WO2014185717A1 - Novel epothilone derivative and use thereof - Google Patents

Novel epothilone derivative and use thereof Download PDF

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WO2014185717A1
WO2014185717A1 PCT/KR2014/004346 KR2014004346W WO2014185717A1 WO 2014185717 A1 WO2014185717 A1 WO 2014185717A1 KR 2014004346 W KR2014004346 W KR 2014004346W WO 2014185717 A1 WO2014185717 A1 WO 2014185717A1
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epoxylon
derivative
derivatives
present
pharmaceutical composition
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PCT/KR2014/004346
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French (fr)
Korean (ko)
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김원곤
박지영
김중수
김현주
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한국생명공학연구원
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

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  • the present invention relates to a novel epoxylon derivative and its use, and more particularly, the present invention relates to a novel epoxylon derivative having improved water solubility due to glucosylation of the epoxylon, a method for preparing the epoxylon derivative, the epoxy
  • the present invention relates to a pharmaceutical composition for treating cancer comprising a cyclone derivative as an active ingredient, a method of treating cancer using the pharmaceutical composition, and a use of the epoxylon derivative used in the manufacture of a pharmaceutical composition for treating cancer.
  • Epothilone is a natural product isolated from soil bacteria ( myxobacterium Sorangium cellulosum strain 90), which is cytotoxic to tumor cells resistant to taxol and is superior to taxol. It is known to exhibit scientific utility.
  • the epoxylon exhibits anticancer activity by acting on a microtubule, an intracellular framework like a thread that is aligned at a specific position in the cell division cycle and then disintegrated, thereby inhibiting cell division, and has a simple chemical structure compared to Taxol.
  • epoxylons In stabilizing microtubules, epoxylons have an effect of 2000 to 5000 times better than taxol, and have partial water solubility as compared to poorly soluble taxol, and have the advantage of easy formulation.
  • taxol and epoxylons are known to differ in chemical structure, epoxylons are known to replace taxol from binding sites and bind to the same active site of microtubules, thus focusing on structural novelty, important biological and interesting mechanisms of action. Active research is in progress.
  • the prepared epoxylon is superior to taxol, but shows partial water solubility, there is a disadvantage in that it is not easy to formulate, and various derivatives have been developed to overcome this disadvantage.
  • ixabepilone (azaepothilone B), BMS-310705, patupilone, epothilone R1645, ZK-EPO, ABJ879 (C20-desmethyl-C20-methylsulfanyl-EpoB), etc.
  • the water solubility is increased while the original anticancer activity is reduced, it was required to develop a new derivative that overcomes these disadvantages.
  • the present inventors earnestly researched to develop an epoxylon derivative that maintains anticancer activity while increasing water solubility. As a result, the present inventors confirmed that the phosphoyl glycosylated derivative exhibited improved water solubility and maintained anticancer activity. It was.
  • One object of the present invention is to provide an epoxylon derivative in which epoxylon is glucosylated.
  • Another object of the present invention is to provide a method for preparing the epoxylon derivative.
  • Another object of the present invention to provide a pharmaceutical composition for treating cancer comprising the epoxylon derivative.
  • Still another object of the present invention is to provide a method of treating cancer using the pharmaceutical composition for treating cancer.
  • Still another object of the present invention is to provide a use of the epoxylon derivative for the preparation of the pharmaceutical composition for treating cancer.
  • the epoxylon derivative of the present invention can be widely used in the formulation of epoxylon since the anti-cancer activity is not lowered even though the water solubility is improved compared to the conventional epoxylon.
  • Figure 1a shows the results of HPLC analysis of the sample before extraction with ethyl acetate.
  • Figure 1b shows the results of HPLC analysis on the ethyl acetate layer.
  • Figure 1c shows the results of HPLC analysis of the remaining water layer.
  • Figure 2a is a graph showing the results of measuring the level of plasma epoxylon derivatives and epoxylon over time after oral administration of the epoxylon derivative of the present invention to the experimental animal
  • Epo-AG is eppo Represents a cyclone derivative
  • Epo-A represents an epoxylon.
  • Figure 2b is a graph showing the results of measuring the level of plasma phosphoyl derivatives and epoxylon over time after administration of the epoxylon derivative of the present invention to experimental animals, Epo-AG is Epoxylon derivatives are shown, and Epo-A represents epoxylons.
  • glycosylation refers to a reaction in which a glycosyl group is transposed to a high molecular compound such as a protein, and the glycosylated high molecular compound may form a complex sugar to perform various functions in vivo. Will be.
  • the inventors expected that the glycosyl groups used for the glycosylation include abundant hydrophilic reactors, so that the compounds having partial water solubility, such as the epoxylon of the present invention, would have excellent water solubility.
  • the present invention provides an epoxylon derivative glucosylated epoxylon.
  • epothilone refers to a natural compound produced from soil bacteria ( myxobacterium Sorangium cellulosum strain 90) and having excellent anticancer activity and partial water solubility.
  • Various derivatives epothilones A to F in which each functional group of the epoxylon is known are known, and the derivatives are known to exhibit pharmacologically active effects on various diseases such as Alzheimer's disease as well as anticancer activity.
  • the eposilone may be epothilones A (EpoA) or epothilones B (EpoB), which are known to exhibit excellent anticancer effects, but in particular, as long as the water solubility is increased by glycosylation, the anticancer activity is maintained.
  • EpoA epothilones A
  • EpoB epothilones B
  • the type of epoxylon is not limited.
  • EpoA epothilones A
  • EpoB epothilones B
  • glucosylation of the present invention is a kind of glycosylation, and means a reaction in which the first carbon of the glucosyl group is bonded to the desired compound to transfer the glucosyl group.
  • glucose group refers to a monovalent reactor in which a hemiacetal hydroxyl group is removed from a glucose molecule as a type of glycosyl group.
  • the glucosylation may be a reaction for transferring a glucosyl group to an epocylon, preferably EpoA or EpoB, but such glucosylation is not particularly limited, but a known chemical method, preferably a biochemical method It can be carried out by, and more preferably by a method using an enzyme, and most preferably can be performed using a glucose transferase.
  • glucose transferase refers to an enzyme capable of performing the glucosylation by catalyzing the transfer of a glucosyl group.
  • the glucose transferase is obtained from a donor of a glucosyl group. It can be understood as an enzyme that performs a catalysis to transfer one glucosyl group to the epoxylon.
  • a donor of the glucosyl group sugar phosphate (Glc-1-P, etc.); Nucleotide glucose (UDP-Glc, etc.); Oligosaccharides (such as sucrose); Polysaccharides and the like can be used alone or in combination.
  • the glucose transferase is not particularly limited as long as it can carry out glucosylation of eposylon, but preferably, MGT (macroside glycosyltransferase) -based glucose transferase or nucleotide glucose is used as a donor of a glucosyl group.
  • the glucose transferase may be used, and more preferably, a MGT-based glucose transferase or UDP derived from B. amyloliquefaciens or a glucose transferase using UDP as a donor of a glucosyl group may be used.
  • Mg-based glucose transferases BmgtB, BL-C (UDP-glucosyltransferase C) derived from the Bacillus strain can be used.
  • glucosylated epoxylon derivative refers to a derivative compound in which a glucosyl group is transferred to a carbon of epoxylon by a glucose transferase and bound (glucosylated).
  • the glucosylated epoxylon derivative is not particularly limited thereto, but preferably carbon of epoxylon may be a glucosylated derivative compound, more preferably carbon 7 of epoxylon. May be a glucosylated derivative compound, and most preferably, carbon 7 of EpoA (7-glucosyl epothilones A) or carbon 7 of EpoB is glucosylated by glucosylating carbon 7 of EpoA.
  • EpoB-G (7-glucosyl epothilones B) which can be represented by the formula (4).
  • recombinant glucose transferase BL-C UDP-glucosyltransferase C
  • Bacillus licheniformis Example 1-1) or BmgtB (Example 1-2)
  • glucosylated carbon 7 of EpoA or EpoB using the prepared BL-C or BmgtB to prepare a glucosylated epoxylon derivative (Table 1 and Table 2), and the prepared epoxylon Derivatives increased water solubility by glucosylation, and the anticancer activity of the derivatives showed anticancer activity against all six cancer cell lines (Table 3).
  • the present invention comprises the steps of (a) adding a glucosyl donor and glucose transferase to the epoxylon compound to perform a glucosylation reaction; And (b) provides a method for producing the glucosylated epoxylon derivative comprising the step of recovering the epoxylon derivative from the reactant.
  • the epoxylon compound, the donor of the glucosyl group and the glucose transferase are the same as described above.
  • the pH conditions for performing the reaction is neutral pH (pH 7.0 to 8.0)
  • the temperature conditions are 25 to 35 °C
  • the time conditions are 10 to 30 hours.
  • the mixing ratio of the donor of the epoxylon compound and the glucosyl group used in the reaction is preferably 1: 1 to 1:10 (w / w), more preferably 1: 1 to 1: 5 (w / w), most preferably 1: 4 (w / w).
  • the step of recovering the epoxylon derivative from the reactant can be carried out by methods known in the art.
  • the method for recovering the epoxylon derivatives is not particularly limited, but centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (eg, ammonium sulfate precipitation), chromatography ( For example, ion exchange, affinity, hydrophobicity and size exclusion).
  • the present invention provides a pharmaceutical composition for treating cancer comprising the epoxylon derivative.
  • the epoxylon derivatives provided by the present invention have improved water solubility compared to the conventional epoxylons, not only maintain anticancer activity but also are converted into epoxylons in the body, the epoxylon derivatives are It can be seen that it can be used as an active ingredient in the form of prodrugs contained in the pharmaceutical composition for cancer treatment.
  • the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable diluent, excipient or carrier.
  • the composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
  • Solid form preparations for oral administration include tablet pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose in one or more compounds. ) And gelatin.
  • lubricants such as magnesium stearate, talc and the like are also used.
  • Liquid preparations for oral administration include suspensions, solution solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • the pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It can have one formulation.
  • the route of administration of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue.
  • the pharmaceutical composition of the present invention may be administered as desired, but is not limited to intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, pulmonary administration, rectal administration.
  • the pharmaceutical composition may be administered by any device in which the active substance may migrate to the target cell.
  • the content of the epoxylon derivative included in the pharmaceutical composition of the present invention is not particularly limited, but may be included in 0.0001 to 50% by weight relative to the total weight of the final composition, preferably in an amount of 0.001 to 10% by weight. It may include.
