WO2015080396A1 - Anticancer composition for oral administration, containing pegylated botulin derivative - Google Patents
Anticancer composition for oral administration, containing pegylated botulin derivative Download PDFInfo
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- WO2015080396A1 WO2015080396A1 PCT/KR2014/010677 KR2014010677W WO2015080396A1 WO 2015080396 A1 WO2015080396 A1 WO 2015080396A1 KR 2014010677 W KR2014010677 W KR 2014010677W WO 2015080396 A1 WO2015080396 A1 WO 2015080396A1
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- 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
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention relates to an anticancer drug composition for oral administration comprising a PEGylated betulin derivative. More specifically, the present invention relates to an anticancer composition for oral administration comprising a pegylated betulin derivative which has excellent anticancer effect against various carcinomas and is particularly useful for the treatment of cancer as a drug with significantly improved oral absorption.
- Betulin and betulinic acid are known to have apoptosis potency.
- Apoptosis refers to the destruction or suicide of cells in eukaryotic cells, and is a basic intracellular process for maintaining homeostasis of an individual, including controlling the normal development of an animal, or removing unnecessary or abnormal cells.
- Apoptosis occurs in response to various external and internal stimuli, with the progression of apoptosis, such as cytoplasmic breakdown, blebbing of cell membranes, changes in the cytoskeleton, cell contraction, chromosome condensation and DNA fragmentation. Distinctive changes occur and, if not activated, cells divide indefinitely and form tumors.
- Betulin represented by the following formula (2) is a lupine-based natural 5-ring triterpene alcohol, betulinol and loop-20 (29) -ene-3 ⁇ , 28-diol (lup). -20 (29) -ene-3 ⁇ , 28-diol). Betulin is present in many bark of tree species, especially in the bark of Betula sp., And methods for separating betulin by extraction from birch bark are known.
- betulinic acid represented by the following formula (3) is isolated by extraction from the cork of the birch ( Betula sp.) Bark or Quercus suber L., or to the direct oxidation of It is known that it can be prepared by the methods.
- betulin (2) was oxidized in the presence of a chromium (VI) catalyst according to the Jones oxidation method disclosed in US Pat. No. 6,280,778 to obtain betunic acid. It is then reported that betulinic acid (3) can be obtained by selective reduction of the obtained beturonic acid with sodium borohydride.
- U. S. Patent No. 5,804, 575 also discloses another method for preparing betulinic acid comprising protecting the 3- ⁇ -hydroxy group of betulin by acetylation and then oxidizing.
- betulin derivatives are a kind of natural biosynthesis produced by plants having a pentacyclic triterpenoid structure, first isolated from evergreen (Ziziphus mauritiana), and are known to be particularly abundant in the bark of white birch. It is known to have an effect of selectively acting on melanoma or neuroectoderm-derived cancer cells to kill cancer cells.
- These anticancer actions of betulin derivatives are known to be through various mechanisms, such as reducing the permeability of mitochondria or deactivating mitochondria, such as cytochrome-C release into the cytoplasm, active oxygen formation, or caspase activation.
- betulin derivatives have been shown to block the activity of HIV-1, showing the possibility of being used as an antiviral agent, and has been reported to be effective in other infectious diseases such as bacteria and malaria.
- betulin derivatives have been reported to have NO production inhibitory activity by interferon- ⁇ , interest in the anti-inflammatory action of betulin is also increasing.
- Betulin derivatives have been studied in addition to the various physiological activities mentioned above, but researches on anticancer agents using them are particularly active due to their excellent anticancer effects based on cell death.
- EP 1309605 and 1124842 disclose that betulin derivatives have inhibitory effects against various malignant tumor cell lines such as sarcomas, oral cancers, breast cancers, lung cancers, colon cancers and the like.
- betulin and its derivatives known to date have problems in the development and commercialization of oral anticancer drugs due to solubility in water and absorption into the body.
- the present inventors have tried to solve the problems of the oral absorption rate of the betulin and its derivatives, as a result, the pegylated betulin derivatives of the general formula (1) is found that the oral absorption is significantly improved and has an excellent anticancer effect and the present invention It was completed.
- X, X ', Y, R 1 and n are as defined in the specification.
- an object of the present invention relates to an anticancer composition for oral administration comprising a pegylated betulin derivative of Formula 1 or a pharmaceutically acceptable salt thereof having an excellent anticancer effect and particularly having improved oral absorption.
- the present invention relates to an anticancer composition for oral administration comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof.
- X is CH 2 or CO
- Y is NR 3 , NH, O, S or -OCO,
- R 1 , R 2 and R 3 are each independently an alkyl group of C 1 -C 6 ,
- n is an integer of 0-800, Preferably it is 10-400, More preferably, it is 40-200.
- the C 1 -C 6 alkyl group means a straight or branched hydrocarbon having 1 to 6 carbon atoms, for example methyl, ethyl, n-propyl, i-propyl, n-butyl, i- Butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, and the like.
- Pegylated betulin derivatives of the present invention include compounds represented by the following Formulas 1a and 1b.
- X ', Y, R 1 and n in Formula 1a are as defined in Formula 1.
- X ', Y, R 1 and n are as defined in Formula 1.
- the polyethylene glycol derivative (5) disclosed in Scheme 2 is an intermediate for single pegylation, and is commercially available or known methods [Ref. Sandler and Karo, Polymer Synthesis , Academic Press, New York, Vol. 3, PP 138-161] may be prepared by ring-opening polymerization of ethylene glycol.
- the PEGylated betulin derivative (6) of the present invention can be prepared by reacting the betulin (2) and methoxy polyethylene glycol propionic acid.
- the PEGylated betulinic acid derivative (8) of the present invention is acetylated betulinic acid (3) to obtain 3- ⁇ -acetoxybutulinic acid (7), and then the polyethylene of Scheme 2 It can be prepared by reacting with a glycol derivative (5).
- the pegylated betulinic acid derivative (8) may be deacetylated to prepare another pegylated betulinic acid derivative (9).
- the anticancer agent compositions of the present invention are not limited thereto, but may be useful for treating various cancers such as prostate cancer, lung cancer, breast cancer, colon cancer, kidney cancer, liver cancer or glioblastoma. It is preferably useful for the inhibition or treatment of prostate cancer.
- an anticancer composition containing the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is administered orally (dose or inhalation). That is, the anticancer composition of the present invention may be administered by formulating in oral form of tablets, capsules, dragees or film coating tablets, solutions or suspensions.
- a diluent for example, lactose, dextrose, sucrose, cellulose, corn starch or potato starch
- a suspending agent for example, Silica, talc, stearic acid, magnesium or calcium stearate and / or polyethylene glycol
- binders e.g. starch, arabic gum, gelatin methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone
- disintegrants e.g. Starch, alginic acid, alginate or sodium starch glycolate
- lecithin polysorbates, laurylsulfate
- pharmacologically inert substances generally used in pharmaceuticals It may contain.
- agents can be prepared by known methods, for example by means of mixing, granulating, tableting, dragging or film-coating processes.
- the active ingredient when formulated in liquid dispersions for oral administration such as syrups, emulsions and suspensions, the active ingredient may contain natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol. .
- the content of a compound of the present invention or a pharmaceutically acceptable salt thereof in the preparation of the medicament depends on the form of the medicament, but is preferably in a concentration of 0.01 to 100% by weight.
- the dosage of the anticancer agent of the present invention varies in a wide range depending on the type of mammal including the person to be treated, the extent of the disease and the judgment of the doctor. In general, however, in the case of oral administration, 0.01 to 50 mg of the active ingredient may be administered per 1 kg of body weight per day.
- the daily dosages described above can be used one at a time or dividedly, and can be arbitrarily changed depending on the extent of the disease and the judgment of the physician.
- Pegylated betulin derivatives according to the present invention can be effectively used as a novel anticancer agent, maximize the efficacy by improving solubility and oral absorption through pegylation can be an effective treatment for various carcinomas.
- it has excellent stability and has both water-soluble and fat-soluble properties, so it can be easily used for various formulation development and complex development.
- the betulin derivatives can be easily used in the formulation development to maximize the convenience of medication by significantly improving oral absorption through pegylation.
- FIG. 1 is a graph showing changes in tumor size for 21 days upon repeated oral administration of betulinic acid and BA-mPEG samples to PC-3 xenograft mice of Test Example 3 according to the present invention.
- Figure 2 is a graph showing the tumor weight of the last day of 21 days when repeated oral administration of betulinic acid, BA-mPEG samples to PC-3 xenograft mice of Test Example 3 according to the present invention.
- Figure 3 is a photograph showing the tumor size of the last day 21 days after repeated oral administration of betulinic acid, BA-mPEG samples to PC-3 xenograft mice of Test Example 3 according to the present invention.
- p-toluenesulfonylchloride (p-toluenesulfonyl) in a solution of 10 g (4.975 mmol) of ⁇ -methoxy- ⁇ -hydroxypoly (ethylene glycol) (MeO-PEG (2K) -OH) dissolved in 300 ml of dry dichloromethane. chloride) 2.46 g (13 mmol), 2 ml (15 mmol) of triethylamine, and 0.18 g (15 mmol) of 4-dimethylaminopyridine were added thereto and stirred at room temperature for 5 hours.
- the reaction solution was diluted with 1 L of dichloromethane, washed sequentially with 800 ml of 1N aqueous hydrochloric acid solution, saturated sodium bicarbonate and saturated sodium chloride aqueous solution, and the organic layer was dried over anhydrous magnesium sulfate and filtered and concentrated under reduced pressure.
- the resulting mixture was subjected to silica gel column chromatography with methanol and dichloromethane (1:30) to give 10.5 g (4.976 mmol) of the title compound (4) as a white solid.
