KR100321844B1 - ACETAL DERIVATIVES OF 4'-O-DEMETHYL-EPIPODOPHYLLOTOXIN-a-D-GLUCOSIDE, PREPARATION METHOD THEREOF AND ANTICANCER COMPOSITION CONTAINING THE SAME - Google Patents
ACETAL DERIVATIVES OF 4'-O-DEMETHYL-EPIPODOPHYLLOTOXIN-a-D-GLUCOSIDE, PREPARATION METHOD THEREOF AND ANTICANCER COMPOSITION CONTAINING THE SAME Download PDFInfo
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- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
Abstract
Description
본 발명은 4'-O-데메틸-에피포도필로톡신-β-D-글루코시드 (4'-O-demethyl-epipodophyllotoxin-β-D-glucoside)의 당 부분인 글루코시드의 4-히드록시기와 6-히드록시기가 아세탈화된, 불포화기 또는 알콕시알킬기 등을 포함하는 새로운 글리코시드 유도체, 이의 제조방법 및 이를 포함하는 항암제 조성물에 관한 것이다.The invention having 4'- O- methyl-epi-podophyllotoxin - 4-hydroxy group of the β -D- glucoside glucoside of the body of (4'- O- demethyl- epi podophyllotoxin- β -D -glucoside) and The present invention relates to a novel glycoside derivative including an unsaturated group, an alkoxyalkyl group, etc., in which a 6-hydroxy group is acetalized, a preparation method thereof, and an anticancer composition comprising the same.
에토포사이드(Etoposide) 및 테니포사이드(Teniposide)는 1960년대 산도즈사(Sandoz Limited)에서 천연물인 포도필룸 펠타툼 엘.(Podophyllum peltatum L.)로부터 분리, 정제한 포도필로톡신(podophyllotoxin)으로부터 반합성적으로 개발된 후 현재 방사선 치료시 병용투여로 폐암, 악성림프종, 백혈병, 고환암 등에 우수한 항암효과를 나타내고 있는 항암제이다(논문[C. Keller-Julsen, M. Kuhn, A. von Wartburg,J. Med. Chem. 1971,14(10), 936-940] 및 US Patent 3,524,844 참조).Etoposide and Teniposide were semisynthetic from podophyllotoxin isolated and purified from Podophyllum peltatum L. , a natural product at Sandoz Limited in the 1960s. It has been developed and is currently an anticancer agent that shows excellent anticancer effects such as lung cancer, malignant lymphoma, leukemia, testicular cancer by combined administration during radiation therapy (C. Keller-Julsen, M. Kuhn, A. von Wartburg, J. Med. Chem) 1971, 14 (10), 936-940], and see US Patent 3,524,844).
이 화합물은 인간 유전자 위상이성질화 II 효소 (Human DNA Topoisomerase II)의 활성을 억제하여 유전자 합성을 저해함으로써 암세포에 대한 효과를 나타내는 것으로 알려져 있다 (논문[J. C. Wang,J. Biol. Chem.,1991,266(11), 6659-62] 참조).This compound is known to have an effect on cancer cells by inhibiting the gene synthesis by inhibiting the activity of human DNA Topoisomerase II (JC Wang, J. Biol. Chem. , 1991 , 266 (11) , 6659-62).
그러나, 상기 에토포사이드 및 테니포사이드는 물에 대해 불용성이고 세포독성이 큰 문제가 있어 이를 개선하기 위하여 에토포사이드의 변형에 의한 새로운 항암제 연구개발이 최근에도 계속적으로 발표되고 있다. 예를 들면, 미합중국의 브리스톨-마이어스사(Bristol-Myers Squibb Co.) 및 일본의 미생물 화학 연구기관(Microbial Chemistry Research Foundation)에서 각각 에토포포스(ETOPOPHOSTM)와 NK-611을 개발하여, 에토포포스는 1997년에 항암제로 승인받았으며, NK-611은 현재 항암제개발을 위한 임상단계에 진입되어 있는 상황이다 (GB 2,207,674 A 및 EP 0,196,618 참조).However, the etoposide and teniposide are insoluble in water and have a large cytotoxic problem. Therefore, new anticancer drug research and development by modification of etoposide has been continuously published in recent years. For example, ETOPOPHOS TM and NK-611 were developed by Bristol-Myers Squibb Co. of the United States and Microbial Chemistry Research Foundation of Japan, respectively. POS was approved as an anticancer drug in 1997, and NK-611 is currently entering a clinical stage for anticancer drug development (see GB 2,207,674 A and EP 0,196,618).
이러한 에토포사이드의 구조 중에서 글루코시드의 4,6-아세탈 작용기가 항암 효과에 가장 직접적인 영향을 주고 있으나 아세탈 작용기 중 불포화 작용기를 갖는 화합물은 이중결합을 갖는 크로톤알데히드(crotonaldehyde) 자체 및 신남알데히드 유도체(cinnamaldehyde)만이 합성되어 보고되어 있는 상황이다 (US Patent 3,524,844 및 논문[C. Keller-Julsen, M. Kuhn, A. von Wartburg,J. Med. Chem. 1971,14(10), 936-940] 참조).Among the structures of etoposide, the 4,6-acetal functional group of glucoside has the most direct effect on the anticancer effect, but the compounds having unsaturated functional groups in the acetal functional group are crotonaldehyde itself having double bonds and cinnamic derivatives (cinnamaldehyde). ) Is synthesized and reported (see US Patent 3,524,844 and in papers C. Keller-Julsen, M. Kuhn, A. von Wartburg, J. Med. Chem. 1971 , 14 (10) , 936-940). .
본 발명자는 현재까지 보고되어 있지 않은 입체적으로 비교적 작은 이중결합, 삼중결합 또는 알콕시알킬의 구조를 갖는 소수성의 그룹을 에토포사이드의 글루코시드 4,6-디올에 도입하여 그 항암효과 및 독성 및 약리학적 개선 효과 등을연구하고자 이 계열의 화합물들을 합성한 후 인간 유전자 위상이성질화 II 효소(Human DNA Topoisomerase II), 생체외 암세포 독성시험(in vitrocytotoxicity), 생체내 항암활성(in vivoanticancer activity)을 시험한 결과, 본 합성화합물들이 비교화합물 에토포사이드보다 우수한 활성을 나타내고 있음을 발견하여, 본 발명을 완성하게 되었다.The present inventors have introduced a hydrophobic group having a three-dimensionally relatively small double bond, triple bond or alkoxyalkyl structure to the glucoside 4,6-diol of etoposide, and its anticancer effect and toxicity and pharmacological Human DNA Topoisomerase II, in vitro cytotoxicity, in vivo anticancer activity As a result of the test, it was found that the present synthetic compounds showed better activity than the comparative compound etoposide, thereby completing the present invention.