  • the pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term "pharmaceutically effective amount" of the present invention, the amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment
  • Effective dose levels are well-known for individual types and severities, age, gender, drug activity, drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other medical fields. It can be determined according to known factors.
  • the pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.
  • the dosage of the pharmaceutical composition for treating cancer containing the epoxylon derivative of the present invention may be used in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as an active ingredient of the patient.
  • One skilled in the art can decide.
  • the pharmaceutical composition of the present invention may be administered at about 0.1 ng to about 100 mg / kg, preferably 1 ng to about 10 mg / kg, per adult, and the frequency of administration of the composition of the present invention is specifically Although not limited, it can be administered once a day or several times in divided doses.
  • the present invention provides a method for treating cancer comprising administering the pharmaceutical composition to a subject having a cancer disease in a pharmaceutically effective amount.
  • the epoxylon derivatives provided in the present invention not only have anticancer activity, but also can be converted into epoxylon in the body, so that the composition can be used to treat cancer.
  • mammals includes, without limitation, mammals, including rats, livestock, humans, and the like, which have or are likely to develop cancer.
  • the route of administration of the pharmaceutical composition may be administered via any general route as long as it can reach the target tissue.
  • the pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonally, or rectally as desired.
  • oral administration may denature the fusion protein by gastric acid
  • oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach.
  • the composition may be administered by any device in which the active substance may migrate to the target cell.
  • the present invention provides the use of the epoxylon derivative for use in the manufacture of the pharmaceutical composition for treating cancer.
  • PCR was performed using the genome DNA of Bacillus licheniformis as a template to obtain BL-C (UDP-glucosyltransferase C, NCBI accession number AAU40842) gene, and the gene was expressed as expression vector pET302 / NT-.
  • a recombinant vector was constructed by cloning his XhoI and BamHI sites. The recombinant vector thus prepared was introduced into Escherichia coli BL21 (DE3) to prepare a transformant.
  • the prepared transformants were inoculated at 50% LB liquid medium at a concentration of 1%, shaken at 37 ° C., and an IPTG (isopropyl-1-thio- ⁇ -D-galactopyranoside when the absorbance value was 0.5 at 600 nm. ) was incubated under aerobic conditions. After the incubation was terminated, the cultured cells were centrifuged to recover the precipitated cells, and 3 ml of the cell grinding buffer (1 mM PMSF, protease inhibitor cocktail, 10 mM imidazole, 100 mM Tris-Cl, pH 8.0) was added to the recovered cells. In addition, a suspension was obtained.
  • IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside when the absorbance value was 0.5 at 600 nm.
  • PCR was performed using the genome DNA of B. amyloliquefaciens subsp . Plantarum AH159-1 as a template to secure a gene encoding BmgtB, a type of glucose transferase, and expressing the gene.
  • the recombinant vector was constructed by cloning into the vector pET28a.
  • the recombinant vector thus prepared was introduced into Escherichia coli BL21 (DE3) to prepare a transformant.
  • the prepared transformants were inoculated at 50% / ml LBA liquid medium containing 50 mg / ml kanamycin at 1% concentration, shaken at 37 ° C., and IPTG was added when the absorbance value was 0.5 to 0.6 at 600 nm.
  • the cultured cells were centrifuged to recover the precipitated cells, and 3 ml of the cell grinding buffer (1 mM PMSF, protease inhibitor cocktail, 10 mM imidazole, 100 mM Tris-Cl, pH 8.0) was added to the recovered cells. In addition, a suspension was obtained. Ultrasonic was added to the suspension to disrupt the cells, the lysate was centrifuged to obtain a supernatant, and the supernatant was subjected to Ni-NTA column chromatography to prepare the desired BmgtB.
  • the cell grinding buffer (1 mM PMSF, protease inhibitor cocktail, 10 mM imidazole, 100 mM Tris-Cl, pH 8.0
  • Epocylon derivatives were prepared using the glucose transferase (BL-C or BmgtB) prepared in Example 1.
  • EpoA-G and EpoB-G were identified by application to NMR analysis and HR-ESI-MS analysis (Tables 1 and 2).
  • each of the epoxylon derivatives (EpoA-G or EpoB-G) prepared in Example 2 was dissolved in DMSO to obtain 0.1, 0.3, 1, 3, 10, 30 and 50 mM solutions, respectively.
  • the prostate cancer cell line PC-3 the lung cancer cell line NCI-H23, the breast cancer cell line MDA-MB-231, the colorectal cancer cell line HCT-15, the kidney cancer cell line ACHN and the gastric cancer cell line RPMI1640, respectively And 10% calf serum.
  • Each cultured cell line was divided into 96-well plates, and the obtained epoxylon derivative solution was added to each of the divided cell lines so that final concentrations were 0.1, 0.3, 1, 3, 10, 30, 50, and 100 uM. Then it was treated for 48 hours. Subsequently, 50 ⁇ l of 50% TCA solution was added to the wells containing each cell line, and each cell line was fixed, left at 4 ° C.
  • the washed 96-well plate was dried, 100 ⁇ l of SRB solution (0.4% sulforhodamine B in 1% acetic acid) was added to each well, and left for 30 minutes. 0.1% acetic acid solution was added thereto, followed by washing, and remaining in each well. The dye was removed.
  • the 96 well plate was dried again, 100 ⁇ l of 10 mM Tris Base (pH 10.5) was added to each well, and the absorbance was measured at 540 nm using a Versa max microplate reader (Molecular Devices). The measured absorbance was applied to Graphpad prism v4.0 software to calculate the growth inhibition index (GI50) value (Table 3).
  • the sample and the ethyl acetate layer before extraction with ethyl acetate contained both epoxylon and its derivatives, it was confirmed that only the epoxylon derivative is present in the remaining water layer.
  • the content of epoxylon derivatives present in the aqueous layer was analyzed to correspond to about 30% of the content of epoxylon derivatives present in the sample before extraction with ethyl acetate.
  • the phosphoylone derivative of the present invention having a water solubility of about 30% is relatively high in water solubility. It was analyzed to have.
  • the epoxylon derivatives prepared in Example 2 were orally administered (20 mg / kg) or intravenously administered (5 mg / kg) to male IRC mice as experimental animals, and the epoxyyl present in plasma over time after administration. Levels of lone derivatives and epoxylons were measured and compared (FIGS. 2A and 2B).
  • the measurement of the level of epoxylon derivatives and epoxylons present in plasma was performed using a triple quadrupole mass spectrometer (3200 Q TRAP LC / MS / MS), and the column was Waters Xterra MS C18 (2.1 x 50 mm, 5 ⁇ m) was used, the acetonitrile concentration gradient (5% to 95%) containing 0.1% formic acid was used, and the flow rate was set to 0.4 ml / min.
  • the plasma levels of orally administered epoxylon derivatives decreased over time and were not detected in plasma after about 5 hours.
  • the plasma levels of epoxylons increased with time, reaching the highest level after about 4 hours, after which the plasma levels gradually decreased, and even after 24 hours, was detected at a level similar to that detected in plasma.
  • the plasma level of intravenous administered epoxylon derivatives decreased over time, but after about 2 hours was not detected in the plasma, compared to oral administration It was confirmed that the plasma was exhausted within a short time. In contrast, the plasma level of epoxylon increased over time and reached the highest level after about 2 hours, after which the level of plasma gradually decreased, and administration was continued after about 8 hours. Immediately after it was confirmed that it was detected at a higher level than that detected in plasma.
  • the level of the epoxylon derivatives decreases over time and in the plasma after a certain time. While undetected, the level of epoxylons has a common point that they are continuously detected in plasma for the measured time, which means that the epoxylon derivatives provided in the present invention are degraded in the body to form epoxylons. It was analyzed.
  • glycoside-derived derivatives are prepared by combining glucose and the like with compounds that simultaneously exhibit anticancer activity and poor solubility, such as epoxylon
  • the derivatives are known to increase water solubility while significantly reducing anticancer activity.
  • the epoxylon derivative of the present invention can be utilized as a prodrug. That is, the phosphoylone derivatives of the present invention have increased water solubility compared to epoxylons and are easily formulated, and thus easy to administer to cancer patients.
  • the epoxylon derivatives of the present invention have a lower level than that of epoxylon, but exhibit anti-cancer activity, and to some cancer cell lines (HCT15 or NUGC-3), adriamycin used as a control. It was confirmed that it shows a relatively good level of anticancer activity.

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Abstract

The present invention relates to a novel epothilone derivative having improved water solubility by glucosylation of epothilone, to a method for preparing an epothilone derivative, to a pharmaceutical composition containing the epothilone derivative as an active ingredient for cancer treatment, to a method for treating cancer using the pharmaceutical composition, and to a use of the epothilone derivative used in the preparation of the pharmaceutical composition for cancer treatment. The epothilone derivative of the present invention has improved water solubility when compared with the conventional epothilone without the deterioration in anticancer activity, and thus can be widely used in the formulation of epothilone.

Description

신규한 에포싸일론 유도체 및 그의 용도Novel epoxylon derivatives and uses thereof
본 발명은 신규한 에포싸일론 유도체 및 그의 용도에 관한 것으로, 보다 구체적으로 본 발명은 에포싸일론이 글루코실화되어 수용성이 향상된 신규한 에포싸일론 유도체, 상기 에포싸일론 유도체의 제조방법, 상기 에포싸일론 유도체를 유효성분으로 포함하는 암치료용 약학 조성물, 상기 약학 조성물을 이용하여 암을 치료하는 방법 및 암치료용 약학 조성물의 제조에 사용되는 상기 에포싸일론 유도체의 용도에 관한 것이다.The present invention relates to a novel epoxylon derivative and its use, and more particularly, the present invention relates to a novel epoxylon derivative having improved water solubility due to glucosylation of the epoxylon, a method for preparing the epoxylon derivative, the epoxy The present invention relates to a pharmaceutical composition for treating cancer comprising a cyclone derivative as an active ingredient, a method of treating cancer using the pharmaceutical composition, and a use of the epoxylon derivative used in the manufacture of a pharmaceutical composition for treating cancer.
