KR100418183B1 - 4-o-[2-(n,n-dialkylamino)-2-deoxy-4,6-o,o-(alkenylidene- or alkynylidene)-beta-d-glucosyl]-4'-o-demethyl-epi-podophyllotoxins, preparation thereof and antitumor composition containing same - Google Patents

Abstract

본 발명은 암세포의 성장 억제 효과가 우수하여 항암제로 사용 가능한 화학식 1의 새로운 4-O-[2-(N,N-디알킬아미노)-2-데옥시-4,6-O,O-(알케닐리덴 또는 알키닐리덴)-β-D-글루코실]-4'-O-데메틸-에피-포도필로톡신 화합물 또는 그의 약제학적으로 허용 가능한 염, 이의 제조방법 및 이를 포함하는 항암제 조성물에 관한 것이다.The present invention is a novel 4- O- [2- ( N, N -dialkylamino) -2-deoxy-4,6- O, O- ( Alkenylidene or alkynylidene) -β-D-glucosyl] -4'- 0 -demethyl-epi-podophyllotoxin compound or a pharmaceutically acceptable salt thereof, a method for preparing the same, and an anticancer composition comprising the same It is about.

(상기식에서, R¹, R²및 R³은 명세서 중에서 정의한 바와 같다.)(Wherein R ¹ , R ² and R ³ are as defined in the specification).

Description

4-Ｏ-[2-(Ｎ,Ｎ-디알킬아미노)-2-데옥시-4,6-Ｏ,Ｏ-(알케닐리덴 또는 알키닐리덴)-β-Ｄ-글루코실]-4'-Ｏ-데메틸-에피-포도필로톡신 화합물, 이의 제조방법 및 이를 포함하는 항암제 조성물{4-O-[2-(N,N-DIALKYLAMINO)-2-DEOXY-4,6-O,O-(ALKENYLIDENE- OR ALKYNYLIDENE)-BETA-D-GLUCOSYL]-4'-O-DEMETHYL-EPI-PODOPHYLLOTOXINS, PREPARATION THEREOF AND ANTITUMOR COMPOSITION CONTAINING SAME}4-O- [2- (N, N-dialkylamino) -2-deoxy-4,6-O, O- (alkenylidene or alkynylidene) -β-D-glucosyl] -4 ' -O-demethyl-epi-podophyllotoxin compound, preparation method thereof and anticancer composition comprising same {4-O- [2- (N, N-DIALKYLAMINO) -2-DEOXY-4,6-O, O- (ALKENYLIDENE- OR ALKYNYLIDENE) -BETA-D-GLUCOSYL] -4'-O-DEMETHYL-EPI-PODOPHYLLOTOXINS, PREPARATION THEREOF AND ANTITUMOR COMPOSITION CONTAINING SAME}

본 발명은 4-O-[2-(N,N-디알킬아미노)-2-데옥시-β-D-글루코실]-4'-O-데메 틸-에피-포도필로톡신{4-O-[2-N,N-(Dialkylamino)-2-deoxy-β-D-glucosyl]-4'-O-demethyl-epi-podophyllotoxin} 화합물의 당 부분인 2-아미노-2-데옥시글루코시드의 4-히드록시기와 6-히드록시기가 불포화기 즉, 이중결합, 삼중결합을 포함하는 새로운 아세탈 구조로 변형된 화합물 및 그의 약제학적으로 허용 가능한 염, 이의 제조방법 및 이를 포함하는 항암제 조성물에 관한 것이다.The present invention relates to 4- O- [2- ( N, N -dialkylamino) -2-deoxy- β -D-glucosyl] -4'- 0 -demethyl-epi-podophyllotoxin {4- O -[2- N, N- (Dialkylamino) -2-deoxy- β- D-glucosyl] -4'- O- demethyl- epi- podophyllotoxin} compound of 2-amino-2-deoxyglucoside The present invention relates to a compound in which a 4-hydroxy group and a 6-hydroxy group are modified with a new acetal structure including an unsaturated group, that is, a double bond and a triple bond, a pharmaceutically acceptable salt thereof, a method for preparing the same, and an anticancer composition comprising the same.

인간 유전자 위상이성질화 II 효소 (Human DNA Topoisomerase II)에 근거한 항암제 개발은 에토포사이드(Etoposide) 및 테니포사이드(Teniposide)를 시작으로많이 연구되고 있다. 에토포사이드 및 테니포사이드는 1960년대 산도즈사(Sandoz Limited)에서 천연물, 포도필룸 펠타툼 L.(Podophyllum peltatum L., 메이 애플(may apple), 맨드레이크(mandrake))로부터 분리, 정제하여 반합성적으로 개발된 후 브리스톨-마이어스-스큅(Bristol-Myers Squibb)사에서 특허권을 인수하여 1980년대 FDA로부터 인증을 받은 화합물로서 현재 방사선 치료시 병용투여함으로써 폐암, 악성림프종, 백혈병, 고환암 등에 우수한 항암효과를 나타내고 있는 항암제이다(문헌[C. Keller-Julsen, M. Kuhn, A. von Wartburg,J. Med. Chem., 1971,14(10), 936-940; 및 C. Keller-Julsen, M. Kuhn, J. Renz, A. von Wartburg, 미국특허 제 3,524,844 호(1970. 8. 18)] 참조).The development of anticancer drugs based on the human DNA Topoisomerase II has been studied a lot, starting with Etoposide and Teniposide. Etoposides and teniposides were semisynthetically developed and isolated from natural products, Podophyllum peltatum L., may apple, and mandrake at Sandoz Limited in the 1960s. After its patent, Bristol-Myers Squibb acquired a patent right and was approved by the FDA in the 1980s and is currently used in combination with radiation therapy, showing excellent anticancer effects in lung cancer, malignant lymphoma, leukemia, and testicular cancer. Anticancer agents (C. Keller-Julsen, M. Kuhn, A. von Wartburg, J. Med. Chem., 1971 , 14 (10) , 936-940; and C. Keller-Julsen, M. Kuhn, J Renz, A. von Wartburg, US Pat. No. 3,524,844 (August 18, 1970).

이 화합물은 그 작용기전이 인간 유전자 위상이성질화 II 효소 (Human DNA Topoisomerase II)의 억제에 의한 유전자 합성을 저해하므로서 암세포에 대한 효과를 나타내고 있다(문헌[J. C. Wang,J. Biol. Chem., 1991,266(11), 6659-62] 참조).This compound has an effect on cancer cells by its mechanism of action inhibiting gene synthesis by inhibition of human DNA Topoisomerase II (JC Wang, J. Biol. Chem., 1991 , 266 (11) , 6659-62).

그러나, 상기 에토포사이드 및 테니포사이드는 물에 불용성이고, 독성이 높다는 단점이 있어, 이를 개선하기 위하여 에토포사이드를 변형시켜 새로운 항암제를 개발하려는 연구가 최근에도 계속 진행되고 있다.However, since the etoposide and teniposide are insoluble in water and have high toxicity, studies to develop new anticancer agents by modifying etoposide to improve them have been continued.

즉, 브리스톨-마이어스-스큅(Bristol-Myers-Squibb Co.) 및 일본의 마이크로바이알 케미스트리 리서치 파운데이션(Microbial Chemistry Research Foundation)에서 각각 에토포포스(Etopophos, 문헌[Mark, G. S. Peter, D. S. John, F. K. GB 2,207,674 A] 참조) 및 NK-611(유럽특허공보 제196,618호(1986.3.26) 참조)을 개발하여, 에토포포스는 1997년 FDA의 인증을 받았으며, NK-611은 현재 항암제 개발을 위한 임상단계에 진입되어 있는 상황이다. NK-611은 에토포사이드의 단점인 낮은 수용성을 개선한 화합물이나 에토포사이드보다 낮은 항암효과 및 독성이 크다는 단점이 있다. 이러한 NK-611의 구조 중에서 글루코시드의 4,6-O,O-아세탈 작용기가 항암효과에 가장 직접적인 영향을 주고 있으나 상기 유럽특허 제196,618호의 실시예 및 명세서에는 단지 저급 알킬기만이 개시되어 있으며 다양한 작용기를 갖는 아세탈기는 포함되어 있지 않다.That is, Etopophos (Mark, GS Peter, DS John, FK GB, respectively) in Bristol-Myers-Squibb Co. and Microbial Chemistry Research Foundation in Japan, respectively. 2,207,674 A] and NK-611 (see European Patent Publication No. 196,618 (1986.3.26)), Etophofos was certified by the FDA in 1997, and NK-611 is currently in the clinical stage for the development of anticancer drugs. The situation is entered. NK-611 is a compound that improves low water solubility, which is a disadvantage of etoposide, but has a lower anticancer effect and greater toxicity than etoposide. Among the structures of NK-611, the 4,6- O, O -acetal functional group of glucoside has the most direct effect on the anticancer effect. However, only the lower alkyl groups are disclosed and described in the examples and specifications of EP 196,618. Acetal groups with functional groups are not included.

본 발명자는 불포화기를 포함하는 새로운 에토포사이드의 아세탈 유도체를 연구한 결과, 우수한 항암 활성 및 개선된 약동력학을 갖고 있는 새로운 에토포사이드 유도체를 기 개발한 바 있다(PCT 국제특허 공개 제WO99/32499호(1999.7.1) 참조). 그러므로 본 발명자는 상기의 연구개발 경험을 NK-611에 적용하여 에토포사이드보다 수용성이 개선됨과 함께, 우수한 항암효과 및 낮은 독성을 갖는 새로운 항암제의 개발에 대한 연구를 하였다.As a result of studying acetal derivatives of etoposide containing unsaturated groups, the present inventors have previously developed new etoposide derivatives having excellent anticancer activity and improved pharmacokinetics (PCT WO 99/32499 ( 1999.7.1). Therefore, the present inventors applied the above research and development experience to NK-611 to improve the water solubility than etoposide, and to study the development of a new anticancer agent having excellent anticancer effect and low toxicity.

따라서, 본 발명의 목적은 4-O-[2-(N,N-디알킬아미노)-2-데옥시-β-D-글루코실]-4'-O-데메틸-에피-포도필로톡신의 당 부분 즉, 아미노글루코스의 4,6-디올에 지금까지 보고되어 있지 않은 이중결합, 삼중결합구조를 포함하는 새로운 아세탈 화합물 및 그의 염, 이를 제조하는 방법 및 이들 화합물을 포함하는 항암제 조성물을 제공하는 것이다.Thus, an object of the present invention is 4- O- [2- ( N, N -dialkylamino) -2-deoxy- β -D-glucosyl] -4'- 0 -demethyl-epi-podophyllotoxin It provides a novel acetal compound and a salt thereof, including a double bond, triple bond structure that has not been reported so far in the sugar portion of the amino glucose, 4,6-diol of aminoglucose, a method for preparing the same and an anticancer composition comprising the compound It is.

상기 목적을 달성하기 위하여, 본 발명에서는 하기 화학식 1의 4-O-[2-(N,N-디알킬아미노)-2-데옥시-4,6-O,O-(알케닐리덴 또는 알키닐리덴)-β-D-글루코실]-4'-O-데메틸-에피-포도필로톡신 화합물 또는 이의 약학적으로 허용 가능한 염을 제공한다.In order to achieve the above object, in the present invention, 4- O- [2- ( N, N -dialkylamino) -2-deoxy-4,6- O, O- (alkenylidene or alkoxy) alkylpiperidinyl) - β -D- glucosyl] -4'- O-de-methyl-epi-podophyllotoxin provides a compound or a pharmaceutically acceptable salt thereof.

화학식 1Formula 1

상기식에서,In the above formula,

R¹및 R²는 각각 서로 독립적으로 수소, C₁-C₁₀알킬 또는 C₅-C₁₀시클로알킬이거나; 또는 상호 연결되어 C₄-C₁₀의 질소원자 함유 고리화합물을 형성하고;R ¹ and R ² are each independently of each other hydrogen, C ₁ -C ₁₀ alkyl or C ₅ -C ₁₀ cycloalkyl; Or are interconnected to form a C ₄ -C ₁₀ nitrogen atom-containing cyclic compound;

R³은 임의로 분쇄상 또는 직쇄상 C₁-C₈알킬, 알콕시, 알콕시알킬, 또는 아릴 치환체를 갖는 비닐, 에티닐, 알릴, C₃-C₁₀시클로알킬, C₅-C₁₀시클로알케닐, 또는 C₅-C₁₀시클로알키닐이다.R ³ is vinyl, ethynyl, allyl, C ₃ -C ₁₀ cycloalkyl, C ₅ -C ₁₀ cycloalkenyl, optionally with pulverized or straight chain C ₁ -C ₈ alkyl, alkoxy, alkoxyalkyl, or aryl substituents, Or C ₅ -C ₁₀ cycloalkynyl.

