KR101638045B1

KR101638045B1 - Novel epothilone derivatives and uses thereof

Info

Publication number: KR101638045B1
Application number: KR1020140058339A
Authority: KR
Inventors: 김원곤; 박지영; 김중수; 김현주
Original assignee: 한국생명공학연구원
Priority date: 2013-05-15
Filing date: 2014-05-15
Publication date: 2016-07-08
Also published as: KR20140135123A; WO2014185717A1

Abstract

The present invention relates to a novel epochilylone derivative in which epothilone is glucosylated to improve water solubility, a process for producing the epothilone derivative, and a pharmaceutical composition for treating cancer comprising the epochilone derivative as an active ingredient. The eprosilon derivatives of the present invention have improved water solubility and lower anticancer activity than conventional epochilones, and thus can be widely used for the formulation of epocylons.

Description

Novel epothilone derivatives and uses thereof < RTI ID = 0.0 >

The present invention relates to a novel epocylon derivative and a use thereof. More particularly, the present invention relates to a novel epothilone derivative which is glucosylated and improved in water solubility, a process for producing said epochilone derivative, To a pharmaceutical composition for treating cancer comprising a cilron derivative as an active ingredient.

Epothilone is a natural product isolated from the bacterial strain ( myxobacterium Sorangium cellulosum strain 90) in soil. It is cytotoxic to taxol-resistant tumor cells and is superior to taxol Which is known to exhibit usefulness. The epocystilon exhibits anticancer activity by acting on a microtubule, a cell internal skeleton such as a thread, which is disassembled after being arranged at a specific position in the cell division cycle, and exhibits anticancer activity. The chemical structure is simple compared to Taxol In stabilizing microtubules, epocylone exhibits an effect of 2000-5000 times better than that of Taxol, and has partial advantages in that it is easier to formulate than Taxol, which is less soluble than taxol. Despite the chemical structural differences between taxol and epocathron, it is known that epocylon replaces taxol from the binding site and binds to the same active site in the microtubule, so that structural novelty, important biological and interesting mechanisms Active research is under way.

For example, since the introduction of the X-ray crystal structure of epocylons, organic chemists have attempted to study the entire synthesis of these compounds and have found that by Nicolaou and Danishefsky, , And then a method of pre-synthesis of epocylone was developed by a number of researchers. A typical prior art synthesis method of epocylons known to date includes (1) solubilizing glycal, which is a derivative of cyclopropane, to ring the ring of cyclopropane to make a gem-dimethyl ; (2) bonding the side chain groups using an alkyl Suzuki coupling reaction; (3) performing an intramolecular-aldol condensation reaction so as to be ring-opened at the C-16 position of epocyllone; And (4) epoxidizing the cis-double bond site in a very stereospecifically manner.

The eprosilon thus prepared is superior to taxol but has partial water solubility and thus has a disadvantage that it is not easy to formulate. Various derivatives have been developed to overcome this disadvantage. Examples thereof include ixabepilone (azaepothilone B), BMS-310705, patupilone, epothilone R1645, ZK-EPO and ABJ879 (C20-desmethyl-C20-methylsulfanyl-EpoB) Or water solubility, while the original anticancer activity is decreased. Thus, it has been required to develop new derivatives overcoming these drawbacks.

Under these circumstances, the inventors of the present invention have made extensive efforts to develop an epochillyron derivative in which the anticancer activity is maintained while increasing the water solubility. As a result, it has been confirmed that the epochillyron-glycocylated derivative exhibits improved water solubility and anticancer activity, Thus completing the present invention.

It is an object of the present invention to provide an epothilone derivative wherein the epothilone is glucosylated.

It is another object of the present invention to provide a method for producing the above epocylon derivative.

It is still another object of the present invention to provide a pharmaceutical composition for treating cancer comprising the above epocylon derivative.

