CN110218174B - Compound and preparation method and application thereof - Google Patents
Compound and preparation method and application thereof Download PDFInfo
- Publication number
- CN110218174B CN110218174B CN201910625803.9A CN201910625803A CN110218174B CN 110218174 B CN110218174 B CN 110218174B CN 201910625803 A CN201910625803 A CN 201910625803A CN 110218174 B CN110218174 B CN 110218174B
- Authority
- CN
- China
- Prior art keywords
- compound
- culture
- iii
- days
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/56—Ring systems containing three or more rings
- C07D209/58—[b]- or [c]-condensed
- C07D209/62—Naphtho [c] pyrroles; Hydrogenated naphtho [c] pyrroles
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/56—Ring systems containing three or more rings
- C07D209/58—[b]- or [c]-condensed
- C07D209/62—Naphtho [c] pyrroles; Hydrogenated naphtho [c] pyrroles
- C07D209/64—Naphtho [c] pyrroles; Hydrogenated naphtho [c] pyrroles with an oxygen atom in position 1
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/56—Ring systems containing three or more rings
- C07D209/58—[b]- or [c]-condensed
- C07D209/62—Naphtho [c] pyrroles; Hydrogenated naphtho [c] pyrroles
- C07D209/66—Naphtho [c] pyrroles; Hydrogenated naphtho [c] pyrroles with oxygen atoms in positions 1 and 3
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
Abstract
The invention provides a compound and a preparation method and application thereof. The compound has a structure shown in formula (I):
wherein R is1Is selected from C1‑C3Alkyl and C1‑C3Alkoxy radical, R2And R3Each independently selected from H, -OH, C1‑C3Alkyl and C1‑C3An alkoxy group; alternatively, an isomer of the compound of formula (I) or a pharmaceutically acceptable salt thereof. The compound of the invention has good anti-tumor activity, can inhibit the proliferation of liver cancer, breast cancer and lung cancer cells, and has c-Met inhibitory activity. The compound is extracted and separated from the fermentation product of the marine actinomycete, has simple preparation process, easy operation and implementation, easy industrial large-scale production and wide application prospect.
Description
Technical Field
The invention relates to the field of medicines, and particularly relates to a compound and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Cancer is a complex disease caused by a variety of factors, also known as malignancy, and is characterized by uncontrolled growth and spread of abnormal cells to other sites, which in turn causes failure of tissues and organs, ultimately leading to death. According to the latest WHO statistics, the number of deaths due to cancer is as high as millions each year, and the number of various cancer patients will reach 2200 million worldwide by 2030, seriously threatening human health and quality of life. Among various biological targets closely related to tumorigenesis and development, the mesenchymal epithelial cell transformation factor (c-Met), which is one of important members of receptor tyrosine kinases, is widely considered as an important target for intervening and treating cancers, and is expected to provide more opportunities for treating various tumors.
The ocean is an open, complex ecosystem that lives hundreds of thousands of animals, plants, and hundreds of millions of microorganisms. The search for new high-activity compounds from marine animals, plants and microorganisms has become a new field of natural product research. The marine actinomycete metabolite is rich, the structure is diversified, the activity is novel, the culture is easy, and the types of compounds which can be produced by the marine actinomycete metabolite relate to alkaloids, macrolides, terpenes, heterocyclic compounds, quinones, peptides, ethers, fatty acids and the like; the structural types and properties of partial compounds of the compound are greatly different from those of the terrestrial actinomycetes.
The inventor finds that the active medicine is found from the metabolic products of marine actinomycetes at present, which is an important research subject at home and abroad, the research is rapidly developed although the research is started later, and a plurality of compounds are already in clinical experiments at present. The research shows that the marine actinomycetes have special characteristics in the physiological characters and genetic backgrounds due to the particularity of marine environment, so that the marine actinomycetes can generate metabolic substances with novel structures certainly, and become important microorganism resources in pharmaceutical industries such as anti-tumor and antibiotics like land actinomycetes.
Disclosure of Invention
Therefore, the object of the present invention is to provide a compound, a preparation method and an application thereof, wherein the compound has a structure shown in formula (I):
wherein R is1Is selected from C1-C3Alkyl and C1-C3Alkoxy radical, R2And R3Each independently selected from H, -OH, C1-C3Alkyl and C1-C3An alkoxy group. The compound of the invention has good anti-tumor activity, can inhibit the proliferation of liver cancer, breast cancer and lung cancer cells, and has c-Met inhibitory activity. The compound is extracted and separated from the fermentation product of the marine actinomycete, has simple preparation process, easy operation and implementation, easy industrial large-scale production and wide application prospect.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the invention, there is provided a compound, or an isomer thereof, or a pharmaceutically acceptable salt thereof, having the structure of formula (I):
wherein R is1Is selected from C1-C3Alkyl and C1-C3Alkoxy radical, R2And R3Each independently selected from H, -OH, C1-C3Alkyl and C1-C3An alkoxy group.