즉, 본 발명의 목적은 항암 활성이 우수한, 새로운 4'-O-데메틸-에피포도필로톡신-β-D-글루코시드의 새로운 아세탈 유도체를 제공하는 것이다.That is, an object of the present invention is to provide a novel acetal derivative of 4'- 0 -demethyl-epipodophyllotoxin- β -D-glucoside having excellent anticancer activity.
상기 목적을 달성하기 위하여 본 발명에서는 하기 일반식 (I)의 새로운 4'-O-데메틸-에피포도필로톡신-β-D-글루코시드 유도체를 제공한다.In order to achieve the above object, the present invention provides a novel 4'- 0 -demethyl-epipodophyllotoxin- β -D-glucoside derivative of the general formula (I).
화학식 1Formula 1
상기식에서,In the above formula,
R1및 R2는 각각 독립적으로 수소원자, 또는 알콕시, 페닐, 치환된 페닐,할로겐, 니트로, 시아노, 히드록시, 카르복실 및 아미노기로 이루어진 군으로부터 선택된 하나 이상의 라디칼로 임의로 치환된 C2-C12알케닐, C2-C12알키닐, C2-C12알콕시알킬 또는 시클로프로필이거나; 이들이 결합된 탄소원자와 함께 상호 연결되어 알콕시, 페닐, 치환된 페닐, 할로겐, 니트로, 시아노, 히드록시, 카르복실 또는 아미노기로 이루어진 군으로부터 선택된 하나 이상의 라디칼로 임의로 치환된 C5-C15의 고리를 형성하며, 단, R1및 R2는 둘다 수소원자는 아니다.R 1 and R 2 are each independently hydrogen or C 2 -optionally substituted with one or more radicals selected from the group consisting of alkoxy, phenyl, substituted phenyl, halogen, nitro, cyano, hydroxy, carboxyl and amino groups. C 12 alkenyl, C 2 -C 12 alkynyl, C 2 -C 12 alkoxyalkyl or cyclopropyl; Of C 5 -C 15 which are interconnected with the carbon atoms to which they are attached and optionally substituted with one or more radicals selected from the group consisting of alkoxy, phenyl, substituted phenyl, halogen, nitro, cyano, hydroxy, carboxyl or amino groups Forming a ring provided that R 1 and R 2 are not both hydrogen atoms.
상기 일반식 (I)의 화합물 중 특히 우수한 항암 활성을 보이는 화합물은 4'-O-데메틸-에피포도필로톡신-β-D-(2-프로페닐리덴)-글루코시드, 4'-O-데메틸-에피포도필로톡신-β-D-(3-부테닐리덴)-글루코시드, 4'-O-데메틸-에피포도필로톡신-β-D-(2-부티닐리덴)-글루코시드, 4'-O-데메틸-에피포도필로톡신-β-D-(3-페닐-2-프로피닐리덴)-글루코시드, 4'-O-데메틸-에피포도필로톡신-β-D-(2-메틸-3-페닐-2-프로페닐리덴)-글루코시드, 4'-O-데메틸-에피포도필로톡신-β-D-(3-사이클로헥세리덴)-글루코시드, 4'-O-데메틸-에피포도필로톡신-β-D-(3-메틸-2-부테닐리덴)-글루코시드, 4'-O-데메틸-에피포도필로톡신-β-D-(2-메틸-2-프로페닐리덴)-글루코시드4'-O-데메틸-에피포도필로톡신-β-D-(3-메톡시-1-부틸리덴)-글루코시드, 4'-O-데메틸-에피포도필로톡신-β-D-(3-에톡시-1-프로필리덴)-글루코시드, 4'-O-데메틸-에피포도필로톡신-β-D-(3-메톡시-1-프로필리덴)-글루코시드, 4'-O-데메틸-에피포도필로톡신-β-D-(시클로프로필메틸리덴)-글루코시드 등이다.Among the compounds of the general formula (I), particularly compounds showing excellent anticancer activity are 4'- 0 -demethyl-epipodophyllotoxin- β -D- (2-propenylidene) -glucoside, 4'- 0- Demethyl-Epipodophyllotoxin- β -D- (3-butenylidene) -glucoside, 4'- 0 -demethyl-epipodophyllotoxin- β -D- (2-butynylidene) -glucoside , 4′- O -demethyl-epipodophyllotoxin- β -D- (3-phenyl-2-propynylidene) -glucoside, 4′- O -demethyl-epipodophyllotoxin- β -D- (2-Methyl-3-phenyl-2-propenylidene) -glucoside, 4'- 0 -demethyl-epipodophyllotoxin- β -D- (3-cyclohexaneidene) -glucoside, 4 ' O -demethyl-epipodophyllotoxin- β -D- (3-methyl-2-butenylidene) -glucoside, 4'- 0 -demethyl-epipodophyllotoxin- β -D- (2- Methyl-2-propenylidene) -glucoside 4'- 0 -demethyl-epipodophyllotoxin- β -D- (3-methoxy-1-butylidene) -glucoside, 4'- 0- de Methyl-Epipodophyllotoxin- β -D- (3-Ethoxy Cy-1-propylidene) -glucoside, 4'- 0 -demethyl-epipodophyllotoxin- β -D- (3-methoxy-1-propylidene) -glucoside, 4'- 0 -demethyl -Epipodophyllotoxin- beta -D- (cyclopropylmethylidene) -glucoside.
또한, 본 발명에서는 4'-O-데메틸-에피포도필로톡신-β-D-글루코시드와 각각의 상응하는 알데히드 또는 아세탈 화합물을 반응시키는 것을 특징으로 하는 상기 일반식(I) 화합물의 제조방법을 제공한다.Also, in the present invention, the method for preparing the compound of formula (I) characterized in that the reaction of 4'- O -demethyl-epipodophyllotoxin- β -D-glucoside with the corresponding aldehyde or acetal compound To provide.
더 나아가서 본 발명에서는 상기 일반식 (I)의 4'-O-데메틸-에피포도필로톡신-β-D-글루코시드 유도체를 포함하는 항암제 조성물을 제공한다.Furthermore, the present invention provides an anticancer composition comprising 4′- O -demethyl-epipodophyllotoxin- β -D-glucoside derivative of the general formula (I).
