Bruker Advance III spectrometer (500 and 125 MHz for 1H and 13C NMR, respectively) equipped with
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    a 1.7 mm TCI MicroCryoProbe ™ probe, using the residual solvent serial as an internal reference (SH 2.50 and 5C 39.5 ppm for DMSO-c / 6). HRESIMS spectra were acquired using a Bruker maXis QTOF mass spectrometer.
    Microorganisms and fermentation conditions
    Strain M-412 (CECT 9281) was isolated from a sample of sediment collected in the Cantabrian Sea at a depth of 2,000 m. 20 Erlenmeyer flasks (250 ml) were prepared, each containing 50 ml of GHSA medium with the following composition: 1% glucose, 1% soybean meal, 0.05% yeast extract, 2.1% MOPS, 0.06% MgS04. 7H20, 0.2% trace element solution of the R5A medium (Fernandez et al. 2015, J. Bacteriol 180: 4929-4937), pH 6.8. After autoclaving, 0.4% of a 5M CaCI2 2H20 solution and 3% DMSO was added to the medium. The flasks were inoculated with spores and incubated on an orbital shaker at 28 ° C and 250 rpm for 7 days.
    Phylogenetic analysis (taxonomy) of the producing microorganism
    Strain M-412 (CECT 9281) was subjected to phylogenetic analysis based on the analysis of the 16S rRNA sequence. The phylogenetic analysis was performed using MEGA version 6.0 after multiple alignment of the data using CLUSTALO. The distances (distance options according to the Kimura two-parameter model) and alignment with the neighbor-joining method were determined using bootstrap values based on 1000 replications.
    Cytotoxic activity of the compound
    The MTT colorimetric assays (3- (4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide), which measure mitochondrial metabolic activity, were performed with five lines of tumor cells and a non-tumor cell line. Ten thousand cells per well (for 72 h analysis) were introduced into 96-well plates with a robotic cell culture system, SelecT (TAP Biosystems, Royston, UK) After 24 h of incubation, the compound was added with a Biomek FX automatic pipettor (Beckman Coulter, Pasadena, CA), and the plates were incubated for an additional 72 hours.The compound was tested in triplicate, using different concentrations, in a V2 dilution curve MTT was added and the plates were read in a Victor2TM Wallac spectrofluorometer (PerkinElmer).
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    Taxonomy of strain M-412
    The 16S rDNA of the producing strain M-412 (CECT 9281) was amplified by polymerase chain reaction (PCR) and sequenced. Sequence analysis demonstrated 100% identity with Micromonospora matsumotoense ('accession number AF152109); therefore, this strain was designated as Micromonospora matsumotoense M-412. The phylogenetic tree generated by the neighborhood-based method based on the 16S rRNA gene sequence clearly revealed the evolutionary relationship of strain M-412 with a group of known Micromonospora species (Figure 1).
    Isolation and purification of paulomycin G
    The cultures were centrifuged and the sediments were extracted with ethyl acetate with 1% formic acid. The supernatants were filtered and applied to a solid phase extraction cartridge (Sep-Pak Vac C18, 10g, Waters). The retained material was eluted using a gradient of methanol and 0.05% trifluoroacetic acid (TFA) in water from 0 to 100% methanol in 60 min, at 5 ml / min. Fractions were taken every 5 min and analyzed by UPLC using previously described chromatographic conditions (Brana et al., 2014, Microb Ecol 69: 512-24). In the fractions collected between 40 and 45 min, a peak corresponding to an unknown paulomycin was observed. These fractions were pooled, partially dried under vacuum and applied to a solid phase extraction cartridge (Sep-Pak Vac C18, 2g, Waters). The cartridge was washed with water and the retained compounds were eluted with methanol and dried in vacuo. The residue was subsequently redissolved in a small volume of acetonitrile and DMSO (2: 1). The same peak of unknown paulomycin was also found in the organic extract of the culture sediments, which was dried and redissolved as indicated above. The desired compound was purified by semi-preparative HPLC using an Atlantis C18 column (10 pm, 10 x 150 mm, Waters). The purification was done in two steps. The mobile phase was a mixture of 50% acetonitrile in water in the first step and 55% methanol and water in the second step, using isoctratic elution at 5 ml / min. In both cases, the solution containing the collected peak was evaporated and finally lyophilized, with a yield of 2.7 mg of pure product.
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    Paulomycin G: pale yellow solid; [a] 1JD + 10.6 ° (c 0.18, MeOH); UV (DAD) A.max 238, 276 and 320 nm; IR (ATR) vmax 3359, 3229, 2979, 2933, 2041, 1736, 1695, 1626, 1571, 1442, 1381, 1260, 1227, 1129, 1025, 909, 751 cm'1; for 1 H and 13 C NMR data see Table 1; HRESIMS m / z 521.0828 [M + Na] + (calcd. For C ^ H ^ OnSNa ", 521.0837) 516.1278 [M + NH4r (calcd. For CzoH ^ NaOnS *. 516.1283), 499.1015 [M + Hf (calcd. tS *. 499.1017).