또한, 본 발명에서는β-D-아미노글루코스의 아미노기가 테트라클로로프탈이미도일기로 보호된 것을 특징으로 하는 ⅰ) 하기 화학식 2의 4'-O-데메틸-에피-포도필로톡신-4-O-[2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]와 하기 화학식 3의 알데히드 또는 화학식 4의 아세탈과 산 촉매하에 반응시켜 하기 화학식 5의 화합물을 얻는 단계; ⅱ) 화학식 5의 아미노 보호기를 탈보호시켜 하기 화학식 6의 화합물을 얻는 단계; ⅲ) 화학식 6의 화합물을 환원제의 존재하에 알킬화 반응시키는 단계를 포함하는 화학식 1 화합물의 제조방법을 제공한다.In the present invention, the amino group of β -D-aminoglucose is protected by a tetrachlorophthalimidoyl group. I) 4'- O -demethyl-epi-podophyllotoxin-4- O -[2-amino-2- (tetrachlorophthalimidoyl) -2-deoxy- β -D-glucoside] is reacted with an aldehyde of formula 3 or an acetal of formula 4 under an acid catalyst to Obtaining a compound; Ii) deprotecting the amino protecting group of formula 5 to obtain a compound of formula 6; Iii) a method of preparing a compound of Formula 1 comprising the step of alkylating the compound of Formula 6 in the presence of a reducing agent.

상기식에서, R¹, R²및 R³은 앞에서 정의한 바와 같다.Wherein R ¹ , R ² and R ³ are as defined above.

또한, 본 발명에서는 상기 화학식 1의 4-O-[2-(N,N-디알킬아미노)-2-데옥시-4,6-O,O-(알케닐리덴 또는 알키닐리덴)-β-D-글루코실]-4'-O-데메틸-에피-포도필로톡신 또는 이의 약학적으로 허용 가능한 염을 활성성분으로서 포함하는 항암제 조성물을 제공한다.In the present invention, 4- O of Formula 1 - [2- (N, N - di-alkylamino) -2-deoxy -4,6- O, O - (alkenyl or alkynyl alkylpiperidinyl alkylpiperidinyl) - β An anticancer composition comprising -D-glucosyl] -4'- O -demethyl-epi-podophyllotoxin or a pharmaceutically acceptable salt thereof as an active ingredient is provided.

이하 본 발명에 대하여 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명의 화학식 1의 4-O-[2-(N,N-디알킬아미노)-2-데옥시-4,6-O,O-(알케닐리덴 또는 알키닐리덴)-β-D-글루코실]-4'-O-데메틸-에피-포도필로톡신 화합물은 하기 반응식 1에 나타낸 방법으로 제조할 수 있다.4- O- [2- ( N, N -dialkylamino) -2-deoxy-4,6- O, O- (alkenylidene or alkynylidene) of the formula 1 of the present invention- β -D- Glucosyl] -4'- 0 -demethyl-epi-podophyllotoxin compounds can be prepared by the method shown in Scheme 1 below.

상기식에서, R¹, R²및 R³은 상기 중에서 정의한 바와 같다.Wherein R ¹ , R ² and R ³ are as defined above.

즉, 상기 반응식 1에서 나타낸 바와 같이, 화학식 2의 4'-O-데메틸-에피-포도필로톡신-4-O-[2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드] 화합물과 과량의 화학식 3의 알데히드 또는 화학식 4의 아세탈을 반응에 참여하지 않는 불활성 유기용매 내에서 또는 용매 없이 산을 촉매로 하여 반응식 1의 4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(알케닐리덴 또는 알키닐리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]을 합성한 후, 반응식 1의 두 번째 반응과 같이 아미노 보호기인 테트라클로로프탈로일기를 일반적으로 잘 알려져 있는 히드라진 혹은 에틸렌디아민으로 비 보호하여 화학식 6의 화합물을 얻는다.That is, as shown in the scheme 1, the 4'- O- methyl having the formula 2-epi-podophyllotoxin -4- O - [2- amino-2- (tetrachloride GRAUFTHAL yimido) -2 having Oxy- β -D-glucoside] and an excess of an aldehyde of Formula 3 or an acetal of Formula 4 in the inert organic solvent that does not participate in the reaction or without the solvent, the acid of 4'- O- Methyl-epi-podophyllotoxin-4- O- [4,6- O, O- (alkenylidene or alkynylidene) -2-amino-2- (tetrachlorophthalimidoyl) -2-deoxy [ beta ] -D-glucoside], and then, as in the second reaction of Scheme 1, the amino protecting group tetrachlorophthaloyl group is unprotected with hydrazine or ethylenediamine, which are generally well known, to obtain a compound of formula 6.

이어서, 화학식 6의 화합물을 환원제의 존재하에 상응하는 알데히드와 반응시키는, 공지된 환원 알킬화 반응을 수행하여 본 발명의 화학식 1의 화합물을 얻을 수 있다.Subsequently, a known reduction alkylation reaction may be carried out in which a compound of formula 6 is reacted with a corresponding aldehyde in the presence of a reducing agent to obtain a compound of formula 1 of the present invention.

상기 반응식 1의 첫 단계를 자세히 설명하면 다음과 같다.The first step of Scheme 1 will be described in detail.

화학식 2의 화합물로부터 화학식 5의 화합물의 합성은 용매 없이 또는 용매 존재 하에 수행될 수 있으며, 사용 가능한 반응용매로서는 니트로메탄, 디클로로메탄, 클로로포름, 디에틸에테르, 테트라히드로푸란, 디메톡시에탄, 아세토니트릴, 디메틸포름아미드와 같은 반응에 참여하지 않는 무수 용매 조건이 바람직하나, 무수 아세토니트릴과 무수 니트로메탄이 가장 좋은 수율을 나타내므로 더욱 바람직하다.Synthesis of the compound of formula (5) from the compound of formula (2) can be carried out without solvent or in the presence of a solvent, and usable reaction solvents include nitromethane, dichloromethane, chloroform, diethyl ether, tetrahydrofuran, dimethoxyethane, acetonitrile Anhydrous solvent conditions that do not participate in the reaction, such as dimethylformamide, are preferred, but anhydrous acetonitrile and anhydrous nitromethane are more preferred since they yield the best yields.

화학식 3의 알데히드 및 화학식 4의 아세탈은 목적하는 화합물 제조시에 원하는 치환기를 제공할 수 있는 것으로 적절히 선택하여 사용할 수 있으며, 상업적으로 구입하거나, 공지된 방법에 따라 합성할 수 있다. 화학식 3 또는 4의 화합물은 화학식 2의 화합물에 대하여, 5 내지 50 당량의 과량으로, 바람직하게는 5 내지15 당량의 양으로 사용된다. 또한, 촉매로 사용하는 산으로서는 황산, p-톨루엔설폰산, 메탄설폰산, 염화아연, 산성 레진 (acidic resin) 등의 무기산 및 유기산 등이 모두 사용가능하나, p-톨루엔설폰산, 메탄설폰산이 가장 적합하다. 촉매는 화학식 2의 화합물을 기준으로 1 내지 20 %(몰 기준), 바람직하게는 5 내지 10 %의 양으로 사용된다. 반응온도는 10 내지 100 ℃, 바람직하게는 20 내지 40 ℃이며, 실온에서 3시간 내지 24시간 동안 반응이 수행된다. 반응의 종결 후 유기염기로서 트리알킬아민, 피리딘류 등을 사용하여 촉매로 첨가된 산을 중화하며, 그 양은 촉매로 첨가된 산보다 약간의 과량을 사용하는 것이 바람직하다.The aldehyde of Formula 3 and the acetal of Formula 4 may be appropriately selected and used to provide a desired substituent in preparing the desired compound, and may be purchased commercially or synthesized according to known methods. The compound of formula 3 or 4 is used in an amount of 5 to 50 equivalents, preferably 5 to 15 equivalents, relative to the compound of formula 2. In addition, examples of the acid used as a catalyst include inorganic acids such as sulfuric acid, p-toluenesulfonic acid, methanesulfonic acid, zinc chloride, and acidic resin, and organic acids, but p-toluenesulfonic acid and methanesulfonic acid Most suitable. The catalyst is used in an amount of 1 to 20% (based on moles), preferably 5 to 10%, based on the compound of formula (2). The reaction temperature is 10 to 100 ℃, preferably 20 to 40 ℃, the reaction is carried out for 3 hours to 24 hours at room temperature. After completion of the reaction, trialkylamine, pyridines and the like are used as organic bases to neutralize the acid added with the catalyst, and the amount is preferably used in a slight excess of the acid added with the catalyst.

반응식 1의 첫 단계를 용매 없이 수행하는 경우, 상응하는 화학식 3의 알데히드 또는 화학식 4의 아세탈을 용매 대신 과량으로 사용하여 산 촉매 하에서 반응시키며, 이때 알데히드 또는 아세탈의 양은 화학식 2의 화합물에 대해 부피비율로 10배 내지 50배가 사용되며 바람직하게는 10 내지 20배가 사용된다. 기타의 반응조건 및 반응 방법은 상기의 용매 존재하에서의 반응과 같다.When the first step of Scheme 1 is carried out without solvent, the reaction is carried out under acid catalyst using the corresponding aldehyde of formula 3 or acetal of formula 4 in excess of solvent, wherein the amount of aldehyde or acetal is proportional to the compound of formula 10 to 50 times are used, preferably 10 to 20 times. Other reaction conditions and reaction methods are the same as those in the presence of the solvent.

반응식 1의 두 번째 단계는 화합물 5의 아미노 보호기를N-탈보호시키는 반응으로서,N-보호기인 테트라클로로프탈이미도일 그룹은 알려진 바와 같이, 약간 과량의 유기염기 즉, 히드라진, 에틸렌디아민을 포함하는 1급 알킬아민에 의해 실온에서 탈보호가 가능하다. 실온 또는 실온이하의 온도에서N-탈보호시키는 반응은 반응식 1의 목화합물 6의 합성을 위해 필수적이며, 그 이유는 1급 아민을 40^oC 이상에서N-탈보호시키기 위하여 사용하였을 때 데메틸-에피포도필로톡신의 트랜스-락톤기가 불안정하여 부반응을 나타내기 때문이다.The second step of Scheme 1 is a N -deprotection of the amino protecting group of compound 5, wherein the N -protecting group tetrachlorophthalimidoyl group, as known, contains a slight excess of organic base, ie hydrazine, ethylenediamine Deprotection at room temperature is possible with primary alkylamines. The reaction of N -deprotection at room temperature or below room temperature is essential for the synthesis of wood compound 6 in Scheme 1, because the primary amine is used for N -deprotection above 40 ^° C. This is because the trans-lactone group of epipodophyllotoxin is unstable and shows side reactions.

반응식 1의 세 번째 단계도 역시 일반적으로 잘 알려진 방법으로서, 환원제로서는 리튬 알루미눔하이드라이드, 소듐 보로하이드라이드, 소듐 시아노보로하이드라이드가 4 내지 7 당량의 양으로 사용될 수 있다. 사용되는 알데히드는 최종 화합물에서 R¹과 R²를 제공할 수 있는 것을 적절히 선택하여 사용할 수 있으며, 포름알데히드, 아세트알데히드가 가장 바람직하다.The third step of Scheme 1 is also generally well known, in which lithium aluminum hydride, sodium borohydride, sodium cyanoborohydride can be used in an amount of 4 to 7 equivalents. The aldehydes used may be appropriately selected from those capable of providing R ¹ and R ² in the final compound, and formaldehyde and acetaldehyde are most preferred.

상기에서 제조된 화학식 1의 화합물은 유기산 또는 무기산을 사용하여 약제학적으로 허용 가능한 염으로 전환시킬 수 있으며, 이때 사용 가능한 유기산으로서는 개미산, 아세트산, 옥살산, 말론산, 젖산, 포도산 등이 있고, 무기산으로는 염산, 브롬산, 황산, 요오드산 등이 있다. 상기 반응은 반응에 참여하지 않는 유기용매 즉, 에테르, 클로로포름, 디클로로메탄 또는 수용액내에서 화학식 1의 화합물을 상응하는 산과 반응시켜 수행할 수 있으며, 이때의 온도는 -20 내지 50 ℃, 바람직하게는, 0 내지 20 ℃이다. 반응 후 생성된 고체의 화합물을 여과하거나, 용매를 감압 증류하여 화학식 1의 화합물의 약학적으로 허용 가능한 염을 제조할 수 있다.The compound of Chemical Formula 1 prepared above may be converted into a pharmaceutically acceptable salt using an organic acid or an inorganic acid, and the usable organic acid may include formic acid, acetic acid, oxalic acid, malonic acid, lactic acid, grape acid, and the like. Examples thereof include hydrochloric acid, bromic acid, sulfuric acid, and iodic acid. The reaction may be carried out by reacting the compound of formula 1 with a corresponding acid in an organic solvent that does not participate in the reaction, ie, ether, chloroform, dichloromethane or aqueous solution, wherein the temperature is -20 to 50 ° C, preferably , 0 to 20 ° C. The compound of the solid produced after the reaction may be filtered or the solvent may be distilled under reduced pressure to prepare a pharmaceutically acceptable salt of the compound of Formula 1.