이하 본 발명에 대하여 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일반식(I)의 신규한 4'-O-데메틸-에피-포도필로톡신-β-D-글루코시드 유도체는 다음 반응식 1 에 나타낸 방법으로 제조할 수 있다.The novel 4′- O -demethyl-epi-podophyllotoxin- β -D-glucoside derivatives of formula (I) of the present invention can be prepared by the method shown in Scheme 1 below.
[반응식 1] [Reaction Scheme 1]
상기식에서, R1및 R2는 앞에서 정의한 바와 같고, R3는 C1-C3의 알킬기이다.Wherein R 1 and R 2 are as defined above and R 3 is an alkyl group of C 1 -C 3 .
상기 반응식에서 보듯이 일반식(I) 화합물은 일반식(II)의 4'-O-데메틸-에피-포도필로톡신-β-D-글루코시드를 각각의 상응하는 약 5 내지 20 당량, 바람직하게는 10 내지 15 당량의 일반식(III)의 알데히드 화합물 또는 일반식 (IV)의 아세탈 화합물과 0℃에서 80℃의 온도 범위, 바람직하게는 실온에서 1 내지 48 시간, 바람직하게는 3 내지 24 시간 동안 반응시켜 합성할 수 있다.As shown in the above scheme, the compound of general formula (I) comprises 4′- O -demethyl-epi-podophyllotoxin- β -D-glucoside of general formula (II), each corresponding to about 5 to 20 equivalents, preferably Preferably 10 to 15 equivalents of an aldehyde compound of formula (III) or an acetal compound of formula (IV) and a temperature range of 0 ° C. to 80 ° C., preferably 1 to 48 hours at room temperature, preferably 3 to 24 hours. It can be synthesized by reacting for a time.
본 발명에 적합하게 사용될 수 있는 용매의 예로는 니트로메탄, 디클로로메탄, 클로로포름, 디에틸에테르, 테트라히드로푸란, 디메톡시에탄, 아세토니트릴, 디메틸포름아미드와 같은 불활성 유기용매가 있고, 이 중에서 무수 아세토니트릴과 무수 니트로메탄이 바람직하다.Examples of solvents that can be suitably used in the present invention include inert organic solvents such as nitromethane, dichloromethane, chloroform, diethyl ether, tetrahydrofuran, dimethoxyethane, acetonitrile and dimethylformamide, among which anhydrous aceto Nitrile and anhydrous nitromethane are preferred.
촉매로는 p-톨루엔술폰산, 메탄술폰산, 염화 아연, 산성 레진 (acidic resin)과 같은 산 촉매가 사용되며p-톨루엔술폰산 또는 염화아연이 바람직하다. 상기 산 촉매는 출발물질, 4'-O-데메틸-에피포도필로톡신-β-D-글루코시드를 기준으로 3 내지 30% 범위, 바람직하게는 5 내지 10 % 범위의 양으로 사용된다.As the catalyst, an acid catalyst such as p-toluenesulfonic acid, methanesulfonic acid, zinc chloride, acidic resin is used, and p- toluenesulfonic acid or zinc chloride is preferable. The acid catalyst is used in an amount ranging from 3 to 30%, preferably from 5 to 10%, based on the starting material, 4'- 0 -demethyl-epipodophyllotoxin- β -D-glucoside.
또한, 과량의 3Å 또는 4Å 분자체(molecular seive)를 반응계에 가하여 반응중에 생성되는 물을 제거할 수도 있다. 일반식(II)의 화합물의 중량을 기준으로 200중량%의 4Å 분자체를 사용하는 것이 바람직하다.In addition, an excess of 3 ′ or 4 ′ molecular sieves may be added to the reaction system to remove water generated during the reaction. Preference is given to using 200% by weight of 4 ′ molecular sieves, based on the weight of the compound of formula (II).
반응의 종결을 위해 유기염기로서 트리알킬아민 또는 피리딘류를 가하여 산촉매를 중화하며 그 양은 촉매로 첨가된 산보다 약간의 과량을 사용하는 것이 바람직하다.To terminate the reaction, trialkylamine or pyridines are added as organic bases to neutralize the acid catalyst, and the amount is preferably used in excess of the acid added as catalyst.
상기 일반식 (II)의 4'-O-데메틸-에피포도필로톡신-β-D-글루코시드는 보고된 문헌[P. Allevi, M. Anastasia, P. Ciuffreda, A. M. Sanvito and P. Macdonald,Tet. Lett. 1992,33,4831-4834; S. Hashimoto, T. Honda, and S. Ikegami,Tet. Lett. 1991,32,1653-1654]에 따라 합성할 수 있다.4′- O -demethyl-epipodophyllotoxin- β -D-glucoside of formula (II) has been reported in [P. Allevi, M. Anastasia, P. Ciuffreda, AM Sanvito and P. Macdonald, Tet. Lett. 1992 , 33, 4831-4834; S. Hashimoto, T. Honda, and S. Ikegami, Tet. Lett. 1991 , 32, 1653-1654.
일반식 (III)의 알데히드 화합물의 구체적인 예로는 시클로프로판카복스알데히드, 페닐프로파길알데히드, 메타크롤레인,α-메틸-트랜스-신남알데히드,1,2,3,6-테트라하이드로벤즈알데히드, 3-메틸-2-부텐알데히드 등이 있고, 일반식 (IV)의 아세탈 화합물의 구체적인 예로는 아크롤레인디메틸아세탈, 3-부텐알디에틸아세탈, 3-메톡시부티르알데히드디메틸아세탈, 2-부틸-1-알데히드디에틸아세탈, 3-에톡시프로피온알데히드디에틸아세탈, 1,1,3-트리메톡시프로판등이 있으며, 시판되고 있는 것을 사용할 수 있다.Specific examples of the aldehyde compound of general formula (III) include cyclopropanecarboxaldehyde, phenylpropargylaldehyde, methacrolein, α -methyl-trans-cinnamaldehyde, 1,2,3,6-tetrahydrobenzaldehyde, 3- Methyl-2-butenealdehyde and the like, and specific examples of the acetal compound of the general formula (IV) include acrolein dimethyl acetal, 3-butene diethyl acetal, 3-methoxybutyraldehyde dimethyl acetal, 2-butyl-1-aldehyde Diethyl acetal, 3-ethoxypropionaldehyde diethyl acetal, 1,1,3-trimethoxypropane, and the like, and commercially available ones can be used.
이와 같이 제조된 본 발명의 일반식(I)의 화합물은 항암효과를 가진다.The compound of formula (I) of the present invention prepared as above has an anticancer effect.