In an embodiment of the invention, R2And R3Not simultaneously the same group.
In an embodiment of the invention, the compound of the invention has a structure represented by formula (II) or formula (III):
wherein R is2And R3Each independently selected from H, -OH, C1-C3Alkyl and C1-C3Alkoxy, but R2And R3Not simultaneously the same group.
In some embodiments of the invention, in the compound of formula (II), R2And R3Each independently selected from H, -OH, C1-C3An alkyl group.
In some embodiments of the invention, in the compound of formula (III), R2And R3Each independently selected from H, -OH, C1-C3An alkoxy group.
In an embodiment of the invention, the compound of the invention is selected from the following structures:
in the embodiment of the present invention, examples of the pharmaceutically acceptable salt include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate and nitrate; organic acid salts such as acetate, propionate, oxalate, succinate, lactate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, benzenesulfonate, p-toluenesulfonate and ascorbate; inorganic base salts such as sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt and aluminum salt; and organic base salts such as arginine salt, benzathine salt, choline salt, diethylamine salt, dialcohol amine salt, glycinate salt, lysine salt, meglumine salt, ethanolamine salt, and tromethamine salt.
And, the pharmaceutically acceptable salts also include zwitterionic (inner) salts, and also quaternary ammonium salts, such as alkylammonium salts.
In an embodiment of the invention, the above pharmaceutically acceptable salts may be obtained directly in the final isolation and purification of the compounds. The compound or an isomer thereof may be appropriately (e.g., an equivalent) mixed with a predetermined amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization.
In a second aspect of the invention, the invention provides a process for the preparation of the above compound isolated from the fermentation product of a marine actinomycete, which is Streptomyces sp (Streptomyces sp.) number 11010, CICC11010 for short, and ordered at the China center for the preservation and management of industrial microbial cultures, with the order site being http:// www.china-cic.
In some embodiments of the present invention, the method comprises inoculating strain cic 11010 in a liquid medium, and performing shake culture to obtain a seed solution; inoculating the seed liquid into a solid culture medium, standing for culture to obtain a solid fermentation product, and then separating and purifying to obtain the compound shown in the formula (I).
In an embodiment of the invention, the liquid medium consists of the following components by weight: soluble starch 15-25g, KNO30.5-1g，K2HPO40.3-0.8g，MgSO4·7H2O 0.5-1g，NaCl0.3-0.8g，FeSO4·7H20.01-0.03g of O, 10-15g of agar, 1L of distilled water and 7-8 of pH.
In some embodiments of the invention, the liquid medium consists of the following components by weight: soluble starch 20.0g, KNO31.0g，K2HPO40.5g，MgSO4·7H2O 0.5g，NaCl 0.5g，FeSO4·7H20.01g of O, 15.0g of agar and 1.0L of distilled water, and the pH value is 7.2-7.4. Sterilizing at 0.06kg/cm2 for 30 min.
In an embodiment of the present invention, the solid medium is a rice solid medium, and the composition thereof is as follows: 500-700g of rice and 1L of distilled water; in some embodiments of the invention, the amount of rice used in 1L of distilled water is 600g, and is obtained by autoclaving.
In an embodiment of the present invention, the conditions of the shake cultivation are that the shake cultivation is performed at 25-30 ℃ and 120-; in some embodiments, the conditions are further at 28 degrees C, 200 rpm/min shaking table culture for 3 days.
In an embodiment of the present invention, the static culture condition is static culture at 25-30 ℃ for 50-80 days, and in some embodiments of the present invention, the condition is further static culture at 28 ℃ for 60 days.
In an embodiment of the present invention, the step of separating and purifying comprises: extracting the fermentation product with organic solvent, concentrating, performing column chromatography, and separating by reversed phase high performance liquid chromatography to obtain compound of formula (I);
preferably, the separation and purification process comprises: leaching the fermentation product with equal volume of ethyl acetate overnight for 2 times, collecting the leaching solution, and concentrating to obtain concentrated extract; subjecting the concentrated extract to silica gel column chromatography, performing gradient elution with petroleum ether-ethyl acetate as eluent, tracking each elution part with thin layer, detecting fractions containing the same compounds, and mixing; separating the obtained fraction by reversed phase high performance liquid chromatography to obtain the compound of formula (I).
In an embodiment of the present invention, the filler of the silica gel column is 200-300 mesh, preferably 300 mesh.