에포싸일론(epothilone)은 토양에서 서식하는 박테리아 균주(myxobacterium Sorangium cellulosum strain 90)로 부터 분리된 천연물로서, 탁솔(taxol)에 내성을 갖는 종양세포에 대하여 세포독성을 나타내며 탁솔(taxol)보다 우수한 임상학적 유용성을 나타낸다고 알려져 있다. 상기 에포싸일론은 세포 분열 주기에서 특정 위치에 정렬했다가 해체되는 실과 같은 세포 내부 골격인 미세소관(microtubule)에 작용하여 세포분열을 억제함으로써, 항암활성을 나타내는데, 탁솔에 비하여 화학적 구조가 단순하고, 미세소관을 안정화함에 있어서 에포싸일론은 탁솔 보다 2000 ∼ 5000배 우수한 효과를 나타내며, 난용성인 탁솔에 비하여 부분적으로 수용성을 나타내어, 제제화가 용이하다는 장점을 가지고 있다. 탁솔과 에포싸일론은 화학 구조적으로 상이함에도 불구하고 에포싸일론이 결합자리로부터 탁솔을 대체하고 미세소관의 같은 활성자리에 결합하는 것으로 알려져 있으므로, 구조적인 신규성, 중요한 생물학적 작용과 흥미로운 작용 기작을 중심으로 활발한 연구가 진행되고 있다. Epothilone is a natural product isolated from soil bacteria ( myxobacterium Sorangium cellulosum strain 90), which is cytotoxic to tumor cells resistant to taxol and is superior to taxol. It is known to exhibit scientific utility. The epoxylon exhibits anticancer activity by acting on a microtubule, an intracellular framework like a thread that is aligned at a specific position in the cell division cycle and then disintegrated, thereby inhibiting cell division, and has a simple chemical structure compared to Taxol. In stabilizing microtubules, epoxylons have an effect of 2000 to 5000 times better than taxol, and have partial water solubility as compared to poorly soluble taxol, and have the advantage of easy formulation. Although taxol and epoxylons are known to differ in chemical structure, epoxylons are known to replace taxol from binding sites and bind to the same active site of microtubules, thus focusing on structural novelty, important biological and interesting mechanisms of action. Active research is in progress.
예를 들어, 에포싸일론의 X-선 결정 구조가 발표된 이래로 유기 화학자들은 이 화합물의 전합성에 관한 연구를 시도하였고, 니콜라우(Nicolaou)와 대니쉐프스키(Danishefsky)에 의해 가장 먼저 전합성되었으며, 이후 다수의 연구자에 의하여 에포싸일론을 전합성하는 방법이 개발되었다. 현재까지 공지되어 있는 에포싸일론의 대표적인 전합성법은 (1) 싸이클로프로판의 유도체인 글리칼(glycal)을 가용매 분해시켜 싸이클로프로판의 고리를 개환하여 제미날 다이메틸기(gem-dimethyl)를 만드는 단계; (2) 알킬 스즈키 결합반응을 이용해서 옆사슬기를 결합시키는 단계; (3) 에포싸일론의 C-16 위치에서 폐환되도록 분자내-알돌축합 반응을 수행하는 단계; 및 (4) 시스-이중결합 위치를 매우 입체 특이적으로 에폭시화하는 단계를 포함한다.For example, since the release of X-ray crystal structures of epoxylons, organic chemists have attempted to study the presynthesis of the compound, the first to be synthesized by Nicolaou and Danishefsky. Since then, a number of researchers have developed a method for presynthesizing epoxylons. Representative electrosynthesis of epoxylon known to date is (1) solvolysis of glycal, a derivative of cyclopropane, to open the ring of cyclopropane to form a gem-dimethyl group (gem-dimethyl). ; (2) bonding the side chain groups using an alkyl Suzuki binding reaction; (3) conducting an intramolecular-aldol condensation reaction such that the ring is closed at the C-16 position of the epoxylon; And (4) very stereospecific epoxidation of the cis-double bond sites.
이같이 제조된 에포싸일론은 탁솔보다는 우수하지만, 부분적인 수용성을 나타내므로, 제제화가 용이하지 않다는 단점이 있어, 이러한 단점을 극복하기 위한 다양한 유도체가 개발되었다. 예를 들어, ixabepilone(azaepothilone B), BMS-310705, patupilone, epothilone R1645, ZK-EPO, ABJ879(C20-desmethyl-C20-methylsulfanyl-EpoB) 등을 들 수 있는데, 이들 유도체는 수용성의 증가폭이 크지 않거나 또는 수용성이 증가된 반면 원래의 항암활성이 감소되는 등의 단점이 있어, 이러한 단점을 극복한 새로운 유도체의 개발이 요구되었다.Although the prepared epoxylon is superior to taxol, but shows partial water solubility, there is a disadvantage in that it is not easy to formulate, and various derivatives have been developed to overcome this disadvantage. For example, ixabepilone (azaepothilone B), BMS-310705, patupilone, epothilone R1645, ZK-EPO, ABJ879 (C20-desmethyl-C20-methylsulfanyl-EpoB), etc. Or there is a disadvantage in that the water solubility is increased while the original anticancer activity is reduced, it was required to develop a new derivative that overcomes these disadvantages.
본 발명자들은 수용성이 증가되면서도 항암활성이 유지되는 에포싸일론 유도체를 개발하고자 예의 연구노력한 결과, 에포싸일론이 글리코실화된 유도체가 향상된 수용성을 나타내면서도 항암활성이 유지됨을 확인하고, 본 발명을 완성하였다. The present inventors earnestly researched to develop an epoxylon derivative that maintains anticancer activity while increasing water solubility. As a result, the present inventors confirmed that the phosphoyl glycosylated derivative exhibited improved water solubility and maintained anticancer activity. It was.
본 발명의 하나의 목적은 에포싸일론이 글루코실화된 에포싸일론 유도체를 제공하는 것이다.One object of the present invention is to provide an epoxylon derivative in which epoxylon is glucosylated.
본 발명의 다른 목적은 상기 에포싸일론 유도체의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for preparing the epoxylon derivative.
본 발명의 또 다른 목적은 상기 에포싸일론 유도체를 포함하는 암치료용 약학 조성물을 제공하는 것이다.Another object of the present invention to provide a pharmaceutical composition for treating cancer comprising the epoxylon derivative.
본 발명의 또 다른 목적은 상기 암치료용 약학 조성물을 이용하여 암을 치료하는 방법을 제공하는 것이다.Still another object of the present invention is to provide a method of treating cancer using the pharmaceutical composition for treating cancer.
본 발명의 또 다른 목적은 상기 암치료용 약학 조성물의 제조를 위한 상기 에포싸일론 유도체의 용도를 제공하는 것이다.Still another object of the present invention is to provide a use of the epoxylon derivative for the preparation of the pharmaceutical composition for treating cancer.
본 발명의 에포싸일론 유도체는 종래의 에포싸일론에 비하여 수용성이 향상되면서도 항암활성이 저하되지 않으므로, 에포싸일론의 제제화에 널리 활용될 수 있을 것이다.The epoxylon derivative of the present invention can be widely used in the formulation of epoxylon since the anti-cancer activity is not lowered even though the water solubility is improved compared to the conventional epoxylon.
도 1a는 에틸아세테이트로 추출하기 이전 시료를 대상으로 한 HPLC 분석결과를 나타낸다.Figure 1a shows the results of HPLC analysis of the sample before extraction with ethyl acetate.
도 1b는 에틸아세테이트 층을 대상으로 한 HPLC 분석결과를 나타낸다.Figure 1b shows the results of HPLC analysis on the ethyl acetate layer.
도 1c는 잔류하는 수층을 대상으로 한 HPLC 분석결과를 나타낸다.Figure 1c shows the results of HPLC analysis of the remaining water layer.
도 2a는 본 발명의 에포싸일론 유도체를 실험동물에 경구투여한 후, 시간의 경과에 따른 혈장내 에포싸일론 유도체 및 에포싸일론의 수준을 측정한 결과를 나타내는 그래프로서, Epo-AG는 에포싸일론 유도체를 나타내고, Epo-A는 에포싸일론을 나타낸다.Figure 2a is a graph showing the results of measuring the level of plasma epoxylon derivatives and epoxylon over time after oral administration of the epoxylon derivative of the present invention to the experimental animal, Epo-AG is eppo Represents a cyclone derivative, and Epo-A represents an epoxylon.
도 2b는 본 발명의 에포싸일론 유도체를 실험동물에 정맥주사투여한 후, 시간의 경과에 따른 혈장내 에포싸일론 유도체 및 에포싸일론의 수준을 측정한 결과를 나타내는 그래프로서, Epo-AG는 에포싸일론 유도체를 나타내고, Epo-A는 에포싸일론을 나타낸다.Figure 2b is a graph showing the results of measuring the level of plasma phosphoyl derivatives and epoxylon over time after administration of the epoxylon derivative of the present invention to experimental animals, Epo-AG is Epoxylon derivatives are shown, and Epo-A represents epoxylons.
본 발명자들은 수용성이 증가되면서도 항암활성이 유지되는 에포싸일론 유도체를 개발하고자 다양한 연구를 수행하던 중, 글리코실화에 주목하게 되었다. 통상적으로, 글리코실화(glycosylation)란 글리코실기(glycosyl group)를 단백질과 같은 고분자 화합물에 전위시키는 반응을 의미하는데, 이처럼 글리코실화된 고분자 화합물은 복합당질을 형성하여 생체내에서 다양한 기능을 수행할 수 있게 된다. 본 발명자들은 상기 글리코실화에 사용되는 글리코실기는 풍부한 친수성 반응기를 포함하기 때문에, 본 발명의 에포싸일론과 같은 부분적인 수용성을 갖는 화합물에 있어서, 우수한 수용성을 갖게 할 것으로 예상하였다. 이에, 에포싸일론의 유도체 화합물로 알려진 에포싸일론 A(EpoA) 및 에포싸일론 B(EpoB)에 글루코스전달효소(glucosyltransferase)를 처리하여 글루코스 잔기가 결합된 에포싸일론 유도체 화합물을 제조하고, 상기 제조된 유도체 화합물의 수용성 및 항암활성을 비교한 결과, 상기 제조된 유도체 화합물은 수용성이 증가되면서도 항암활성이 크게 변화되지 않음을 알 수 있었다. 또한, 상기 에포싸일론 유도체의 수용성을 평가한 결과, 약 30%의 수용성을 나타내고, 상기 에포싸일론 유도체는 체내에서 에포싸일론을 형성할 수 있음을 확인하였다.The present inventors came to pay attention to glycosylation while conducting various studies to develop an epoxylon derivative that maintains anticancer activity while increasing water solubility. Typically, glycosylation refers to a reaction in which a glycosyl group is transposed to a high molecular compound such as a protein, and the glycosylated high molecular compound may form a complex sugar to perform various functions in vivo. Will be. The inventors expected that the glycosyl groups used for the glycosylation include abundant hydrophilic reactors, so that the compounds having partial water solubility, such as the epoxylon of the present invention, would have excellent water solubility. Thus, by treating glucose transferase (glucosyltransferase) to the epoxylon A (EpoA) and epoxylon B (EpoB) known as derivative compounds of the epoxylon to prepare a phosphoylone derivative compound with a glucose moiety, As a result of comparing the water solubility and anticancer activity of the prepared derivative compounds, it was found that the anticancer activity was not significantly changed while the water solubility was increased. In addition, as a result of evaluating the water solubility of the epoxylon derivative, it was confirmed that the water soluble of about 30%, the epoxylon derivative can form epoxylon in the body.