The present inventors have focused on glycosylation while carrying out various studies to develop eprosilon derivatives which maintain the anticancer activity while increasing water solubility. Typically, glycosylation refers to the reaction of a glycosyl group to a polymeric compound such as a protein. Such a glycosylated polymeric compound forms a complex saccharide, which can perform various functions in vivo . Since the glycosyl groups used in the glycosylation include an abundant hydrophilic reactor, the present inventors have anticipated that the compounds having partial water solubility such as the epothilone of the present invention will have excellent water solubility. Thus, eprosilon A (EpoA) and epo-Cylon B (EpoB), known as epocylon derivative compounds, are treated with a glucosyltransferase to prepare an eprosilon derivative compound having a glucose residue bound thereto, As a result of comparing the water solubility and anticancer activity of the prepared derivative compounds, it was found that the prepared derivative compounds did not significantly change the anticancer activity while increasing the water solubility. Further, as a result of evaluating the water solubility of the epocylon derivative, it was confirmed that the aqueous solution had a water solubility of about 30%, and that the epocylon derivative could form epocathron in the body.

In one embodiment for achieving the above object, the present invention provides eprosilon derivatives in which epocylons are glucosylated.

The term "epothilone" of the present invention means soil bacteria ( myxobacterium Sorangium cellulosum strain 90) and have excellent anticancer activity and partial water solubility. Various derivatives (epothilones A to F) substituted with respective functional groups of the above epocylone are known, and it is known that the above derivatives exhibit a pharmacological activity effect for various diseases such as Alzheimer as well as anticancer activity.

In the present invention, the epocylone may be epothilones A (EpoA) or epothilones B (EpoB), which are known to exhibit excellent anti-cancer effects. However, The types of epocylons are not limited.

The term " epothilones A (EpoA) "of the present invention means a compound having one of the epocylon derivative compounds having a molecular weight of 656.3101, a formula of C ₃₂ H ₅₀ NO ₁₁ S and a structure of the following formula do.

The term " epothilones B (EpoB) "of the present invention means a compound having one of the epocylon derivative compounds having a molecular weight of 692.3077, a formula of C ₃₂ H ₅₁ NO ₁₁ NaS and a structure of the following formula (2) do.

The term "glucosylation" of the present invention is a kind of glycosylation, and refers to a reaction in which a carbon atom of a glucosyl group is bound to a desired compound to thereby transfer a glucosyl group.

The term "glucosyl group" of the present invention is a kind of glycosyl group, which means a monovalent reactant in which a hemiacetal hydroxyl group is removed from a glucose molecule.

In the present invention, the glucosylation may be a reaction of delivering a glucosyl group to EpocA or EpoB, which is not particularly limited, but may be a known chemical method, preferably a biochemical method , More preferably by an enzyme-based method, and most preferably by using a glucose transporter.

The term "glucosyltransferase " of the present invention means an enzyme capable of carrying out the glucosylation by catalyzing the transfer of a glucosyl group. For the purpose of the present invention, the glucose transporting enzyme is obtained from a donor of a glucosyl group And can be understood as an enzyme that performs a catalytic reaction of transferring one glucosyl group to epochylon. At this time, examples of the donor of the glucosyl group include sugar phosphate (Glc-1-P, etc.); Nucleotide glucose (UDP-Glc and the like); Oligosaccharides (such as sucrose); Polysaccharide and the like may be used singly or in combination.

In the present invention, the glucose transporting enzyme is not particularly limited as long as it can perform glucosylation of epocylone, but it is preferable to use a glucose transporter enzyme or nucleotide glucose MGT (macroside glycosyltransferase) as a donor of a glucosyl group More preferably, a glucose transporting enzyme using a MGT-based glucose transporter or UDP derived from B. amyloliquefaciens as a donor of a glucosyl group can be used, and most preferably, a glucose transporter BmgtB and BL-C (UDP-glucosyltransferase C), which are glucose transport enzymes of MGT series derived from Bacillus strains, can be used.

The term "glucosylated epopycolon derivative " of the present invention means a derivative compound (glucosylated form) in which a glucosyl group is transferred to a carbon atom of epoclidone by a glucose transport enzyme to bind (glucosylated).

In the present invention, the glucosylated epopycolon derivative is not particularly limited, but it is preferable that the carbon of the epothilone can be a glucosylated derivative compound, more preferably the 7th carbon of epocylron May be a glucosylated derivative compound and most preferably the 7th carbon of EpoA is glucosylated to give the 7 carbon of EpoA-G (7-glucosyl epothilones A) or EpoB, which may be represented by formula 3, (7-glucosyl epothilones B) which can be represented by the formula (4).