In an embodiment of the present invention, the mass ratio of the concentrated extract to the filler is 1: 5-20, preferably 1: 10.
In an embodiment of the present invention, the column chromatography gradient elution is performed in a volume ratio of: a gradient of 80:1 to 5:1, 2 times.
In an embodiment of the present invention, the chromatographic conditions of the reversed-phase high performance liquid are as follows:
chromatographic column, Sepax Amethyl C-18, 5 μm, 21.2 × 250 mm;
detection wavelength: 254 nm;
mobile phase: acetonitrile-water solution system, which contains 0.2-0.5 wt% of trifluoroacetic acid, and isocratic elution, wherein the volume ratio of acetonitrile/water is 20:80, and the flow rate is 5 mL/min.
In certain embodiments of the invention, a method of making a compound of the invention comprises: inoculating CICC11010 into a 500mL conical flask, wherein each flask contains 250mL of liquid culture medium, and performing shake culture at 28 ℃ for 3 days at 200rpm to obtain a fermentation culture medium inoculated with CICC 11010; inoculating 15ml of the fermentation medium into a solid medium, and standing and culturing at 28 ℃ for 60 days to obtain a fermentation product;
leaching the fermentation product with equal volume of ethyl acetate overnight for 2 times, collecting the leaching solution, and concentrating to obtain concentrated extract. Performing silica gel column chromatography on the obtained concentrated extract, filling the extract with 300 meshes, eluting the extract and the filler according to the gradient of the petroleum ether-ethyl acetate as an eluent in the volume ratio of 80:1, 40:1, 20:1, 10:1 and 5:1, tracking each eluted part by a thin layer, identifying fractions containing the same compound, and combining the fractions;
separating the obtained fraction by reversed phase high performance liquid chromatography under the following chromatographic conditions:
chromatographic column, Sepax Amethyl C-18, 5 μm, 21.2 × 250 mm;
detection wavelength: 254 nm.
Mobile phase: acetonitrile-water solution system, containing 0.2 wt% o trifluoroacetic acid, isocratic elution, acetonitrile/water volume ratio of 20:80, flow rate of 5 mL/min;
collecting chromatographic peaks of 20-23min, 25-28min, 32-35min, 40-43min, 43-46min and 52-55min, and recovering the solvent to obtain the compounds II-1, II-2, II-3, III-1, III-2 and III-3 of the invention respectively.
In a third aspect of the invention, the invention provides a composition or pharmaceutical formulation comprising a compound as described in the first aspect above and at least one pharmaceutically acceptable adjuvant or carrier.
The compositions of the present invention are generally safe, non-toxic and biologically desirable, and therefore the pharmaceutically acceptable carriers or excipients of the present invention are non-toxic and safe, as well as combinations thereof with the compounds of the present invention. The pharmaceutically acceptable carriers and excipients of the present invention are generally well known to those skilled in the art or can be determined by those skilled in the art based on the actual circumstances.
The composition or pharmaceutical preparation of the compound of the present invention can be administered by any means selected by those skilled in the art as appropriate: oral, aerosol inhalation, rectal, nasal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, with oral, intramuscular, intraperitoneal or intravenous administration being preferred.
The compounds of the present invention or pharmaceutical compositions or formulations containing these compounds may be administered in unit dosage form. The administration dosage form can be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or mixed suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.
The pharmaceutical combination or pharmaceutical preparation of the present invention may further comprise a conventional carrier, wherein the pharmaceutically acceptable carrier includes but is not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of 1% to 98% by weight, typically about 80% by weight. For convenience, the local anesthetic, preservative, buffer, etc. may be dissolved directly in the vehicle.
Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated by methods known in the art of pharmacy.
The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dried product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.
Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.
For parenteral administration, liquid dosage forms are generally prepared from the compound and a sterile carrier. The carrier is preferably water. The compound can be dissolved in the carrier or made into suspension solution according to the concentration of the carrier and the drug, and the compound is firstly dissolved in water when made into the solution for injection, filtered and sterilized and then filled into a sealed bottle or ampoule.
It will be appreciated that the optimum dosage and interval for administration of a compound of the invention will be determined by the nature of the compound and external conditions, such as the form, route and condition of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It should also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over a nominal period of time, may be determined by methods known in the art.
In a fourth aspect, the present invention also provides the use of a compound of the first aspect as described above in the preparation of an anti-cancer or anti-tumor medicament.
Wherein the cancer or tumor is selected from the group consisting of liver cancer, breast cancer, and lung cancer (such as non-small cell lung cancer).
The invention also provides the use of a compound as described in the first aspect above in the manufacture of a medicament for the treatment of a c-Met kinase inhibitor.