또한, 화학식 2의 화합물은 유럽특허공보 제196,618호 및 문헌[J. S. Debenham, S. D. Debenham, and B. Freiser-Reid,Bioorg. Med. Chem., 1996,4, 1909참조]에 기재된 방법에 따라 제조될 수 있다.Compounds of formula (2) are also described in EP 196,618 and JS Debenham, SD Debenham, and B. Freiser-Reid, Bioorg. Med. Chem., 1996 , 4 , 1909].

이와 같이 제조된 본 발명의 화학식 1의 화합물은 항암효과를 가진다.The compound of Formula 1 of the present invention thus prepared has an anticancer effect.

본 발명에서는 화학식 1의 화합물 또는 이의 약학적으로 허용 가능한 염을 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, the anticancer composition may be prepared by mixing the compound of Formula 1 or a pharmaceutically acceptable salt thereof with an 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 100㎎/㎏체중, 바람직하게는 1 내지 20㎎/㎏ 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 a day, preferably in the range of 1 to 100 mg / kg body weight, preferably 1 to 20 mg / kg body weight. Can be administered dividedly once to three times. 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.The present invention will be described in more detail with reference to the following examples.

단, 본 발명의 범위가 하기 실시예 만으로 한정되는 것은 아니다.However, the scope of the present invention is not limited only to the following Examples.

제조예 1:Preparation Example 1: 4'-4'- O-O- 데메틸-에피-포도필로톡신-4-Demethyl-epi-podophyllotoxin-4- OO -[4,6--[4,6- O,OO, O -(알케닐리덴 또는 알키닐리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시--(Alkenylidene or alkynylidene) -2-amino-2- (tetrachlorophthalimidoyl) -2-deoxy- ββ -D-글루코시드] (화학식 5의 화합물)를 유기용매 내에서 산 촉매하에 합성하는 방법-D-glucoside] (compound of formula 5) is synthesized in an organic solvent under an acid catalyst.

화학식 2의 4'-O-데메틸-에피-포도필로톡신-4-O-[2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드](1 mmol)을 질소 기류하에서 무수 아세토니트릴 (50 mL)에 녹이고 무수p-톨루엔설폰산 (60 mg)과 각각의 상응하는 화학식3의 알데히드 또는 화학식 4의 아세탈 (5 mmol)[아크롤레인 디메틸아세탈(4a), 3-부테날디에틸아세탈(4b), 프로피올알데히드 디에틸아세탈(4c), 2-부텐-1-알데히드 디에틸아세탈(4d), 시클로프로판카복스알데히드(3e), 2-부틴-1-알데히드 디에틸아세탈(4f), 1,2,5,6-테트라하이드로 벤즈알데히드(3g), 트랜스-신남알데히드(3h)]을 넣고 실온에서 교반하였다.4'- of Formula 2O-Demethyl-epi-podophyllotoxin-4-O-[2-amino-2- (tetrachlorophthalimidoyl) -2-deoxy-β-D-glucoside] (1 mmol) was dissolved in anhydrous acetonitrile (50 mL) under a stream of nitrogen. myriadp-Toluenesulfonic acid (60 mg) and the corresponding aldehyde of formula 3 or acetal of formula 4 (5 mmol) [acrolein dimethylacetal (4a), 3-butenaldiethylacetal (4b), propioaldehyde diethylacetal (4c), 2-butene-1-aldehyde diethylacetal (4d), cyclopropanecarboxaldehyde (3e), 2-butyn-1-aldehyde diethylacetal (4f), 1,2,5,6-tetra Hydrobenzaldehyde (3 g) and trans-cinnaaldehyde (3h)] were added and stirred at room temperature.

TLC로 반응 완결을 확인한 후, 첨가된 산에 대하여 약간 과량의 트리에틸아민으로 반응을 종결시켰다. 아세토니트릴을 감압증류한 후 생성된 고체를 클로르포름(200 mL)에 녹여 염화나트륨의 포화수용액(10 mL)으로 씻어주고 유기층을 증류수(30 mL x 2 회)로 다시 씻어준 후, 무수 MgSO₄로 건조시키고 여과하였다. 여액을 감압증류하고 관 크로마토그라피(용출액: 클로르포름:메탄올 = 40:1)로 분리하여 각각의 상응하는 화합물 5a 내지 5h를 얻었다.After completion of the reaction by TLC, the reaction was terminated with a slight excess of triethylamine for the added acid. After distilling acetonitrile under reduced pressure, the resulting solid was dissolved in chloroform (200 mL), washed with a saturated aqueous solution of sodium chloride (10 mL), and the organic layer was washed again with distilled water (30 mL x 2 times), followed by anhydrous MgSO ₄ . Dried and filtered. The filtrate was distilled under reduced pressure and separated by column chromatography (eluent: chloroform: methanol = 40: 1) to give the corresponding compounds 5a to 5h.

화학식 5의 화합물 각각의 물성은 하기와 같다.Physical properties of each compound of Formula 5 are as follows.

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(프로페닐리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]:4'-O-Demethyl-epi-podophyllotoxine-4-O-[4,6-O,O-(propenylidene)-2-amino-2-(tetrachlorophthalimidoyl)-2-deoxy-β-D-glucoside (화합물 5a).4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - ( alkylidene-propenyl) -2-amino-2- (tetrachloride GRAUFTHAL yimido yl) -2- deoxy -β-D- glucoside]: 4'- O -Demethyl- epi -podophyllotoxine- 4- O - [4,6- O, O- (propenylidene) -2-amino-2- (tetrachlorophthalimidoyl) -2 -deoxy- β- D-glucoside (Compound 5a).

수율 = 88 %Yield = 88%

mp 228-229 ℃;mp 228-229 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.46(s, 1H), 6.23(s, 1H), 6.12(s, 2H),5.88(ddd,J=4.6, 10.6, 12.8Hz, 1H) 5.74(s, 2H), 5.44(bd,J=16.3Hz, 1H), 5.35(m, 2H), 5.04(d,J=4.5Hz, 1H), 4.80(m, 1H), 4.68(d,J=2.9Hz, 1H), 4.45(d,J=5.3Hz, 1H), 4.39~4.06(m, 4H), 3.72(s, 6H), 3.68~3.44(m, 3H), 3.24(dd,J=5.3, 14.3Hz, 1H), 2.72(m, 1H), 2.61(br, 1H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.46 (s, 1H), 6.23 (s, 1H), 6.12 (s, 2H), 5.88 (ddd, J = 4.6, 10.6, 12.8Hz, 1H) 5.74 (s , 2H), 5.44 (bd, J = 16.3 Hz, 1H), 5.35 (m, 2H), 5.04 (d, J = 4.5 Hz, 1H), 4.80 (m, 1H), 4.68 (d, J = 2.9 Hz , 1H), 4.45 (d, J = 5.3 Hz, 1H), 4.39-4.06 (m, 4H), 3.72 (s, 6H), 3.68-3.44 (m, 3H), 3.24 (dd, J = 5.3, 14.3 Hz, 1H), 2.72 (m, 1H), 2.61 (br, 1H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(3-부테닐리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]: 4'-O-Demethyl-epi-podophyllotoxine-4-O-[4,6-O,O-(3-butenylidene)-2-amino-2-(tetrachlorophthalimidoyl)-2-deoxy-β-D-glucoside (화합물 5b).4'- O- demethyl-epi-podophyllotoxin-4- O- [4,6- O, O- (3-butenylidene) -2-amino-2- (tetrachlorophthalimidoyl)- 2-deoxy - β -D- glucoside]: 4'- O -Demethyl- epi -podophyllotoxine- 4- O - [4,6- O, O- (3-butenylidene) -2-amino-2- ( tetrachlorophthalimidoyl) -2-deoxy- β- D-glucoside (Compound 5b).

수율 = 76 %Yield = 76%

mp 231-232℃;mp 231-232 ° C;

¹H NMR (200 MHz, CDCl₃) δ6.45(s, 1H), 6.22(s, 1H), 6.12(s, 2H), 5.80(m, 1H), 5.74(s, 2H), 5.38(s, 1H), 5.32(d,J=8.1Hz, 1H), 5.14(m, 2H), 4.72(m, 1H), 4.65(d,J=5.1Hz, 1H), 4.45(d,J=5.7Hz, 1H), 4.36(d,J=9.6Hz, 1H), 4.25~4.04(m, 3H), 3.71(s, 6H), 3.64~3.32(m, 3H), 3.23(dd,J=5.4, 14.2Hz, 1H), 2.78(m, 1H), 2.26(m, 2H). ¹ H NMR (200 MHz, CDCl ₃ ) δ6.45 (s, 1H), 6.22 (s, 1H), 6.12 (s, 2H), 5.80 (m, 1H), 5.74 (s, 2H), 5.38 (s , 1H), 5.32 (d, J = 8.1 Hz, 1H), 5.14 (m, 2H), 4.72 (m, 1H), 4.65 (d, J = 5.1 Hz, 1H), 4.45 (d, J = 5.7 Hz , 1H), 4.36 (d, J = 9.6 Hz, 1H), 4.25-4.04 (m, 3H), 3.71 (s, 6H), 3.64-3.32 (m, 3H), 3.23 (dd, J = 5.4, 14.2 Hz, 1H), 2.78 (m, 1H), 2.26 (m, 2H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(프로피닐리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]: 4'-O-Demethyl-epi-podophyllotoxine-4-O-[4,6-O,O-(propynylidene)-2-amino-2-(tetrachlorophthalimidoyl)-2-deoxy-β-D-glucoside (화합물 5c).4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - ( propionyl alkylpiperidinyl) -2-amino-2- (tetrachloride GRAUFTHAL yimido yl) -2- deoxy - β -D- glucoside]: 4'- O -Demethyl- epi -podophyllotoxine- 4- O - [4,6- O, O- (propynylidene) -2-amino-2- (tetrachlorophthalimidoyl) -2 -deoxy- β- D-glucoside (Compound 5c).

수율 = 46 %Yield = 46%

mp 205-206 ℃;mp 205-206 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.50(s, 1H), 6.29(s, 1H), 6.15(s, 2H), 5.78(s, 1H) 5.71(s, 1H), 5.39(m, 3H), 4.73(d,J=3.1Hz, 1H), 4.52(m, 1H), 4.46(d,J=5.1, 2H), 4.24(m, 1H), 4.08(m, 2H), 3.93~3.76(m, 2H), 3.72(s, 6H), 3.64(m, 2H), 3.19(dd,J=5.4, 14.0Hz, 1H), 2.80(m, 1H), 2.64(q,J=1.6Hz, 1H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.50 (s, 1H), 6.29 (s, 1H), 6.15 (s, 2H), 5.78 (s, 1H) 5.71 (s, 1H), 5.39 (m, 3H ), 4.73 (d, J = 3.1 Hz, 1H), 4.52 (m, 1H), 4.46 (d, J = 5.1, 2H), 4.24 (m, 1H), 4.08 (m, 2H), 3.93-3.76 ( m, 2H), 3.72 (s, 6H), 3.64 (m, 2H), 3.19 (dd, J = 5.4, 14.0 Hz, 1H), 2.80 (m, 1H), 2.64 (q, J = 1.6 Hz, 1H ).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(2-부테닐리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]:4'-O-Demethyl-epi-podophyllotoxine-4-O-[4,6-O,O-(2-butenylidene)-2-amino-2-(tetrachlorophthalimidoyl)-2-deoxy-β-D-glucoside (화합물 5d).4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - (2- butenyl alkylpiperidinyl) -2-amino-2- (tetrachloride GRAUFTHAL yimido yl) - 2-deoxy - β -D- glucoside]: 4'- O -Demethyl- epi -podophyllotoxine- 4- O - [4,6- O, O- (2-butenylidene) -2-amino-2- ( tetrachlorophthalimidoyl) -2-deoxy- β- D-glucoside (Compound 5d).