상기 목적을 달성하기 위한 일 실시양태로서, 본 발명은 에포싸일론이 글루코실화된 에포싸일론 유도체를 제공한다.As one embodiment for achieving the above object, the present invention provides an epoxylon derivative glucosylated epoxylon.
본 발명의 용어 "에포싸일론(epothilone)"이란, 토양 박테리아(myxobacterium Sorangium cellulosum strain 90)로부터 생산되고, 우수한 항암활성과 부분적인 수용성을 갖는 천연 화합물을 의미한다. 상기 에포싸일론의 각 기능기가 치환된 다양한 유도체(epothilones A 내지 F)가 공지되어 있으며, 상기 유도체는 항암활성 뿐만 아니라, 알츠하이머 등의 다양한 질환에 대한 약리활성 효과를 나타낸다고 알려져 있다.The term "epothilone" of the present invention refers to a natural compound produced from soil bacteria ( myxobacterium Sorangium cellulosum strain 90) and having excellent anticancer activity and partial water solubility. Various derivatives (epothilones A to F) in which each functional group of the epoxylon is known are known, and the derivatives are known to exhibit pharmacologically active effects on various diseases such as Alzheimer's disease as well as anticancer activity.
본 발명에 있어서, 상기 에포싸일론은 우수한 항암효과를 나타낸다고 알려진 epothilones A(EpoA) 또는 epothilones B(EpoB)가 될 수 있으나, 글리코실화에 의하여 수용성이 증가하면서도 항암활성이 유지되는 특성을 나타내는 한 특별히 에포싸일론의 종류가 제한되지는 않는다.In the present invention, the eposilone may be epothilones A (EpoA) or epothilones B (EpoB), which are known to exhibit excellent anticancer effects, but in particular, as long as the water solubility is increased by glycosylation, the anticancer activity is maintained. The type of epoxylon is not limited.
본 발명의 용어 "에포싸일론 A(epothilones A, EpoA)"란, 에포싸일론 유도체 화합물 중의 하나로서, 분자량 656.3101, C32H50NO11S의 화학식 및 하기 화학식 1의 구조를 가지는 화합물을 의미한다. As used herein, the term "epothilones A (EpoA)" refers to a compound having a chemical formula of molecular weight 656.3101, C 32 H 50 NO 11 S and a structure represented by the following Chemical Formula 1 as one of the epoxylon derivative compounds. do.
화학식 1
Figure PCTKR2014004346-appb-C000001
 Formula 1
Figure PCTKR2014004346-appb-C000001
본 발명의 용어 "에포싸일론 B(epothilones B, EpoB)"란, 에포싸일론 유도체 화합물 중의 하나로서, 분자량 692.3077, C32H51NO11NaS의 화학식 및 하기 화학식 2의 구조를 가지는 화합물을 의미한다. The term "epothilones B (EpoB)" as used herein refers to a compound having a chemical formula of molecular weight 692.3077, C 32 H 51 NO 11 NaS and a structure represented by the following Chemical Formula 2, as one of the epoxylon derivative compounds. do.
화학식 2
Figure PCTKR2014004346-appb-C000002
 Formula 2
Figure PCTKR2014004346-appb-C000002
본 발명의 용어 "글루코실화(glucosylation)"란, 글리코실화(glycosylation)의 일종으로서, 목적하는 화합물에 글루코실기의 1번 탄소를 결합시켜서, 글루코실기를 전이시키는 반응을 의미한다. The term "glucosylation" of the present invention is a kind of glycosylation, and means a reaction in which the first carbon of the glucosyl group is bonded to the desired compound to transfer the glucosyl group.
본 발명의 용어 "글루코실기(glucosyl group)"란, 글리코실기(glycosyl group)의 일종으로서, 글루코스 분자에서 헤미아세탈 수산기를 제거한 1가(價)의 반응기를 의미한다. As used herein, the term "glucosyl group" refers to a monovalent reactor in which a hemiacetal hydroxyl group is removed from a glucose molecule as a type of glycosyl group.
본 발명에 있어서, 상기 글루코실화는 에포싸일론 바람직하게는 EpoA 또는 EpoB에 글루코실기를 전달하는 반응이 될 수 있는데, 이러한 글루코실화는 특별히 제한되지 않으나, 공지된 화학적 방법, 바람직하게는 생화학적 방법에 의해 수행될 수 있고, 보다 바람직하게는 효소를 이용한 방법에 의해 수행될 수 있고, 가장 바람직하게는 글루코스전달효소를 이용하여 수행될 수 있다.In the present invention, the glucosylation may be a reaction for transferring a glucosyl group to an epocylon, preferably EpoA or EpoB, but such glucosylation is not particularly limited, but a known chemical method, preferably a biochemical method It can be carried out by, and more preferably by a method using an enzyme, and most preferably can be performed using a glucose transferase.
본 발명의 용어 "글루코스전달효소(glucosyltransferase)"란, 글루코실기의 전달을 촉매하여 상기 글루코실화를 수행할 수 있는 효소를 의미하는데, 본 발명의 목적상 상기 글루코스전달효소는 글루코실기의 공여체로부터 수득한 글루코실기를 에포싸일론에 전달하는 촉매반응을 수행하는 효소로 이해될 수 있다. 이때, 상기 글루코실기의 공여체로서는 당인산(Glc-1-P 등); 뉴클레오티드 글루코오스(UDP-Glc 등); 올리고당(수크로오스 등); 다당 등을 단독으로 또는 혼합하여 사용할 수 있다.The term "glucosyltransferase" of the present invention refers to an enzyme capable of performing the glucosylation by catalyzing the transfer of a glucosyl group. For the purposes of the present invention, the glucose transferase is obtained from a donor of a glucosyl group. It can be understood as an enzyme that performs a catalysis to transfer one glucosyl group to the epoxylon. In this case, as a donor of the glucosyl group, sugar phosphate (Glc-1-P, etc.); Nucleotide glucose (UDP-Glc, etc.); Oligosaccharides (such as sucrose); Polysaccharides and the like can be used alone or in combination.
본 발명에 있어서, 상기 글루코스전달효소는 에포싸일론의 글루코실화를 수행할 수 있는 한 특별히 제한되지 않으나, 바람직하게는 MGT(macroside glycosyltransferase) 계열의 글루코스전달효소 또는 뉴클레오티드 글루코오스를 글루코실기의 공여체로 사용하는 글루코스전달효소를 사용할 수 있고, 보다 바람직하게는 바실러스 균주(B. amyloliquefaciens)로부터 유래된 MGT 계열의 글루코스전달효소 또는 UDP를 글루코실기의 공여체로 사용하는 글루코스전달효소를 사용할 수 있으며, 가장 바람직하게는 바실러스 균주로부터 유래된 MGT 계열의 글루코스전달효소인 BmgtB, BL-C(UDP-glucosyltransferase C) 등을 사용할 수 있다.In the present invention, the glucose transferase is not particularly limited as long as it can carry out glucosylation of eposylon, but preferably, MGT (macroside glycosyltransferase) -based glucose transferase or nucleotide glucose is used as a donor of a glucosyl group. The glucose transferase may be used, and more preferably, a MGT-based glucose transferase or UDP derived from B. amyloliquefaciens or a glucose transferase using UDP as a donor of a glucosyl group may be used. Mg-based glucose transferases BmgtB, BL-C (UDP-glucosyltransferase C) derived from the Bacillus strain can be used.
본 발명의 용어 "글루코실화된 에포싸일론 유도체"란, 글루코스전달효소에 의하여 에포싸일론의 탄소에 글루코실기가 전달되어 결합된(글루코실화된) 형태의 유도체 화합물을 의미한다. As used herein, the term "glucosylated epoxylon derivative" refers to a derivative compound in which a glucosyl group is transferred to a carbon of epoxylon by a glucose transferase and bound (glucosylated).
본 발명에 있어서, 상기 글루코실화된 에포싸일론 유도체는 특별히 이에 제한되지 않으나, 바람직하게는 에포싸일론의 탄소가 글루코실화된 유도체 화합물이 될 수 있고, 보다 바람직하게는 에포싸일론의 7번 탄소가 글루코실화된 유도체 화합물이 될 수 있으며, 가장 바람직하게는 EpoA의 7번 탄소가 글루코실화되어 화학식 3으로 표시될 수 있는 EpoA-G(7-glucosyl epothilones A) 또는 EpoB의 7번 탄소가 글루코실화되어 화학식 4로 표시될 수 있는 EpoB-G(7-glucosyl epothilones B)가 될 수 있다.In the present invention, the glucosylated epoxylon derivative is not particularly limited thereto, but preferably carbon of epoxylon may be a glucosylated derivative compound, more preferably carbon 7 of epoxylon. May be a glucosylated derivative compound, and most preferably, carbon 7 of EpoA (7-glucosyl epothilones A) or carbon 7 of EpoB is glucosylated by glucosylating carbon 7 of EpoA. To be EpoB-G (7-glucosyl epothilones B) which can be represented by the formula (4).