    Structural elucidation
    Paulomycin G has the molecular formula C20H22N2OHS according to its ESI-TOF MS data (m / z 499.1015 [M + H] +, calcd. For CjqHssNjOhS4, 499.1017). Its UV spectrum shows maximum at 238, 276 and 320 nm, in accordance with a paulomycin-like structure. An intense absorption band at 2041 cm "1 in its IR spectrum confirmed the presence in the molecule of an isothiocyanate group, present in the Paulic acid residue of all paulomycins. NMR spectra (Table 1) confirmed the presence of this Paulic acid residue, with signals for a methyl group (4i 1.89, Sc 14.6 ppm) coupled to a sp2 proton (4i 6.71, <5c 136.9 ppm) in the COZY spectrum. The HMBC correlations of the last proton to carbons at & 159.8 (carbonyl group a, p-unsaturated C1 "), 122.3 ppm (sp2 C2" quaternary carbon), 141.6 ppm (C5 "isothiocyanate carbon, 4-link correlation distance), and 14.6 ppm (C4" methyl) completed the structural assignment of the residue of paulic acid.
    In addition, signals for an aliphatic methylene at Su 3.22 and 3.17 that correlates in the HMBC spectrum with carbon signals at 159.4 (C3), 188.8 (C4), 77.5 (C6 and C8), and 197.5 (C7), and the presence of two additional signals at Sc 169.0 and 99.1 ppm indicated the presence in the substructure molecule of the substituted 2-amino-5-hydroxy-3,6-dioxcyclohex-1-enecarboxylic acid in position C6, present in the family of compounds of Paulomycins Additionally, signals for five oxygenated metins at A 3.69, 361, 5.29, 4.51, and 3.79 ppm, and an aliphatic methyl group at 0.88 ppm formed a spin system, which according to the correlations observed in the COZY spectrum, represent the ring B in the structure of the molecule. The connection between carbon C6 of ring A and carbon C8 in ring B was further confirmed by the HMBC correlations observed between H8 and C5, C6, and C7. Finally, signals for acetyl functionality (<) h 2.10 ppm, Sc 170.1 and 20.8 ppm) were observed. This acetyl group was located at C10 based on HMBC correlations of H2 'and H10 to carbonyl carbon CT and the low-field chemical shift of
    H10 The Paulic acid residue was similarly located at C11 based on an HMBC correlation observed between H11 and C1 ". The relative configuration proposed around the chiral centers in ring B was based on coupling constants and correlations observed in the NOESY spectrum, and the absolute configuration was assumed to be the same as in other compounds of the paulomycin series.
    Table 1. NMR data (S in ppm) for paulomycin G (DMSO-d6, 500 MHz, 24 ° C).
     Pos.  5 (13C) 5 (H), (mult, Jen Hz)
     one  169.0
     2  99.1
     3  159.4
     4  188.8
     5  47.7 3.22 (d, 16.0), 3.17 (d, 16.0)
     6  77.5
     7  197.5
     8  77.5 3.69 (d, 9.9)
     9  67.0 3.61 (brdt, 9.7, 3.3)
     10  69.8 5.29 (dd, 2.6, 2.6)
     eleven  73.4 4.51 (dd, 9.9, 2.6)
     12  69 9 3.79 (dq, 9.9, 6.2)
     13  16.3 0.88 (d, 6.2)
     V  170.1
     2'  20.8 2.10 (s)
     one"  159.8
     3"  136.9 6.71 (quart., 7.1)
     4"  14.6 1.89 (d, 7.1)
     5"  141.6
     NH2 (3)    9.71 (brs), 9.35 (br s)
     OH (6)    5.43 (s)
     OH (9)    5.78 (d, 4.4)
    one
    HOOC
    image4
    Cytotoxic activity of paulomycin G
    Cytotoxic activity was observed for the compound at the concentrations tested against human breast adenocarcinoma cell lines (MCF-7),
    pancreatic adenocarcinoma (MiaPaca_2) and hepatocellular carcinoma (HepG2) (Table 2). Paulomycin B showed no cytotoxic activity against these three cell lines when tested in parallel.
    Table 2. Cytotoxic activity against different tumor cell lines.
    Paulomicina G Paulomicina B
     (ED50 hM) (EDS0 nM)
     HepG2  4.3> 36
     MCF-7  1.6> 36
     MiaPaca 2  2.7> 36
    10 In conclusion, a new natural product, paulomycin G, was isolated and characterized from a marine actinobacteria, Micromonospora matsumotoense M-412, obtained from a sediment collected at 2,000 m depth in the underwater Canon of Avilds, at sea Cantabrian. This compound exhibits important cytotoxic activities against human tumor cell lines. These results are an example of the relevance of marine natural products as candidates in antitumor chemotherapy.