본 발명에서는 일반식(I)의 화합물을 유효성분으로서 약제학적으로 허용되는 담체와 혼합하여 항암제 조성물을 제조할 수 있다. 상기 조성물은 통상적으로 사용되는 부형제, 붕해제, 감미제, 활탁제, 향미제 등을 추가로 포함할 수 있으며, 통상적인 방법에 의해 경구투여용 제제(예: 정제, 캡슐제 등) 또는 비경구 투여용 제제(예: 주사제, 현탁액)와 같은 단위 투여형 또는 수회 투여형 약제학적 제제로 제형화될 수 있다. 이중에서 주사제가 바람직하다.In the present invention, an anticancer composition may be prepared by mixing the compound of Formula (I) with a pharmaceutically acceptable carrier as an active ingredient. The composition may further include conventionally used excipients, disintegrants, sweeteners, suspending agents, flavoring agents and the like, oral administration (e.g. tablets, capsules, etc.) or parenteral administration by conventional methods It may be formulated in a unit dosage form or in multiple dosage forms such as a dosage form (eg, injection, suspension). Of these, injection is preferred.
본 발명의 약학 조성물은, 목적하는 바에 따라 비경구 투여하거나 경구 투여할 수 있고, 하루에 1 내지 100mg/kg체중, 바람직하게는 1 내지 20mg/kg 체중 범위에서 1회 내지 수회, 바람직하게는, 1회 내지 3회에 나누어 투여할 수 있다. 특정 환자에 대한 투여 용량은 환자의 체중, 연령, 성별, 건강 상태, 식이, 투여 시간, 투여 방법, 배설율, 질환의 중증도 등에 따라 변화될 수 있다.The pharmaceutical composition of the present invention may be parenterally or orally administered as desired, and may be 1 to several times, preferably, in the range of 1 to 100 mg / kg body weight, preferably 1 to 20 mg / kg body weight per day. It can be administered in one to three divided doses. Dosages for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of the disease, and the like.
이하 실시예를 통하여 본 발명을 더욱 상세히 설명한다. 단, 본 발명의 범위가 하기 실시예만으로 한정되는 것은 아니다.The present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited only to the following Examples.
실시예 1Example 1
4'-O-데메틸-에피포도필로톡신-β-D-(알케닐리덴 또는 알키닐리덴)-글루코시드 계열 유도체(화합물 1 내지 12)의 합성Synthesis of 4′- O -demethyl-epipodophyllotoxin- β -D- (alkenylidene or alkynylidene) -glucoside derivatives (compounds 1 to 12)
무수 아세토니트릴 (20 mL)에 4'-O-데메틸-에피포도필로톡신-β-D-글루코시드 (200 mg, 0.36 mmol)을 녹인 용액에p-톨루엔술폰산 (20 mg)과 각 각의 해당하는 과량의 아크로레인 디알킬 알데히드 또는 아세탈 유도체 (acrolein dialkyl aldehyde or acetal derivatives, 2.0 - 3.5 mmol)를 넣고 실온, 질소기류하에서 3 시간 내지 24 시간 동안 교반하였다. TLC 로 반응의 완결을 확인한 후 트리에틸아민 (0.2 mL)을 가하여 반응을 중단시킨 후 아세토니트릴을 감압증류하여 생성된 고체를 클로로포름 (100 mL x 2 회)에 녹여 유기층을 증류수 (10 mL)로 씻어준 후 무수 MgSO4로 탈수시켰다. MgSO4를 여과한 후 여액을 감압증류하고 생성물을 관 크로마토그라피 (클로로포름:메탄올 = 40:1)로 분리하여 각각의 순수한 화합물을 얻었다. 분리된 화합물이 불순물을 포함한 경우에는 관 크로마토그라피 (헥산:에틸아세테이트 = 1:1) 로 다시 분리하여 하기 화합물들을 순수한 상태로 얻었다.Anhydrous acetonitrile (20 mL) to the 4'- O- methyl-epi-podophyllotoxin - β -D- glucoside (200 mg, 0.36 mmol) was dissolved in a solution of p- toluenesulfonic acid (20 mg) and respectively Corresponding excess of acrolein dialkyl aldehyde or acetal derivatives (acrolein dialkyl aldehyde or acetal derivatives, 2.0-3.5 mmol) was added thereto, and the mixture was stirred for 3 to 24 hours at room temperature and under a nitrogen stream. After completion of the reaction by TLC, triethylamine (0.2 mL) was added to terminate the reaction. Acetonitrile was distilled under reduced pressure, and the resulting solid was dissolved in chloroform (100 mL x 2 times), and the organic layer was diluted with distilled water (10 mL). After washing, it was dehydrated with anhydrous MgSO 4 . After filtering MgSO 4 , the filtrate was distilled under reduced pressure and the product was separated by column chromatography (chloroform: methanol = 40: 1) to obtain each pure compound. When the separated compound contained impurities, the residue was separated by column chromatography (hexane: ethyl acetate = 1: 1) to obtain the following compounds in a pure state.
화합물 1 : 4'-O-데메틸-에피포도필로톡신-β-D-(2-프로페닐리덴)-글루코시드Compound 1: 4′- O -demethyl-epipodophyllotoxin- β -D- (2-propenylidene) -glucoside
[화학식 2] [ Formula 2 ]
화합물 2 : 4'-O-데메틸-에피포도필로톡신-β-D-(3-부테닐리덴)-글루코시드Compound 2: 4'- 0 -demethyl-epipodophyllotoxin- β -D- (3-butenylidene) -glucoside
[화학식 3] [ Formula 3 ]
화합물 3 : 4'-O-데메틸-에피포도필로톡신-β-D-(2-부티닐리덴)-글루코시드Compound 3: 4'- 0 -demethyl-epipodophyllotoxin- β -D- (2-butynylidene) -glucoside
[화학식 4] [ Formula 4 ]
화합물 4 : 4'-O-데메틸-에피포도필로톡신-β-D-(3-페닐-2-프로피닐리덴)-글루코시드Compound 4: 4′- O -demethyl-epipodophyllotoxin- β -D- (3-phenyl-2-propynylidene) -glucoside
[화학식 5] [ Formula 5 ]
화합물 5 : 4'-O-데메틸-에피포도필로톡신-β-D-(2-메틸-3-페닐-2-프로페닐리덴)-글루코시드;Compound 5: 4'- 0 -demethyl-epipodophyllotoxin- β -D- (2-methyl-3-phenyl-2-propenylidene) -glucoside;
[화학식 6] [ Formula 6 ]
화합물 6 : 4'-O-데메틸-에피포도필로톡신-β-D-(3-사이클로헥세닐리덴)-글루코시드.Compound 6: 4'- 0 -demethyl-epipodophyllotoxin- β -D- (3-cyclohexenylidene) -glucoside.