In an embodiment of the present invention, the compounds II-1 to II-3, III-1 to III-3 of the present invention are human hepatoma cells HepG2 (IC)505-10 μ M), human breast cancerCell MCF-7 (IC)504-12 μ M) and human non-small cell lung carcinoma cell A549 (IC)505-13 mu M), and particularly the inhibitory activity of the compounds II-3 and III-1 to III-3 is obviously superior to that of a positive control drug 5-FU.
In the embodiment of the invention, the compounds II-1 to II-3 and III-1 to III-3(5 mu M) have effective inhibition effect on c-Met, and especially the inhibition activity of the compounds III-1 to III-3 on the c-Met can reach 88 percent at most.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
The fermentation bacteria of the invention adopts the strain number 11010 of Streptomyces sp, which is sold by China Industrial microorganism culture Collection (CICC), and is abbreviated as CICC11010 in the following embodiments.
Example 1
Strain: streptomyces sp. CICC11010, which is purchased from China center for Industrial culture Collection of microorganisms (CICC) and is hereinafter referred to as CICC 11010.
Culture medium:
1. liquid culture medium: soluble starch 20.0g, KNO31.0g，K2HPO40.5g，MgSO4·7H2O 0.5g，NaCl 0.5g，FeSO4·7H20.01g of O, 15.0g of agar and 1.0L of distilled water, and the pH value is 7.2-7.4. 0.06kg/cm2Sterilizing for 30 min.
2. Solid medium: 600g of rice and 1L of distilled water, and sterilizing for 30 min.
The preparation method comprises the following steps: and inoculating the CICC11010 into 500mL conical flasks, wherein each flask contains 250mL of liquid culture medium, and performing shake culture at the temperature of 28 ℃ for 3 days at 200rpm to obtain a fermentation culture medium inoculated with the CICC 11010. Taking 15ml of the fermentation culture medium, inoculating the fermentation culture medium into a solid culture medium, and standing and culturing for 60 days at 28 ℃ to obtain a fermentation product.
Leaching the fermentation product with equal volume of ethyl acetate overnight for 2 times, collecting the leaching solution, and concentrating to obtain concentrated extract. Subjecting the obtained concentrated extract (ethyl acetate part) to silica gel column (filler 300 mesh) chromatography. The mass ratio of the extract to the filler is 1: 10. Eluting with petroleum ether-ethyl acetate as eluent at volume ratio of 80:1, 40:1, 20:1, 10:1, and 5:1, tracking each eluate with thin layer, detecting fractions containing the same compounds, and mixing.
Separating the obtained fraction by reversed phase high performance liquid chromatography under the following chromatographic conditions:
chromatographic column Sepax Amethyl C-18, 5 μm, 21.2 × 250 mm.
Detection wavelength: 254 nm.
Mobile phase: acetonitrile-water solution system (containing trifluoroacetic acid, 0.2wt per mill), isocratic elution, acetonitrile/water volume ratio of 20:80, flow rate of 5 mL/min.
Collecting chromatographic peaks of 20-23min, 25-28min, 32-35min, 40-43min, 43-46min and 52-55min, and recovering the solvent to obtain the compounds II-1, II-2, II-3, III-1, III-2 and III-3 respectively.
Identification of Compounds II-1 to II-3, III-1 to III-3:
II-1: 8- (dimethylamino) -1-methyl-4, 9-dioxo-6-phenyl-4, 9-dihydro-2H-benzo [ f ] isoindole-2-carboxylic acid methyl ester
methyl 8-(dimethylamino)-1-methyl-4,9-dioxo-6-phenyl-4,9-dihydro-2H-benzo[f]isoindole-2-carboxylate