수율 = 82 %Yield = 82%

mp 212-213 ℃;mp 212-213 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.43(s, 1H), 6.23(s, 1H), 6.13(s, 2H), 6.00(dd,J=6.7, 15.5Hz, 1H), 5.72(s, 2H), 5.54(m, 1H), 5.35(d,J=8.3Hz, 1H), 5.02(d,J=5.5Hz, 1H), 4.79(m, 1H), 4.67(d,J=3.1Hz, 2H), 4.46(d,J=5.7Hz, 2H), 4.37(bd,J=10.6Hz, 1H), 4.26~4.07(m, 2H), 3.72(s, 6H), 3.55(m, 2H), 3.24(dd,J=5.4, 14.3Hz, 1H), 2.78(m, 1H), 1.76(d,J=6.7Hz, 3H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.43 (s, 1H), 6.23 (s, 1H), 6.13 (s, 2H), 6.00 (dd, J = 6.7, 15.5Hz, 1H), 5.72 (s, 2H), 5.54 (m, 1H), 5.35 (d, J = 8.3 Hz, 1H), 5.02 (d, J = 5.5 Hz, 1H), 4.79 (m, 1H), 4.67 (d, J = 3.1 Hz, 2H), 4.46 (d, J = 5.7 Hz, 2H), 4.37 (bd, J = 10.6 Hz, 1H), 4.26-4.07 (m, 2H), 3.72 (s, 6H), 3.55 (m, 2H), 3.24 (dd, J = 5.4, 14.3 Hz, 1H), 2.78 (m, 1H), 1.76 (d, J = 6.7 Hz, 3H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(시클로프로필메틸리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]: 4'-O-Demethyl-epi-podophyllotoxine-4-O-[4,6-O,O-(cyclopropylmethylidene)-2-amino-2-(tetrachlorophthalimidoyl)-2-deoxy-β-D-glucoside (화합물 5e).4'- O- to methyl-epi-podophyllotoxin -4- O - [O 4,6-, O - (cyclopropyl-methylidene) -2-amino-2- (tetrachloride GRAUFTHAL yimido days) -2 - deoxy - β -D- glucoside]: 4'- O -Demethyl- epi -podophyllotoxine- 4- O - [4,6- O, O- (cyclopropylmethylidene) -2-amino-2- (tetrachlorophthalimidoyl) - 2-deoxy- β- D-glucoside (Compound 5e).

수율 = 67 %Yield = 67%

mp 199-200 ℃;mp 199-200 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.46(s, 1H), 6.22(s, 1H), 6.12(s, 2H), 5.74(s, 2H), 5.38(d,J=8.3Hz, 1H), 5.33(d,J=8.4Hz, 1H), 4.78(m, 1H), 4.67(d,J=2.9Hz, 1H), 4.45(d,J=5.3Hz, 1H), 4.39~4.04(m, 4H), 3.71(s, 6H), 3.62~3.39(m, 3H), 3.23(dd,J=5.2, 14.2Hz, 2H), 2.84(m, 1H), 1.18(m, 1H), 0.55(m, 4H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.46 (s, 1H), 6.22 (s, 1H), 6.12 (s, 2H), 5.74 (s, 2H), 5.38 (d, J = 8.3Hz, 1H) , 5.33 (d, J = 8.4 Hz, 1H), 4.78 (m, 1H), 4.67 (d, J = 2.9 Hz, 1H), 4.45 (d, J = 5.3 Hz, 1H), 4.39-4.04 (m, 4H), 3.71 (s, 6H), 3.62-3.39 (m, 3H), 3.23 (dd, J = 5.2, 14.2 Hz, 2H), 2.84 (m, 1H), 1.18 (m, 1H), 0.55 (m , 4H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(2-부티닐리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]: 4'-O-Demethyl-epi-podophyllotoxine-4-O-[4,6-O,O-(2-butynylidene)-2-amino-2-(tetrachlorophthalimidoyl)-2-deoxy-β-D-glucoside (화합물 5f).4'- O- demethyl-epi-podophyllotoxin-4- O- [4,6- O, O- (2-butynylidene) -2-amino-2- (tetrachlorophthalimidoyl)- 2-deoxy - β -D- glucoside]: 4'- O -Demethyl- epi -podophyllotoxine- 4- O - [4,6- O, O- (2-butynylidene) -2-amino-2- ( tetrachlorophthalimidoyl) -2-deoxy- β- D-glucoside (Compound 5f).

수율 = 54 %Yield = 54%

mp 205-206 ℃;mp 205-206 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.48(s, 1H), 6.24(s, 1H), 6.14(s, 2H), 5.75(s, 2H), 5.39(s, 1H), 5.31(bd,J=10.2Hz, 2H), 4.78(m, 1H), 4.68(d,J=2.9Hz, 1H), 4.47(d,J=5.3Hz, 1H), 4.39~3.95(m, 5H), 3.73(s, 6H), 3.56(m, 2H), 3.25(m, 1H), 2.82(m, 1H), 1.94(d,J=13.2Hz, 3H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.48 (s, 1H), 6.24 (s, 1H), 6.14 (s, 2H), 5.75 (s, 2H), 5.39 (s, 1H), 5.31 (bd, J = 10.2 Hz, 2H), 4.78 (m, 1H), 4.68 (d, J = 2.9 Hz, 1H), 4.47 (d, J = 5.3 Hz, 1H), 4.39-3.95 (m, 5H), 3.73 ( s, 6H), 3.56 (m, 2H), 3.25 (m, 1H), 2.82 (m, 1H), 1.94 (d, J = 13.2 Hz, 3H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(3-시클로헥센-1-메틸리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]:4'-O-Demethyl-epi-podophyllotoxine-4-O-[4,6-O,O-(3-cyclohexen-1-methylidene)-2-amino-2-(tetrachlorophthalimidoyl)-2-deoxy-β-D-glucoside (화합물 5g).4'-O-Demethyl-epi-podophyllotoxin-4-O-[4,6-O, O-(3-cyclohexene-1-methylidene) -2-amino-2- (tetrachlorophthalimidoyl) -2-deoxy-β-D-glucoside]: 4'-O-Demethyl-epi-podophyllotoxine-4-O-[4,6-O, O-(3-cyclohexen-1-methylidene) -2-amino-2- (tetrachlorophthalimidoyl) -2-deoxy-β-D-glucoside (compound 5g).

수율 = 66 %Yield = 66%

mp 197-198 ℃;mp 197-198 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.46(s, 1H), 6.23(s, 1H), 6.12(s, 2H), 5.74(s, 2H), 5.67(s, 1H), 5.33(d,J=8.3Hz, 1H), 4.75(m, 1H), 4.68(d,J=2.7Hz, 1H), 4.45(d,J=2.3Hz, 1H), 4.38(d,J=10.4Hz, 2H), 4.26~4.06(m, 3H), 3.72(s, 6H), 3.56(dd,J=9.1, 19.2Hz, 1H), 3.47~3.31(m, 2H), 3.24(dd,J=5.4. 13.9Hz, 1H), 2.78(m, 1H), 2.04~1.88(br, 6H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.46 (s, 1H), 6.23 (s, 1H), 6.12 (s, 2H), 5.74 (s, 2H), 5.67 (s, 1H), 5.33 (d, J = 8.3 Hz, 1H), 4.75 (m, 1H), 4.68 (d, J = 2.7 Hz, 1H), 4.45 (d, J = 2.3 Hz, 1H), 4.38 (d, J = 10.4 Hz, 2H) , 4.26 to 4.06 (m, 3H), 3.72 (s, 6H), 3.56 (dd, J = 9.1, 19.2 Hz, 1H), 3.47 to 3.31 (m, 2H), 3.24 (dd, J = 5.4.13.9 Hz , 1H), 2.78 (m, 1H), 2.04-1.88 (br, 6H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(트랜스-신나밀리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]: 4'-O-Demethyl-epi-podophyllotoxine-4-O-[4,6-O,O-(trans-cinnamylidene)-2-amino-2-(tetrachlorophthalimidoyl)-2-deoxy-β-D-glucoside (화합물 5h).4'-O-Demethyl-epi-podophyllotoxin-4-O-[4,6-O, O-(Trans-cinnamylidene) -2-amino-2- (tetrachlorophthalimidoyl) -2-deoxy-β-D-glucoside]: 4'-O-Demethyl-epi-podophyllotoxine-4-O-[4,6-O, O-(trans-cinnamylidene) -2-amino-2- (tetrachlorophthalimidoyl) -2-deoxy-β-D-glucoside (compound 5h).

수율 = 58 %Yield = 58%

mp 194-195 ℃;mp 194-195 ° C;

¹H NMR (200 MHz, CDCl₃) δ 7.34~7.26(m, 5H), 6.80(d,J=16.1Hz, 1H), 6.47(s, 1H), 6.22(s, 1H), 6.18(d,J=11.6Hz, 1H), 6.12(s, 2H), 5.74(s, 2H), 5.36(d,J=9.0Hz, 2H), 5.23(d,J=4.7Hz, 1H), 4.86(m, 1H), 4.65(d,J=3.1Hz, 1H), 4.46(d,J=5.3Hz, 2H), 4.40~4.09(m, 3H), 3.72(s, 6H), 3.58(m, 1H), 3.25(dd,J=5.5. 14.1Hz, 1H), 2.82(m, 1H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 7.34-7.26 (m, 5H), 6.80 (d, J = 16.1 Hz, 1H), 6.47 (s, 1H), 6.22 (s, 1H), 6.18 (d, J = 11.6 Hz, 1H), 6.12 (s, 2H), 5.74 (s, 2H), 5.36 (d, J = 9.0 Hz, 2H), 5.23 (d, J = 4.7 Hz, 1H), 4.86 (m, 1H), 4.65 (d, J = 3.1Hz, 1H), 4.46 (d, J = 5.3Hz, 2H), 4.40 ~ 4.09 (m, 3H), 3.72 (s, 6H), 3.58 (m, 1H), 3.25 (dd, J = 5.5 14.1 Hz, 1 H), 2.82 (m, 1 H).

제조예 2: 4'-Preparation Example 2 4'- O-O- 데메틸-에피-포도필로톡신-4-Demethyl-epi-podophyllotoxin-4- OO -[(4,6--[(4,6- O,OO, O -알케닐리덴 또는 알키닐리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-Alkenylidene or alkynylidene) -2-amino-2- (tetrachlorophthalimidoyl) -2-deoxy- ββ -D-글루코시드] (화학식 5의 화합물)를 유기용매를 사용하지 않고 합성하는 방법-D-glucoside] (compound of formula 5) is synthesized without using an organic solvent

4'-O-데메틸-에피-포도필로톡신-4-O-[2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드](화합물 2, 0.10 mmol)와 각각의 해당하는 과량의 화학식 3의 알데히드 또는 화학식 4의 아세탈(1.8-3.0 mmol)[아크롤레인 디메틸아세탈(4a), 3-부테날디에틸아세탈(4b), 프로피올알데히드 디에틸아세탈(4c), 2-부텐-1-알데히드 디에틸아세탈(4d), 시클로프로판카복스알데히드(3e), 2-부틴-1-알데히드 디에틸아세탈(4f), 1,2,5,6-테트라하이드로 벤즈알데히드(3g), 트랜스-신남알데히드(3h)]을 질소 기류하에서 넣고 반응용액의 온도를 0 ℃ 로 낮춘다. 진한 황산 1 방울 또는, 메탄설포닐산(30 mg)을 넣고 0 ℃에서 한 시간 동안 교반 후 실온에서 3 시간 내지 24 시간 동안 교반하였다.4'- O- to methyl-epi-podophyllotoxin -4- O - [2- amino-2- (tetrachloride GRAUFTHAL yimido yl) -2-deoxy-β -D- glucoside (compound 2, 0.10 mmol) and each corresponding excess of aldehyde of formula 3 or acetal of formula 4 (1.8-3.0 mmol) [acrolein dimethylacetal (4a), 3-butenaldiethylacetal (4b), propioaldehyde diethylacetal ( 4c), 2-butene-1-aldehyde diethylacetal (4d), cyclopropanecarboxaldehyde (3e), 2-butyn-1-aldehyde diethylacetal (4f), 1,2,5,6-tetrahydro Benzaldehyde (3 g) and trans-cinnaaldehyde (3 h)] were added under a nitrogen stream, and the temperature of the reaction solution was lowered to 0 deg. One drop of concentrated sulfuric acid or methanesulfonyl acid (30 mg) was added thereto, stirred at 0 ° C. for 1 hour, and then stirred at room temperature for 3 to 24 hours.