[화학식 7] [ Formula 7 ]
화합물 7 : 4'-O-데메틸-에피포도필로톡신-β-D-(3-메틸-2-부테닐리덴)-글루코시드;Compound 7: 4'- 0 -demethyl-epipodophyllotoxin- β -D- (3-methyl-2-butenylidene) -glucoside;
[화학식 8] [ Formula 8 ]
화합물 8: 4'-O-데메틸-에피포도필로톡신-β-D-(2-메틸-2-프로페닐리덴)-글루코시드;Compound 8: 4'- 0 -demethyl-epipodophyllotoxin- β -D- (2-methyl-2-propenylidene) -glucoside;
[화학식 9] [ Formula 9 ]
실시예 2Example 2
4'-O-데메틸-에피포도필로톡신-β-D-(알콕시알킬리덴)-글루코시드 유도체(화합물 9 내지 11)의 합성Synthesis of 4′- O -demethyl-epipodophyllotoxin- β -D- (alkoxyalkylidene) -glucoside derivatives (compounds 9 to 11)
4'-O-데메틸-에피포도필로톡신-β-D-글루코시드(170 mg, 0.30 mmol)을 무수 니트로메탄 (20 mL)에 녹인 용액에 ZnCl2(20 mg)과 각각의 해당하는 과량의 알콕시알킬알데히드 또는 아세탈 화합물(1.8-3.0 mmol)을 질소기류하에서 넣고 실온에서 3 시간 내지 24 시간 동안 교반하였다. 반응의 완결을 TLC로 확인하였으며 니트로메탄올 감압 증류하고 생성된 고체를 클로로포름(100 mL)에 녹여 유기층을 증류수(10 mL x 2회)로 씻어준 후 무수 MgSO4로 건조시키고 여과한 다음 여액을 감압증류하고 관 크로마토그라피 (클로로포름:메탄올 = 40:1)로 분리하여 하기 화합물들을 순수한 형태로 얻었다.ZnCl 2 (20 mg) and each corresponding excess in a solution of 4'- O -demethyl-epipodophyllotoxin- β -D-glucoside (170 mg, 0.30 mmol) in anhydrous nitromethane (20 mL) Alkoxyalkylaldehyde or acetal compound (1.8-3.0 mmol) was added under a nitrogen stream and stirred at room temperature for 3 to 24 hours. The completion of the reaction was confirmed by TLC, distillation under reduced pressure of nitromethanol, the resulting solid was dissolved in chloroform (100 mL), the organic layer was washed with distilled water (10 mL x 2 times), dried over anhydrous MgSO 4 , filtered and the filtrate was decompressed Distillation and separation by column chromatography (chloroform: methanol = 40: 1) gave the following compounds in pure form.
화합물 9 :4'-O-데메틸-에피포도필로톡신-β-D-(3-메톡시-1-부틸리덴)-글루코시드Compound 9: 4'- 0 -demethyl-epipodophyllotoxin- β -D- (3-methoxy-1-butylidene) -glucoside
[화학식 10] [ Formula 10 ]
화합물 10 : 4'-O-데메틸-에피포도필로톡신-β-D-(3-에톡시-1-프로필리덴)-글루코시드Compound 10: 4'- 0 -demethyl-epipodophyllotoxin- β -D- (3-ethoxy-1-propylidene) -glucoside
[화학식 11] [ Formula 11 ]
화합물 11 : 4'-O-데메틸-에피포도필로톡신-β-D-(3-메톡시-1-프로필리덴)-글루코시드Compound 11: 4'- 0 -demethyl-epipodophyllotoxin- β -D- (3-methoxy-1-propylidene) -glucoside
[화학식 12] [ Formula 12 ]
실시예 12Example 12
4'-O-데메틸-에피포도필로톡신-β-D-(시클로프로필메틸리덴)-글루코시드(화합물 12)의 합성Synthesis of 4′- O -demethyl-epipodophyllotoxin- β -D- (cyclopropylmethylidene) -glucoside (Compound 12)
[화학식 13] [ Formula 13 ]
4'-O-데메틸-에피포도필로톡신-β-D-글루코시드(100 mg, 0.18 mmol)을 질소기류하에서 무수 아세토니트릴 (10 mL)에 녹이고p-톨루엔술폰산(10 mg)과 시클로프로판카르복스알데히드(0.12 mL)를 넣고 실온에서 24 시간 교반하였다. TLC로 반응 완결을 확인하고 트리에틸아민으로 반응을 중단시켰다. 아세토니트릴을 감압증류한 후 생성된 고체를 클로로포름(20 mL)에 녹여 염화나트륨의 포화수용액(10 mL)로 씻어주고 유기층을 증류수(10 mL x 2 회)로 다시 씻어준 후 무수 MgSO4로 건조시키고 여과하였다. 여액을 감압증류하고 관 크로마토그라피(클로로포름:메탄올 = 40:1)로 1차, 관 크로마토그라피(헥산:에틸아세테이트=1:1)로 2차 분리하여 표제 화합물을 얻었다.4'- O -demethyl-epipodophyllotoxin- β -D-glucoside (100 mg, 0.18 mmol) was dissolved in anhydrous acetonitrile (10 mL) under nitrogen stream and p- toluenesulfonic acid (10 mg) and cyclopropane Carboxaldehyde (0.12 mL) was added and stirred at room temperature for 24 hours. The reaction was complete by TLC and the reaction was stopped with triethylamine. After distilling acetonitrile under reduced pressure, the resulting solid was dissolved in chloroform (20 mL), washed with a saturated aqueous solution of sodium chloride (10 mL), the organic layer was washed again with distilled water (10 mL x 2 times), and dried over anhydrous MgSO 4 . Filtered. The filtrate was distilled under reduced pressure and separated first by column chromatography (chloroform: methanol = 40: 1) and second by column chromatography (hexane: ethyl acetate = 1: 1) to obtain the title compound.
수율 : 72 %Yield: 72%
mp. 229 - 232 ℃mp. 229-232 ℃
MS, m/z [M]+= 614MS, m / z [M] + = 614
IR(KBr, cm-1) 3448, 2884, 1769, 1609, 1508, 1482.IR (KBr, cm −1 ) 3448, 2884, 1769, 1609, 1508, 1482.
합성된 대표적인 일반식 (I)의 화합물을 표 1 에 요약하였다.Representative compounds of formula (I) synthesized are summarized in Table 1.