Chemical Formula:C23H20N2O4
1H-NMR(400MHz,CDCl3)7.89(s,1H),7.50-7.54(m,2H),7.35-7.48(m,4H),7.03(s,1H),3.75(s,3H)，3.30(s,6H),2.05(s,3H).m/z:389.15,[M+H]+。13C-NMR(100MHz,CDCl3)182.5,181.0,152.0,144.3,141.4,136.8,126.2,125.8,124.9,124.6,122.1,120.1,119.5,118.6,115.6,107.8,103.3,55.3,45.4,9.3。
II-2: 5- (dimethylamino) -1-hydroxy-3-methyl-4, 9-dioxo-7-phenyl-4, 9-dihydro-2H-benzo [ f ] isoindole-2-carboxylic acid methyl ester
methyl 5-(dimethylamino)-1-hydroxy-3-methyl-4,9-dioxo-7-phenyl-4,9-dihydro-2H-benzo[f]isoindole-2-carboxylate
Chemical Formula:C23H20N2O5
1H-NMR(400MHz,CDCl3)7.93(s,1H),7.52-7.55(m,2H),7.30-7.42(m,4H),3.89(s,3H)，3.28(s,6H),2.11(s,3H).m/z:405.14,[M+H]+。13C-NMR(100MHz,CDCl3)182.2,181.3,153.6,145.1,144.2,138.4,136.5,129.3,126.1,127.8,127.1,120.4,116.4,111.5,112.2,110.2,104.6,54.5,45.9,9.8。
II-3: 5- (dimethylamino) -1-hydroxy-4, 9-dioxo-7-phenyl-4, 9-dihydro-2H-benzo [ f ] isoindole-2-carboxylic acid methyl ester
methyl 5-(dimethylamino)-1-hydroxy-4,9-dioxo-7-phenyl-4,9-dihydro-2H-benzo[f]isoindole-2-carboxylate
Chemical Formula:C22H18N2O5
1H-NMR(400MHz,CDCl3)7.78(s,1H),7.50-7.58(m,2H),7.36-7.44(m,4H),6.98(s,1H),4.05(s,3H),3.38(s,6H)。m/z:391.13,[M+H]+。13C-NMR(100MHz,CDCl3)182.1,181.2,151.6,143.7,141.4,140.8,129.8,129.2,127.9,127.6,124.3,120.1,118.8,116.6,109.9,104.3,54.2,45.4。
III-1: 8- (dimethylamino) -1-methoxy-6-phenyl-2-propyl-2H-benzo [ f ] isoindole-4, 9-dione
8-(dimethylamino)-1-methoxy-6-phenyl-2-propionyl-2H-benzo[f]isoindole-4,9-dione
Chemical Formula:C24H22N2O4
1H-NMR(400MHz,CDCl3)7.75(s,1H),7.45-7.56(m,2H),7.30-7.45(m,4H),6.94(s,1H),4.21(s,3H),3.31(s,6H),2.54(q,J＝6.8Hz,2H),1.13(t,J＝6.8Hz,3H)。m/z:403.17,[M+H]+。13C-NMR(100MHz,CDCl3)181.0,179.5,152.1,143.7,141.4,140.8,129.6,129.2,127.9,126.0,125.5,123.1,117.6,116.5,109.8,109.9,104.3,54.8,45.6,29.1,8.9。
III-2: 5- (dimethylamino) -1-hydroxy-3-methoxy-7-phenyl-2-propyl-2H-benzo [ f ] isoindole-4, 9-dione
5-(dimethylamino)-1-hydroxy-3-methoxy-7-phenyl-2-propionyl-2H-benzo[f]isoindole-4,9-dione
Chemical Formula:C24H22N2O5
1H-NMR(400MHz,CDCl3)7.65(s,1H),7.43-7.50(m,2H),7.35-7.44(m,4H),3.98(s,3H),3.34(s,6H),2.58(q,J＝6.8Hz,2H),1.03(t,J＝6.8Hz,3H)。m/z:419.16,[M+H]+。13C-NMR(100MHz,CDCl3)182.1,179.6,159.3,143.7,141.4,140.8,129.8,129.2,127.9,127.6,122.3,120.1,116.6,112.4,111.3,109.9,104.3,54.8,45.4,29.4,8.2。
III-3: 5- (dimethylamino) -1-hydroxy-7-phenyl-2-propyl-2H-benzo [ f ] isoindole-4, 9-dione
5-(dimethylamino)-1-hydroxy-7-phenyl-2-propionyl-2H-benzo[f]isoindole-4,9-dione
Chemical Formula:C23H20N2O4
1H-NMR(400MHz,CDCl3)7.78(s,1H),7.38-7.46(m,2H),7.28-7.36(m,4H),6.99(s,1H),3.26(s,6H),2.62(q,J＝6.8Hz,2H),1.05(t,J＝6.8Hz,3H)。m/z:389.15,[M+H]+。13C-NMR(100MHz,CDCl3)182.1,180.3,151.9,143.7,141.4,140.8,129.8,127.9,129.2,127.6,120.1,118.8,116.6,115.3,113.4,109.9,104.3,45.4,29.1。
Experimental example 1Experiment for inhibiting tumor cell proliferation in vitro
Experimental materials: human liver cancer cell HepG2, human breast cancer cell MCF-7 and human non-small cell lung cancer cell A549, 96-well plate, cell counting plate, Countstar automatic cell counter, DMEM/High Glucose culture medium, fetal bovine serum, penicillin-streptomycin mixed solution, pancreatin, PBS buffer solution, CCK-8, dimethyl sulfoxide (DMSO), multifunctional enzyme labeling instrument (BioTek), positive control: 5-FU, Experimental drugs: compounds II-1, II-2, II-3, III-1, III-2, III-3 prepared in example 1.