반응의 완결을 TLC로 확인한 후, 알데히드 또는 아세탈을 감압 증류하고 생성물을 클로르포름(100 mL)에 녹여 유기층을 증류수(10 mL x 2회)로 씻어준 후 무수 MgSO₄로 건조시키고 여과한 다음 여액을 감압증류하고 관 크로마토그라피 (용출액 : 클로르포름:메탄올 = 40:1)로 분리하여 각각의 순수한 화합물 5a 내지 5h를 얻었으며 그 물성은 상기 대표적 제조예 1에서 얻은 화합물들과 같았다.After completion of the reaction by TLC, the aldehyde or acetal was distilled under reduced pressure, the product 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 purified by distillation under reduced pressure and the residue was separated by column chromatography (eluent: chloroform: methanol = 40: 1) to obtain the pure compounds 5a to 5h, and the physical properties thereof were the same as those obtained in the representative Preparation Example 1.

제조예 3: 4'-Preparation Example 3 4'- O-O- 데메틸-에피-포도필로톡신-4-Demethyl-epi-podophyllotoxin-4- OO -[4,6--[4,6- O,OO, O -(알케닐리덴 또는 알키닐리덴)-2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시--(Alkenylidene or alkynylidene) -2-amino-2- (tetrachlorophthalimidoyl) -2-deoxy- ββ -D-글루코시드](화학식 5의 화합물)의 탈보호 반응-D-glucoside] (compound of formula 5)

각각의 화합물 5a 내지 5h(0.10 mmol)를 메탄올 (20 mL)에 녹인 후 히드라진(0.2 mmol)을 넣고 실온에서 1 시간 내지 10 시간 동안 교반하였다. TLC로 반응의 완결을 확인한 후, 메탄올을 감압 증류하고 생성된 고체를 클로르포름(100 mL)에 녹여 관 크로마토그라피 (용출액 : 클로르포름:메탄올 = 20:1)로 분리하여 각각의 순수한 화합물 6a 내지 6h를 얻었으며, 이들의 물성은 하기와 같다.Each compound 5a to 5h (0.10 mmol) was dissolved in methanol (20 mL), hydrazine (0.2 mmol) was added thereto, and the mixture was stirred at room temperature for 1 to 10 hours. After confirming the completion of the reaction by TLC, methanol was distilled under reduced pressure, and the resulting solid was dissolved in chloroform (100 mL), separated by column chromatography (eluent: chloroform: methanol = 20: 1), and each pure compound 6a to 6h was obtained, and their physical properties are as follows.

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(프로페닐리덴)-2-아미노-2-데옥시-β-D-글루코시드]: 4'-O-demethyl-epi-podophyllotoxin-4-O-[4,6-O,O-(propenylidene)-2-amino-2-deoxy-β-D-glucoside] (화합물 6a).4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - ( alkylidene-propenyl) -2-amino-2-deoxy-β -D- glucoside]: 4'- O -demethyl- epi -podophyllotoxin-4- O - [4,6- O, O- (propenylidene) -2-amino-2-deoxy- β -D-glucoside] ( compound 6a).

수율 = 88 %Yield = 88%

mp 205-207 ℃;mp 205-207 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.83(s, 1H), 6.53(s, 1H), 6.25(s, 2H), 5.98(s, 1H), 5.96(s, 1H), 5.87(ddd,J=4.6, 10.7, 12.7Hz, 1H) 5.51(bd,J=17.3Hz, 1H), 5.35(bd,J=10.4Hz, 1H), 5.01(d,J=4.3Hz, 1H), 4.90(d,J=3.1Hz, 1H), 4.59(d,J=6.7Hz, 1H), 4.42(m, 1H), 4.21(m, 3H), 3.75(s, 6H), 3.7-~3.44(m, 2H), 3.40~3.38(m, 2H), 2.24(dd,J=5.2, 14.1Hz, 1H), 2.82(m, 1H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.83 (s, 1H), 6.53 (s, 1H), 6.25 (s, 2H), 5.98 (s, 1H), 5.96 (s, 1H), 5.87 (ddd, J = 4.6, 10.7, 12.7 Hz, 1H) 5.51 (bd, J = 17.3 Hz, 1H), 5.35 (bd, J = 10.4 Hz, 1H), 5.01 (d, J = 4.3 Hz, 1H), 4.90 (d , J = 3.1 Hz, 1H), 4.59 (d, J = 6.7 Hz, 1H), 4.42 (m, 1H), 4.21 (m, 3H), 3.75 (s, 6H), 3.7--3.44 (m, 2H ), 3.40-3.38 (m, 2H), 2.24 (dd, J = 5.2, 14.1 Hz, 1H), 2.82 (m, 1H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(3-부테닐리덴)-2-아미노-2-데옥시-β-D-글루코시드]:4'-O-demethyl-epi-podophyllotoxin-4-O-[4,6-O,O-(3-butenylidene)-2-amino-2-deoxy-β-D-glucoside] (화합물 6b).4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - (3- butenyl alkylpiperidinyl) -2-amino-2-deoxy-β -D- glucoside ]: 4'- O -demethyl- epi -podophyllotoxin- 4- O - [4,6- O, O- (3-butenylidene) -2-amino-2-deoxy- β -D-glucoside] ( compound 6b) .

수율 = 92 %Yield = 92%

mp 201-202 ℃;mp 201-202 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.83(s, 1H), 6.53(s, 1H), 6.24(s, 2H), 5.98(s, 1H), 5.96(s, 1H), 5.83(m, 1H), 5.13(m, 2H), 4.90(d,J=3.5Hz, 1H), 4.65~4.58(m, 3H), 4.45(m, 1H), 4.24~4.16(m, 3H), 3.75(s, 6H), 3.60~3.52(m, 2H), 3.43~3.30(m, 2H), 3.24(dd,J=5.3, 14.0Hz, 1H), 2.85(m, 1H), 2.45(m, 2H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.83 (s, 1H), 6.53 (s, 1H), 6.24 (s, 2H), 5.98 (s, 1H), 5.96 (s, 1H), 5.83 (m, 1H), 5.13 (m, 2H), 4.90 (d, J = 3.5 Hz, 1H), 4.65 to 4.58 (m, 3H), 4.45 (m, 1H), 4.24 to 4.16 (m, 3H), 3.75 (s , 6H), 3.60-3.52 (m, 2H), 3.43-3.30 (m, 2H), 3.24 (dd, J = 5.3, 14.0 Hz, 1H), 2.85 (m, 1H), 2.45 (m, 2H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(프로피닐리덴)-2-아미노-2-데옥시-β-D-글루코시드]:4'-O-demethyl-epi-podophyllotoxin-4-O-[4,6-O,O-(propynylidene)-2-amino-2-deoxy-β-D-glucoside] (화합물 6c).4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - ( propionyl alkylpiperidinyl) -2-amino-2-deoxy-β -D- glucoside]: 4'- O -demethyl- epi -podophyllotoxin-4- O - [4,6- O, O- (propynylidene) -2-amino-2-deoxy- β -D-glucoside] ( compound 6c).

수율 = 78 %Yield = 78%

mp 153-155 ℃;mp 153-155 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.89(s, 1H), 6.53(s, 1H), 6.26(s, 2H), 6.00(s, 2H) 5.33(d,J=1.63, 1H), 4.92(br, 1H), 4.58(d,J=5.1Hz, 1H), 4.51(d,J=7.9Hz, 2H), 4.35(m, 1H), 3.90~3.81(m, 2H), 3.76(s, 6H), 3.65~3.40(m, 4H), 3.23(dd,J=5.4, 14.2Hz, 1H), 2.70(m, 1H), 2.61(q,J=1.6Hz, 1H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.89 (s, 1H), 6.53 (s, 1H), 6.26 (s, 2H), 6.00 (s, 2H) 5.33 (d, J = 1.63, 1H), 4.92 (br, 1H), 4.58 (d, J = 5.1 Hz, 1H), 4.51 (d, J = 7.9 Hz, 2H), 4.35 (m, 1H), 3.90-3.81 (m, 2H), 3.76 (s, 6H), 3.65-3.40 (m, 4H), 3.23 (dd, J = 5.4, 14.2 Hz, 1H), 2.70 (m, 1H), 2.61 (q, J = 1.6 Hz, 1H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(2-부테닐리덴)-2-아미노-2-데옥시-β-D-글루코시드]:4'-O-demethyl-epi-podophyllotoxin-4-O-[4,6-O,O-(2- butenylidene)-2-amino-2-deoxy-β-D-glucoside] (화합물 6d).4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - (2- butenyl alkylpiperidinyl) -2-amino-2-deoxy-β -D- glucoside ]: 4'- O -demethyl- epi -podophyllotoxin- 4- O - [4,6- O, O- (2- butenylidene) -2-amino-2-deoxy- β -D-glucoside] ( compound 6d) .

수율 = 81 %Yield = 81%

mp 208-209 ℃;mp 208-209 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.84(s, 1H), 6.51(s, 1H), 6.24(s, 2H), 5.96(dd,J=6.7, 15.5Hz, 1H), 5.92(s, 2H), 5.54(m, 1H), 5.35(d,J=8.3Hz, 1H), 5.02(d,J=5.5Hz, 1H), 4.79(m, 1H), 4.67(d,J=3.1Hz, 2H), 4.46(d,J=5.7Hz, 2H), 4.37(bd,J=10.6Hz, 1H), 4.26~4.07(m, 2H), 3.72(s, 6H), 3.55(m, 2H), 3.24(dd,J=5.4, 14.3Hz, 1H), 2.78(m, 1H), 1.76(d,J=6.7Hz, 3H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.84 (s, 1H), 6.51 (s, 1H), 6.24 (s, 2H), 5.96 (dd, J = 6.7, 15.5Hz, 1H), 5.92 (s, 2H), 5.54 (m, 1H), 5.35 (d, J = 8.3 Hz, 1H), 5.02 (d, J = 5.5 Hz, 1H), 4.79 (m, 1H), 4.67 (d, J = 3.1 Hz, 2H), 4.46 (d, J = 5.7 Hz, 2H), 4.37 (bd, J = 10.6 Hz, 1H), 4.26-4.07 (m, 2H), 3.72 (s, 6H), 3.55 (m, 2H), 3.24 (dd, J = 5.4, 14.3 Hz, 1H), 2.78 (m, 1H), 1.76 (d, J = 6.7 Hz, 3H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(시클로프로필메틸리덴)-2-아미노-2-데옥시-β-D-글루코시드]:4'-O-demethyl-epi-podophyllotoxin-4-O-[4,6-O,O-(cyclopropylmethylidene)-2-amino-2-deoxy-β-D-glucoside] (화합물 6e).4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - ( cyclopropyl-methylidene) -2-amino-2-deoxy-β -D- glucoside; : 4'- O -demethyl- epi -podophyllotoxin-4- O - [4,6- O, O- (cyclopropylmethylidene) -2-amino-2-deoxy- β -D-glucoside] ( compound 6e).