[표 1] TABLE 1
시험예 2 : 급성 독성시험Test Example 2: Acute Toxicity Test
5 주령의 특정병원균 부재(specific pathogen free) ICR 마우스로서 체중28.0-31.5g 의 숫컷 12마리를 온도 23±3 ℃, 습도 50±10 %, 조도 150-300 Lux.(12시간, 오전 7시-오후 7시)의 동물실 내에서 사육하였다. 마우스는 실험에 사용되기 전 7일 정도 순화시켰다. 실험동물용 고형사료(공급처: 제일사료 주식회사) 및 음료수를 멸균한 후 공급하였으며 자유 섭취시켰다.Twelve males weighing 28.0-31.5 g body weight were 23 ± 3 ° C., 50 ± 10% humidity, and 150-300 Lux. (12 hours, 7:00 am–) as 5 week-old specific pathogen free ICR mice. It was raised in an animal room of 7 pm). Mice were allowed to acclimate for 7 days before being used for the experiment. Solid feed for experimental animals (source: Cheil Feed Co., Ltd.) and beverages were sterilized and supplied freely.
본 발명의 화합물 번호 1을 멸균 생리식염수에 녹여 마우스의 체중 당 50 mg/kg, 100 mg/kg, 150 mg/kg의 투여량으로 각각 10 ml/kg의 투여액량으로 복강투여 하였다. 시료는 1회 복강 투여하였으며 투여 후 14일 동안 다음과 같이 부작용 또는 치사여부를 관찰하였다. 즉, 투여당일은 투여 후 1시간에서 6시간까지 매시간마다 그리고 투여익일부터 14일째까지는 매일 1회 이상씩 일반 증상의 변화 및 사망동물의 유무를 관찰하였다. 또한, 투여 14일째에 동물을 치사시켜 해부한 후 육안으로 내부장기를 검사하였다. 또한, 투여 당일부터 1일 간격으로 체중의 변화를 측정하여 각 동물의 체중감소현상을 관찰하였다.Compound No. 1 of the present invention was dissolved in sterile saline solution and administered intraperitoneally at a dose of 10 ml / kg at doses of 50 mg / kg, 100 mg / kg, and 150 mg / kg, respectively, per mouse body weight. Samples were administered intraperitoneally once and observed for side effects or lethality for 14 days after administration. That is, on the day of administration, changes in general symptoms and the presence or absence of dead animals were observed every hour from 1 hour to 6 hours after administration and at least once daily from the day following administration. In addition, at 14 days after administration, the animals were killed and dissected, and the internal organs were visually examined. In addition, the weight loss was measured at intervals of 1 day from the day of administration, and the weight loss phenomenon of each animal was observed.
본 발명의 화합물 번호 1의 은 LD50값은 50.88 mg/kg으로 계산되었다.The silver LD 50 value of compound No. 1 of the present invention was calculated to be 50.88 mg / kg.
시험예 2 : 생체외 항암활성Test Example 2: In vitro anticancer activity
단계 1 : 세포 배양Step 1: Cell Culture
항암활성 측정에 사용한 세포들은 A-549(비소형 세포 폐암), SK-OV-3(선암, 난소 악성 복수증), SK-MEL-2(악성흑색종, 대퇴부 피부로의 전이), XF498(중추신경계 종양) 및 HCT15(결장선암)이며, 이들 암세포는 모두 인간 유래의 종양세포주들로서 미국의 국립암연구소(NCI)로부터 분양받아 한국화학연구소에서 계대 배양중인것을 사용하였다. 이 세포들은 모두 배양액으로서 5 % 소의 태아 혈청으로 보강된 RPMI 1640 배양액을 사용하여, 37 ℃ 항온 항습 5 % CO2, 인큐베이터에서 배양하였다. 세포의 계대는 3-4일에 1회씩 하였으며, 세포를 부착면으로부터 분리하기위하여 PBS(인산염 완충 식염수) 용액에 0.25 % 트립신과 3 mM 트란스-1,2-디아미노사이클로헥산-N,N,N,N-테트라아세트산(CDTA)을 용해시킨 용액을 사용하였다.The cells used to measure anticancer activity were A-549 (non-small cell lung cancer), SK-OV-3 (adenocarcinoma, ovarian malignant asymptomatic), SK-MEL-2 (malignant melanoma, metastasis to the femoral skin), XF498 ( Central nervous system tumor) and HCT15 (colon adenocarcinoma), all of these cancer cells were human-derived tumor cell lines obtained from the National Cancer Institute (NCI) of the United States and used in subculture. All of these cells were cultured in a 37 ° C. constant temperature and 5% CO 2 , incubator using RPMI 1640 culture supplemented with 5% fetal bovine serum as culture. Cells were passaged once every 3-4 days, and 0.25% trypsin and 3 mM trans-1,2-diaminocyclohexane- N, N, in PBS (phosphate buffered saline) solution to separate the cells from the adherent surface . A solution in which N, N- tetraacetic acid (CDTA) was dissolved was used.
단계 2 : 생체외 항암 활성Step 2: in vitro anticancer activity
1989년 미국의 국립암연구소에서 약물의 생체외 항암활성을 측정하기위하여 개발된 SRB분석법(sulforhodamine B assay method)을 사용하였다. 즉, 계대중인 세포들을 실험에 사용하기 위하여 트립신-CDTA 용액을 이용하여 세포들을 착면으로부터 분리시키고, 플레이트(96 well microplate, Falcon사 제품)에 웰(well)당 세포수가 5 x 103(A549, HCT15), 1 x 104(SK-MEL-2, XF498), 2 x 104(SK-OV-3)이 되도록 분주하였다. 분주된 세포들은 CO2인큐베이터내에서 24 시간 배양하여 바닥에 부착시킨 후, 아스피레이터로 배양액을 제거하고, 6 농도의 로그 투여량(log dose)으로 배양액에 희석된 화합물(화합물 번호 1 내지 12) 용액들을 세포가 들어 있는 웰에 각각 100 ml 씩 3배수로 넣어주고, 48 시간동안 더 배양하였다. 화합물을 용해시키기 위하여 필요에 따라 디메틸설폭사이드(DMSO)를 사용하였다. 또한 이렇게 희석한 화합물 용액은 세포에 가하기 전에 0.22 ml 필터로 여과하여 실험의 무균 상태를 유지하였다.In 1989, the US National Cancer Institute used the sulforhodamine B assay method developed to measure the in vitro anticancer activity of drugs. That is, cells were passaged from the implant using trypsin-CDTA solution to use passaged cells for experiments, and the number of cells per well in a plate (96 well microplate, manufactured by Falcon) was 5 x 10 3 (A549, HCT15), 1 × 10 4 (SK-MEL-2, XF498), 2 × 10 4 (SK-OV-3). The aliquoted cells were incubated in a CO 2 incubator for 24 hours and attached to the bottom, followed by removal of the culture solution with an aspirator, and a compound diluted in the culture solution at a log dose of 6 concentrations (Compound Nos. 1 to 12). ) The solutions were added in triplicates of 100 ml each to the wells containing cells, and further incubated for 48 hours. Dimethyl sulfoxide (DMSO) was used as needed to dissolve the compound. In addition, the diluted compound solution was filtered through a 0.22 ml filter prior to addition to the cells to maintain the sterility of the experiment.