According to one embodiment of the present invention, a recombinant glucose transporter (BL-C) (UDP-glucosyltransferase C) (Example 1-1) derived from Bacillus licheniformis or BmgtB (Example 1-2) And the 7th carbon of EpoA or EpoB was glycosylated using the prepared BL-C or BmgtB to produce a glucosylated epopycolon derivative (Table 1 and Table 2), and the epocylon The derivatives were increased in water solubility by glucosylation, and their anticancer activities were measured. As a result, it was confirmed that all of the six types of cancer cells exhibited anticancer activity (Table 3). Further, it was confirmed that the water-solubility of the epothilone derivative was evaluated to be about 30% (Figs. 1A to 1C), and when the epothilone derivative was administered into the body by the oral or intravenous injection method, It was confirmed that plasma levels of the rhodamine derivatives were rapidly decreased, and plasma levels of epocyllone were increased and then gradually decreased (Figs. 2A and 2B).

According to another aspect of the present invention, there is provided a method for preparing a pharmaceutical composition, comprising the steps of: (a) carrying out a glucosylation reaction by adding a glucosyl group donor and a glucose transporting enzyme to an epothilone compound; And (b) recovering the epocylon derivative from the reactant. The present invention also provides a method for producing the glucosylated epothilone derivative.

At this time, the epoclonion compound, the donor of the glucosyl group, and the glucose transport enzyme are the same as described above. In addition, the pH condition for carrying out the reaction is neutral pH (pH 7.0 to 8.0), the temperature condition is 25 to 35 占 폚, and the time condition is 10 to 30 hours. The mixing ratio of the epoxystilone compound and the donor of 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), and most preferably 1: 4 (w / w).

In addition, the step of recovering the epochilyl derivative from the reaction can be carried out by methods known in the art. Specifically, the method for recovering an epochilyl derivative is not particularly limited, but may be performed by centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (for example, ammonium sulfate precipitation) For example, ion exchange, affinity, hydrophobicity and size exclusion).

In another aspect of the present invention, the present invention provides a pharmaceutical composition for treating cancer comprising the above epocylon derivative.

The glucosylated epothilone derivative of the present invention has improved water solubility as compared with the conventional epochilone, and it has been confirmed that not only the anticancer activity is maintained but also the epothilone derivative is converted into the epochilone in the body, It can be used as an active ingredient in the form of a prodrug contained in a 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 can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablet pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose ), Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories It can have one formulation.

In the present invention, the administration route of the pharmaceutical composition may be administered through any ordinary route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonarily, rectally, but not exclusively, as desired. In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell.

The content of the epochilyl derivative contained in the pharmaceutical composition of the present invention is not particularly limited but may be in the range of 0.0001 to 50% by weight, preferably 0.001 to 10% by weight, based on the total weight of the final composition. .

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment The effective dose level refers to the type and severity of the disease, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, May be determined according to known factors. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

The dosage of the pharmaceutical composition for treating cancer comprising the epochiloin derivative of the present invention may be appropriately selected depending on the purpose of use, the degree of poisoning of the disease, the age, body weight, sex, history of the patient, Can be determined by those skilled in the art. 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 particularly It is not limited, but it can be administered once a day or divided into several doses.

In another aspect of the present invention, the present invention provides a method for treating cancer, comprising administering the pharmaceutical composition to a subject suffering from a cancer disease in a pharmaceutically effective amount.

As described above, since the epocylon derivatives provided in the present invention have not only anticancer activity but also can be converted into epopycolone in the body, the composition can be used to treat cancer.

The term "individual" as used herein includes, without limitation, mammals including, but not limited to, rats, cattle, humans,

In the method of treating cancer of the present invention, the administration route of the pharmaceutical composition may be administered through any conventional route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intracorporally, or rectally, as desired, though not particularly limited thereto. However, since the fusion protein can be denatured by gastric acid upon oral administration, the oral composition should be formulated so as to coat the active agent or protect it from decomposition at the top. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

As another embodiment for achieving the above object, the present invention provides the use of epocylon derivatives for use in the production of the pharmaceutical composition for treating cancer.

The eprosilon derivatives of the present invention have improved water solubility and lower anticancer activity compared to conventional epochilones, and thus can be widely used for the formulation of epocylons.