- Example 1-2 ⁇ -methoxy- ⁇ -aminopoly (ethylene glycol) (MeO-PEG (2K) -NH 2 Manufacture of 5
- the reaction solution was diluted with 100 ml of dichloromethane, washed with 300 mL of 0.1 N aqueous hydrochloric acid solution, and then the separated organic layer was washed with 300 mL of saturated aqueous sodium hydrogen carbonate solution. Then, the organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The resulting mixture was precipitated by adding 400 ml of diethyl ether. The precipitate was filtered while washing sufficiently with diethyl ether to give 20.3 g of the title compound (6) as a white solid.
- the reaction solution was diluted with 500 ml of dichloromethane, washed sequentially with 5000 ml of 1N aqueous hydrochloric acid solution, saturated sodium bicarbonate and saturated sodium chloride aqueous solution, and the organic layer was dried over anhydrous magnesium sulfate and filtered and concentrated under reduced pressure.
- the resulting mixture was subjected to silica gel column chromatography with methanol and dichloromethane (1:30) to give 2.4 g (0.968 mmol) of the title compound (8) as a white solid.
- SNU354 was distributed by the Korea Cell Line Bank, and the remaining cell lines used in the experiment were purchased from ATCC. All cell lines were used within 10 passages. Cell lines were cultured containing RPMI1640 and 10% calf serum.
- Test Example 1-2 Human-derived Cancer Cell Line
- Samples were dissolved 30 mM using DMSO and diluted again 30, 10, 3, 1, 0.3, 0.1, 0.03 mM.
- the diluted stock solution was diluted 1000-fold at each dose using RPMI-1640 culture media, and then each sample was treated with final 30, 10, 3, 1, 0.3, 0.1, and 0.03 uM.
- Test Example 1-4 Measurement method and result of cell growth inhibition activity
- the loading of cancer cell lines was applied differently depending on the growth rate of the cell line.
- the working solution was treated to a final concentration of 30, 10, 3, 1, 0.3, 0.1, 0.03 (uM).
- the drug-treated plate was fixed by adding 50 ⁇ l / well in 50% TCA. The plate was fixed for 60 minutes at 4 °C and then washed 4 to 5 times with tap water (tap water). After washing, the plate was dried, and 100 ⁇ l / well of SRB solution (0.4% sulforhodamine B in 1% acetic acid) was added and left for 30 minutes.
- Unbound dyeing reagent was washed by adding 0.1% acetic acid, and after drying, the dyeing reagent was dissolved by adding 100 ⁇ l / well of 10 mM Tris Base (pH 10.5). Absorbance was measured at 540 nm using a Versa max microplate reader (Molecular Devices), and the measured absorbance was calculated as a percentage of the solvent treated group. The GI 50 value of the test material was calculated using Graphpad prism v4.0 software.
- Table 1 shows the GI 50 values of the two betulin derivatives and the comparative substances for eight human-derived cancer cell lines.
- Test Example 2 Evaluation of anticancer drug efficacy by repeated oral administration in PC-3 xenograft model of human-derived prostate cancer
- PC-3 prostate cancer
- Test Example 2-2 Cancer Cell Culture
- the cells were thawed and thawed in the liquid nitrogen and then cultured. Culture of the cells was incubated for a suitable period of time in a CO 2 incubator (Forma, USA) at a temperature of 37 °C and 5% CO 2 concentration.
- a CO 2 incubator Forma, USA
- SPF Specific pathogen free nude mouse of BALB / C strain, 5w, female
- Test Example 2-5 Test Substance
- Test Example 2-7 Preparation and Administration Method of Sample
- Betulinic acid was dissolved at concentrations of 0.5, 2 mg / ml and 3.13, 12.52 mg / ml (molecular weight considerations) using EtOH 10% + [20% HPBCD (in distilled water) 90% solvent, respectively.
- mPEG was used after dissolving at a concentration of 2.925, 11.7 mg / ml and 4.285, 17.14 mg / ml (considering molecular weight) using sterile distilled water, respectively.
- mice were orally administered to the mice five times a week at 0.2 ml per 20 g (days 0-4, 7-11, 14-18).
- Positive control substance Dox.hcl
- mice were orally administered to the mice five times a week at 0.2 ml per 20 g (days 0-4, 7-11, 14-18).
- Positive control substance Dox.hcl
- mice were orally administered to the mice five times a week at 0.2 ml per 20 g (days 0-4, 7-11, 14-18).
- Positive control substance Dox.hcl
- mice was dissolved in physiological saline at the concentration of 0.2 mg / ml, and then in mice every 10 days with 10 ml / kg fluid (days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20) repeated intraperitoneal administration.
- Test Example 2-8 Observation and Inspection Items
- Test Example 2-8-1 General Symptoms and Weight Change
- the average tumor size of each group from 56.6 mm 3 to 21 days was measured in three directions using a vernier caliper, and then expressed as a formula of length X width X height / 2.
- Test Example 2-8-3 Last Day Autopsy (Tumor Weighing, Photography, Fixation)
- mice On day 21 after the start of drug administration, mice were killed by CO 2 gas, and tumors were separated and weighed on a chemical balance. After taking pictures, the tumors were fixed in formalin.
- Test Example 2-8-4 Statistical Test Method
- Test Example 2-9 Results of Anticancer Drug Evaluation by Repeated Oral Administration
- Test Example 2-9-1 General Symptoms and Weight Change
- PC-3 tumors were excised and weighed 21 days after the initiation of drug administration and showed 2.2% and 6.3% for betulinic acid and BA-mPEG 20 mg / kg, respectively; Tumor weight reductions of 1.2% and 42.0% (p ⁇ 0.001) were noted. Positive control (Dox.hcl) had a tumor weight reduction of 60.8% (p ⁇ 0.001) (Table 2, Figure 2).
- ⁇ ⁇ t Vt-Vo, Measurement of the tumor volume (Vt), Initial tumor volume (Vo)
- the new oral anticancer drugs containing pegylated betulin derivatives according to the present invention have confirmed excellent efficacy with a PC-3 xenograft model, a hormone refractory prostate cell line.
- the efficacy of cell-based analysis was confirmed through experiments on various representative carcinomas including prostate cancer, and can be widely used as a novel oral anticancer agent related to apoptosis.
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Abstract
The present invention relates to an anticancer composition for oral administration, containing a pegylated botulin derivative.
Description
본 발명은 페길레이션된 베튤린 유도체를 포함하는 경구 투여용 항암제 조성물에 관한 것이다. 보다 구체적으로는 다양한 암종에 대하여 우수한 항암효과를 가지며 특히 경구흡수가 현저히 개선된 약물로서 암의 치료에 유용하게 사용될 수 있는 페길레이션된 베튤린 유도체를 포함하는 경구 투여용 항암제 조성물에 관한 것이다.The present invention relates to an anticancer drug composition for oral administration comprising a PEGylated betulin derivative. More specifically, the present invention relates to an anticancer composition for oral administration comprising a pegylated betulin derivative which has excellent anticancer effect against various carcinomas and is particularly useful for the treatment of cancer as a drug with significantly improved oral absorption.
베튤린과 베튤린산은 세포사멸(apoptosis) 효능을 갖는 것으로 알려져 있다. 세포사멸은 진핵세포에서 나타나는 세포의 파괴 또는 자살을 의미하는 것으로, 동물의 정상적인 발생을 조절하거나, 불필요하거나, 비정상적인 세포를 제거하는 등을 포함하는 개체의 항상성 유지를 위한 기본적인 세포 내 과정이다. 세포사멸은 다양한 외부 및 내부 자극에 반응하여 발생하게 되는데, 세포사멸의 진행과 함께, 세포질의 파괴, 세포막의 기포 형성(blebbing), 세포 골격의 변화, 세포 수축, 염색체의 응축 및 DNA 분절화 등과 같은 특징적인 변화가 일어나며, 작동되지 않으면 세포는 무한분열 하면서 종양을 형성하게 된다.Betulin and betulinic acid are known to have apoptosis potency. Apoptosis refers to the destruction or suicide of cells in eukaryotic cells, and is a basic intracellular process for maintaining homeostasis of an individual, including controlling the normal development of an animal, or removing unnecessary or abnormal cells. Apoptosis occurs in response to various external and internal stimuli, with the progression of apoptosis, such as cytoplasmic breakdown, blebbing of cell membranes, changes in the cytoskeleton, cell contraction, chromosome condensation and DNA fragmentation. Distinctive changes occur and, if not activated, cells divide indefinitely and form tumors.
하기 화학식 2로 나타내는 베튤린(betulin)은 루판(lupine) 계의 천연 5환 트리테르펜(triterpene) 알코올로서, 베튤리놀(betulilnol) 및 루프-20(29)-엔-3β,28-디올(lup-20(29)-ene-3β,28-diol)로서도 알려져 있다. 베튤린은 몇몇 나무종의 나무껍질, 특히 자작나무(Betula sp.) 껍질에 많이 존재하며, 자작나무 껍질로부터 추출에 의해 베튤린을 분리하는 방법들이 공지되어 있다.Betulin represented by the following formula (2) is a lupine-based natural 5-ring triterpene alcohol, betulinol and loop-20 (29) -ene-3β, 28-diol (lup). -20 (29) -ene-3β, 28-diol). Betulin is present in many bark of tree species, especially in the bark of Betula sp., And methods for separating betulin by extraction from birch bark are known.
[화학식 2][Formula 2]
한편, 하기 화학식 3으로 나타내는 베튤린산(betulinic acid)은 자작나무 (Betula sp.) 껍질 또는 황병나무(Quercus suber L.)의 코르크로부터 추출에 의해 분리하거나, 베튤린 또는 자작나무 껍질의 직접 산화에 의한 방법들에 의해 제조할 수 있는 것으로 알려져 있다. 예를 들어 하기 반응식 1에 도시된 바와 같이, 미국특허 제6,280,778호에 개시된 존스(Jones) 산화 방법에 따라 베튤린(2)을 산화크롬(VI) 촉매의 존재하에 산화시켜 베튤로닉산을 수득한 다음, 수득된 베튤로닉산을 수소화붕소나트륨으로 선택적 환원 반응시켜 베튤린산(3)을 수득할 수 있는 것으로 보고되어 있다.On the other hand, betulinic acid (betulinic acid) represented by the following formula (3) is isolated by extraction from the cork of the birch ( Betula sp.) Bark or Quercus suber L., or to the direct oxidation of It is known that it can be prepared by the methods. For example, as shown in Scheme 1, betulin (2) was oxidized in the presence of a chromium (VI) catalyst according to the Jones oxidation method disclosed in US Pat. No. 6,280,778 to obtain betunic acid. It is then reported that betulinic acid (3) can be obtained by selective reduction of the obtained beturonic acid with sodium borohydride.