The experimental method comprises the following steps: tumor cells in logarithmic growth phase are taken, trypsinized to prepare single cell suspension, counted on a Countstar automatic cell counter, diluted by 4000 cells per well, and evenly inoculated on a 96-well plate, wherein each well is 100 mu L. And additionally setting blank control: only the same volume of medium; negative control: only cell suspension was added, no drug was added. Adding compounds (II-1-II-3, III-1-III-3) to be tested with different concentrations into the experimental wells, setting 4 multiple wells for each concentration, and adding 100 μ L culture medium into the negative control well and the blank control well respectivelyAnd (5) nutrient base. At 37 ℃ 5% CO2After incubation for 72 hours, 100. mu.L of CCK-8 was added to each well, and after incubation for 1.5 hours at 37 ℃ in an incubator, the OD at 450nm was measured using a multifunctional microplate reader (BioTek), and data processing was performed.
IC calculation Using GraphPad Prism 5 software50The value, inhibition ratio formula is as follows:
the experimental results are as follows: see table 1.
TABLE 1 proliferation inhibitory Activity of Compounds on tumor cells
aMean values of three experiments. + -. Standard Deviation (SD)
Experimental results show that the compounds II-1-II-3 and III-1-III-3 prepared in the embodiment 1 of the invention have good proliferation inhibition activity on human liver cancer cells HepG2, human breast cancer cells MCF-7 and human non-small cell lung cancer cells A549, and especially the inhibition activity of the compounds II-3 and III-1-III-3 is obviously superior to that of a positive control drug 5-FU.
Experimental example 2In vitro inhibition of c-Met kinase
Experimental materials: MET kinase, substrate peptide FAM-P2, ATP, DMSO, EDTA, 384-well plate, kinase reaction buffer 50mM HEPES (pH 7.5), 0.0015% Brij-35; kinase reaction stop solution: 100mM HEPES (pH 7.5), 0.015% Brij-35, 50mM EDTA; a positive control drug Staurosporine; the test compounds (i.e., compounds II-1 to II-3, and III-1 to III-3).
The experimental method comprises the following steps: test compounds were first dissolved in 100% DMSO, respectively, at an initial concentration of 50. mu.M each, and then diluted to 1. mu.M.
The test samples were divided into a negative control group (blank group without compound), a positive control group (Crizotinib as positive control, CR for short) and a test compound group.
Kinase reaction: (1) preparing 2.5 times of kinase solution, namely adding kinase into 1 time of kinase buffer solution to form 2.5 times of kinase solution. (2) Preparing 2.5 times of substrate solution, namely adding FAM-labeled substrate peptide and ATP into 1 time of kinase buffer solution to form 2.5 times of substrate solution. (3) To a 384-well reaction plate containing 5. mu.L of the compound solution, 10. mu.L of a 2.5-fold kinase solution was added, and incubated at room temperature for 10 minutes. (4) To a 384-well reaction plate, 10. mu.L of a 2.5-fold substrate solution was added, and after a reaction time of a specific duration at 28 ℃ 25. mu.L of a stop solution was added to terminate the reaction.
Conversion data were copied from Caliper and then converted to inhibition:
the inhibition ratio (%) (max-conversion)/(max-min) × 100%
Wherein: "conversion" represents the conversion of the compound group; "max" represents DMSO control without compound added; "min" represents the low control.
The experimental results are as follows: see table 2.