Figure 1A shows the results of HPLC analysis on samples before extraction with ethyl acetate.
Figure 1B shows the results of HPLC analysis on an ethyl acetate layer.
Figure 1C shows the results of HPLC analysis on the remaining aqueous layer.
FIG. 2A is a graph showing the results of measuring levels of epopycolone derivatives and epocystilone in plasma after oral administration of the epothilone derivative of the present invention to experimental animals, Cyclone derivatives, and Epo-A stands for epocylons.
FIG. 2b is a graph showing the results of measuring the levels of epopycolinium derivatives and epocystilone in plasma after the intravenous injection of the epocylon derivative of the present invention into an experimental animal, wherein Epo-AG Epo-A represents an epocylon.

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.

Example 1: Recombination Glucose Transporter Enzyme Produce

Example 1-1: BL -C ( UDP - glucosyltransferase C)

(UDP-glucosyltransferase C, NCBI accession number AAU40842) gene was obtained by performing PCR using the genomic DNA of Bacillus licheniformis as a template, and the gene was cloned into the expression vector pET302 / NT- His were cloned into the XhoI and BamHI sites to construct a recombinant vector. The prepared recombinant vector was introduced into Escherichia coli BL21 (DE3) to prepare a transformant. The transformant prepared above was inoculated in 50 ml of LB liquid medium at a concentration of 1% and incubated at 37 ° C with shaking. When the absorbance value reached 0.5 at 600 nm, IPTG (isopropyl-1-thio-β-D-galactopyranoside ) Were added and cultured under aerobic conditions. After completion of the culturing, the culture was centrifuged to collect the precipitated cells, and 3 ml of a cell lysis buffer (1 mM PMSF, protease inhibitor cocktail, 10 mM imidazole, 100 mM Tris-Cl, pH 8.0) Was added, and a suspension was obtained. The supernatant was applied to Ni-NTA (Ni-NTA) column chromatography (Qiagen), and the desired BL-C was eluted from the supernatant by centrifugation. .

Example 1-2: Preparation of BmgtB

Bacillus amyloliquefaciens ( B. amyloliquefaciens subsp . plantarum AH159-1) as a template to obtain a gene encoding BmgtB, which is a kind of glucose transduction enzyme, and the gene was cloned into an expression vector pET28a to prepare a recombinant vector. The prepared recombinant vector was introduced into Escherichia coli BL21 (DE3) to prepare a transformant. The transformant was inoculated at a concentration of 1% in 50 ml of LB liquid medium containing 50 mg / ml of kanamycin and incubated at 37 ° C with shaking. When the absorbance at 600 nm reached 0.5 to 0.6, IPTG was added And cultured at 20 DEG C for 15 hours. After completion of the culturing, the culture was centrifuged to collect the precipitated cells, and 3 ml of a cell lysis buffer (1 mM PMSF, protease inhibitor cocktail, 10 mM imidazole, 100 mM Tris-Cl, pH 8.0) Was added, and a suspension was obtained. Ultrasonic waves were applied to the suspension to disrupt the cells, and the lysate was centrifuged to obtain a supernatant. The supernatant was applied to Ni-NTA column chromatography to prepare the desired BmgtB.

Example 2: Epocylon Preparation of derivatives

An eprosilon derivative was prepared using the glucose transporter (BL-C or BmgtB) prepared in Example 1 above.

Specifically, 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 ㎍ glucose transporting enzyme (BL-C or BmgtB) Lt; 0 > C for 18 hours.

Then, the same amount of ethyl acetate was added to the reaction solution, followed by vigorous mixing and fractional extraction, and then an ethyl acetate layer was obtained. The obtained ethyl acetate layer was concentrated under reduced pressure to remove ethyl acetate, and the residue was dissolved in methanol. The residue was applied to silica gel TLC to be glycosylated with a glucosylated epopycolon derivative (EpoA-G or EpoB-G) And separated epocylons. At this time, as a TLC developing solvent, a mixed solvent in which chloroform and methanol were mixed at a ratio of 10: 1 (v / v) was used. As a result of applying the above TLC, EpoA-G 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 NMR analysis and HR-ESI-MS analysis (Tables 1 and 2).

Example 3: Epocylon Measurement of antitumor activity of derivatives

To confirm whether each of the epocylon derivatives prepared in Example 2 maintains anticancer activity.

First, each of the epocylon 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.