수율 = 91 %Yield = 91%

mp 188-190 ℃;mp 188-190 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.83(s, 1H), 6.54(s, 1H), 6.25(s, 2H), 5.99(s, 1H), 5.97(s, 1H), 4.90(d,J=2.9Hz, 1H), 4.58(m, 2H), 4.37(m, 1H), 4.17(m, 3H), 3.80(m, 1H), 3.63~3.42 (m, 2H), 3.35~3.19(m, 3H), 2.83(m, 1H), 1.20(m, 1H), 0.58~0.49(m, 4H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.83 (s, 1H), 6.54 (s, 1H), 6.25 (s, 2H), 5.99 (s, 1H), 5.97 (s, 1H), 4.90 (d, J = 2.9 Hz, 1H), 4.58 (m, 2H), 4.37 (m, 1H), 4.17 (m, 3H), 3.80 (m, 1H), 3.63-3.42 (m, 2H), 3.35-3.19 (m , 3H), 2.83 (m, 1H), 1.20 (m, 1H), 0.58-0.49 (m, 4H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(2-부티닐리덴)-2-아미노-2-데옥시-β-D-글루코시드]:4'-O-demethyl-epi-podophyllotoxin-4-O-[4,6-O,O-(2- butynylidene)-2-amino-2-deoxy-β-D-glucoside (화합물 6f)4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - (2- butynyl alkylpiperidinyl) -2-amino-2-deoxy-β -D- glucoside ]: 4'- O -demethyl- epi -podophyllotoxin- 4- O - [4,6- O, O- (2- butynylidene) -2-amino-2-deoxy- β -D-glucoside ( compound 6f)

수율 = 87 %Yield = 87%

mp 210-212 ℃;mp 210-212 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.84(s, 1H), 6.53(s, 1H), 6.25(s, 2H), 5.99(s, 1H), 5.94(s, 1H), 5.70(d,J=2.0Hz, 1H), 5.23(d,J=1.6Hz, 1H), 4.88(d,J=3.3Hz, 1H), 4.59(m, 2H), 4.43(m, 1H), 4.21(d,J=7.1Hz,1H), 4.15~3.81(m, 2H), 3.75(s, 6H), 3.54(m, 2H), 3.32(dd,J=6.5, 15.9Hz, 1H), 3.22(dd,J=4.3, 9.4Hz, 1H), 2.82(m, 1H), 1.91(dd,J=1.7, 6.6Hz, 3H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.84 (s, 1H), 6.53 (s, 1H), 6.25 (s, 2H), 5.99 (s, 1H), 5.94 (s, 1H), 5.70 (d, J = 2.0 Hz, 1H), 5.23 (d, J = 1.6 Hz, 1H), 4.88 (d, J = 3.3 Hz, 1H), 4.59 (m, 2H), 4.43 (m, 1H), 4.21 (d, J = 7.1 Hz, 1H), 4.15-3.81 (m, 2H), 3.75 (s, 6H), 3.54 (m, 2H), 3.32 (dd, J = 6.5, 15.9 Hz, 1H), 3.22 (dd, J = 4.3, 9.4 Hz, 1H), 2.82 (m, 1H), 1.91 (dd, J = 1.7, 6.6 Hz, 3H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(3-시클로헥센-1-메틸리덴)-2-아미노-2-데옥시-β-D-글루코시드]:4'-O-demethyl-epi-podophyllotoxin-4-O-[4,6-O,O-(3-cyclohxen-1-methylidene)-2-amino-2-deoxy-β-D-glucoside] (화합물 6g).4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - (3- cyclohexene-1-methylidene) -2-amino-2-deoxy-β- D- glucoside]: 4'- O -demethyl- epi -podophyllotoxin- 4- O - [4,6- O, O- (3-cyclohxen-1-methylidene) -2-amino-2-deoxy- β - D-glucoside] (compound 6 g).

수율 = 84 %Yield = 84%

mp 199-200 ℃;mp 199-200 ° C;

¹H NMR (200 MHz, CDCl₃) δ 6.83(s, 1H), 6.54(s, 1H), 6.25(s, 2H), 5.99(s, 1H), 5.97(s, 1H), 5.66(s, 2H), 4.90(d,J=3.3Hz, 1H), 4.60~4.56(m, 2H), 4.47~4.37(m, 2H), 4.21(m, 2H), 3.75(s, 6H), 3.57~3.32(m, 2H), 3.31~3.20(m, 3H), 2.89(m, 1H), 2.76(m, 1H), 2.17~1.88(m, 4H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.83 (s, 1H), 6.54 (s, 1H), 6.25 (s, 2H), 5.99 (s, 1H), 5.97 (s, 1H), 5.66 (s, 2H), 4.90 (d, J = 3.3 Hz, 1H), 4.60-4.56 (m, 2H), 4.47-4.37 (m, 2H), 4.21 (m, 2H), 3.75 (s, 6H), 3.57-3.32 (m, 2H), 3.31-3.20 (m, 3H), 2.89 (m, 1H), 2.76 (m, 1H), 2.17-1.88 (m, 4H).

4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(트랜스-신나밀리덴)-2-아미노-2-데옥시-β-D-글루코시드]: 4'-O-demethyl-epi-podophyllotoxin-4-O-[4,6-O,O-(trans-cinnamylidene)-2-amino-2-deoxy-β-D-glucoside (화합물 6h)4'- O- to methyl-epi-podophyllotoxin -4- O - [4,6- O, O - ( trans-cinnamic mm den) -2-amino-2-deoxy-β -D- glucoside ]: 4'- O -demethyl- epi -podophyllotoxin- 4- O - [4,6- O, O- (trans -cinnamylidene) -2-amino-2-deoxy- β -D-glucoside ( compound 6h)

수율 = 78 %Yield = 78%

mp 197-198 ℃;mp 197-198 ° C;

¹H NMR (200 MHz, CDCl₃) δ 7.41~7.29(m, 5H), 6.86(s, 1H), 6.82(d,J=16.3Hz, 1H), 6.54(s, 2H), 6.20(dd,J=4.7, 10.9Hz 1H), 5.99(s, 1H), 5.98(s, 1H), 5.21(d,J=4.9Hz, 1H), 4.88(d,J=3.5Hz, 1H), 4.62(m, 2H), 4.42(m, 1H), 4.31~3.86(m, 2H), 3.76(s, 6H), 3.65(m, 1H), 3.44(m, 2H), 3.24(dd,J=5.1, 14.2Hz, 1H), 2.81(m, 1H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 7.41-7.29 (m, 5H), 6.86 (s, 1H), 6.82 (d, J = 16.3 Hz, 1H), 6.54 (s, 2H), 6.20 (dd, J = 4.7, 10.9 Hz 1H), 5.99 (s, 1H), 5.98 (s, 1H), 5.21 (d, J = 4.9 Hz, 1H), 4.88 (d, J = 3.5 Hz, 1H), 4.62 (m , 2H), 4.42 (m, 1H), 4.31-3.86 (m, 2H), 3.76 (s, 6H), 3.65 (m, 1H), 3.44 (m, 2H), 3.24 (dd, J = 5.1, 14.2 Hz, 1H), 2.81 (m, 1H).

실시예 1: 화합물 1의 합성Example 1 Synthesis of Compound 1

메탄올 (20 mL)에 각각의 상응하는 화학식 6의 화합물 4'-O-데메틸-에피-포도필로톡신-4-O-[4,6-O,O-(알케닐리덴 혹은 알키닐리덴)-2-아미노-2-데옥시-β-D-글루코시드] (0.83 mmol)을 용해시킨 후, 생성된 용액에 포름알데히드 수용액 (37 wt. % 수용액, 0.70 ml)와 소듐시아노보로하이드라이드(160 mg)을 넣고 실온에서 교반하였다.Methanol (20 mL) each of the corresponding compound of formula (6) to the 4'- O- methyl-epi-podophyllotoxin -4- O - [4,6- O, O - ( alkenyl or alkynyl alkylpiperidinyl alkylpiperidinyl) -2-amino-2-deoxy- β -D-glucoside] (0.83 mmol), and then an aqueous formaldehyde solution (37 wt.% Aqueous solution, 0.70 ml) and sodium cyanoborohydride were added to the resulting solution. (160 mg) was added and stirred at room temperature.

4시간 반응 후 메탄올을 감압증류하고, 물과 디클로로메탄을 넣어 수용액 층을 디클로로메탄으로 씻고 디클로로메탄 층을 감압 증류하였다. 생성물을 실리카겔 칼럼크로마토그래피 (디클로로메탄/아세톤, 10:1)로 분리하여 각각의 상응하는 화합물 1a 내지 1h를 얻었다. 이들의 물성은 아래와 같다.After 4 hours of reaction, methanol was distilled under reduced pressure, water and dichloromethane were added, the aqueous layer was washed with dichloromethane, and the dichloromethane layer was distilled under reduced pressure. The product was separated by silica gel column chromatography (dichloromethane / acetone, 10: 1) to give each corresponding compound 1a-1h. Their physical properties are as follows.

4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(프로페닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신: 4-O-[2-(N,N-dimethylamino)-2-deoxy- 4,6-O,O-(propenylidene)-β-D-glucosyl]-4'-O-demethyl-epi-podophyllotoxin (화합물 1a).4-O-[2-(N, N-Dimethylamino) -2-deoxy-4,6-O, O-(Propenylidene)-β-D-glucosyl] -4'-O-Demethyl- epipodophyllotoxin: 4-O-[2-(N, N-dimethylamino) -2-deoxy-4,6-O, O-(propenylidene)-β-D-glucosyl] -4'-O-demethyl-epi-podophyllotoxin (compound 1a).

수율 = 88 %Yield = 88%

mp 166-167 ℃;mp 166-167 ° C;

IR (KBr) 3462, 2877, 1776, 1612, 1484, 1331, 1231, 1090 ㎝^-1;IR (KBr) 3462, 2877, 1776, 1612, 1484, 1331, 1231, 1090 cm ^-1 ;

¹H NMR (200 MHz, CDCl₃) δ 6.75(s, 1H), 6.58(s, 1H), 6.23(s, 2H), 6.01(s, 2H), 5.91(ddd,J=4.5, 10.7, 13.0Hz, 1H), 5.52(bd,J=17.5Hz, 1H),5.34(bd,J=10.7Hz, 1H), 5.05(d,J=5.1Hz, 1H), 5.00(d,J=2.9Hz,1H), 4.86(d,J=8.3Hz, 1H), 4.64(d,J=5.5Hz, 1H), 4.37~4.30(m, 3H), 3.76(s, 6H), 3.69~3.60(m, 2H), 3.50~3.29(m, 3H), 2.92(m, 1H), 2.41(m, 1H), 2.27(s, 6H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.75 (s, 1H), 6.58 (s, 1H), 6.23 (s, 2H), 6.01 (s, 2H), 5.91 (ddd, J = 4.5, 10.7, 13.0 Hz, 1H), 5.52 (bd, J = 17.5 Hz, 1H), 5.34 (bd, J = 10.7 Hz, 1H), 5.05 (d, J = 5.1 Hz, 1H), 5.00 (d, J = 2.9 Hz, 1H), 4.86 (d, J = 8.3 Hz, 1H), 4.64 (d, J = 5.5 Hz, 1H), 4.37-4.30 (m, 3H), 3.76 (s, 6H), 3.69-3.60 (m, 2H ), 3.50-3.29 (m, 3H), 2.92 (m, 1H), 2.41 (m, 1H), 2.27 (s, 6H).

4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(3-부테닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신:4-O-[2-(N,N-dimethylamino)-2-deoxy-4,6-O,O-(3-butenylidene)-β-D-glucosyl]-4'-O-demethyl-epi-podophyllotoxin (화합물 1b). 4- O - [2- (N, N - dimethylamino) -2-deoxy -4,6- O, O - (3- butenyl alkylpiperidinyl) - β -D- glucosyl] -4'- O - to methyl-epi-podophyllotoxin: 4- O - [2- (N , N -dimethylamino) -2-deoxy-4,6- O, O- (3-butenylidene) - β -D-glucosyl] -4 ' -O -demethyl- epi -podophyllotoxin (Compound 1b).

수율 = 91 %Yield = 91%

mp 163-164 ℃;mp 163-164 ° C;

IR (KBr) 3456, 2919, 1775, 1613, 1484, 1231, 1110, 1035 ㎝^-1;IR (KBr) 3456, 2919, 1775, 1613, 1484, 1231, 1110, 1035 cm ⁻¹ ;

¹H NMR (200 MHz, CDCl₃) δ 6.74(s, 1H), 6.58(s, 1H), 6.23(s, 2H), 6.0(s, 2H), 6.01(s, 2H), 5.77(m, 1H), 5.55(dd,J=2.4, 3.2Hz, 1H), 5.08(m, 1H), 4.99(d,J=2.7Hz, 2H), 4.85(d,J=8.3Hz, 1H), 4.64(d,J=5.5Hz, 1H), 4.43~4.19(m, 3H), 3.76(s, 1H), 3.73~3.56(m, 2H), 3.43~3.27(m, 3H), 2.92(m, 1H), 2.44(m, 1H), 2.26(s, 6H), 1.73(dd,J=1.4, 6.3Hz, 2H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.74 (s, 1H), 6.58 (s, 1H), 6.23 (s, 2H), 6.0 (s, 2H), 6.01 (s, 2H), 5.77 (m, 1H), 5.55 (dd, J = 2.4, 3.2 Hz, 1H), 5.08 (m, 1H), 4.99 (d, J = 2.7 Hz, 2H), 4.85 (d, J = 8.3 Hz, 1H), 4.64 ( d, J = 5.5 Hz, 1H), 4.43-4.19 (m, 3H), 3.76 (s, 1H), 3.73-3.56 (m, 2H), 3.43-3.27 (m, 3H), 2.92 (m, 1H) , 2.44 (m, 1H), 2.26 (s, 6H), 1.73 (dd, J = 1.4, 6.3 Hz, 2H).