[반응식 1]Scheme 1
또한 미국 특허 제5,804,575호에는 베튤린의 3-β-히드록시기를 아세틸화에 의해 보호한 다음, 산화반응시키는 단계를 포함하는 베튤린산의 다른 제조방법이 개시되어 있다.U. S. Patent No. 5,804, 575 also discloses another method for preparing betulinic acid comprising protecting the 3-β-hydroxy group of betulin by acetylation and then oxidizing.
이러한 베튤린 유도체들은 펜타사이클릭 트리테르페노이드 구조를 가진 식물에서 생합성되는 천연물의 일종으로, 상록수(Ziziphus mauritiana)에서 최초로 분리되었으며, 특히 흰 자작나무의 껍질에 풍부한 것으로 알려져 있다. 이는 흑색종이나 신경외배엽 유래 암세포에 선택적으로 작용하여 암세포를 사멸시키는 효과를 가진 것으로 알려져 있다. 베튤린 유도체들의 이러한 항암작용은 미토콘드리아의 투과성을 감소시키거나 세포질 내로 사이토크롬-C 방출 등의 미토콘드리아의 기능 저하를 비롯하여 활성산소 형성, 또는 케스페이즈 활성화 등과 같은 다양한 기전을 통하여 이루어지는 것으로 알려져 있다. 또한 베튤린 유도체들은 HIV-1의 활성을 차단하는 효과를 보여, 항바이러스 제제로 활용될 가능성을 보여 주었으며, 그 외 박테리아 및 말라리아와 같은 감염성 질환에도 효과가 있는 것으로 보고되어 있다. 더불어, 이들 베튤린 유도체들이 인터페론-γ에 의한 NO 생성 저해 활성을 가지는 것으로 보고됨에 따라, 베튤린의 항 염증 작용에 관한 관심도 높아지고 있다.These betulin derivatives are a kind of natural biosynthesis produced by plants having a pentacyclic triterpenoid structure, first isolated from evergreen (Ziziphus mauritiana), and are known to be particularly abundant in the bark of white birch. It is known to have an effect of selectively acting on melanoma or neuroectoderm-derived cancer cells to kill cancer cells. These anticancer actions of betulin derivatives are known to be through various mechanisms, such as reducing the permeability of mitochondria or deactivating mitochondria, such as cytochrome-C release into the cytoplasm, active oxygen formation, or caspase activation. In addition, betulin derivatives have been shown to block the activity of HIV-1, showing the possibility of being used as an antiviral agent, and has been reported to be effective in other infectious diseases such as bacteria and malaria. In addition, since these betulin derivatives have been reported to have NO production inhibitory activity by interferon-γ, interest in the anti-inflammatory action of betulin is also increasing.
베튤린 유도체들은 위에 언급한 다양한 생리활성 작용과 더불어 많은 연구가 진행되고 있으나 특히 세포사멸에 기반을 둔 뛰어난 항암효과로 이를 활용한 항암제 관련 연구가 활발히 진행되고 있다.Betulin derivatives have been studied in addition to the various physiological activities mentioned above, but researches on anticancer agents using them are particularly active due to their excellent anticancer effects based on cell death.
특히 베튤린산의 경우 경부암 세포주인 KB세포를 이용하여 세포증식을 감소시키고 세포사멸을 유도하는 것을 확인하고 세포증식 억제 효과는 SP1과 SP1의 표적 단백질인 Mcl-1 그리고 suvivin의 감소 때문인 것으로 확인한 예(참고문헌: J. Fd Hyg., 26, No. 2, pp. 150~153 (2011))가 보고되어 있으며, 호르몬 불응성 전립선 암종인 PC-3 세포주에 있어서도 NFkB 저해를 통한 Bax/Bcl-2 감소를 유의성 있게 보여주는 연구결과도 보고되어 있다(참고문헌: Mol carcinog, 47, No. 12, pp. 964-973 (2008)).Especially in case of betulinic acid, it was confirmed that KB cell, which is a cervical cancer cell line, reduced cell proliferation and induced apoptosis. References: J. Fd Hyg. , 26, No. 2, pp. 150-153 (2011)), and Bax / Bcl-2 through NFkB inhibition in PC-3 cell line, a hormone-resistant prostate carcinoma There have also been reports of studies showing significant reductions ( Mol carcinog , 47, No. 12, pp. 964-973 (2008)).
유럽 공개특허 제1309605호 및 제1124842호에는 베튤린 유도체들이 육종, 구강암, 유방암, 폐암, 대장암 등의 다양한 악성 종양 세포주에 대한 억제효능을 갖는 것으로 기재되어 있다.EP 1309605 and 1124842 disclose that betulin derivatives have inhibitory effects against various malignant tumor cell lines such as sarcomas, oral cancers, breast cancers, lung cancers, colon cancers and the like.
그러나 현재까지 공지된 베튤린 및 그의 유도체는 물에 대한 용해도, 생체내로의 흡수 문제로 경구화 항암제의 개발 및 상용화가 어려운 문제점이 있다.However, betulin and its derivatives known to date have problems in the development and commercialization of oral anticancer drugs due to solubility in water and absorption into the body.
본 발명자들은 상기한 베튤린 및 그의 유도체의 경구흡수율의 문제점을 해결하고자 연구 노력한 결과, 하기 화학식 1의 페길레이션된 베튤린 유도체가 경구흡수가 현저히 개선되고 우수한 항암효과를 가짐을 발견하고 본 발명을 완성하게 되었다.The present inventors have tried to solve the problems of the oral absorption rate of the betulin and its derivatives, as a result, the pegylated betulin derivatives of the general formula (1) is found that the oral absorption is significantly improved and has an excellent anticancer effect and the present invention It was completed.
[화학식 1][Formula 1]
상기 식에서, X, X', Y, R1 및 n은 명세서 중에서 정의한 바와 같다.In the above formula, X, X ', Y, R 1 and n are as defined in the specification.
따라서 본 발명의 목적은 우수한 항암효과를 가지며 특히 경구흡수가 현저히 개선된 상기 화학식 1의 페길레이션된 베튤린 유도체 또는 그의 약제학적으로 허용되는 염을 포함하는 경구 투여용 항암제 조성물에 관한 것이다.Accordingly, an object of the present invention relates to an anticancer composition for oral administration comprising a pegylated betulin derivative of Formula 1 or a pharmaceutically acceptable salt thereof having an excellent anticancer effect and particularly having improved oral absorption.
본 발명은 하기 화학식 1의 화합물 또는 그의 약제학적으로 허용되는 염을 포함하는 경구 투여용 항암제 조성물에 관한 것이다.The present invention relates to an anticancer composition for oral administration comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof.
[화학식 1][Formula 1]
상기 식에서,Where
X는 CH2 또는 CO이고,X is CH 2 or CO,
X'는 H, CH2R2 또는 COR2이며,X 'is H, CH 2 R 2 or COR 2 ,
Y는 NR3, NH, O, S 또는 -OCO이며,Y is NR 3 , NH, O, S or -OCO,
R1, R2 및 R3은 각각 독립적으로 C1-C6의 알킬기이며,R 1 , R 2 and R 3 are each independently an alkyl group of C 1 -C 6 ,
n은 0 내지 800, 바람직하게는 10 내지 400, 보다 바람직하게는 40 내지 200의 정수이다.n is an integer of 0-800, Preferably it is 10-400, More preferably, it is 40-200.
본 발명의 화합물에서 C1-C6의 알킬기는 탄소수 1 내지 6개로 구성된 직쇄형 또는 분지형 탄화수소를 의미하며, 예를 들어 메틸, 에틸, n-프로필, i-프로필, n-부틸, i-부틸, s-부틸, t-부틸, n-펜틸, n-헥실 등이 포함되나 이에 한정되는 것은 아니다.In the compound of the present invention, the C 1 -C 6 alkyl group means a straight or branched hydrocarbon having 1 to 6 carbon atoms, for example methyl, ethyl, n-propyl, i-propyl, n-butyl, i- Butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, and the like.
본 발명의 페길레이션된 베튤린 유도체는 하기 화학식 1a 및 1b로 표시되는 화합물을 포함한다.Pegylated betulin derivatives of the present invention include compounds represented by the following Formulas 1a and 1b.
[화학식 1a][Formula 1a]
상기 화학식 1a에서 X', Y, R1 및 n은 화학식 1에서 정의한 바와 같다.X ', Y, R 1 and n in Formula 1a are as defined in Formula 1.
[화학식 1b][Formula 1b]
상기 화학식 1b에서 X', Y, R1 및 n은 화학식 1에서 정의한 바와 같다.In Formula 1b, X ', Y, R 1 and n are as defined in Formula 1.
본 발명의 화합물의 제조방법을 하기 반응식 2 내지 5에 나타내었다. 하기 반응식에 기재된 방법은 대표적으로 사용된 방법을 예시한 것일 뿐 단위조작의 순서, 반응시약, 반응조건 등은 경우에 따라 얼마든지 변경될 수 있다.Methods for preparing the compounds of the present invention are shown in Schemes 2-5. The methods described in the following reaction schemes are merely illustrative of the methods used, and the order of unit operations, reaction reagents, reaction conditions, and the like may be changed as much as the case may be.