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 renal cancer cell line ACHN and the gastric cancer cell line NUGC- And 10% calf serum. Each of the cultured cell lines was dispensed into a 96-well plate. To each of the cell lines thus obtained, the resulting epcystilorn derivative solution was added so that the final concentrations were 0.1, 0.3, 1, 3, 10, 30, Then, it was treated for 48 hours. Next, 50 占 퐇 of 50% TCA solution was added to each well containing each cell line, each cell line was fixed, left at 4 占 폚 for 60 minutes, and then washed 4-5 times with tap water. The washed 96-well plate was dried and 100 μl of SRB solution (0.4% sulforhodamine B in 1% acetic acid) was added to each well. Then, the plate was allowed to stand for 30 minutes and 0.1% acetic acid solution was added thereto. 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 GI50 (growth inhibition index) (Table 3).

The growth inhibitory activity of epocylon derivatives for each cancer cell line Cancer cell line GI50 (uM) of EpoA-G < RTI ID = 0.0 & GI50 (uM) of EpoB-G PC-3
NCI-H23
MDA-MB-231
HCT-15
ACHN
NUGC-3 33.21
3.286
7.589
0.926
2.713
0.988 31.20
4.821
6.425
0.830
2.395
0.605

As shown in Table 3, it can be seen that each of the epocylon derivatives prepared in Example 2 maintains anticancer activity.

Example 4: Epocylon Evaluation of water solubility of derivatives

To evaluate the water solubility of the epothilone derivative prepared in Example 2 above. Specifically, the sample before extraction with ethyl acetate produced in the preparation of the epochilyl 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 was removed HPLC analysis was performed on the subject (Figs. 1A-1C).

As shown in FIGS. 1A to 1C, it was confirmed that only the epocylon derivative was present in the residual aqueous layer while the sample before ethyl acetate extraction and the ethyl acetate layer contained both epocylone and its derivatives. The content of the epothilone derivative present in the water layer was analyzed to be about 30% of the epocylron derivative content present in the sample before extraction with ethyl acetate.

Given that derivative compounds in the form of glycosides linked to sugars typically exhibit different levels of water solubility depending on the type of sugar bound, the epothilone derivatives of the present invention, which exhibit a water solubility of about 30%, have relatively high levels of water solubility Respectively.

Example 5: In vivo Epocylon Changes in derivatives

(20 mg / kg) or an intravenous injection (5 mg / kg) to the male IRC mouse as an experimental animal, and the epocylon derivative prepared in Example 2 was administered intravenously The levels of the rhone derivatives and epocathrons were measured and compared (Figures 2a and 2b). At this time, the level of the epoclonoid derivative and the epoclonol present in the plasma was measured using a triple quadrupole mass spectrometer (3200 Q TRAP LC / MS / MS). The column was a Waters Xterra MS C18 (2.1 x 50 mm, (5% to 95%) containing 0.1% formic acid was used as the mobile phase, and the flow rate was set to 0.4 ml / min.

First, as shown in FIG. 2A, plasma levels of orally administered epochillyron derivatives decreased with the lapse of time, and were not detected in plasma after about 5 hours. In contrast, the plasma level of epocyllone increased with time and reached its maximum level after about 4 hours, after which the plasma level gradually decreased. After about 24 hours, Was detected at a level similar to that detected in plasma.

Next, as shown in FIG. 2B, plasma levels of intravenous epothilone derivatives decreased with the lapse of time. After about 2 hours, they were not detected in plasma, It was confirmed that plasma was consumed within a short time. In contrast, plasma levels of epocyllone increased with the passage of time and reached a maximum level after about 2 hours, after which plasma levels gradually decreased, and even after about 8 hours Was detected at a level higher than that immediately detected in plasma.

As shown in FIGS. 2A and 2B, although the residence time of the epocylon derivative in the plasma was varied depending on the administration method, the level of the epocylon derivative decreased with time, and after a certain time, While levels of epocylons are continuously detected in the plasma for the measured time, indicating that the epocylon derivatives provided in the present invention are decomposed in the body to form epocathylons Respectively.