TABLE 2 inhibitory Activity of c-Met kinase in vitro (5. mu.M)
| Inhibition ratio (%) | +/-SD (Standard deviation) | |
| Compound II-1 | 66.7 | 1.4 |
| Compound II-2 | 59.9 | 8.3 |
| Compound (I)II-3 | 74.6 | 0.5 |
| Compound III-1 | 80.9 | 1.6 |
| Compound III-2 | 88.1 | 0.4 |
| Compound III-3 | 77.3 | 2.2 |
| CR | 78.9 | 4.3 |
The compounds II-1 to II-3 and III-1 to III-3 prepared in the example 1 have certain inhibitory action on c-Met, and especially the inhibitory activity of the compounds III-1 to III-3 on c-Met is equivalent to or even better than that of the positive control drug crizotinib.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A compound, or a pharmaceutically acceptable salt thereof, selected from the structures:
2. a process for the preparation of a compound as claimed in claim 1, which is isolated from the fermentation product of Streptomyces sp.CICC 11010 species, which comprises inoculating the CICC11010 species in a liquid medium, shake culturing to obtain a seed solution; inoculating the seed liquid into a solid culture medium, standing for culture to obtain a fermentation product, and then separating and purifying to obtain a compound;
wherein the liquid culture medium comprises the following components in parts by weight: soluble starch 15-25g, KNO30.5-1g，K2HPO40.3-0.8g，MgSO4·7H2O 0.5-1g，NaCl 0.3-0.8g，FeSO4·7H20.01-0.03g of O, 10-15g of agar, 1L of distilled water and 7-8 of pH;
the solid culture medium is a rice solid culture medium and comprises the following components: 500-700g of rice and 1L of distilled water;
the condition of shaking table culture is that the shaking table is cultured for 2 to 5 days at the temperature of between 25 and 30 ℃ and at the speed of 120-;
the static culture condition is that the static culture is carried out for 50-80 days at 25-30 ℃;
the steps of separating and purifying comprise: leaching the fermentation product with equal volume of ethyl acetate overnight for 2 times, collecting the leaching solution, and concentrating to obtain concentrated extract; subjecting the concentrated extract to silica gel column chromatography, performing gradient elution with petroleum ether-ethyl acetate as eluent, tracking each elution part with thin layer, detecting fractions containing the same compounds, and mixing; separating the fractions by reverse phase high performance liquid chromatography to obtain the compounds of claim 1;
wherein, the filler of the silica gel column is 200-300 meshes;
the mass ratio of the concentrated extract to the filler is 1: 5-20 parts of;
the volume ratio of the column chromatography gradient elution is as follows: gradient of 80: 1-5: 1, 2 times;
the chromatographic conditions of the reversed-phase high-performance liquid phase are as follows:
chromatographic column, Sepax Amethyl C-18, 5 μm, 21.2 × 250 mm;
detection wavelength: 254 nm;
mobile phase: acetonitrile-water solution system, which contains 0.2-0.5 wt% of trifluoroacetic acid, and isocratic elution, wherein the volume ratio of acetonitrile/water is 20:80, and the flow rate is 5 mL/min.
3. The method according to claim 2, wherein the liquid medium is composed of the following components by weight: soluble starch 20.0g, KNO31.0g，K2HPO40.5g，MgSO4·7H2O 0.5g，NaCl 0.5g，FeSO4·7H2O0.01g, agar 15.0g, distilled water 1.0L, pH 7.2-7.4.
4. The method according to claim 2, wherein the shake culture is carried out at 28 ℃ and 200rpm for 3 days.
5. The method according to claim 2, wherein the static culture condition is static culture at 28 ℃ for 60 days.
6. The method for preparing according to any one of claims 2 to 5, characterized in that it comprises: inoculating CICC11010 into a 500mL conical flask, wherein each flask contains 250mL of liquid culture medium, and performing shake culture at 28 ℃ for 3 days at 200rpm to obtain a fermentation culture medium inoculated with CICC 11010; inoculating 15ml of the fermentation medium into a solid medium, and standing and culturing at 28 ℃ for 60 days to obtain a fermentation product;
leaching the fermentation product with equal volume of ethyl acetate overnight for 2 times, collecting the leaching solution, concentrating to obtain concentrated extract, performing silica gel column chromatography on the obtained concentrated extract, filling with 300 meshes of filler, wherein the mass ratio of the extract to the filler is 1:10, eluting with petroleum ether-ethyl acetate as eluent according to the gradient of the volume ratio of 80:1, 40:1, 20:1, 10:1 and 5:1, tracking each eluted part by a thin layer, detecting fractions containing the same compounds, and combining;
separating the obtained fraction by reversed phase high performance liquid chromatography under the following chromatographic conditions:
chromatographic column, Sepax Amethyl C-18, 5 μm, 21.2 × 250 mm;
detection wavelength: 254 nm;
mobile phase: acetonitrile-water solution system, containing 0.2 wt% o trifluoroacetic acid, isocratic elution, acetonitrile/water volume ratio of 20:80, flow rate of 5 mL/min;
collecting chromatographic peaks of 20-23min, 25-28min, 32-35min, 40-43min, 43-46min and 52-55min, and recovering solvent to obtain compounds II-1, II-2, II-3, III-1, III-2 and III-3.
7. A pharmaceutical composition or formulation comprising a compound of claim 1 and at least one pharmaceutically acceptable adjuvant or carrier.
8. The use of a compound of claim 1 for the manufacture of an anti-cancer medicament;
and/or, the use of a compound of claim 1 for the manufacture of a medicament for the treatment of c-Met kinase inhibitors.