[반응식 2] Scheme 2
상기 반응식 2에 개시된 폴리에틸렌글리콜 유도체(5)는 단일 페길레이션을 위한 중간체이며, 상업적으로 입수하거나 공지된 방법[참고문헌: Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, PP 138-161]에 따라 에틸렌글리콜을 개환 중합반응시켜 제조할 수 있다.The polyethylene glycol derivative (5) disclosed in Scheme 2 is an intermediate for single pegylation, and is commercially available or known methods [Ref. Sandler and Karo, Polymer Synthesis , Academic Press, New York, Vol. 3, PP 138-161] may be prepared by ring-opening polymerization of ethylene glycol.
[반응식 3]Scheme 3
상기 반응식 3에 나타나 있듯이, 본 발명의 페길레이션된 베튤린 유도체(6)는 베튤린(2)와 메톡시폴리에틸렌글리콜프로피온산을 반응시켜 제조할 수 있다.As shown in Scheme 3, the PEGylated betulin derivative (6) of the present invention can be prepared by reacting the betulin (2) and methoxy polyethylene glycol propionic acid.
[반응식 4]Scheme 4
상기 반응식 4에 개시된 바와 같이, 본 발명의 페길레이션된 베튤린산 유도체(8)은 베튤린산(3)을 아세틸화하여 3-β-아세톡시베튤린산(7)을 얻은 후, 상기 반응식 2의 폴리에틸렌글리콜 유도체(5)와 반응시켜 제조할 수 있다. 또한, 상기 페길레이션된 베튤린산 유도체(8)를 탈아세틸화하여 다른 페길레이션된 베튤린산 유도체(9)를 제조할 수 있다.As disclosed in Scheme 4, the PEGylated betulinic acid derivative (8) of the present invention is acetylated betulinic acid (3) to obtain 3-β-acetoxybutulinic acid (7), and then the polyethylene of Scheme 2 It can be prepared by reacting with a glycol derivative (5). In addition, the pegylated betulinic acid derivative (8) may be deacetylated to prepare another pegylated betulinic acid derivative (9).
본 발명의 항암제 조성물은 이들에 한정되는 것은 아니지만, 전립선암, 폐암, 유방암, 대장암, 신장암, 간암 또는 교모세포종 등의 다양한 암을 치료하는데 유용할 수 있다. 바람직하게는 전립선암의 억제 또는 치료에 유용하다.The anticancer agent compositions of the present invention are not limited thereto, but may be useful for treating various cancers such as prostate cancer, lung cancer, breast cancer, colon cancer, kidney cancer, liver cancer or glioblastoma. It is preferably useful for the inhibition or treatment of prostate cancer.
본 발명의 화합물 또는 그의 약제학적으로 허용되는 염을 활성성분으로 함유하는 항암제 조성물은 경구적으로(복용 또는 흡입) 투여된다. 즉, 본 발명의 항암제 조성물은 정제, 캡슐, 당의정 또는 필름 피막 정제, 액제 또는 현탁제의 경구 형태로 제형화하여 투여될 수 있다.An anticancer composition containing the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is administered orally (dose or inhalation). That is, the anticancer composition of the present invention may be administered by formulating in oral form of tablets, capsules, dragees or film coating tablets, solutions or suspensions.
본 발명의 항암제 조성물을 경구용 고형제로 제형화하는 경우, 활성 성분과 함께 희석제(예를 들면, 락토즈, 덱스트로즈, 자당, 셀룰로즈, 옥수수 전분 또는 감자 전분), 활탁제(예를 들면, 실리카, 탈크, 스테아린산, 마그네슘 또는 칼슘 스테아레이트 및/또는 폴리에틸렌 글리콜), 결합제(예를 들면, 전분, 아라빅 검, 젤라틴 메틸셀룰로즈, 카르복시메틸셀룰로즈 또는 폴리비닐 피롤리돈), 붕해제(예를 들면, 전분, 알긴산, 알기네이트 또는 나트륨 전분 글리콜레이트), 포르말 혼합물, 염료, 감미제, 습윤제(예를 들면, 레시틴, 폴리솔베이트, 라우릴설페이트) 및 일반적으로 약제에 사용되는 약물학적 불활성 물질을 함유할 수 있다. 이들 약제는 공지된 방법, 예를 들면 혼합, 과립화, 타정, 당의 또는 필름-피복 공정의 수단에 의해 제조할 수 있다.When the anticancer composition of the present invention is formulated as an oral solid, a diluent (for example, lactose, dextrose, sucrose, cellulose, corn starch or potato starch) with an active ingredient, a suspending agent (for example, Silica, talc, stearic acid, magnesium or calcium stearate and / or polyethylene glycol), binders (e.g. starch, arabic gum, gelatin methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone), disintegrants (e.g. Starch, alginic acid, alginate or sodium starch glycolate), formal mixtures, dyes, sweeteners, humectants (e.g. lecithin, polysorbates, laurylsulfate) and pharmacologically inert substances generally used in pharmaceuticals It may contain. These agents can be prepared by known methods, for example by means of mixing, granulating, tableting, dragging or film-coating processes.
또한, 시럽, 유화액 및 현탁액 등의 경구 투여용 액체 분산액으로 제형화하는 경우, 활성성분과 함께 천연 검, 한천, 나트륨 알기네이트, 펙틴, 메틸셀룰로즈, 카르복시메틸셀룰로즈 또는 폴리비닐 알코올을 함유할 수 있다.In addition, when formulated in liquid dispersions for oral administration such as syrups, emulsions and suspensions, the active ingredient may contain natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol. .
상기 약제의 제조에 있어서 본 발명의 화합물 또는 그의 약제학적으로 허용되는 염의 함량은 약제의 형태에 따라 다르지만, 바람직하게는 0.01 내지 100 중량%의 농도이다.The content of a compound of the present invention or a pharmaceutically acceptable salt thereof in the preparation of the medicament depends on the form of the medicament, but is preferably in a concentration of 0.01 to 100% by weight.
본 발명의 항암제의 투여량은 치료되는 사람을 포함한 포유동물의 종류, 질환의 정도 및 의사의 판단 등에 따라 넓은 범위에서 다양하게 변화된다. 그러나, 일반적으로 경구투여의 경우에는 체중 1kg당 하루에 활성성분 0.01 내지 50 mg이 투여될 수 있다. 상술한 일일 투여량은 한번에 또는 나누어서 사용될 수 있으며, 질환의 정도 및 의사의 판단에 따라 임의로 변화될 수 있다.The dosage of the anticancer agent of the present invention varies in a wide range depending on the type of mammal including the person to be treated, the extent of the disease and the judgment of the doctor. In general, however, in the case of oral administration, 0.01 to 50 mg of the active ingredient may be administered per 1 kg of body weight per day. The daily dosages described above can be used one at a time or dividedly, and can be arbitrarily changed depending on the extent of the disease and the judgment of the physician.
본 발명에 따른 페길레이션된 베튤린 유도체들은 신규 항암제로서 효과적으로 사용될 수 있고, 페길레이션을 통하여 용해도 증가와 경구흡수를 향상시킴으로써 효능을 극대화시키며 다양한 암종에 대하여 효과적인 치료를 할 수 있다. 뿐만 아니라 안정성이 우수하고 수용성, 지용성의 양쪽 성질을 모두 가지고 있어 다양한 제형 개발과 복합제 개발에 용이하게 사용될 수 있다. 특히 상기 베튤린 유도체들은 페길레이션을 통해 경구흡수를 현저히 개선시켜 복약 편의성이 극대화된 제형 개발에 용이하게 사용될 수 있다.Pegylated betulin derivatives according to the present invention can be effectively used as a novel anticancer agent, maximize the efficacy by improving solubility and oral absorption through pegylation can be an effective treatment for various carcinomas. In addition, it has excellent stability and has both water-soluble and fat-soluble properties, so it can be easily used for various formulation development and complex development. In particular, the betulin derivatives can be easily used in the formulation development to maximize the convenience of medication by significantly improving oral absorption through pegylation.
도 1은 본 발명에 따른 시험예 3의 PC-3 이종 이식 마우스에 베튤린산, BA-mPEG 시료들을 반복 경구 투여 시 21일 동안의 종양 크기에 대한 변화를 나타낸 그래프이다.1 is a graph showing changes in tumor size for 21 days upon repeated oral administration of betulinic acid and BA-mPEG samples to PC-3 xenograft mice of Test Example 3 according to the present invention.
도 2는 본 발명에 따른 시험예 3의 PC-3 이종 이식 마우스에 베튤린산, BA-mPEG 시료들을 반복 경구 투여 시 21일 최종일의 종양 무게를 측정하여 나타낸 그래프이다.Figure 2 is a graph showing the tumor weight of the last day of 21 days when repeated oral administration of betulinic acid, BA-mPEG samples to PC-3 xenograft mice of Test Example 3 according to the present invention.
도 3은 본 발명에 따른 시험예 3의 PC-3 이종 이식 마우스에 베튤린산, BA-mPEG 시료들을 반복 경구 투여 후 21일 최종일의 종양의 크기를 확인할 수 있는 사진이다.Figure 3 is a photograph showing the tumor size of the last day 21 days after repeated oral administration of betulinic acid, BA-mPEG samples to PC-3 xenograft mice of Test Example 3 according to the present invention.