In general, when derivatives of glycoside form are produced by binding glucose or the like to a compound that simultaneously exhibits anticancer activity and poorly-soluble property such as epocyllone, it is known that the above-mentioned derivatives have increased water solubility but greatly reduced anticancer activity It can be seen from the above results that the eprosilon derivative of the present invention can be utilized as a prodrug. That is, the epochillylone derivative of the present invention has an increased water solubility as compared with epocylone, and is easy to formulate and is easy to administer to cancer patients, and once the epochillylone derivative is administered into the body, Epothilone was converted to epothilone and exhibited the original anticancer activity. Therefore, it was analyzed that the epocylon derivative could be used as a prodrug.

Example 6: Epocylon Derivatives and Epocylone Comparison of antitumor activity

The anticancer activities of the conventional epocystilone and the epocylon derivatives provided in the present invention were compared.

Specifically, the present invention provides a method for screening for a cancer cell, comprising the steps of: (a) culturing a cell line selected from the group consisting of 6 kinds of cancer cell lines: kidney cancer cell line (ACHN), colon cancer cell line HCT15, breast cancer cell line MDA- (PC-3) was treated with epocylron A, glucosylated epocylon A derivative, epocyllon B and glucosylated epocylron B derivative, respectively, and the growth of each cancer cell was reduced by 50% The concentrations (GI50, mu M) were measured and the cytotoxicity of each compound was compared (Table 4). At this time, as a control group, a cancer cell line treated with a known anticancer drug adriamycin was used.

Comparison of anticancer activity between epocylon and epocylon derivatives (GI50, μM) Cancer cell line Control group Epocylon Epocylon derivative A B A B ACHN
HCT-15
MDA-MB-231
NCI-H23
NUGC-3
PC-3 0.5
1.1
0.6
1.1
1.1
0.9 0.01
0.0007
0.01
0.02
0.007
0.03 0.003
0.002
0.01
0.01
0.003
0.03 2.7
0.9
7.5
3.2
0.9
33.2 2.3
0.8
6.4
4.8
0.6
31.2

As shown in Table 4, the epothilone derivative of the present invention is lower in level than that of epocylone but exhibits anticancer activity. Adramycin, which is used as a control for some cancer cell lines (HCT15 or NUGC-3) It was confirmed that the anticancer activity was relatively good.

Claims

A pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, an epothilone derivative having a glucosyl group bonded to carbon number 7 of epothilones, wherein the epothilone derivative is water-soluble Wherein the glucosylated site is degraded upon administration to the body and converted to epocylon.

The method according to claim 1,
Wherein said epocystilone is epocylon A or epocylon B. < RTI ID = 0.0 > 8. < / RTI >

The method according to claim 1,
Wherein said epocylon derivative acts as a prodrug.

An eprosilon derivative having a structure of the following formula (3) by glucosylation to carbon number 7 of epocylon A.
(3)

An eprosilon derivative having a structure of the following formula (4) by glucosylation to carbon number 7 of epocyllon B.
[Chemical Formula 4]

(a) performing a glucosylation reaction in which a glucosyl group donor and a glucose transporting enzyme BL-C (UDP-glucosyltransferase C) or BmgtB are added to epothilone to bind a glucosyl group to carbon number 7 of epoclonyl; And
(b) recovering from the reaction product an epothilone derivative having a glucosyl group bound to carbon number 7 of epothilone and glucosylated, the glucosyl group is bonded to carbon number 7 of the epothilone, A method for producing epocylon derivatives.

The method according to claim 6,
Wherein the epcystilone is epocylon A or epocylon B. < RTI ID = 0.0 >

The method according to claim 6,
Wherein the donor of the glucosyl group is selected from the group consisting of sugar phosphate, nucleotide glucose, oligosaccharides, polysaccharides, and combinations thereof.

The method according to claim 6,
Wherein the ratio of the epocystilone to the donor of the glucosyl group is from 1: 1 to 1:10 (w / w).

The method according to claim 6,
The reaction is carried out at pH 7.0 to 8.0, 25 to 35? And 10 to 30 hours.

An epocylon derivative according to claim 4 or 5; 10. A pharmaceutical composition for preventing or treating cancer, comprising an epopycialone derivative prepared by the method of any one of claims 6 to 10 as an active ingredient.

12. The method of claim 11,
Wherein said epocylon derivative acts as a prodrug.

12. The method of claim 11,
Wherein said epocylon derivative is increased in water solubility as compared to epocylone and is converted into epocystilone upon degradation of the glucosylated site upon administration to the body.