화학식 3
Figure PCTKR2014004346-appb-C000003
 Formula 3
Figure PCTKR2014004346-appb-C000003
화학식 4
Figure PCTKR2014004346-appb-C000004
 Formula 4
Figure PCTKR2014004346-appb-C000004
본 발명의 일 실시예에 따르면, 바실러스 리체니포르미스(Bacillus licheniformis) 유래의 재조합 글루코스전달효소인 BL-C(UDP-glucosyltransferase C)(실시예 1-1) 또는 BmgtB(실시예 1-2)를 제조하고, 상기 제조된 BL-C 또는 BmgtB을 이용하여 EpoA 또는 EpoB의 7번 탄소를 글루코실화시켜서 글루코실화된 에포싸일론 유도체를 제조하였으며(표 1 및 표 2), 상기 제조된 에포싸일론 유도체는 글루코실화에 의해 수용성이 증가하였으며, 그의 항암활성을 측정한 결과, 6종류의 암세포주에 대하여 모두 항암활성을 나타냄을 확인하였다(표 3). 또한, 상기 에포싸일론 유도체의 수용성을 평가한 결과 약 30%의 수용성을 나타냄을 확인하였고(도 1a 내지 1c), 상기 에포싸일론 유도체를 경구 또는 정맥주사 방법으로 체내에 투여한 경우, 에포싸일론 유도체의 혈장내 수준이 급속하게 저하되고, 에포싸일론의 혈장내 수준이 증가한 후, 서서히 감소함을 확인하였다(도 2a 및 2b).According to an embodiment of the present invention, recombinant glucose transferase BL-C (UDP-glucosyltransferase C) derived from Bacillus licheniformis (Example 1-1) or BmgtB (Example 1-2) And glucosylated carbon 7 of EpoA or EpoB using the prepared BL-C or BmgtB to prepare a glucosylated epoxylon derivative (Table 1 and Table 2), and the prepared epoxylon Derivatives increased water solubility by glucosylation, and the anticancer activity of the derivatives showed anticancer activity against all six cancer cell lines (Table 3). In addition, as a result of evaluating the water solubility of the epoxylon derivative, it was confirmed that the water solubility was about 30% (FIGS. 1A to 1C), and when the epoxylon derivative was administered to the body by oral or intravenous injection method, epoxyyl It was confirmed that the plasma levels of the Ron derivatives were rapidly lowered, and the plasma levels of the epoxylons were increased and then gradually decreased (FIGS. 2A and 2B).
상기 목적을 달성하기 위한 다른 실시양태로서, 본 발명은 (a) 에포싸일론 화합물에 글루코실기의 공여체 및 글루코스전달효소를 가하여 글루코실화 반응을 수행하는 단계; 및 (b) 상기 반응물로부터 에포싸일론 유도체를 회수하는 단계를 포함하는 상기 글루코실화된 에포싸일론 유도체의 제조방법을 제공한다.As another embodiment for achieving the above object, the present invention comprises the steps of (a) adding a glucosyl donor and glucose transferase to the epoxylon compound to perform a glucosylation reaction; And (b) provides a method for producing the glucosylated epoxylon derivative comprising the step of recovering the epoxylon derivative from the reactant.
이때, 상기 에포싸일론 화합물, 글루코실기의 공여체 및 글루코스전달효소로는 상술한 바와 동일하다. 또한, 상기 반응을 수행하기 위한 pH 조건은 중성 pH(pH 7.0 내지 8.0)이고, 온도 조건은 25 내지 35℃이며, 시간 조건은 10 내지 30시간이다. 아울러, 상기 반응시에 사용되는 에포싸일론 화합물 및 글루코실기의 공여체 의 혼합비는 바람직하게는 1:1 내지 1:10(w/w), 보다 바람직하게는 1:1 내지 1:5(w/w), 가장 바람직하게는 1:4(w/w) 이다.At this time, the epoxylon compound, the donor of the glucosyl group and the glucose transferase are the same as described above. In addition, the pH conditions for performing the reaction is neutral pH (pH 7.0 to 8.0), the temperature conditions are 25 to 35 ℃, the time conditions are 10 to 30 hours. In addition, the mixing ratio of the donor of the epoxylon compound and the glucosyl group used in the reaction is preferably 1: 1 to 1:10 (w / w), more preferably 1: 1 to 1: 5 (w / w), most preferably 1: 4 (w / w).
아울러, 반응물로부터 에포싸일론 유도체를 회수하는 단계는 당업계에 공지된 방법에 의해 수행될 수 있다. 구체적으로, 에포싸일론 유도체 회수 방법은 특별히 이에 제한되지 않으나, 원심분리, 여과, 추출, 분무, 건조, 증발, 침전, 결정화, 전기영동, 분별용해(예를 들면 암모늄 설페이트 침전), 크로마토그래피(예를 들면 이온 교환, 친화성, 소수성 및 크기배제) 등의 방법을 사용함이 바람직하다.In addition, the step of recovering the epoxylon derivative from the reactant can be carried out by methods known in the art. Specifically, the method for recovering the epoxylon derivatives is not particularly limited, but centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (eg, ammonium sulfate precipitation), chromatography ( For example, ion exchange, affinity, hydrophobicity and size exclusion).
상기 목적을 달성하기 위한 또 다른 실시양태로서, 본 발명은 상기 에포싸일론 유도체를 포함하는 암치료용 약학 조성물을 제공한다.As another embodiment for achieving the above object, the present invention provides a pharmaceutical composition for treating cancer comprising the epoxylon derivative.
본 발명에서 제공하는 글루코실화된 에포싸일론 유도체는 종래의 에포싸일론에 비하여 수용성이 향상되는 한편, 항암활성을 유지하였을 뿐만 아니라 체내에서 에포싸일론으로 전환됨을 확인하였으므로, 상기 에포싸일론 유도체는 암치료용 약학 조성물에 포함되는 프로드러그 형태의 유효성분으로 사용될 수 있음을 알 수 있었다.Since the glucosylated epoxylon derivatives provided by the present invention have improved water solubility compared to the conventional epoxylons, not only maintain anticancer activity but also are converted into epoxylons in the body, the epoxylon derivatives are It can be seen that it can be used as an active ingredient in the form of prodrugs contained in the pharmaceutical composition for cancer treatment.
상기 본 발명의 약학적 조성물은 약학적으로 허용 가능한 희석제, 부형제 또는 담체를 추가로 포함할 수 있다. 약학적으로 허용 가능한 담체를 포함하는 상기 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable diluent, excipient or carrier. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablet pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose in one or more compounds. ) And gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solution solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
상기 약학적 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제 및 좌제로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있다.The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It can have one formulation.
본 발명에 있어서, 상기 약학 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여도 투여될 수 있다. 본 발명의 약학 조성물은 목적하는 바에 따라 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 비내 투여, 폐내 투여, 직장내 투여될 수 있으나, 이에 제한되지는 않는다. 또한, 상기 약학 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.In the present invention, the route of administration of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered as desired, but is not limited to intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, pulmonary administration, rectal administration. In addition, the pharmaceutical composition may be administered by any device in which the active substance may migrate to the target cell.
본 발명의 상기 약학 조성물에 포함된 에포싸일론 유도체의 함량은 특별히 이에 제한되지 않으나, 최종 조성물 총 중량에 대하여 0.0001 내지 50 중량%로 포함할 수 있고, 바람직하게는 0.001 내지 10 중량%의 함량으로 포함할 수 있다. The content of the epoxylon derivative included in the pharmaceutical composition of the present invention is not particularly limited, but may be included in 0.0001 to 50% by weight relative to the total weight of the final composition, preferably in an amount of 0.001 to 10% by weight. It may include.
상기 본 발명의 약학 조성물은 약학적으로 유효한 양으로 투여될 수 있는데, 본 발명의 용어 "약학적으로 유효한 양"이란, 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하다.The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term "pharmaceutically effective amount" of the present invention, the amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment Effective dose levels are well-known for individual types and severities, age, gender, drug activity, drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other medical fields. It can be determined according to known factors. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.
본 발명의 에포싸일론 유도체를 포함하는 암치료용 약학 조성물의 투여량은 사용목적, 질환의 중독도, 환자의 연령, 체중, 성별, 기왕력, 또는 유효성분으로서 사용되는 물질의 종류 등을 고려하여 당업자가 결정할 수 있다. 예를 들어, 본 발명의 약학 조성물은 성인 1인당 약 0.1 ng 내지 약 100 mg/kg, 바람직하게는 1 ng 내지 약 10 mg/kg로 투여할 수 있고, 본 발명의 조성물의 투여빈도는 특별히 이에 제한되지 않으나, 1일 1회 투여하거나 또는 용량을 분할하여 수회 투여할 수 있다.The dosage of the pharmaceutical composition for treating cancer containing the epoxylon derivative of the present invention may be used in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as an active ingredient of the patient. One skilled in the art can decide. For example, the pharmaceutical composition of the present invention may be administered at about 0.1 ng to about 100 mg / kg, preferably 1 ng to about 10 mg / kg, per adult, and the frequency of administration of the composition of the present invention is specifically Although not limited, it can be administered once a day or several times in divided doses.
상기 목적을 달성하기 위한 또 다른 실시양태로서, 본 발명은 상기 약학 조성물을 약제학적으로 유효한 양으로 암질환이 발병된 개체에 투여하는 단계를 포함하는 암을 치료하는 방법을 제공한다.As another embodiment for achieving the above object, the present invention provides a method for treating cancer comprising administering the pharmaceutical composition to a subject having a cancer disease in a pharmaceutically effective amount.
상술한 바와 같이, 본 발명에서 제공하는 에포싸일론 유도체는 항암활성을 가질 뿐만 아니라, 체내에서 에포싸일론으로 전환될 수 있으므로, 상기 조성물은 암을 치료하는데 사용될 수 있다.As described above, the epoxylon derivatives provided in the present invention not only have anticancer activity, but also can be converted into epoxylon in the body, so that the composition can be used to treat cancer.
본 발명에서 용어 "개체"란 암이 발병될 가능성이 있거나 또는 발병된 쥐, 가축, 인간 등을 포함하는 포유동물을 제한 없이 포함한다.As used herein, the term "individual" includes, without limitation, mammals, including rats, livestock, humans, and the like, which have or are likely to develop cancer.
본 발명의 암을 치료하는 방법에 있어서, 상기 약학 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여도 투여될 수 있다. 본 발명의 약학 조성물은 특별히 이에 제한되지 않으나, 목적하는 바에 따라 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 비내 투여, 폐내 투여, 직장내 투여될 수 있다. 다만, 경구 투여시에는 위산에 의하여 상기 융합단백질이 변성될 수 있기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 되어야 한다. 또한, 상기 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다. In the method of treating cancer of the present invention, the route of administration of the pharmaceutical composition may be administered via any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonally, or rectally as desired. However, since oral administration may denature the fusion protein by gastric acid, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.