이하, 실시예에 의해 본 발명을 보다 구체적으로 설명하고자 한다. 이들 실시예는 오직 본 발명을 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업자에게 있어서 자명하다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
실시예 1: 폴리에틸렌글리콜 유도체의 제조Example 1 Preparation of Polyethylene Glycol Derivatives
실시예 1-1: 메톡시폴리(에틸렌글리콜)토실레이트(MeO-PEG(2K)-OTs) (4)의 제조Example 1-1 Preparation of Methoxypoly (ethyleneglycol) tosylate (MeO-PEG (2K) -OTs) (4)
α-메톡시-ω-하이드록시폴리(에틸렌글리콜)(MeO-PEG(2K)-OH) 10 g(4.975 mmol)을 건조 디클로로메탄 300 ml에 용해한 용액에 p-톨루엔설포닐클로라이드(p-toluenesulfonyl chloride) 2.46 g(13 mmol), 트리에틸아민 2 ml(15 mmol), 4-디메틸아미노피리딘(4-dimethylaminopyridine) 0.18 g(15 mmol)을 가하고 실온에서 5시간 동안 교반하였다. 반응 용액을 디클로로메탄 1 L로 희석시키고, 1N 염산 수용액, 포화 탄산수소나트륨 및 포화 염화나트륨 수용액 각각 800 ml로 순차적으로 세척하고, 유기층을 무수 황산마그네슘으로 건조한 후 여과하여 감압농축 하였다. 생성된 혼합물을 메탄올과 디클로로메탄(1:30)으로 실리카겔 컬럼 크로마토그래피하여 백색 고형의 표제 화합물(4) 10.5 g(4.976 mmol)을 수득하였다.p-toluenesulfonylchloride (p-toluenesulfonyl) in a solution of 10 g (4.975 mmol) of α-methoxy-ω-hydroxypoly (ethylene glycol) (MeO-PEG (2K) -OH) dissolved in 300 ml of dry dichloromethane. chloride) 2.46 g (13 mmol), 2 ml (15 mmol) of triethylamine, and 0.18 g (15 mmol) of 4-dimethylaminopyridine were added thereto and stirred at room temperature for 5 hours. The reaction solution was diluted with 1 L of dichloromethane, washed sequentially with 800 ml of 1N aqueous hydrochloric acid solution, saturated sodium bicarbonate and saturated sodium chloride aqueous solution, and the organic layer was dried over anhydrous magnesium sulfate and filtered and concentrated under reduced pressure. The resulting mixture was subjected to silica gel column chromatography with methanol and dichloromethane (1:30) to give 10.5 g (4.976 mmol) of the title compound (4) as a white solid.
1H NMR 400 MHz (CDCl3): δ 7.93(d, 2H), 7.41(d, 2H), 3.82-3.46 (m,175H), 3.38(s, 3H), 2.49(s, 3H) 1 H NMR 400 MHz (CDCl 3 ): δ 7.93 (d, 2H), 7.41 (d, 2H), 3.82-3.46 (m, 175H), 3.38 (s, 3H), 2.49 (s, 3H)
실시예 1-2: α-메톡시-ω-아미노폴리(에틸렌글리콜)(MeO-PEG(2K)-NHExample 1-2: α-methoxy-ω-aminopoly (ethylene glycol) (MeO-PEG (2K) -NH
22
) (5)의 제조Manufacture of 5
MeO-PEG(2K)-OTs(4) 10.5 g(4.976 mmol)을 30% 암모니아수 800 ml에 가하고 실온에서 12시간 동안 교반하였다. 반응 용액을 디클로로메탄 500 ml로 추출하여 포화 염화나트륨 용액 500 ml로 세척하고, 유기층을 무수 황산마그네슘으로 건조한 후 여과하여 감압농축 하였다. 생성된 혼합물을 디메틸에테르에 침전시켜 백색 고형의 표제 화합물(5) 10 g(4.96 mmol)을 수득하였다. 10.5 g (4.976 mmol) of MeO-PEG (2K) -OTs (4) was added to 800 ml of 30% aqueous ammonia and stirred at room temperature for 12 hours. The reaction solution was extracted with 500 ml of dichloromethane and washed with 500 ml of saturated sodium chloride solution. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The resulting mixture was precipitated in dimethyl ether to give 10 g (4.96 mmol) of the title compound (5) as a white solid.
1H NMR 400 MHz (CDCl3): δ 3.82-3.46 (m, 175H), 3.38(s, 3H), 2.92(t, 3H) 1 H NMR 400 MHz (CDCl 3 ): δ 3.82-3.46 (m, 175H), 3.38 (s, 3H), 2.92 (t, 3H)
실시예 2: 페길레이션된 베튤린 유도체의 제조Example 2: Preparation of Pegylated Betulin Derivatives
실시예 2-1: 화학식 6의 페길레이션된 베튤린 유도체 (BET-mPEG)의 제조Example 2-1 Preparation of Pegylated Bettulin Derivatives of Formula 6 (BET-mPEG)
베튤린(2) 1.7 g (3.8 mmol)과 메톡시폴리에틸렌글리콜프로피온산 19.2 g (3.8 mmol)을 건조 디클로로메탄 200 mL에 용해하고 4-디메틸아미노피리딘(4-dimethylaminopyridine) 0.47 g(0.38 mmol)을 첨가하였다. 그런 다음, 반응 용액에 N-(3-디메틸아미노프로필)-N-(에틸카보디이미드) 염산 (N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride) 0.95 g(4.6 mmol)을 첨가한 후 실온에서 12 시간 동안 교반하였다. 반응 용액을 디클로로메탄 100 ml로 희석시키고, 0.1 N 염산 수용액 300 mL로 세척한 다음, 분리된 유기층을 포화 탄산수소나트륨 수용액 300 mL로 세척하였다. 그런 다음, 유기층을 무수 황산마그네슘으로 건조한 후 여과하여 감압 농축하였다. 생성된 혼합물은 디에틸에테르 400 ml를 가하여 침전시켰다. 침전물을 디에틸에테르로 충분히 세척하면서 여과하여 백색 고형의 표제 화합물(6) 20.3 g을 수득하였다.1.7 g (3.8 mmol) of betulin (2) and 19.2 g (3.8 mmol) of methoxypolyethylene glycol propionic acid were dissolved in 200 mL of dry dichloromethane, and 0.47 g (0.38 mmol) of 4-dimethylaminopyridine was added. It was. Then, the reaction solution N- (3- dimethylaminopropyl) -N- (ethylcarbodiimide) hydrochloride (N - (3-dimethylaminopropyl) - N -ethylcarbodiimide hydrochloride) 0.95 g (4.6 mmol) was added and stirred at room temperature for 12 hours. The reaction solution was diluted with 100 ml of dichloromethane, washed with 300 mL of 0.1 N aqueous hydrochloric acid solution, and then the separated organic layer was washed with 300 mL of saturated aqueous sodium hydrogen carbonate solution. Then, the organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The resulting mixture was precipitated by adding 400 ml of diethyl ether. The precipitate was filtered while washing sufficiently with diethyl ether to give 20.3 g of the title compound (6) as a white solid.
1H NMR 400 MHz (CDCl3): δ 4.69(ds, 29-1H), 4.59(s, 29-1H), 4.29(d, 28-1Ha), 3.88(d, 28-1Hb), 3.82-3.46 (m, PEG back-bone), 3.38(s, CH
3 O-PEG, 3H), 3.19(t, 3-1H), 2.62(t, -CH
2 COO-, 2H), 2.44(m, 19-1H), 1.68(s, 30-3H), 1.03(s, 26-3H), 0.97(s, 23-3H), 0.96(s, 24-3H), 0.82(s, 25-3H), 0.76(s, 27-3H), 2.02-0.65(m, betulin-24H) 1 H NMR 400 MHz (CDCl 3 ): δ 4.69 (ds, 29-1H), 4.59 (s, 29-1H), 4.29 (d, 28-1Ha), 3.88 (d, 28-1Hb), 3.82-3.46 (m, PEG back-bone), 3.38 (s, CH 3 O-PEG, 3H), 3.19 (t, 3-1 H), 2.62 (t, -CH 2 COO-, 2H), 2.44 (m, 19- 1H), 1.68 (s, 30-3H), 1.03 (s, 26-3H), 0.97 (s, 23-3H), 0.96 (s, 24-3H), 0.82 (s, 25-3H), 0.76 ( s, 27-3H), 2.02-0.65 (m, betulin-24H)
실시예 3: 페길레이션된 베튤린산 유도체의 제조Example 3: Preparation of PEGylated Betulinic Acid Derivatives
실시예 3-1: 3β-아세톡시베튤린산(3-Ac-BA) (7)의 제조Example 3-1 Preparation of 3β-Acetoxyvetulinic Acid (3-Ac-BA) (7)
베튤린산(3) 21 g (0.046 mol)을 피리딘 15.2 ml (0.189 mol)과 무수아세트산 186.6 ml (1.98 mol)에 넣고, 실온에서 12 시간 동안 교반하였다. 반응 용액을 얼음물 300 ml에 붓고 30 분간 교반한 후 여과하여 얻은 고형물을 에틸아세테이트:헥산(1:1)으로 실리카겔 컬럼 크로마토그래피하여 백색 고형의 표제 화합물(7) 15.6 g을 수득하였다.21 g (0.046 mol) of betulinic acid (3) was added to 15.2 ml (0.189 mol) of pyridine and 186.6 ml (1.98 mol) of acetic anhydride, and stirred at room temperature for 12 hours. The reaction solution was poured into 300 ml of ice water, stirred for 30 minutes, and the resulting solid was filtered through silica gel column chromatography with ethyl acetate: hexane (1: 1) to give 15.6 g of the title compound (7) as a white solid.