세포를 약물과 48시간 배양한 후 각 웰의 배양액을 제거하고 10 % 트리클로로아세트산(TCA)를 100 ml씩 가하여 4 ℃에서 1 시간 동안 방치하여 세포들을 플레이트의 바닥면에 고정시켰다. 세포의 고정이 끝난 후 플레이트를 물로 5-6회 세척하여 남아있는 TCA용액을 완전히 제거하고, 남은 물기가 없도록 실온에서 건조시켰다. 완전히 건조된 플레이트는 웰당 100 ml의 1 % 아세트산 용액에 0.4 % SRB를 용해시킨 염색용액을 가하여 30 분간 세포를 염색하고, 다시 1% 아세트산 용액으로 5-6회 세척하여 세포에 결합하지 않은 SRB를 제거하였다. 이렇게 염색된 셀 플레이트(cell plate)들은 다시 실온에서 건조시킨 후 웰당 100 ml의 10 mM 완충되지 않은 트리스마 염기 용액(unbuffered trisma base solution)을 가하여 진탕기(titer plate shaker)로 10 분간 흔들어 염색약을 용출시킨후 마이크로 플레이트 리더(microplate reader)를 사용하여 520 nm에서 흡광도를 측정하였다.After incubating the cells with the drug for 48 hours, the culture solution of each well was removed, and 100 ml of 10% trichloroacetic acid (TCA) was added thereto and left at 4 ° C. for 1 hour to fix the cells on the bottom of the plate. After fixation of the cells, the plate was washed 5-6 times with water to completely remove the remaining TCA solution, and dried at room temperature so that no remaining water was left. Completely dried plates were stained with 30 ml of 1% acetic acid solution per well in 0.4% SRB and stained cells for 30 minutes, and again washed 5-6 times with 1% acetic acid solution to remove SRB that did not bind to cells. Removed. The cell plates thus stained are dried at room temperature and then added with 100 ml of 10 mM unbuffered trisma base solution per well and shaken for 10 minutes with a titer plate shaker. After elution, the absorbance was measured at 520 nm using a microplate reader.
암세포들에 대한 약물의 효과를 계산하기 위하여 약물을 가할때의 세포수(Tz)와, 약물이 들어있지 않은 배양액을 가하여 48시간 배양하였을때의 세포수(C) 및 각 농도의 약물과 함께 48시간 배양했을때의 세포수(T) 등을 측정하여 다음의 수식에 의하여 계산하였다:To calculate the effect of the drug on cancer cells, the cell number (Tz) at the time of drug addition, the cell number (C) at the time of incubation for 48 hours with the drug-free medium, and the drug at each concentration 48 The cell number (T) and the like at time incubation were measured and calculated by the following formula:
[수학식 1] [ Equation 1 ]
[수학식 2] [ Equation 2 ]
이렇게 계산된 값들로부터 로터스 프로그램(LOTUS program)의 데이터회귀(data regression)를 이용하여 약물이 암세포의 성장을 50 % 억제하는 농도 즉, ED50를 계산하여 각 약물의 항암활성도를 비교하였다.Using the data regression of the LOTUS program from the calculated values, the concentrations at which the drug inhibits the growth of cancer cells by 50%, that is, ED 50 was calculated to compare the anticancer activity of each drug.
이상과 같은 시험관내의 활성측정 결과의 일부를 세계적으로 사용하고 있는 항암물질인 에토포사이드(Etoposide, 미국 BMS사 제품)와 비교하였다.Some of the above in vitro activity measurement results were compared with etoposide (BMS, USA), an anticancer substance used worldwide.
시험예 3: 인간 유전자 위상이성질화 II 효소 저해능 측정Test Example 3: Determination of human gene phase isomerization II enzyme inhibition
단계 1 : 유전자 위상이성질화 II 효소 조제Stage 1: Gene Phase Isozyme II Enzyme Preparation
본 실험에 사용한 세포주는 ATCC(American Type Culture Collection)로부터 구입한 HeLa 셀을 사용하였다. 즉 HeLa 셀을 10% 소 태아 혈청이 포함된 RPMI 1640 배지에서 배양한 다음 HeLa 셀을 모아 3배 용량의 1 mM의 에틸렌디아민 사초산(EDTA), 1 mM 머캅토에탄올, 0.5 mM 페닐메틸술포닐클로라이드(PMSF) 및 10 % 글리세롤을 포함하는 50 mM KH2PO4(pH 7.0) 완충액을 가하여 빙욕중에서 펄리트론으로 30초간 균질화하고 여기에 2 M KH2(PO4)(pH 7.0) 완충액을 가하여 최종농도가 0.3 M이 되게 한 후 다시 30초간 균질화하여 1시간 동안 빙욕중에 방치한 다음 30,000 x g에서 30 분간 원심분리하여 얻은 상청액을 포스포셀룰로스 크로마토그라피 등의 방법으로 부분정제하여 사용한다.The cell lines used in this experiment were HeLa cells purchased from the American Type Culture Collection (ATCC). That is, HeLa cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, and then HeLa cells were collected to obtain a three-fold volume of 1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM mercaptoethanol, and 0.5 mM phenylmethylsulfonyl. 50 mM KH 2 PO 4 (pH 7.0) buffer containing chloride (PMSF) and 10% glycerol was added to homogenize with Peritron for 30 seconds in an ice bath and 2 M KH 2 (PO 4 ) (pH 7.0) buffer was added thereto. The final concentration is 0.3 M, homogenized again for 30 seconds, left in an ice bath for 1 hour, and the supernatant obtained by centrifugation at 30,000 xg for 30 minutes is partially purified by a method such as phosphocellulose chromatography.