상기 목적을 달성하기 위한 또 다른 실시양태로서, 본 발명은 상기 암 치료용 약학 조성물의 제조에 사용하기 위한 에포싸일론 유도체의 용도를 제공한다.As another embodiment for achieving the above object, the present invention provides the use of the epoxylon derivative for use in the manufacture of the pharmaceutical composition for treating cancer.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1: 재조합 글루코스전달효소의 제조Example 1 Preparation of Recombinant Glucose Transferase
실시예 1-1: BL-C(UDP-glucosyltransferase C)의 제조Example 1-1 Preparation of BL-C (UDP-glucosyltransferase C)
바실러스 리체니포르미스(Bacillus licheniformis)의 제노믹 DNA를 주형으로 한 PCR을 수행하여, BL-C(UDP-glucosyltransferase C, NCBI accession number AAU40842) 유전자를 확보하고, 상기 유전자를 발현벡터 pET302/NT-his의 XhoI과 BamHI 부위에 클로닝하여 재조합 벡터를 제작하였다. 상기 제작된 재조합 벡터를 대장균 BL21(DE3)에 도입하여 형질전환체를 제조하였다. 상기 제조된 형질전환체를 LB 액체배지 50㎖에 1% 농도로 접종하고, 37℃에서 진탕배양하였으며, 600㎚에서 흡광도 값이 0.5가 되었을 때 IPTG(isopropyl-1-thio-β-D-galactopyranoside)를 첨가하여 호기적인 조건으로 배양하였다. 상기 배양이 종료된 후, 배양물을 원심분리하여 침전된 균체를 회수하고, 회수된 균체에 균체 분쇄용 완충액(1mM PMSF, protease inhibitor cocktail, 10mM imidazole, 100mM Tris-Cl, pH 8.0) 3㎖를 가하여, 현탁액을 수득하였다. 상기 현탁액에 초음파를 가하여 균체를 파쇄하고, 상기 파쇄물을 원심분리하여 상등액을 수득하였으며, 상기 상등액을 Ni-NTA(nickel-nitriloriacetic acid) 컬럼 크로마토그래피(Qiagen)에 적용하여, 목적하는 BL-C를 제조하였다.PCR was performed using the genome DNA of Bacillus licheniformis as a template to obtain BL-C (UDP-glucosyltransferase C, NCBI accession number AAU40842) gene, and the gene was expressed as expression vector pET302 / NT-. A recombinant vector was constructed by cloning his XhoI and BamHI sites. The recombinant vector thus prepared was introduced into Escherichia coli BL21 (DE3) to prepare a transformant. The prepared transformants were inoculated at 50% LB liquid medium at a concentration of 1%, shaken at 37 ° C., and an IPTG (isopropyl-1-thio-β-D-galactopyranoside when the absorbance value was 0.5 at 600 nm. ) Was incubated under aerobic conditions. After the incubation was terminated, the cultured cells were centrifuged to recover the precipitated cells, and 3 ml of the cell grinding buffer (1 mM PMSF, protease inhibitor cocktail, 10 mM imidazole, 100 mM Tris-Cl, pH 8.0) was added to the recovered cells. In addition, a suspension was obtained. Ultrasonic was added to the suspension to disrupt the cells, and the lysate was centrifuged to obtain a supernatant. The supernatant was subjected to nickel-nitriloriacetic acid (Ni-NTA) column chromatography (Qiagen) to obtain the desired BL-C. Prepared.
실시예 1-2: BmgtB의 제조Example 1-2 Preparation of BmgtB
바실러스 아밀로리퀴에파시엔스(B. amyloliquefaciens subsp. plantarum AH159-1)의 제노믹 DNA를 주형으로 한 PCR을 수행하여, 글루코스전달효소의 일종인 BmgtB를 코딩하는 유전자를 확보하고, 상기 유전자를 발현벡터 pET28a에 클로닝하여 재조합 벡터를 제작하였다. 상기 제작된 재조합 벡터를 대장균 BL21(DE3)에 도입하여 형질전환체를 제조하였다. 상기 제조된 형질전환체를 50 mg/㎖ 카나마이신이 포함된 LB 액체배지 50㎖에 1% 농도로 접종하고, 37℃에서 진탕배양하였으며, 600㎚에서 흡광도 값이 0.5 내지 0.6이 되었을 때 IPTG를 첨가하여 20℃에서 15시간 동안 배양하였다. 상기 배양이 종료된 후, 배양물을 원심분리하여 침전된 균체를 회수하고, 회수된 균체에 균체 분쇄용 완충액(1mM PMSF, protease inhibitor cocktail, 10mM imidazole, 100mM Tris-Cl, pH 8.0) 3㎖를 가하여, 현탁액을 수득하였다. 상기 현탁액에 초음파를 가하여 균체를 파쇄하고, 상기 파쇄물을 원심분리하여 상등액을 수득하였으며, 상기 상등액을 Ni-NTA 컬럼 크로마토그래피에 적용하여, 목적하는 BmgtB를 제조하였다.PCR was performed using the genome DNA of B. amyloliquefaciens subsp . Plantarum AH159-1 as a template to secure a gene encoding BmgtB, a type of glucose transferase, and expressing the gene. The recombinant vector was constructed by cloning into the vector pET28a. The recombinant vector thus prepared was introduced into Escherichia coli BL21 (DE3) to prepare a transformant. The prepared transformants were inoculated at 50% / ml LBA liquid medium containing 50 mg / ml kanamycin at 1% concentration, shaken at 37 ° C., and IPTG was added when the absorbance value was 0.5 to 0.6 at 600 nm. Incubated at 20 ℃ for 15 hours. After the incubation was terminated, the cultured cells were centrifuged to recover the precipitated cells, and 3 ml of the cell grinding buffer (1 mM PMSF, protease inhibitor cocktail, 10 mM imidazole, 100 mM Tris-Cl, pH 8.0) was added to the recovered cells. In addition, a suspension was obtained. Ultrasonic was added to the suspension to disrupt the cells, the lysate was centrifuged to obtain a supernatant, and the supernatant was subjected to Ni-NTA column chromatography to prepare the desired BmgtB.
실시예 2: 에포싸일론 유도체의 제조Example 2: Preparation of Epoxylon Derivatives
상기 실시예 1에서 제조된 글루코스전달효소(BL-C 또는 BmgtB)를 이용하여 에포싸일론 유도체를 제조하였다.Epocylon derivatives were prepared using the glucose transferase (BL-C or BmgtB) prepared in Example 1.
구체적으로, 50 mM Tris(pH8.0), 1mM MgCl2, 2mM UDP-glucose, 0.5mM EpoA(또는 EpoB) 및 20 ㎍ 글루코스전달효소(BL-C 또는 BmgtB)를 포함한 반응액을 이용하여, 30℃에서 18시간 동안 반응시켰다.Specifically, using a reaction solution containing 50 mM Tris (pH 8.0), 1 mM MgCl 2 , 2 mM UDP-glucose, 0.5 mM EpoA (or EpoB) and 20 μg glucose transferase (BL-C or BmgtB), The reaction was carried out at 18 ° C. for 18 hours.
이어, 상기 반응액에 동량의 에틸아세테이트를 가하고 격렬히 혼합하여 분별 추출한 다음, 에틸아세테이트 층을 수득하였다. 상기 수득한 에틸아세테이트 층을 감압농축하여 에틸아세테이트를 제거하고, 잔존물을 메탄올에 용해시킨 다음, silica gel TLC에 적용하여 글루코실화된 에포싸일론 유도체(EpoA-G 또는 EpoB-G)와 글루코실화되지 않은 에포싸일론을 분리하였다. 이때, TLC 전개용매로는 클로로포름과 메탄올이 10:1(v/v)로 혼합된 혼합용매를 사용하였다. 상기 TLC에 적용한 결과, Rf=0.65에서 EpoA, Rf=0.15에서 EpoA-G, Rf=0.60에서 EpoB, Rf=0.15에서 EpoB-G를 각각 수득하였다. 상기 수득한 EpoA-G 및 EpoB-G를 NMR 분석 및 HR-ESI-MS 분석에 적용하여 동정하였다(표 1 및 2).Subsequently, an equal amount of ethyl acetate was added to the reaction solution, mixed vigorously, and fractionated extraction was performed to obtain an ethyl acetate layer. The obtained ethyl acetate layer was concentrated under reduced pressure to remove ethyl acetate, the residue was dissolved in methanol, and then subjected to silica gel TLC to prevent glucosylation with glucosylated epoxylon derivatives (EpoA-G or EpoB-G). Phosphoylone was removed. In this case, a mixed solvent in which chloroform and methanol were mixed at 10: 1 (v / v) was used as the TLC developing solvent. As a result of the TLC, EpoA was obtained at Rf = 0.65, EpoA-G at Rf = 0.15, EpoB at Rf = 0.60, and EpoB-G at Rf = 0.15, respectively. The obtained EpoA-G and EpoB-G were identified by application to NMR analysis and HR-ESI-MS analysis (Tables 1 and 2).
표 1
Figure PCTKR2014004346-appb-T000001
 Table 1
Figure PCTKR2014004346-appb-T000001
표 2
Figure PCTKR2014004346-appb-T000002
 TABLE 2
Figure PCTKR2014004346-appb-T000002
실시예 3: 에포싸일론 유도체의 항암활성 측정Example 3: Determination of anticancer activity of epoxylon derivative
상기 실시예 2에서 제조한 각각의 에포싸일론 유도체가 항암활성을 유지하는지의 여부를 확인하고자 하였다.It was intended to confirm whether each of the epoxylon derivatives prepared in Example 2 maintains anticancer activity.
먼저, 상기 실시예 2에서 제조한 각각의 에포싸일론 유도체(EpoA-G 또는 EpoB-G)를 DMSO에 용해시켜서, 각각 0.1, 0.3, 1, 3, 10, 30 및 50mM 용액을 수득하였다.First, each of the epoxylon derivatives (EpoA-G or EpoB-G) prepared in Example 2 was dissolved in DMSO to obtain 0.1, 0.3, 1, 3, 10, 30 and 50 mM solutions, respectively.