1H NMR 400 MHz (CDCl3): δ 4.74(ds, 29-1H), 4.61(s, 29-1H), 4.47(dt, 3-1H), 2.99(m, 19-1H), 2.30-2.16(m, 2-2H), 2.04(s, CH
3 CO-3H), 2.01-1.93(m, 21-2H), 1.69(s, 30-3H), 0.97(s, 26-3H), 0.94(s, 23-3H), 0.85(s, 24-3H), 0.84(s, 25-3H), 0.83(s, 27-3H), 1.74-0.77(m, 20H) 1 H NMR 400 MHz (CDCl 3 ): δ 4.74 (ds, 29-1H), 4.61 (s, 29-1H), 4.47 (dt, 3-1H), 2.99 (m, 19-1H), 2.30-2.16 (m, 2-2H), 2.04 (s, CH 3 CO-3H), 2.01-1.93 (m, 21-2H), 1.69 (s, 30-3H), 0.97 (s, 26-3H), 0.94 ( s, 23-3H), 0.85 (s, 24-3H), 0.84 (s, 25-3H), 0.83 (s, 27-3H), 1.74-0.77 (m, 20H)
실시예 3-2: 화학식 8의 페길레이션된 베튤린산 유도체(Ac-BA-mPEG)의 제조Example 3-2 Preparation of Pegylated Betulinic Acid Derivatives (Ac-BA-mPEG) of Formula 8
실시예 3-1에서 수득한 3-Ac-BA(7) 2.1 g(4.211 mmol)을 벤젠 40 ml에 용해하고 옥살릴클로라이드 2.2 ml(25.263 mmol)을 가하여 실온에서 3시간 교반하였다. 반응 용액을 감압 농축하여 남아있는 옥살릴클로라이드를 제거하여 노란 고체 형태의 화합물을 수득한 후 건조 디클로로메탄 80 ml에 용해하고 실시예 1-2 에서 수득한 PEG2000-mono NH2 (5) 7.03 g(3.79 mmol)을 가하여 실온에서 3시간 교반하였다. 반응 용액을 디클로로메탄 500 ml로 희석시키고, 1N 염산 수용액, 포화 탄산수소나트륨 및 포화 염화나트륨 수용액 각각 5000 ml로 순차적으로 세척하고, 유기층을 무수 황산마그네슘으로 건조한 후 여과하여 감압농축 하였다. 생성된 혼합물은 메탄올과 디클로로메탄(1:30)으로 실리카겔 컬럼 크로마토그래피하여 백색 고형의 표제 화합물(8) 2.4 g(0.968 mmol)을 수득하였다.2.1 g (4.211 mmol) of 3-Ac-BA (7) obtained in Example 3-1 was dissolved in 40 ml of benzene, and 2.2 ml (25.263 mmol) of oxalyl chloride were added and stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure to remove the remaining oxalyl chloride. After obtaining the compound in the form of a yellow solid, dissolved in 80 ml of dry dichloromethane, 7.03 g (3.79 mmol) of PEG2000-mono NH 2 (5) obtained in Example 1-2 was added and stirred at room temperature for 3 hours. The reaction solution was diluted with 500 ml of dichloromethane, washed sequentially with 5000 ml of 1N aqueous hydrochloric acid solution, saturated sodium bicarbonate and saturated sodium chloride aqueous solution, and the organic layer was dried over anhydrous magnesium sulfate and filtered and concentrated under reduced pressure. The resulting mixture was subjected to silica gel column chromatography with methanol and dichloromethane (1:30) to give 2.4 g (0.968 mmol) of the title compound (8) as a white solid.
1H NMR 400 MHz (CDCl3): δ 6.13(t, 1H), 4.75(s, 1H), 4.60 (s, 1H), 3.83(t, 2H), 3.82-3.4 (m, 175H), 3.40(s, 3H), 3.18(t, 2H), 2.43(bs, 3H), 2.02(s, 3H), 2.0-1.2(m, 25H), 1.67(s, 3H), 0.97(s, 3H), 0.94(s, 3H), 0.84(m, 9H) 1 H NMR 400 MHz (CDCl 3 ): δ 6.13 (t, 1H), 4.75 (s, 1H), 4.60 (s, 1H), 3.83 (t, 2H), 3.82-3.4 (m, 175H), 3.40 ( s, 3H), 3.18 (t, 2H), 2.43 (bs, 3H), 2.02 (s, 3H), 2.0-1.2 (m, 25H), 1.67 (s, 3H), 0.97 (s, 3H), 0.94 (s, 3H), 0.84 (m, 9H)
실시예 3-3: 화학식 9의 페길레이션된 베튤린산 유도체(BA-mPEG)의 제조Example 3-3 Preparation of Pegylated Betulinic Acid Derivatives of Formula 9 (BA-mPEG)
수산화나트륨 0.5 g (11.5 mmol)을 물 2.5 ml에 용해한 용액을 실시예 3-2에서 수득한 3β-아세틸-28-메톡시폴리에틸렌글리콜 베튤린산 아미드(8) 5.0 g (2.02 mmol)을 메탄올 7.5 ml와 테트라히드로퓨란 10 ml에 용해한 용액에 가하고, 실온에서 12 시간 동안 교반하였다. 반응 용액에 1.0 N 염산 수용액을 pH 2가 될 때까지 천천히 가하고, 디클로로메탄 100 ml로 추출하였다. 유기층을 무수 황산마그네슘으로 건조한 후 여과시키고, 감압 농축한 다음 디에틸에테르 100 ml를 가하여 침전시켰다. 침전물을 디에틸에테르로 충분히 세척하면서 여과하여 백색 고형의 표제 화합물(9) 4.3 g을 수득하였다.5.0 g (2.02 mmol) of 3β-acetyl-28-methoxypolyethylene glycol betulinic acid amide (8) obtained in Example 3-2 was dissolved in a solution of 0.5 g (11.5 mmol) of sodium hydroxide in 2.5 ml of water. Was added to a solution dissolved in 10 ml of tetrahydrofuran and stirred at room temperature for 12 hours. A 1.0 N aqueous hydrochloric acid solution was slowly added to the reaction solution until the pH was 2, and extracted with 100 ml of dichloromethane. The organic layer was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and precipitated by adding 100 ml of diethyl ether. The precipitate was filtered off with sufficient washing with diethyl ether to give 4.3 g of the title compound (9) as a white solid.
1H NMR 400 MHz (CDCl3): δ 6.07(t, -NHCO-, 1H), 4.70(ds, 29-1H), 4.56(s, 29-1H), 3.79(t, -NHCH2
CH
2 O-), 3.72-3.39(m, PEG back-bone), 3.38(s, CH
3 O-PEG, 3H), 3.20-3.05(m, 3, 19-2H), 2.41(dt, 2-1H), 1.67(s, 30-3H), 0.99(s, 26,23-6H), 0.93(s, 24-3H), 0.79(s, 25-3H), 0.72(s, 27-3H), 1.93-0.63(m, Betulinic acid-22H) 1 H NMR 400 MHz (CDCl 3 ): δ 6.07 (t, - NH CO-, 1H), 4.70 (ds, 29-1H), 4.56 (s, 29-1H), 3.79 (t, -NHCH 2 CH 2 O-), 3.72-3.39 (m, PEG back-bone), 3.38 (s, CH 3 O-PEG, 3H), 3.20-3.05 (m, 3, 19-2H), 2.41 (dt, 2-1H) , 1.67 (s, 30-3H), 0.99 (s, 26,23-6H), 0.93 (s, 24-3H), 0.79 (s, 25-3H), 0.72 (s, 27-3H), 1.93- 0.63 (m, Betulinic acid-22H)
시험예 1: 인간 유래 암세포주 8종에 대한 세포기반 저해효능 시험Test Example 1 Test of Cell-Based Inhibitory Effect on Eight Human Cancer Cell Lines
시험예 1-1: 세포 배양Test Example 1-1: Cell Culture
SNU354는 한국 세포주은행에서 분양받았으며, 실험에 사용된 나머지 세포주는 ATCC에서 구입하였다. 모든 세포주는 10 passage내에서 사용하였다. 세포주는 RPMI1640 과 10%의 송아지 혈청을 함유한 배양액을 사용하였다.SNU354 was distributed by the Korea Cell Line Bank, and the remaining cell lines used in the experiment were purchased from ATCC. All cell lines were used within 10 passages. Cell lines were cultured containing RPMI1640 and 10% calf serum.
시험예 1-2: 인간 유래 암세포주Test Example 1-2: Human-derived Cancer Cell Line
전립선암(Prostate cancer): PC-3, LN Cap Prostate cancer: PC-3, LN Cap
폐암(Lung cancer): NCI-H23 Lung cancer: NCI-H23
유방암(Breast cancer): MDA-MB-231Breast cancer: MDA-MB-231
대장암(Colon cancer): HCT-15Colon cancer: HCT-15
신장암(Renal cancer): ACHNRenal cancer: ACHN
간암(Liver cancer): SNU354Liver cancer: SNU354
교모세포종(Glioblastoma cancer): U87MGGlioblastoma cancer: U87MG
시험예 1-3: 시료의 제조Test Example 1-3: Preparation of Sample
시료는 DMSO를 이용하여 30mM 용해한 후 다시 30, 10, 3, 1, 0.3, 0.1, 0.03 mM 희석하였다. 희석된 스톡 용액(stock solution)을 RPMI-1640 배양 배지(culture media)를 이용하여 각 dose별로 1000배 희석한 후 각 시료를 final 30, 10, 3, 1, 0.3, 0.1, 0.03 uM 처리하였다. Samples were dissolved 30 mM using DMSO and diluted again 30, 10, 3, 1, 0.3, 0.1, 0.03 mM. The diluted stock solution was diluted 1000-fold at each dose using RPMI-1640 culture media, and then each sample was treated with final 30, 10, 3, 1, 0.3, 0.1, and 0.03 uM.
시험예 1-4: 세포성장저해 활성 측정법 및 결과 Test Example 1-4: Measurement method and result of cell growth inhibition activity
암세포주의 로딩(loading)하는 농도는 세포주의 성장속도에 따라서 다르게 적용하였다. 각각의 세포주를 96-well plate 에 로딩을 한 후에 워킹 용액(working solution)을 최종농도가 30, 10, 3, 1, 0.3, 0.1, 0.03(uM)이 되도록 처리하였다. 48시간 배양 후 약물을 처리한 plate를 50% TCA로 50㎕/well씩을 넣어서 고정하였다. 배양고정한 plate는 4℃에서 60분간 방치한 뒤 수돗물(tap water)로 4~5번 정도 세척을 하였다. 세척한 plate는 건조한 후 SRB solution (0.4% sulforhodamine B in 1% acetic acid) 을 100 ㎕/well을 가하고 30분 정도를 방치하였다. 결합하지 않은 염색 시약은 0.1% 아세트산(acetic acid)를 가하여 세척하였으며, 다시 건조를 한 후에 10 mM Tris Base (pH 10.5)를 100 ㎕/well를 가하여 염색시약을 용해시켰다. 흡광도는 Versa max microplate reader (Molecular Devices)를 사용하여 540 nm에서 측정하였으며, 측정한 흡광도는 용매 처리군에 대한 백분율로 계산하였다. 시험물질의 GI50 값은 Graphpad prism v4.0 software을 이용하여 계산하였다.The loading of cancer cell lines was applied differently depending on the growth rate of the cell line. After loading each cell line into a 96-well plate, the working solution was treated to a final concentration of 30, 10, 3, 1, 0.3, 0.1, 0.03 (uM). After 48 hours of incubation, the drug-treated plate was fixed by adding 50 μl / well in 50% TCA. The plate was fixed for 60 minutes at 4 ℃ and then washed 4 to 5 times with tap water (tap water). After washing, the plate was dried, and 100 μl / well of SRB solution (0.4% sulforhodamine B in 1% acetic acid) was added and left for 30 minutes. Unbound dyeing reagent was washed by adding 0.1% acetic acid, and after drying, the dyeing reagent was dissolved by adding 100 μl / well of 10 mM Tris Base (pH 10.5). Absorbance was measured at 540 nm using a Versa max microplate reader (Molecular Devices), and the measured absorbance was calculated as a percentage of the solvent treated group. The GI 50 value of the test material was calculated using Graphpad prism v4.0 software.