9. The use according to claim 8, wherein said cancer is selected from the group consisting of liver cancer, breast cancer and lung cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910625803.9A CN110218174B (en) | 2019-07-11 | 2019-07-11 | Compound and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910625803.9A CN110218174B (en) | 2019-07-11 | 2019-07-11 | Compound and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110218174A CN110218174A (en) | 2019-09-10 |
| CN110218174B true CN110218174B (en) | 2020-07-07 |
Family
ID=67813295
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910625803.9A Active CN110218174B (en) | 2019-07-11 | 2019-07-11 | Compound and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110218174B (en) |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4494547A (en) * | 1981-03-30 | 1985-01-22 | North Carolina Central University | 2H-isoindolediones, their synthesis and use as radiosensitizers |
| WO2011006803A1 (en) * | 2009-07-14 | 2011-01-20 | Nerviano Medical Sciences S.R.L. | 3-0x0-2, 3, -dihydro-lh-isoindole-4-carboxamides with selective parp-1 inhibition |
| EP2701511A4 (en) * | 2011-04-29 | 2015-06-10 | Univ Akron | Azolium and purinium salt anticancer and antimicrobial agents |
| CN102757908B (en) * | 2012-05-11 | 2014-05-14 | 中国科学院南海海洋研究所 | Streptomyces sp., indole sesquiterpenoids, and preparation method and application of indole sesquiterpenoids to preparation of antibacterial and antitumor medicament |
| CN108017625B (en) * | 2017-11-23 | 2019-05-21 | 浙江大学 | Aphthothiophenes cyclohexadione compounds and its preparation method and application |
-
2019
- 2019-07-11 CN CN201910625803.9A patent/CN110218174B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN110218174A (en) | 2019-09-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| BRPI0017067B1 (en) | cell division inhibitor, dehydrogenase, method for producing a cell division inhibitor and compound | |
| CN107353274B (en) | Selenolonic acid I from penicillium oxalicum and application thereof in preparation of human esophageal cancer resistant medicine | |
| JPH08175990A (en) | Pi3 kinase-inhibiting agent and its production | |
| US9827232B2 (en) | Protein tyrosine phosphatase inhibitor, preparation method and uses thereof | |
| CN113387872A (en) | Preparation method and application of compound | |
| CN109553600B (en) | Isocoumarin compound in mangrove endophytic fungi and preparation method and application thereof | |
| WO2021098593A1 (en) | Secoemestrin c preparation method and use therof | |
| CN107298669B (en) | Selenolonic acid I from penicillium oxalicum and application of medicine for resisting human oral epidermoid carcinoma | |
| CN110218174B (en) | Compound and preparation method and application thereof | |
| CN110407792B (en) | Secalonic acid J compound derived from penicillium oxalicum and preparation method thereof | |
| CN101538247B (en) | Preparation method of alkaloid compound | |
| CN109364045B (en) | Heat shock protein 90 inhibitor and application thereof in liver cancer treatment | |
| CN110407797B (en) | Secalonic acid K compound derived from penicillium oxalicum and preparation method thereof | |
| CN110407794B (en) | Selenolonic acid K derived from penicillium oxalicum and application thereof in inhibiting cancer cell proliferation | |
| US20100240887A1 (en) | New methods of producing HHT | |
| CN113082019A (en) | Application of guaiane type sesquiterpene compound in preparation of medicine for treating pancreatic cancer | |
| JP3719689B2 (en) | Novel substance triprostatin, process for producing the same, cell cycle inhibitor and antitumor agent | |
| CN109134417B (en) | Selenolonic acid I from penicillium oxalicum and application of medicine for resisting human cervical cancer | |
| CN107746422B (en) | Ergosta-7, 22-diene-3-ketaminothiohydrazone, preparation method thereof and application thereof in preparation of antibacterial drugs | |
| CN111499649A (en) | Benzodifuranone compound with anti-tumor activity, preparation method and application thereof | |
| CN110698441A (en) | 2-methyl-4- (1-glycerol) -furan compounds and preparation method and application thereof | |
| CN113582863B (en) | Aminoethyl biphenyl compound and preparation method and application thereof | |
| CN115990162B (en) | Application of 4-hydroxy-2-pyridone alkaloid in preparation of medicines for treating gastric cancer | |
| CN108546247B (en) | Application of alkaloid compound in preparation of anti-obesity drugs | |
| CN115105488B (en) | Application of Dankesterone A in preparation of medicines for treating hepatic fibrosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230830 Address after: No. 1, South Fuqian Road, Xuecheng Chemical Industry Park, Xuecheng District, Zaozhuang, Shandong 277000 Patentee after: Shandong Kairui Chemical Co.,Ltd. Address before: Zaozhuang College, No.1 Bei'an Road, Shizhong District, Zaozhuang City, Shandong Province, 277160 Patentee before: ZAOZHUANG University Patentee before: SHANDONG ENGINEERING AND TECHNOLOGY INSTITUTE OF LUNAN COAL CHEMICAL ENGINEERING |