4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(프로파질리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신:4-O-[2-(N,N-dimethylamino)-2-deoxy-4,6-O,O-(propagylidene)-β-D-glucosyl]-4'-O-demethyl-epi-podophyllotoxin (화합물 1c). 4- O - [2- (N, N - dimethylamino) -2-deoxy -4,6- O, O - (profile tired den) - β -D- glucosyl] -4'- O - having methyl-epi-podophyllotoxin: 4- O - [2- (N, -dimethylamino N) -2-deoxy-4,6- O, O- (propagylidene)-β -D-glucosyl] -4'- O - demethyl- epi -podophyllotoxin (compound 1c).

수율 = 78 %Yield = 78%

mp 153-154 ℃;mp 153-154 ° C;

IR (KBr) 3465, 2938, 2365, 2267, 1778, 1638, 1496, 1235, 1097, 1042 ㎝^-1;IR (KBr) 3465, 2938, 2365, 2267, 1778, 1638, 1496, 1235, 1097, 1042 cm ⁻¹ ;

¹H NMR (200 MHz, CDCl₃) δ 6.88(s, 1H), 6.56(s, 1H), 6.24(s, 1H), 6.00(s, 1H), 5.37(d,J=1.6Hz, 1H), 5.05(d,J=2.9Hz, 1H), 4.72(d,J=8.1Hz, 1H), 4.63(d,J=4.6Hz, 1H), 4.44(bt,J=9.8Hz, 1H), 4.29(bt,J=8.2Hz, 1H), 3.95(bd,J=3.7Hz, 1H), 3.83(m, 1H), 3.77(s, 6H), 3.68~3.60(m, 2H), 3.55(dd,J=4.5, 12.0Hz, 1H), 3.40(dd,J=5.7, 14.2Hz, 1H), 2.90(m, 1H), 2.60(dd,J=1.6, 4.5Hz, 1H), 2.37(dd,J=8.4, 10.0Hz, 1H), 2.28(s, 6H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.88 (s, 1H), 6.56 (s, 1H), 6.24 (s, 1H), 6.00 (s, 1H), 5.37 (d, J = 1.6Hz, 1H) , 5.05 (d, J = 2.9 Hz, 1H), 4.72 (d, J = 8.1 Hz, 1H), 4.63 (d, J = 4.6 Hz, 1H), 4.44 (bt, J = 9.8 Hz, 1H), 4.29 (bt, J = 8.2 Hz, 1H), 3.95 (bd, J = 3.7 Hz, 1H), 3.83 (m, 1H), 3.77 (s, 6H), 3.68-3.60 (m, 2H), 3.55 (dd, J = 4.5, 12.0 Hz, 1H), 3.40 (dd, J = 5.7, 14.2 Hz, 1H), 2.90 (m, 1H), 2.60 (dd, J = 1.6, 4.5 Hz, 1H), 2.37 (dd, J = 8.4, 10.0 Hz, 1 H), 2.28 (s, 6 H).

4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(2-부테닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신:4-O-[2-(N,N-dimethylamino)-2-deoxy-4,6-O,O-(2-butenylidene)-β-D-glucosyl]-4'-O-demethyl-epi-podophyllotoxin (화합물 1d). 4- O - [2- (N, N - dimethylamino) -2-deoxy -4,6- O, O - (2- butenyl alkylpiperidinyl) - β -D- glucosyl] -4'- O - to methyl-epi-podophyllotoxin: 4- O - [2- (N , N -dimethylamino) -2-deoxy-4,6- O, O- (2-butenylidene) - β -D-glucosyl] -4 ' -O -demethyl- epi -podophyllotoxin (Compound 1d).

수율 = 85 %Yield = 85%

mp 167-168 ℃;mp 167-168 ° C;

IR (KBr) 3458, 2923, 1778, 1615, 1486, 1233, 1115, 1038 ㎝^-1;IR (KBr) 3458, 2923, 1778, 1615, 1486, 1233, 1115, 1038 cm ⁻¹ ;

¹H NMR (200 MHz, CDCl₃) δ 6.74(s, 1H), 6.57(s, 1H), 6.22(s, 2H), 6.00(s, 2H), 5.94(bd,J=6.7Hz, 1H), 5.59(dd,J=1.1, 4.2Hz, 1H), 5.27(d,J=3.3Hz, 1H), 4.99(d,J=2.4Hz, 1H), 4.84(d,J=5.3Hz, 1H), 4.43~4.20(m, 2H), 3.76(s, 6H), 3.72~3.58(m, 2H), 3.48~3.33(m, 3H), 2.89(m, 1H), 2.40(bt,J=9.1Hz, 1H), 2.26(s, 6H), 1.73(d,J=5.5Hz, 3H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.74 (s, 1H), 6.57 (s, 1H), 6.22 (s, 2H), 6.00 (s, 2H), 5.94 (bd, J = 6.7 Hz, 1H) , 5.59 (dd, J = 1.1, 4.2 Hz, 1H), 5.27 (d, J = 3.3 Hz, 1H), 4.99 (d, J = 2.4 Hz, 1H), 4.84 (d, J = 5.3 Hz, 1H) , 4.43-4.20 (m, 2H), 3.76 (s, 6H), 3.72-3.58 (m, 2H), 3.48-3.33 (m, 3H), 2.89 (m, 1H), 2.40 (bt, J = 9.1 Hz , 1H), 2.26 (s, 6H), 1.73 (d, J = 5.5 Hz, 3H).

4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(시클로프로필메틸리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신:4-O-[2-(N,N-dimethylamino)-2-deoxy-4,6-O,O-(cyclopropylmethylidene)-β-D-glucosyl]-4'-O-demethyl-epi-podophyllotoxin (화합물 1e).4- O- [2- ( N, N -dimethylamino) -2-deoxy-4,6- O, O- (cyclopropylmethylidene) -β -D-glucosyl] -4'- O -dec methyl-epi-podophyllotoxin: 4- O - [2- (N , N -dimethylamino) -2-deoxy-4,6- O, O- (cyclopropylmethylidene) - β -D-glucosyl] -4'- O - demethyl- epi -podophyllotoxin (Compound 1e).

수율 = 89 %Yield = 89%

mp 168-169 ℃;mp 168-169 ° C;

IR (KBr) 3450, 2876, 2357, 1775, 1614, 1485, 1331, 1231, 1121, 1034 ㎝^-1;IR (KBr) 3450, 2876, 2357, 1775, 1614, 1485, 1331, 1231, 1121, 1034 cm ^-1 ;

¹H NMR (200 MHz, CDCl₃) δ 6.74(s, 1H), 6.57(s, 1H), 6.22(s, 2H), 6.00(s, 2H), 4.99(d,J=5.0Hz, 1H), 4.83(d,J=8.1Hz, 1H), 4.64(d,J=5.1Hz, 1H), 4.38(dd,J=8.8, 10.6Hz, 1H), 4.28~4.16(m, 4H), 3.76(s, 8H), 3.75~3.43(m, 2H), 3.40~3.24(m, 3H), 2.92(m, 1H), 2.38(dd,J=8.3, 10.1Hz, 1H), 1.20(m, 1H), 0.69(m, 4H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.74 (s, 1H), 6.57 (s, 1H), 6.22 (s, 2H), 6.00 (s, 2H), 4.99 (d, J = 5.0Hz, 1H) , 4.83 (d, J = 8.1 Hz, 1H), 4.64 (d, J = 5.1 Hz, 1H), 4.38 (dd, J = 8.8, 10.6 Hz, 1H), 4.28-4.16 (m, 4H), 3.76 ( s, 8H), 3.75-3.43 (m, 2H), 3.40-3.24 (m, 3H), 2.92 (m, 1H), 2.38 (dd, J = 8.3, 10.1 Hz, 1H), 1.20 (m, 1H) , 0.69 (m, 4 H).

4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(2-부티닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신:4-O-[2-(N,N-dimethylamino)-2-deoxy-4,6-O,O-(2-butynylidene)-β-D-glucosyl]-4'-O-demethyl-epi-podophyllotoxin (화합물1f). 4- O - [2- (N, N - dimethylamino) -2-deoxy -4,6- O, O - (2- butynyl alkylpiperidinyl) - β -D- glucosyl] -4'- O - to methyl-epi-podophyllotoxin: 4- O - [2- (N , N -dimethylamino) -2-deoxy-4,6- O, O- (2-butynylidene) - β -D-glucosyl] -4 ' -O -demethyl- epi -podophyllotoxin (Compound 1f).

수율 = 90 %Yield = 90%

mp 173-174 ℃;mp 173-174 ° C;

IR (KBr) 3462, 2935, 2360, 2263, 1774, 1487, 1231, 1092, 1038 ㎝^-1;IR (KBr) 3462, 2935, 2360, 2263, 1774, 1487, 1231, 1092, 1038 cm ⁻¹ ;

¹H NMR (200 MHz, CDCl₃) δ 6.74(s, 1H), 6.58(s, 1H), 6.23(s, 2H), 6.01(s, 2H), 5.72(d, 1H), 5.24(d,J=1.8Hz, 1H), 4.97(d,J=3.1Hz, 1H), 4.85(d,J=8.1Hz, 1H), 4.64(d,J=5.1Hz, 1H), 4.40(dd,J=10.8, 20.4Hz, 1H), 4.35(dd,J=3.8, 6.4Hz, 1H), 4.00(dd,J=6.5, 9.6Hz, 1H), 3.76(s, 6H), 3.76~3.54(m, 2H), 3.45~3.24(m, 3H), 2.91(m, 1H), 2.45(dd,J=9.1, 12.1Hz, 1H), 2.26(s, 6H), 1.91(dd,J=1.8, 10.2Hz, 3H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.74 (s, 1H), 6.58 (s, 1H), 6.23 (s, 2H), 6.01 (s, 2H), 5.72 (d, 1H), 5.24 (d, J = 1.8 Hz, 1H), 4.97 (d, J = 3.1 Hz, 1H), 4.85 (d, J = 8.1 Hz, 1H), 4.64 (d, J = 5.1 Hz, 1H), 4.40 (dd, J = 10.8, 20.4 Hz, 1H), 4.35 (dd, J = 3.8, 6.4 Hz, 1H), 4.00 (dd, J = 6.5, 9.6 Hz, 1H), 3.76 (s, 6H), 3.76-3.54 (m, 2H ), 3.45 to 3.24 (m, 3H), 2.91 (m, 1H), 2.45 (dd, J = 9.1, 12.1 Hz, 1H), 2.26 (s, 6H), 1.91 (dd, J = 1.8, 10.2 Hz, 3H).

4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(3-시클로헥센-1-메틸리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신:4-O-[2-(N,N-dimethylamino)-2-deoxy-4,6-O,O-(3-cyclohexen-1-methylidene)-β-D-glucosyl]-4'-O-demethyl-epi-podophyllotoxin (화합물 1g).4- O- [2- ( N, N -dimethylamino) -2-deoxy-4,6- O, O- (3-cyclohexene-1-methylidene) -β -D-glucosyl] -4 '- O-de-methyl-epi-podophyllotoxin: 4- O - [2- (N , N -dimethylamino) -2-deoxy-4,6- O, O- (3-cyclohexen-1-methylidene) - β -D-glucosyl] -4'- O -demethyl- epi -podophyllotoxin (compound 1 g).

수율 = 85 %Yield = 85%

mp 169-170 ℃;mp 169-170 ° C;

IR (KBr) 3455,2920, 2359, 1776, 1614, 1505, 1231, 1112, 1035 ㎝^-1;IR (KBr) 3455,2920, 2359, 1776, 1614, 1505, 1231, 1112, 1035 cm ⁻¹ ;

¹H NMR (200 MHz, CDCl₃) δ 6.74(s, 1H), 6.58(s, 1H), 6.23(s, 2H),6.01(s, 2H), 5.66(br. s, 2H), 5.00(d,J=3.1Hz, 1H), 4.85(d,J=8.3Hz, 1H), 3.46(d,J=5.3Hz, 1H), 4.44(br. s, 2H), 4.34~4.18(m, 2H), 3.76(s, 6H), 3.72~3.54(m, 2H), 3.44~3.23(m, 2H), 2.90(m, 2H), 2.39(bt, J=9.2Hz, 2H), 2.27(s, 6H), 2.01(m, 1H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 6.74 (s, 1H), 6.58 (s, 1H), 6.23 (s, 2H), 6.01 (s, 2H), 5.66 (br. S, 2H), 5.00 ( d, J = 3.1Hz, 1H) , 4.85 (d, J = 8.3Hz, 1H), 3.46 (d, J = 5.3Hz, 1H), 4.44 (br. s, 2H), 4.34 ~ 4.18 (m, 2H ), 3.76 (s, 6H), 3.72-3.54 (m, 2H), 3.44-3.23 (m, 2H), 2.90 (m, 2H), 2.39 (bt, J = 9.2 Hz, 2H), 2.27 (s, 6H), 2.01 (m, 1 H).