8종의 인간 유래 암세포주에 대한 베튤린 유도체 2종과 비교물질들의 GI50 값은 표 1에 나타내었다.Table 1 shows the GI 50 values of the two betulin derivatives and the comparative substances for eight human-derived cancer cell lines.
[표 1]TABLE 1
8종의 인간 유래 암세포주에 대한 베튤린 유도체 2종과 비교물질의 GI50 값GI 50 Values of Two Betulin Derivatives and Comparative Substances on Eight Human-derived Cancer Cell Lines
화합물 6과 화합물 9의 경우 전구물질로 활성이 떨어지며, 그의 활성형인 베튤린과 베튤린산의 경우 수 uM 수준의 활성을 보여 유의성 있는 결과를 나타내었다.In the case of compounds 6 and 9, the activity was reduced as a precursor, and the active forms of betulin and betulinic acid showed activity of several uM levels, which showed a significant result.
시험예 2: 인체유래 전립선암 PC-3 이종 이식 모델에서 반복 경구투여에 의한 항암 약효 평가Test Example 2: Evaluation of anticancer drug efficacy by repeated oral administration in PC-3 xenograft model of human-derived prostate cancer
시험예 2-1: 암세포 종류Test Example 2-1: Cancer Cell Types
인체유래 전립선암세포주 PC-3(prostate cancer)Human prostate cancer cell line PC-3 (prostate cancer)
시험예 2-2: 암세포 배양Test Example 2-2: Cancer Cell Culture
액체 질소 속에서 냉동보관 중이던 암세포를 해동(thawing)한 후 세포 배양을 실시하였다. 세포의 배양은 CO2 인큐베이터(incubator) (Forma, USA) 내에서 온도 37℃와 CO2 농도 5%로 맞춰서 적절한 기간 동안 배양하였다.The cells were thawed and thawed in the liquid nitrogen and then cultured. Culture of the cells was incubated for a suitable period of time in a CO 2 incubator (Forma, USA) at a temperature of 37 ℃ and 5% CO 2 concentration.
시험예 2-3: 시험동물Test Example 2-3: Test Animal
BALB/C 계통의 특정병원체 부재(SPF) 누드 마우스, 5w, female (공급원: Nara Biotech Co.)Specific pathogen free (SPF) nude mouse of BALB / C strain, 5w, female (Source: Nara Biotech Co.)
시험예 2-4: 암세포 이식Test Example 2-4: Cancer Cell Transplantation
배양 최종일에 모든 암세포를 수거하여 계수하고 무혈청 배지(serum free media)를 이용하여 세포 농도를 3 x 107 cells/ml로 조절하였다. 이렇게 조절된 세포 배양액을 마우스당 0.3 ml(9 x 106 cells/mouse)씩 우측의 견갑부와 흉벽 사이의 액와 부위 피하에 주입하였다.All cancer cells were harvested and counted on the last day of culture and the cell concentration was adjusted to 3 × 10 7 cells / ml using serum free media. The cell cultures thus adjusted were injected subcutaneously between the scapula and the chest wall at 0.3 ml (9 x 10 6 cells / mouse) per mouse.
시험예 2-5: 시험물질Test Example 2-5: Test Substance
(1) 명칭: 베튤린산(Betulinic acid), BA-mPEG(1) Name: Betulinic acid, BA-mPEG
(2) 외관 색상 및 성상: 흰색, 옅은 노란색(베튤린산) 및 결정성 분말(2) Appearance color and appearance: white, pale yellow (betulinic acid) and crystalline powder
(3) 보관조건: 냉장(3) Storage condition: refrigerated
시험예 2-6: 양성대조물질Test Example 2-6: Positive Control
(1) 명칭: 염산 독소루비신(Doxorubicin hydrochloride)(1) Name: Doxorubicin hydrochloride
(2) 외관 색상 및 성상: 선홍색 및 결정성 분말(2) Appearance color and appearance: cerise and crystalline powder
(3) 제품번호: D1515(3) Model number: D1515
(4) 로트번호: Lot# SLBF1340VLot number: Lot # SLBF1340V
(5) 보관조건: 냉장보관(5) Storage conditions: Refrigerated storage
(6) 공급자: SIGMA-ALDRICH®
(6) Supplier: SIGMA-ALDRICH ®
시험예 2-7: 시료의 조제 및 투여방법Test Example 2-7: Preparation and Administration Method of Sample
베튤린산은 EtOH 10% + [20% HPBCD(in distilled water)] 90%의 용매를 이용하여 각각 0.5, 2 mg/ml와 3.13, 12.52 mg/ml(분자량 고려)의 농도로 용해하였으며, BA-mPEG는 멸균증류수를 이용하여 각각 2.925, 11.7 mg/ml와 4.285, 17.14 mg/ml(분자량 고려)의 농도로 용해한 후 사용하였다.Betulinic acid was dissolved at concentrations of 0.5, 2 mg / ml and 3.13, 12.52 mg / ml (molecular weight considerations) using EtOH 10% + [20% HPBCD (in distilled water) 90% solvent, respectively. mPEG was used after dissolving at a concentration of 2.925, 11.7 mg / ml and 4.285, 17.14 mg / ml (considering molecular weight) using sterile distilled water, respectively.
조제된 시료들은 마우스에 20g 당 0.2 ml씩 주 5회(days 0-4, 7-11, 14-18) 반복 경구투여하였다. 양성대조물질(Dox.hcl)은 생리식염수를 이용하여 0.2 mg/ml의 농도로 용해한 후 마우스에 10 ml/kg 액량으로 2일에 1회씩 (days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20) 반복 복강투여하였다.The prepared samples were orally administered to the mice five times a week at 0.2 ml per 20 g (days 0-4, 7-11, 14-18). Positive control substance (Dox.hcl) was dissolved in physiological saline at the concentration of 0.2 mg / ml, and then in mice every 10 days with 10 ml / kg fluid ( days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20) repeated intraperitoneal administration.
시험예 2-8: 관찰 및 검사항목Test Example 2-8: Observation and Inspection Items
시험예 2-8-1: 일반증상 및 체중 변화Test Example 2-8-1: General Symptoms and Weight Change
모든 동물에 대하여 투여 개시시 및 시험기간 중 투여 직전 일반증상 관찰 및 체중 측정을 하였다.All animals were observed for general symptoms and weighed at the start of administration and immediately prior to administration.
시험예 2-8-2: 종양크기 변화Test Example 2-8-2: Change in tumor size
암세포 이식 후 군별 평균 종양크기가 56.6 mm3 도달시부터 21일째까지 총 10회 개체별로 버니어 캘리퍼(vernier caliper)를 이용하여 3방향을 측정한 후 length X width X height/2의 계산식으로 표현하였다.After cancer cell transplantation, the average tumor size of each group from 56.6 mm 3 to 21 days was measured in three directions using a vernier caliper, and then expressed as a formula of length X width X height / 2.
시험예 2-8-3: 최종일 부검(종양조직 무게 측정, 사진촬영, 고정)Test Example 2-8-3: Last Day Autopsy (Tumor Weighing, Photography, Fixation)
약물투여 개시 후 21일째 CO2 가스를 이용하여 마우스를 치사시킨 뒤 종양을 분리하여 화학 저울(chemical balance)에 무게를 측정하였으며, 사진 촬영 후 종양은 포르말린에 고정하였다.On day 21 after the start of drug administration, mice were killed by CO 2 gas, and tumors were separated and weighed on a chemical balance. After taking pictures, the tumors were fixed in formalin.
시험예 2-8-4: 통계학적 검사 방법Test Example 2-8-4: Statistical Test Method
모든 측정 항목의 값은 t-TEST 통계법을 사용하여 실험 대조군과 약물투여군을 비교하여 통계학적인 유의성을 검사하였다.The values of all measurement items were tested for statistical significance by comparing the experimental control group and the drug administration group using the t-TEST statistical method.
시험예 2-9: 반복 경구투여에 의한 항암 약효 평가 결과Test Example 2-9: Results of Anticancer Drug Evaluation by Repeated Oral Administration
시험예 2-9-1: 일반증상 및 체중변화Test Example 2-9-1: General Symptoms and Weight Change
PC-3 이식 누드 마우스에 베튤린산 및 BA-mPEG 시료들을 각각 5 및 20 mg/kg 용량으로 반복 경구 투여시 독성 정도를 알아보기 위해 투여기간 동안 동물의 일반증상 및 체중변화를 관찰하였다. 그 결과 시험기간 동안 특이한 일반증상은 없었으며, 투여 기간 동안 통계학적으로 유의한 체중 감소도 관찰되지 않았다. 양성대조물질(Dox.hcl)은 여윔 증상과 최종일 33.3%(p<0.001)의 통계학적으로 유의한 체중 감소가 나타났다.The general symptoms and body weight of the animals were observed during the administration period to determine the toxicity of repeated doses of betulinic acid and BA-mPEG samples at 5 and 20 mg / kg doses in PC-3 transplanted nude mice, respectively. As a result, there were no general symptoms during the trial period, and no statistically significant weight loss was observed during the administration period. The positive control (Dox.hcl) showed extraordinary symptoms and statistically significant weight loss of 33.3% (p <0.001) at the last day.