    权利要求:
    Claims (16)
    [1]
    1. Bacterial strain of Micromonospora matsumotoense M-412 deposited in the Spanish Type Culture Collection under the access number CECT 9281.
    5 2. Supernatant of a strain culture according to claim 1.
    [3]
    3. Use of the strain according to claim 1 for the production of the compound of formula (I) or any of its salts or tautomers:
    image 1
    (I)
    10 where,
    R1 is selected from hydrogen, substituted or unsubstituted C ^ Ce alkyl, substituted or unsubstituted C2-C6 alkenyl or substituted or unsubstituted C2-C6 alkynyl;
    R2 is selected from hydrogen, substituted or unsubstituted Ct-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted Ci-C6 alkylaryl, substituted or unsubstituted benzyl or benzoyl substituted or unsubstituted;
    R3 and R4 are independently selected from hydrogen, CORa, COORa, substituted or unsubstituted CrCe alkyl, substituted or unsubstituted C2-C6 alkenyl or substituted or unsubstituted C2-C6 alkynyl, where Ra is selected from hydrogen, substituted Cr6 C6 alkyl or unsubstituted, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group.
    [4]
    4. Method for obtaining a compound of general formula (I), or any of its salts or tautomers, described in claim 3 comprising:
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    to. cultivating the strain according to claim 1 in a culture medium under fermentation conditions, and
    b. purify the compound of formula (I) produced by the culture strain of step (a).
    [5]
    5. Compound of general formula (I):
    image2
    (I)
    or any of its salts or tautomers, where,
    R1 is selected from hydrogen, substituted or unsubstituted C ^ Cg alkyl, substituted or unsubstituted C2-C6 alkenyl or substituted or unsubstituted C2-C6 alkynyl;
    R2 is selected from hydrogen, substituted or unsubstituted Ci-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C ^ Cg alkylaryl, substituted or unsubstituted benzyl or substituted benzoyl or unsubstituted;
    R3 and R4 are independently selected from hydrogen, CORa, COORa, substituted or unsubstituted C ^ Cg alkyl, substituted or unsubstituted C2-C6 alkenyl or substituted or unsubstituted C2-C6 alkynyl where Ra is selected from hydrogen, C ^ Cg alkyl substituted or unsubstituted, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted aryl or a substituted or unsubstituted heterocyclic group. 6
    [6]
    6. A compound according to claim 5 wherein the group R1 is selected from hydrogen, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted or unsubstituted iso-propyl, n-butyl
    substituted or unsubstituted, substituted or unsubstituted tert-butyl, substituted or unsubstituted vinyl, substituted or unsubstituted allyl or substituted or unsubstituted ethynyl.
    [7]
    7. A compound according to claim 5 wherein the group R1 is selected from hydrogen and methyl.
    A compound according to claim 5 wherein the R1 group is hydrogen.
    [9]
    9. A compound according to any of claims 5-8 wherein the group R2 is selected from hydrogen, substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-propyl, substituted iso-propyl or unsubstituted, n-butyl substituted or unsubstituted, substituted or unsubstituted tert-butyl,
    10 substituted or unsubstituted vinyl, substituted or unsubstituted allyl, substituted or unsubstituted ethynyl, substituted or unsubstituted acetyl, substituted or unsubstituted benzyl and substituted or unsubstituted benzoyl.
    [10]
    10. A compound according to claim 9 wherein the R2 group is selected from hydrogen, acetyl, benzyl and benzoyl.
    11. A compound according to claim 9 wherein the R2 group is hydrogen.
    [12]
    12. A compound according to any of claims 5-11 wherein the groups R3 and R4 are independently selected from hydrogen, CORa, COORa and substituted or unsubstituted CrC6 alkyl, wherein Ra is selected from hydrogen and substituted or unsubstituted CrC6 alkyl .
    13. A compound according to claim 12 wherein Ra is selected from
    methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, tert-butyl, sec-butyl and iso-butyl.
    [14]
    14. Compound according to claim 12 wherein the R3 and R4 groups are hydrogen.
    15. The compound of claim 5 with the following formula (II):
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    image3
    [16]
    16. Composition comprising the supernatant according to claim 2 or the compound according to claims 5 to 15.
    [17]
    17. Composition according to claim 16, which further comprises another antitumor agent.
    [18]
    18. Composition according to any of claims 16 or 17, wherein the composition is a pharmaceutical or cosmetic composition and also comprises a pharmaceutically or cosmetically acceptable excipient or vehicle.
    [19]
    19. Use of the supernatant according to claim 2, of the compound according to claims 5 to 15 or of the composition according to any of claims 16 or 17, for the preparation of a medicament.
    [20]
    20. Use of the supernatant according to claim 2, of the compound according to claims 5 to 15 or of the composition according to any of claims 16 or 17, for the preparation of a medicament for the treatment of tumors.
    [21]
    21. Use of claim 20, wherein the tumors are pancreatic or breast or liver.
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