다음으로, 전립선암 세포주인 PC-3, 폐암 세포주인 NCI-H23, 유방암 세포주인 MDA-MB-231, 대장암 세포주인 HCT-15, 신장암 세포주인 ACHN 및 위암 세포주인 NUGC-3를 각각 RPMI1640 과 10%의 송아지 혈청을 함유한 배양액에서 배양하였다. 상기 배양된 각 세포주를 96웰 플레이트에 분주하고, 이에 최종 농도가 0.1, 0.3, 1, 3, 10, 30, 50 및 100uM이 되도록 상기 분주된 각 세포주에 상기 수득한 에포싸일론 유도체 용액을 가한 다음, 48시간 동안 처리하였다. 이어, 각 세포주를 포함하는 웰에 50% TCA용액 50㎕를 가하여, 각 세포주를 고정시키고, 4℃에서 60분간 방치한 다음, tap water로 4-5회 세척하였다. 상기 세척된 96웰 플레이트를 건조시키고, 각 웰에 SRB 용액(0.4% sulforhodamine B in 1% acetic acid) 100 ㎕를 가한 다음 30분 동안 방치하였으며, 이에 0.1% 초산용액을 가하고 세척하여 각 웰에서 잔류하는 염색약을 제거하였다. 상기 96웰 플레이트를 다시 건조시키고, 각 웰에 10 mM Tris Base (pH 10.5) 100 ㎕를 가한 다음, Versa max microplate reader (Molecular Devices)를 사용하여 540 nm에서 흡광도를 측정하였다. 측정된 흡광도는 Graphpad prism v4.0 software에 적용하여 GI50(growth inhibition index) 값을 산출하였다(표 3).Next, the prostate cancer cell line PC-3, the lung cancer cell line NCI-H23, the breast cancer cell line MDA-MB-231, the colorectal cancer cell line HCT-15, the kidney cancer cell line ACHN and the gastric cancer cell line RPMI1640, respectively And 10% calf serum. Each cultured cell line was divided into 96-well plates, and the obtained epoxylon derivative solution was added to each of the divided cell lines so that final concentrations were 0.1, 0.3, 1, 3, 10, 30, 50, and 100 uM. Then it was treated for 48 hours. Subsequently, 50 µl of 50% TCA solution was added to the wells containing each cell line, and each cell line was fixed, left at 4 ° C. for 60 minutes, and then washed 4-5 times with tap water. The washed 96-well plate was dried, 100 μl of SRB solution (0.4% sulforhodamine B in 1% acetic acid) was added to each well, and left for 30 minutes. 0.1% acetic acid solution was added thereto, followed by washing, and remaining in each well. The dye was removed. The 96 well plate was dried again, 100 μl of 10 mM Tris Base (pH 10.5) was added to each well, and the absorbance was measured at 540 nm using a Versa max microplate reader (Molecular Devices). The measured absorbance was applied to Graphpad prism v4.0 software to calculate the growth inhibition index (GI50) value (Table 3).
표 3 각 암세포주에 대한 에포싸일론 유도체의 성장 저해 활성
암세포주 EpoA-G의 GI50(μM) EpoB-G의 GI50(μM)
PC-3 33.21 31.20
NCI-H23 3.286 4.821
MDA-MB-231 7.589 6.425
HCT-15 0.926 0.830
ACHN 2.713 2.395
NUGC-3 0.988 0.605
TABLE 3 Growth Inhibitory Activity of Eposylon Derivatives on Each Cancer Cell Line
Cancer cell line GI50 (μM) from EpoA-G GI50 (μM) on EpoB-G
PC-3 33.21 31.20
NCI-H23 3.286 4.821
MDA-MB-231 7.589 6.425
HCT-15 0.926 0.830
ACHN 2.713 2.395
NUGC-3 0.988 0.605
상기 표 3에서 보듯이, 상기 실시예 2에서 제조된 각각의 에포싸일론 유도체가 항암활성을 유지하고 있음을 알 수 있었다.As shown in Table 3, it can be seen that each of the epoxylon derivatives prepared in Example 2 maintains anticancer activity.
실시예 4: 에포싸일론 유도체의 수용성 평가Example 4 Evaluation of Water Solubility of Epothylon Derivatives
상기 실시예 2에서 제조된 에포싸일론 유도체의 수용성을 평가하고자 하였다. 구체적으로, 상기 실시예 2에서 에포싸일론 유도체를 제조하면서 생성되는 에틸아세테이트로 추출하기 이전 시료, 상기 시료를 에틸아세테이트로 추출하여 수득한 에틸아세테이트 층 및 에틸아세테이트 층이 제거된 후 잔류하는 수층을 대상으로 HPLC 분석을 수행하였다(도 1a 내지 1c). To evaluate the water solubility of the epoxylon derivative prepared in Example 2. Specifically, the sample before extraction with ethyl acetate produced in the preparation of the epoxylon derivative in Example 2, the ethyl acetate layer obtained by extracting the sample with ethyl acetate and the aqueous layer remaining after the ethyl acetate layer is removed HPLC analysis was performed on the subject (FIGS. 1A-1C).
도 1a 내지 1c에서 보듯이, 에틸아세테이트로 추출하기 이전 시료와 에틸아세테이트 층에는 에포싸일론과 그의 유도체가 모두 포함된 반면, 잔류하는 수층에는 에포싸일론 유도체 만이 존재함을 확인하였다. 상기 수층에 존재하는 에포싸일론 유도체의 함량은 에틸아세테이트로 추출하기 이전 시료에 존재하는 에포싸일론 유도체 함량의 약 30% 에 해당하는 것으로 분석되었다.As shown in Figure 1a to 1c, the sample and the ethyl acetate layer before extraction with ethyl acetate contained both epoxylon and its derivatives, it was confirmed that only the epoxylon derivative is present in the remaining water layer. The content of epoxylon derivatives present in the aqueous layer was analyzed to correspond to about 30% of the content of epoxylon derivatives present in the sample before extraction with ethyl acetate.
통상적으로, 당이 결합된 배당체 형태의 유도체 화합물은 결합된 당의 종류에 따라서 서로 다른 수준의 수용성을 나타냄을 감안한다면, 약 30%의 수용성을 나타내는 본 발명의 에포싸일론 유도체는 비교적 높은 수준의 수용성을 갖는 것으로 분석되었다.Generally, considering that the derivative compound in the form of a glycoside in which the sugar is bound shows different levels of water solubility depending on the type of sugar to which the sugar is bound, the phosphoylone derivative of the present invention having a water solubility of about 30% is relatively high in water solubility. It was analyzed to have.
실시예 5: 생체내에서 에포싸일론 유도체의 변화Example 5 Changes in Epothylon Derivatives in Vivo
상기 실시예 2에서 제조된 에포싸일론 유도체를 실험동물인 웅성 IRC 마우스에 경구투여(20mg/kg) 또는 정맥주사투여(5mg/kg)하고, 투여후 시간의 경과에 따라 혈장내에 존재하는 에포싸일론 유도체 및 에포싸일론의 수준을 측정하고, 비교하였다(도 2a 및 2b). 이때, 혈장내에 존재하는 에포싸일론 유도체 및 에포싸일론의 수준 측정은 Triple quadrupole 질량분석기(3200 Q TRAP LC/MS/MS)를 이용하여 수행하였는데, 컬럼은 Waters Xterra MS C18 (2.1 x 50 mm, 5㎛)를 사용하였고, 이동상은 0.1% 포름산을 포함하는 아세토나이트릴 농도구배(5% 내지 95%)를 사용하였으며, 유속은 0.4㎖/min로 설정하였다.The epoxylon derivatives prepared in Example 2 were orally administered (20 mg / kg) or intravenously administered (5 mg / kg) to male IRC mice as experimental animals, and the epoxyyl present in plasma over time after administration. Levels of lone derivatives and epoxylons were measured and compared (FIGS. 2A and 2B). At this time, the measurement of the level of epoxylon derivatives and epoxylons present in plasma was performed using a triple quadrupole mass spectrometer (3200 Q TRAP LC / MS / MS), and the column was Waters Xterra MS C18 (2.1 x 50 mm, 5 μm) was used, the acetonitrile concentration gradient (5% to 95%) containing 0.1% formic acid was used, and the flow rate was set to 0.4 ml / min.
먼저, 도 2a에서 보듯이, 경구투여된 에포싸일론 유도체의 혈장내 수준은 시간의 경과에 따라 감소하고, 약 5시간이 경과된 후에는 혈장에서 검출되지 않았다. 이에 반하여, 에포싸일론의 혈장내 수준은 시간의 경과에 따라 증가하여 약 4시간이 경과된 후에는 최고 수준을 나타내었는데 이후에는 혈장내 수준이 서서히 감소하였으며, 약 24시간이 경과된 후에도 투여 직후에 혈장에서 검출된 것과 유사한 수준으로 검출됨을 확인하였다.First, as shown in FIG. 2A, the plasma levels of orally administered epoxylon derivatives decreased over time and were not detected in plasma after about 5 hours. In contrast, the plasma levels of epoxylons increased with time, reaching the highest level after about 4 hours, after which the plasma levels gradually decreased, and even after 24 hours, Was detected at a level similar to that detected in plasma.
다음으로, 도 2b에서 보듯이, 정맥주사 투여된 에포싸일론 유도체의 혈장내 수준은 시간의 경과에 따라 감소하였는데, 약 2시간이 경과된 후에는 혈장에서 검출되지 않았으므로, 경구투여된 것에 비하여 단시간 내에 혈장에서 소진됨을 확인하였다. 이에 반하여, 에포싸일론의 혈장내 수준은 시간의 경과에 따라 증가하여 약 2시간이 경과된 후에는 최고 수준을 나타내었는데, 이후에는 혈장내 수준이 서서히 감소하였으며, 약 8시간이 경과된 후에도 투여 직후에 혈장에서 검출된 것보다 높은 수준으로 검출됨을 확인하였다.Next, as shown in Figure 2b, the plasma level of intravenous administered epoxylon derivatives decreased over time, but after about 2 hours was not detected in the plasma, compared to oral administration It was confirmed that the plasma was exhausted within a short time. In contrast, the plasma level of epoxylon increased over time and reached the highest level after about 2 hours, after which the level of plasma gradually decreased, and administration was continued after about 8 hours. Immediately after it was confirmed that it was detected at a higher level than that detected in plasma.
상기 도 2a 및 2b에서 보듯이, 투여방법에 따라 에포싸일론 유도체의 혈장내 잔류시간이 달라지기는 하였으나, 상기 에포싸일론 유도체의 수준이 시간의 경과에 따라 감소하여 일정시간 후에는 혈장내에서 검출되지 않은 반면, 에포싸일론의 수준은 측정된 시간동안 계속해서 혈장에서 검출된다는 공통점을 나타내고, 이는 본 발명에서 제공하는 에포싸일론 유도체가 체내에서 분해되어 에포싸일론을 형성함을 나타냄을 의미하는 것으로 분석되었다.As shown in FIGS. 2a and 2b, although the plasma residence time of the epoxylon derivatives varies depending on the administration method, the level of the epoxylon derivatives decreases over time and in the plasma after a certain time. While undetected, the level of epoxylons has a common point that they are continuously detected in plasma for the measured time, which means that the epoxylon derivatives provided in the present invention are degraded in the body to form epoxylons. It was analyzed.