시험예 2-9-2: 종양크기 변화Test Example 2-9-2: Tumor Size Change
최종일(day 21) 결과를 보면 베튤린산 및 BA-mPEG 5, 20 mg/kg 투여군에서 각각 4.0%, 5.4%; 4.6%, 45.2%(p<0.001)의 종양성장 억제가 관찰되었다. 양성대조물질(Dox.hcl)은 62.2%(p<0.001)의 종양성장 억제가 있었다(표 2 및 도 1).Day 21 results showed 4.0% and 5.4% for betulinic acid and BA- mPEG 5 and 20 mg / kg, respectively; Tumor growth inhibition of 4.6% and 45.2% (p <0.001) was observed. Positive control (Dox.hcl) had a tumor growth inhibition of 62.2% (p <0.001) (Table 2 and Figure 1).
시험예 2-9-3: 최종일 종양무게Test Example 2-9-3: Last Day Tumor Weight
약물투여 개시 후 21일째 PC-3 종양을 절제하여 그 무게를 측정한 결과, 베튤린산 및 BA-mPEG 20 mg/kg 투여군에서 각각 2.2%, 6.3%; 1.2%, 42.0%(p<0.001)의 종양무게 감소가 나타났다. 양성대조물질(Dox.hcl)은 60.8%(p<0.001)의 종양무게 감소가 있었다(표 2, 도 2).PC-3 tumors were excised and weighed 21 days after the initiation of drug administration and showed 2.2% and 6.3% for betulinic acid and BA-mPEG 20 mg / kg, respectively; Tumor weight reductions of 1.2% and 42.0% (p <0.001) were noted. Positive control (Dox.hcl) had a tumor weight reduction of 60.8% (p <0.001) (Table 2, Figure 2).
[표 2]TABLE 2
종양크기 변화 및 최종일(day 21) 종양무게Tumor size change and last day (day 21) tumor weight
significant figures(t-TEST): * p<0.05, ** p<0.01, *** p<0.001(vs Vehicle Control)significant figures (t-TEST): * p <0.05, ** p <0.01, *** p <0.001 (vs Vehicle Control)
† △t=Vt-Vo, Vt(Measurement of the tumor volume), Vo(Initial tumor volume) † Δt = Vt-Vo, Measurement of the tumor volume (Vt), Initial tumor volume (Vo)
‡ Inhibition Rate(vs Vehicle Control) ‡ Inhibition Rate (vs Vehicle Control)
본 발명에 따른 페길레이션된 베튤린 유도체를 포함하는 신규 경구용 항암제 약물들은 호르몬 불응성 전립선 세포주인 PC-3 이종 이식 동물모델(Xenograft model)로 뛰어난 효능을 확인하였다. 또한 본 발명에서 베튤린 유도체들에 대하여 전립선암을 포함한 다양한 대표 암종들에 대하여 세포기반 분석을 실험을 통하여 효능을 확인하였으며, 세포사멸 기전과 관련된 신규 경구용 항암제로서 폭넓게 사용될 수 있다.The new oral anticancer drugs containing pegylated betulin derivatives according to the present invention have confirmed excellent efficacy with a PC-3 xenograft model, a hormone refractory prostate cell line. In addition, in the present invention, the efficacy of cell-based analysis was confirmed through experiments on various representative carcinomas including prostate cancer, and can be widely used as a novel oral anticancer agent related to apoptosis.
Claims (13)
- 하기 화학식 1의 화합물 또는 그의 약제학적으로 허용되는 염을 포함하는 경구 투여용 항암제 조성물:An anticancer composition for oral administration comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof:[화학식 1][Formula 1]상기 식에서,WhereX는 CH2 또는 CO이고,X is CH 2 or CO,X'는 H, CH2R2 또는 COR2이며,X 'is H, CH 2 R 2 or COR 2 ,Y는 NR3, NH, O, S 또는 -OCO이며,Y is NR 3 , NH, O, S or -OCO,R1, R2 및 R3은 각각 독립적으로 C1-C6의 알킬기이며,R 1 , R 2 and R 3 are each independently an alkyl group of C 1 -C 6 ,n은 0 내지 800의 정수이다.n is an integer of 0 to 800.
- 제1항에 있어서, 화학식 1의 화합물은 하기 화학식 1a로 표시되는 화합물인 것을 특징으로 하는 경구 투여용 항암제 조성물:[Claim 2] The anticancer composition for oral administration according to claim 1, wherein the compound of Formula 1 is a compound represented by the following Formula 1a:[화학식 1a][Formula 1a]상기 화학식 1a에서, X', Y, R1 및 n은 제1항의 화학식 1에 대해 정의한 바와 같다.In Formula 1a, X ', Y, R 1 and n are as defined for Formula 1 of claim 1.
- 제1항에 있어서, 화학식 1의 화합물은 하기 화학식 1b로 표시되는 화합물인 것을 특징으로 하는 경구 투여용 항암제 조성물:According to claim 1, wherein the compound of Formula 1 is an oral anticancer composition, characterized in that the compound represented by the formula (1b):[화학식 1b][Formula 1b]상기 화학식 1b에서, X', Y, R1 및 n은 제1항의 화학식 1에 대해 정의한 바와 같다.In Formula 1b, X ', Y, R 1 and n are as defined for Formula 1 of claim 1.
- 제1항에 있어서, R1는 메틸, 에틸, n-프로필, i-프로필, n-부틸, i-부틸, s-부틸, t-부틸, n-펜틸 및 n-헥실로 이루어진 군에서 선택되는 것을 특징으로 하는 경구 투여용 항암제 조성물.The compound of claim 1, wherein R 1 is selected from the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl and n-hexyl Anticancer composition for oral administration, characterized in that.
- 제4항에 있어서, R1는 메틸인 것을 특징으로 하는 경구 투여용 항암제 조성물.The anticancer composition for oral administration according to claim 4, wherein R 1 is methyl.
- 제2항에 있어서, X'가 H이고, Y가 -OCO인 것을 특징으로 하는 경구 투여용 항암제 조성물.The anticancer composition for oral administration according to claim 2, wherein X 'is H and Y is -OCO.
- 제3항에 있어서, X'가 H 또는 CH3CO이고, Y가 NH인 것을 특징으로 하는 경구 투여용 항암제 조성물.The anticancer composition for oral administration according to claim 3, wherein X 'is H or CH 3 CO, and Y is NH.
- 제6항 또는 제7항에 있어서, R1는 메틸인 것을 특징으로 하는 경구 투여용 항암제 조성물.8. The anticancer composition for oral administration according to claim 6 or 7, wherein R 1 is methyl.
- 제1항에 있어서, n은 10 내지 400의 정수인 것을 특징으로 하는 경구 투여용 항암제 조성물.The anticancer composition for oral administration according to claim 1, wherein n is an integer of 10 to 400.
- 제1항에 있어서, n은 40 내지 200의 정수인 것을 특징으로 하는 경구 투여용 항암제 조성물.The anticancer composition for oral administration according to claim 1, wherein n is an integer of 40 to 200.
- 제1항에 있어서, 전립선암, 폐암, 유방암, 대장암, 신장암, 간암 또는 교모세포종에 적용되는 것을 특징으로 하는 경구 투여용 항암제 조성물.The anticancer composition for oral administration according to claim 1, which is applied to prostate cancer, lung cancer, breast cancer, colon cancer, kidney cancer, liver cancer or glioblastoma.
- 제11항에 있어서, 전립선암에 적용되는 것을 특징으로 하는 경구 투여용 항암제 조성물.The anticancer composition for oral administration according to claim 11, which is applied to prostate cancer.
- 제1항에 있어서, 경구용 고형제 또는 경구용 액체 분산액으로 제형화되는 것을 특징으로 하는 경구 투여용 항암제 조성물.The anticancer composition for oral administration according to claim 1, which is formulated as an oral solid or oral liquid dispersion.
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KR20010080507A (en) * | 1998-11-18 | 2001-08-22 | 추후보정 | Novel betulinic acid derivatives, processes for preparing such derivatives and its use as cancer growth inhibitors |
JP2004517045A (en) * | 2000-08-18 | 2004-06-10 | ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ イリノイ | Prodrugs of betulinic acid derivatives for treatment of cancer and HIV |
KR100578382B1 (en) * | 2004-07-16 | 2006-05-11 | 나재운 | Water soluble chitosan nanoparticle for delivering a anticance agent and preparing method thereof |
JP2009530405A (en) * | 2006-03-23 | 2009-08-27 | アドバンスト ライフ サイエンシズ インコーポレイテッド | Synthetic pentacyclic triterpenoids and derivatives of betulinic acid and betulin |
KR20100109618A (en) * | 2009-04-01 | 2010-10-11 | 주식회사 페라온 | Pegylated betulin derivatives and cosmetic composition including the same |
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KR20010080507A (en) * | 1998-11-18 | 2001-08-22 | 추후보정 | Novel betulinic acid derivatives, processes for preparing such derivatives and its use as cancer growth inhibitors |
JP2004517045A (en) * | 2000-08-18 | 2004-06-10 | ザ ボード オブ トラスティーズ オブ ザ ユニバーシティ オブ イリノイ | Prodrugs of betulinic acid derivatives for treatment of cancer and HIV |
KR100578382B1 (en) * | 2004-07-16 | 2006-05-11 | 나재운 | Water soluble chitosan nanoparticle for delivering a anticance agent and preparing method thereof |
JP2009530405A (en) * | 2006-03-23 | 2009-08-27 | アドバンスト ライフ サイエンシズ インコーポレイテッド | Synthetic pentacyclic triterpenoids and derivatives of betulinic acid and betulin |
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