단계 2 : P4 언노팅(unknotting) 분석Step 2: P4 unknotting analysis
합성한 Topo II억제제의 IC50결정을 위해 언노팅 분석을 행한다. 즉, 50 mM N-[2-히드록시에틸]피페라진-N'-[2-에탄술폰산](HEPES)(pH 7.0), 50 mM KCl, 0.1mM EDTA, 100 mM NaCl, 10 mM MgCl2, 100 ㎍/ml 소 태아 혈청 알부민, 1 mM 아데노신 트리포스페이트(ATP), 0.4 ㎍ P4노팅된(knotted) DNA와 효소를 가하여 총반응액 량이 20 ㎕가 되게 한 후 37 ℃에서 30 분간 반응시킨다. 반응은 최종농도가 1 % 되게 나트륨 도데실설페이트(SDS)를 가하여 정지시키고 50 mM 트리스 붕산염 (pH 8.3), 2.5 mM EDTA용액으로 평형화된 1 % 아가로스 겔에 가하여 전기영동한 후 0.5 ㎍/ml의 에티듐 브로마이드 용액에서 염색, 자외선하에서 사진을 찍어 활성을 측정한다. 효소활성은 0.4 ㎍의 노팅된 P4DNA의 50 %가 언노팅된 것을 1 유니트로 한다. 이상과 같은 방법으로 결정한 IC50를 기존의 Topo II 억제제인 에토포사이드와 비교하였다.Unnotification analysis is performed to determine the IC 50 of the synthesized Topo II inhibitor. Namely, 50 mM N- [2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid] (HEPES) (pH 7.0), 50 mM KCl, 0.1 mM EDTA, 100 mM NaCl, 10 mM MgCl 2 , 100 μg / ml fetal bovine serum albumin, 1 mM adenosine triphosphate (ATP), 0.4 μg P 4 knotted DNA and enzyme are added to make the total reaction solution 20 μl and reacted at 37 ° C. for 30 minutes. The reaction was stopped by addition of sodium dodecyl sulfate (SDS) to a final concentration of 1%, electrophoresis on 1% agarose gel equilibrated with 50 mM tris borate (pH 8.3), 2.5 mM EDTA solution, and 0.5 ㎍ / ml Dye in ethidium bromide solution, take photos under ultraviolet light and measure the activity. Enzymatic activity is 1 unit in which 50% of 0.4 µg of notched P 4 DNA is unnoted. IC 50 determined by the above method was compared with etoposide, a conventional Topo II inhibitor.
상기 시험예 1 및 2 에 따른 결과를 나타내었다.Results according to Test Examples 1 and 2 are shown.
[표 2] TABLE 2
상기 표 2에서 보듯이 본 발명의 일반식(I) 화합물들은 시험한 5가지 암세포 주에서 비교화합물 에토포사이드와 비슷하거나 우수한 암세포독성을 나타내었다. 특히, 화합물 1의 경우 250배 내지 3배 더 강한 암세포독성을 나타내고 있으며 또한, 인간 유전자 이성질화 II 효소에 대해서도 화합물 번호 1은 5배 강한 저해활성을 나타내고 있다. 화합물 3, 5, 6, 7, 9, 10, 11, 12번의 경우에도 에토포사이드보다 낮은 농도에서 이 효소를 저해하고 있음을 알 수 있다.As shown in Table 2, the general formula (I) compounds of the present invention showed similar or superior cancer cytotoxicity to the comparative compound etoposide in the five cancer cell lines tested. In particular, Compound 1 shows 250-fold to 3 times stronger cancer cytotoxicity, and Compound No. 1 shows 5-fold stronger inhibitory activity against human gene isomerization II enzyme. Compounds 3, 5, 6, 7, 9, 10, 11 and 12 also inhibited this enzyme at lower concentrations than etoposide.
시험예 4: 생체내 항암활성 측정Test Example 4: In vivo anticancer activity measurement
6주령 BDF1 생쥐 8마리를 한 그룹으로 하여 DBA/2 mouse에서 계대중인 류케미아 L1210 세포를 각각의 생쥐에 1 × 105셀/0.1 ml 씩 복강내로 이식하였다.A group of eight 6-week-old BDF1 mice were implanted intraperitoneally with 1 × 10 5 cells / 0.1 ml of leuchemia L1210 cells passaged in DBA / 2 mice.
시험약물은 1, 2, 3, 4, 5 일에 각각의 농도로서 복강내로 투여하였다.Test drugs were administered intraperitoneally at respective concentrations at 1, 2, 3, 4 and 5 days.
생쥐를 매일 관찰하면서 생존시간을 측정하고 각 실험군의 평균 생존일으로부터 대조군에 대한 투여군의 평균 생존일의 증가된 비율(T/C %)을 계산하여 항암 효과를 판정하였다. 그 결과를 하기 표 3 에 나타내었다.The survival time was measured while the mice were observed daily, and the anticancer effect was determined by calculating the increased ratio (T / C%) of the average survival days of the administration group to the control group from the average survival days of each experimental group. The results are shown in Table 3 below.
[표 3] TABLE 3
생체내 항암활성 시험 결과, 표 3에서 알 수 있듯이, 기준화합물 에토포사이드의 경우 최고 활성농도(20 mg/kg)에서 60일 이상 생존한 쥐가 5마리(T/C 〉508.0%)였으나 화합물 1의 경우 이보다 2배 낮은 투여농도(10 mg/kg)에서도 시험한 8마리의 쥐가 모두 생존(T/C 〉657.2 %)하였다. 또한 에토포사이드와 같은 농도(20 mg/kg)로 시험하였을때에도 화합물 1은 시험한 8마리의 쥐가 또한 모두 생존(T/C 〉657.2 %)하였다. 이와같은 결과를 살펴볼 때 본 일반식 (I)의 화합물은 그 항암 효과가 비교화합물보다 매우 우수함을 알 수 있었다.As a result of in vivo anticancer activity test, as shown in Table 3, 5 rats (T / C> 508.0%) survived more than 60 days at the highest active concentration (20 mg / kg) for the reference compound etoposide. All mice survived (T / C> 657.2%) even at 2 times lower dose (10 mg / kg). In addition, when tested at the same concentration (20 mg / kg) as etoposide, Compound 1 also survived (T / C> 657.2%) in all eight rats tested. Looking at the results, it was found that the compound of the general formula (I) showed much better anticancer effect than the comparative compound.
본 발명의 일반식 (I)의 4'-O-데메틸-에피포도필로톡신-β-D-글루코시드 유도체는 인간 유전자 이성질화 II 효소에 대한 활성 및 암세포독성에서 기준화합물 에토포사이드와 비슷하거나 우수한 활성을 갖고 있으며 매우 우수한 생체내 항암효과를 나타낸다.4'- O -demethyl-epipodophyllotoxin- β -D-glucoside derivatives of the general formula (I) of the present invention are similar to the reference compound etoposide in activity against human gene isomerization II enzyme and in cancer cytotoxicity. It has excellent activity and shows very good anticancer effect in vivo.
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