통상적으로, 에포싸일론과 같이 항암활성과 난용성을 동시에 나타내는 화합물에 글루코스 등을 결합시켜서 배당체 형태의 유도체를 제조할 경우, 상기 유도체는 수용성이 증가하는 반면 항암활성이 대폭 감소되는 것으로 알려져 있다는 점을 감안한다면, 상기 결과로부터 본 발명의 에포싸일론 유도체가 프로드러그로서 활용될 수 있음을 알 수 있었다. 즉, 본 발명의 에포싸일론 유도체는 에포싸일론에 비하여 수용성이 증가하여 제제화가 용이하여, 암환자에게 투여하기가 용이하고, 일단 상기 에포싸일론 유도체가 체내로 투여되면, 체내에서 짧은 시간내에 에포싸일론으로 전환되어 원래의 항암활성을 나타내므로, 상기 에포싸일론 유도체는 프로드러그로서 사용할 수 있을 것으로 분석되었다.In general, when glycoside-derived derivatives are prepared by combining glucose and the like with compounds that simultaneously exhibit anticancer activity and poor solubility, such as epoxylon, the derivatives are known to increase water solubility while significantly reducing anticancer activity. In view of the above, it can be seen from the above results that the epoxylon derivative of the present invention can be utilized as a prodrug. That is, the phosphoylone derivatives of the present invention have increased water solubility compared to epoxylons and are easily formulated, and thus easy to administer to cancer patients. Once the epoxylon derivatives are administered to the body, within a short time in the body It was analyzed that the epoxylon derivative could be used as a prodrug because it was converted to epoxylon and exhibited original anticancer activity.
실시예 6: 에포싸일론 유도체와 에포싸일론의 항암활성 비교Example 6 Comparison of Anticancer Activity between Epothylon Derivatives and Epothylon
종래의 에포싸일론과 본 발명에서 제공하는 에포싸일론 유도체의 항암활성을 비교하였다.The anticancer activity of the conventional epoxylon and epoxylon derivatives provided by the present invention was compared.
구체적으로, 6종의 암세포주인 신장암세포주(ACHN), 대장암세포주(HCT15), 유방암세포주(MDA-MB-231), 폐암세포주(NCI-H23), 위암세포주(NUGC-3) 및 전립선암세포주(PC-3)에, 에포싸일론 A, 글루코실화된 에포싸일론 A 유도체, 에포싸일론 B 및 글루코실화된 에포싸일론 B 유도체를 각각 처리하고, 상기 각 암세포주의 성장을 50% 감소시키는 농도(GI50, μM)을 측정하여, 상기 각 화합물의 세포독성을 비교하였다(표 4). 이때, 대조군으로는 공지된 항암제인 아드리아마이신을 처리한 암세포주를 사용하였다.Specifically, six cancer cell lines, renal cancer cell line (ACHN), colon cancer cell line (HCT15), breast cancer cell line (MDA-MB-231), lung cancer cell line (NCI-H23), gastric cancer cell line (NUGC-3) and prostate cancer cells Week (PC-3) was treated with epoxylon A, glucosylated epoxylon A derivatives, epoxylon B and glucosylated epoxylon B derivatives, respectively, which reduced the growth of each cancer cell line by 50%. Concentrations (GI50, μM) were measured to compare the cytotoxicity of each compound (Table 4). In this case, a cancer cell line treated with adriamycin, a known anticancer agent, was used as a control.
표 4 에포싸일론과 에포싸일론 유도체의 항암활성 비교(GI50, μM)
암세포주 대조군 에포싸일론 에포싸일론 유도체
A B A B
ACHN 0.5 0.01 0.003 2.7 2.3
HCT-15 1.1 0.0007 0.002 0.9 0.8
MDA-MB-231 0.6 0.01 0.01 7.5 6.4
NCI-H23 1.1 0.02 0.01 3.2 4.8
NUGC-3 1.1 0.007 0.003 0.9 0.6
PC-3 0.9 0.03 0.03 33.2 31.2
Table 4 Comparison of anticancer activity between epoxylon and epoxylon derivatives (GI50, μM)
Cancer cell line Control Epoxylon Epoxylon derivatives
A B A B
ACHN 0.5 0.01 0.003 2.7 2.3
HCT-15 1.1 0.0007 0.002 0.9 0.8
MDA-MB-231 0.6 0.01 0.01 7.5 6.4
NCI-H23 1.1 0.02 0.01 3.2 4.8
NUGC-3 1.1 0.007 0.003 0.9 0.6
PC-3 0.9 0.03 0.03 33.2 31.2
상기 표 4에서 보듯이, 본 발명의 에포싸일론 유도체는 에포싸일론의 것에 비하여는 낮은 수준이지만, 항암활성을 나타내며, 일부 암세포주(HCT15 또는 NUGC-3)에 대하여는 대조군으로 사용된 아드리아마이신에 비하여 상대적으로 우수한 수준의 항암활성을 나타냄을 확인하였다.As shown in Table 4, the epoxylon derivatives of the present invention have a lower level than that of epoxylon, but exhibit anti-cancer activity, and to some cancer cell lines (HCT15 or NUGC-3), adriamycin used as a control. It was confirmed that it shows a relatively good level of anticancer activity.

Claims (15)

  1. 에포싸일론(epothilones)이 글루코실화된 에포싸일론 유도체.Epothylon derivatives in which epothilones are glucosylated.
  2. 제1항에 있어서,The method of claim 1,
    상기 에포싸일론은 에포싸일론 A 또는 에포싸일론 B인 것인 유도체.Derivatives of the epoxylon is epoxylon A or epoxylon B.
  3. 제1항에 있어서,The method of claim 1,
    상기 글루코실화는 에포싸일론의 7번 탄소에 글루코실기가 결합되는 것인 유도체.The glucosylation is a derivative in which a glucosyl group is bonded to carbon number 7 of the epoxylon.
  4. 제1항에 있어서,The method of claim 1,
    상기 에포싸일론 유도체는 에포싸일론 A의 7번 탄소에 글루코실화되어 화학식 3의 구조를 가지는 것인 유도체.The epoxylon derivative is a glucosylated at carbon number 7 of epoxylon A to have a structure of formula (3).
    [화학식 3][Formula 3]
    Figure PCTKR2014004346-appb-I000001
    Figure PCTKR2014004346-appb-I000001
  5. 제1항에 있어서,The method of claim 1,
    상기 에포싸일론 유도체는 에포싸일론 B의 7번 탄소에 글루코실화되어 화학식 4의 구조를 가지는 것인 유도체.The epoxylon derivative is glucosylated at carbon number 7 of epoxylon B to have a structure of formula (4).
    [화학식 4][Formula 4]
    Figure PCTKR2014004346-appb-I000002
    Figure PCTKR2014004346-appb-I000002
  6. (a) 에포싸일론에 글루코실기의 공여체 및 글루코스전달효소를 가하여 글루코실화 반응을 수행하는 단계; 및 (a) adding a glucosyl donor and glucose transferase to the epoxylon to perform a glucosylation reaction; And
    (b) 상기 반응물로부터 에포싸일론 유도체를 회수하는 단계를 포함하는 에포싸일론 유도체의 제조방법.(b) recovering the epoxylon derivative from the reactant.
  7. 제6항에 있어서,The method of claim 6,
    상기 에포싸일론은 에포싸일론 A 또는 에포싸일론 B인 것인 방법.Said epoxylon is epoxylon A or epoxylon B.
  8. 제6항에 있어서,The method of claim 6,
    상기 글루코실기의 공여체는 당인산, 뉴클레오티드 글루코오스, 올리고당, 다당 및 이들의 조합으로 구성된 군으로부터 선택되는 것인 방법.The donor of the glucosyl group is selected from the group consisting of glycophosphate, nucleotide glucose, oligosaccharides, polysaccharides and combinations thereof.
  9. 제6항에 있어서,The method of claim 6,
    상기 글루코스전달효소는 BL-C(UDP-glucosyltransferase C) 또는 BmgtB인 것인 방법.Wherein said glucose transferase is BL-C (UDP-glucosyltransferase C) or BmgtB.
  10. 제6항에 있어서,The method of claim 6,
    상기 에포싸일론과 글루코실기의 공여체의 비율은 1:1 내지 1:10(w/w)인 것인 방법.The ratio of the donor of the epoxylon and glucosyl group is 1: 1 to 1:10 (w / w).
  11. 제6항에 있어서,The method of claim 6,
    상기 반응은 pH 7.0 내지 8.0, 25 내지 35℃ 및 10 내지 30시간 동안 수행되는 것인 방법.Wherein the reaction is performed at pH 7.0-8.0, 25-35 ° C. and 10-30 hours.
  12. 제1항 내지 제5항 중 어느 한 항의 에포싸일론 유도체 또는 제6항 내지 제11항 중 어느 한 항의 방법으로 제조된 에포싸일론 유도체를 유효성분으로 포함하는 암치료용 약학 조성물.A pharmaceutical composition for treating cancer, comprising an epoxylon derivative of any one of claims 1 to 5 or an epoxylon derivative prepared by the method of any one of claims 6 to 11 as an active ingredient.
  13. 제12항에 있어서,The method of claim 12,
    상기 에포싸일론 유도체는 프로드러그로서 작용하는 것인 조성물.Wherein said epoxylon derivative acts as a prodrug.
  14. 제12항의 약학 조성물을 약제학적으로 유효한 양으로 암질환이 발병된 개체에 투여하는 단계를 포함하는 암을 치료하는 방법.A method of treating cancer comprising administering the pharmaceutical composition of claim 12 to a subject having a cancer disease in a pharmaceutically effective amount.
  15. 암치료용 약학 조성물의 제조를 위한, 제1항의 에포싸일론 유도체의 용도.Use of the epoxylon derivative of claim 1 for the manufacture of a pharmaceutical composition for cancer treatment.
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CN111205343A (en) * 2020-01-08 2020-05-29 山东大学 Nitrogen acetyl glucoside or galactoside compound of epothilone B, and enzymatic preparation and application thereof
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