4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(트랜스-신나밀리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신:4-O-[2-(N,N-dimethylamino)-2-deoxy-4,6-O,O-(trans-cinnamylidene)-β-D-glucosyl]-4'-O-demethyl-epi-podophyllotoxin (화합물 1h).4- O- [2- ( N, N -dimethylamino) -2-deoxy-4,6- O, O- (trans-cinnamylidene) -β -D-glucosyl] -4'- O- to methyl-epi-podophyllotoxin: 4- O - [2- (N , N -dimethylamino) -2-deoxy-4,6- O, O- (trans -cinnamylidene) - β -D-glucosyl] -4 ' -O -demethyl- epi -podophyllotoxin (compound 1h).

수율 = 91 %Yield = 91%

mp 175-176 ℃;mp 175-176 ° C;

IR (KBr) 3452, 2876, 2359, 1773, 1484, 1231, 1090, 1044 ㎝^-1;IR (KBr) 3452, 2876, 2359, 1773, 1484, 1231, 1090, 1044 cm ^-1 ;

¹H NMR (200 MHz, CDCl₃) δ 7.42~7.32(m, 5H), 6.83(d,J=16.5Hz, 1H), 6.76(s, 1H), 6.58(s, 1H), 6.24(s, 2H), 6.22(dd,J=4.5, 16.3Hz,1H), 6.01(s, 2H), 5.23(d,J=4.5Hz, 1H), 5.01(d,J=3.1Hz, 1H), 4.87(d,J=3.8Hz, 1H), 4.65(d,J=4.1Hz, 1H), 4.45~4.23(m, 4H), 3.77(s, 6H), 3.68(m, 2H), 3.56~3.35(m, 3H), 2.93(m, 1H), 2.43(dd,J=8.3, 9.7Hz, 1H), 2.28(s, 6H). ¹ H NMR (200 MHz, CDCl ₃ ) δ 7.42 to 7.32 (m, 5H), 6.83 (d, J = 16.5 Hz, 1H), 6.76 (s, 1H), 6.58 (s, 1H), 6.24 (s, 2H), 6.22 (dd, J = 4.5, 16.3 Hz, 1H), 6.01 (s, 2H), 5.23 (d, J = 4.5 Hz, 1H), 5.01 (d, J = 3.1 Hz, 1H), 4.87 ( d, J = 3.8 Hz, 1H), 4.65 (d, J = 4.1 Hz, 1H), 4.45-4.23 (m, 4H), 3.77 (s, 6H), 3.68 (m, 2H), 3.56-3.35 (m , 3H), 2.93 (m, 1H), 2.43 (dd, J = 8.3, 9.7 Hz, 1H), 2.28 (s, 6H).

시험예 1: 세포독성의 측정Test Example 1 Measurement of Cytotoxicity

암세포배양Cancer 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 배양중인 것을 사용하였다.The cells used to measure anticancer activity were A-549 (non-small cell lung cancer), SK-OV-3 (adenocarcinoma, ovarian malignant ascites), SK-MEL-2 (malignant melanoma, metastasis to the femoral skin), XF498 ( Central nervous system tumor) and HCT15 (colon adenocarcinoma), and these cancer cells were all human-derived tumor cell lines obtained from the National Cancer Institute of America (NCI) and passaged at the Korea Research Institute of Chemical Technology.

이 세포들은 모두 배양액으로서 5 % 소의 태아 혈청으로 보강된 RPMI 1640 배양액을 사용하여, 37 ℃ 항온 항습 5 % CO₂, 인큐베이터에서 배양되었다. 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.These cells were all 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.

세포독성측정Cytotoxicity Measurement

1989년 미국의 국립암연구소에서 약물의 생체외 항암활성을 측정하기위하여 개발된 SRB분석법(sulforhodamine B assay method)을 사용하였다.In 1989, the US National Cancer Institute used the sulforhodamine B assay method developed to measure the in vitro anticancer activity of drugs.

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 제거하였다.Cells were separated from the implant using trypsin-CDTA solution to use passaged cells for experiments, and the cells per well in plates (96 well microplate, manufactured by Falcon) were 5 x 10 ³ (A549, HCT15). , 1 × 10 ⁴ (SK-MEL-2, XF498), 2 × 10 ⁴ (SK-OV-3). The aliquoted cells were incubated for 24 hours in a CO ₂ incubator and attached to the bottom, followed by removal of the culture with an aspirator.

본 발명의 화합물 1a 내지 1h 및 에토포사이드(BMS, USA)를 시험화합물로 사용하였다. 시험화합물 각각을 배양액에 용해시켜 시험용액을 제조하였다. 화합물을 용해시키기 위하여 필요에 따라 디메틸설폭사이드(DMSO)를 사용하였다. 이어서, 상기 시험용액을 6 농도의 로그 투여량(log dose)으로, 즉, 1, 10^-1, 10^-2, 10^-3, 10^-4및 10^-5의 비율로 배양액에 희석하였다. 희석된 시험 화합물 용액을 0.22 ㎕ 필터로 여과한 후, 세포가 들어 있는 웰에 각각 100 ㎕ 씩 3 배수로 넣어주고, 48 시간 동안 더 배양하였다.Compounds 1a to 1h of the present invention and etoposide (BMS, USA) were used as test compounds. Each test compound was dissolved in a culture solution to prepare a test solution. Dimethyl sulfoxide (DMSO) was used as needed to dissolve the compound. Subsequently, the test solution was diluted in the culture solution at a log dose of 6 concentrations, that is, at a ratio of 1, 10 ⁻¹ , 10 ⁻² , 10 ⁻³ , 10 ^−4, and 10 ⁻⁵ . The diluted test compound solution was filtered through a 0.22 μl filter, and then 100 μl each of the wells containing the cells were added in 3 multiples, and further incubated for 48 hours.

세포를 약물과 48시간 배양한 후 각 웰의 배양액을 제거하고 10 % 트리클로로아세트산(TCA)를 100 ㎕씩 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.After incubating the cells with the drug for 48 hours, the culture solution of each well was removed, and 100 μl 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.

완전히 건조된 플레이트에 웰당 1 % 아세트산 용액에 0.4 % SRB를 용해시킨 염색용액 100 ㎕를 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 ㎕의 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.100 μl of a staining solution in which 0.4% SRB was dissolved in 1% acetic acid solution per well was added to a completely dried plate, and the cells were stained for 30 minutes, and washed again 5 times with 1% acetic acid solution to remove SRBs not bound to the cells. It was. The stained cell plates are then dried at room temperature and then shaken for 10 minutes with a titer plate shaker by adding 100 μl of 10 mM unbuffered trisma base solution per well. 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 수학식 1 또는 2에 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 the time of incubation were measured and calculated by the following Equation 1 or 2:

이렇게 계산된 값들로부터 로터스 프로그램(LOTUS program)의 데이터 회귀(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 drugs inhibit 50% of cancer cell growth, that is, ED ₅₀ was calculated to compare the anticancer activity of each drug.

시험예 2: 인간 유전자 위상이성질화 II 효소 저해능 측정Test Example 2: Determination of Human Gene Phase Isomerization II Enzyme Inhibition

유전자 위상이성질화 II 효소 조제Gene phase isomerization II enzyme preparation

본 실험에 사용한 세포주는 ATCC로부터 구입한 HeLa 셀을 사용하였다. HeLa 셀을 10% 소 태아 혈청이 포함된 RPMI 1640 배지에서 배양한 다음 HeLa 셀을 모아 3배 용량의 1 mM의 EDTA, 1 mM 머캅토에탄올, 0.5 mM PMSF 및 10 % 글리세롤을 포함하는 50 mM KH₂PO₄(pH 7.0) 완충액을 가하여 빙욕중에서 펄리트론(Polytron)으로 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 관 크로마토그라피 등의 방법으로 부분정제하여 사용한다.The cell line used in this experiment used HeLa cells purchased from ATCC. HeLa cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, then HeLa cells were collected and 50 mM KH containing three volumes of 1 mM EDTA, 1 mM mercaptoethanol, 0.5 mM PMSF and 10% glycerol. ₂ PO ₄ (pH 7.0) buffer was added, homogenized with Polytron for 30 seconds in an ice bath, and 2 M KH ₂ (PO ₄ ) (pH 7.0) buffer was added to make final concentration to 0.3 M. Supernatant obtained by homogenizing for 1 hour and standing in an ice bath for 1 hour and then centrifuged at 30,000 xg for 30 minutes is used after partial purification by phosphocellulose tube chromatography.

PP ₄₄ 언노팅(unknotting) 분석Unnotting Analysis

위상이성질화 II (Topo II)에 대한 시험화합물의 IC₅₀값 결정을 위해 언노팅 분석을 행하였다.Unnotification analysis was performed to determine the IC ₅₀ value of the test compound for topoisomerization II (Topo II).

50 mM KCl, 0.1 mM EDTA, 100 mM NaCl, 10 mM MgCl₂, 100 ㎍/ml 소 태아 혈청 알부민 및 1 mM ATP을 함유하는 50 mM HEPES(N-[2-하이드록시에틸]-피페라진-N'-[2-에탄설폰산])(pH 7.0) 완충액에 0.4 ㎍ P₄노팅된(knotted) DNA 및 상기 제조한 Topo II 효소를 가하여 총반응액의 부피가 20 ㎕가 되게 한 후 37 ℃에서 30 분간 반응시켰다. 최종농도가 1 %되게 SDS를 가하여 반응을 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.50 mM HEPES (N- [2-hydroxyethyl] -piperazine-N containing 50 mM KCl, 0.1 mM EDTA, 100 mM NaCl, 10 mM MgCl ₂ , 100 μg / ml Fetal Bovine Albumin and 1 mM ATP 0.4 μg P ₄ knotted DNA and the prepared Topo II enzyme were added to '-[2-ethanesulfonic acid]) (pH 7.0) buffer to make the total reaction solution 20 μl and then at 37 ° C. The reaction was carried out for 30 minutes. The reaction was stopped by adding SDS to a final concentration of 1%, followed by electrophoresis on 1% agarose gel equilibrated with 50 mM tris borate (pH 8.3) and 2.5 mM EDTA solution, followed by electrophoresis of 0.5 μg / ml of ethidium bromide solution. Dyeing, taking photos under ultraviolet light and measuring 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.

합성된 화합물의 세포독성 및 인간 위상 이성질화 II 효소에 대한 억제 활성을 다음 표 1에 나타내었다.The cytotoxicity and inhibitory activity of the synthesized compounds against human phase isomerization II enzymes are shown in Table 1 below.

상기 표에서 나타난 바와 같이 합성된 화학식 1의 화합물은 대조화합물 에토포사이드와 비교할 때, 시험한 다섯 가지 인간 암세포에 대하여 대부분 2 내지 3 배 정도 더 강한 세포독성을 지니며, 인간 유전자 위상이성질화 II 효소에 대하여 2배 정도 더 강한 효소저해 효과를 보인다.Compounds of formula (1) synthesized as shown in the table above have two to three times stronger cytotoxicity against the five human cancer cells tested when compared to the control compound etoposide, and human gene phase isomerization II It shows about 2 times stronger enzyme inhibition.

상기 화합물 중 특히 우수한 세포독성 및 인간 유전자 위상 이성질화 효소에 대한 저해능을 갖는 화합물은 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(프로페닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1a), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(3-부테닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1b), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(2-부테닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1d), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(시클로프로필메틸리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1e), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(2-부티닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1f), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(3-시클로헥세닐메틸리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1g), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(트랜스-신나밀리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1h)이다.Among the above compounds, compounds having particularly good cytotoxicity and inhibitory ability against human gene phase isomerase are 4- O- [2- ( N, N -dimethylamino) -2-deoxy-4,6- O, O- . (Propenylidene) -β -D-glucosyl] -4'- 0 -demethyl-epipodophyllotoxin (Compound 1a), 4- O- [2- ( N, N -dimethylamino) -2-de oxy -4,6- O, O - (3- butenyl alkylpiperidinyl) - β -D- glucosyl] -4'- O-de-methyl-epi-podophyllotoxin (compound 1b), 4- O - [2- (N, N-dimethylamino) -2-deoxy -4,6- O, O - (2- butenyl alkylpiperidinyl) - β -D- glucosyl] -4'- O-de-methyl-epi-podophyllotoxin (Compound 1d), 4- O- [2- ( N, N -dimethylamino) -2-deoxy-4,6- O, O- (cyclopropylmethylidene) -β -D-glucosyl] -4 ' -O -Demethyl-epipodophyllotoxin (Compound 1e), 4- O- [2- ( N, N -dimethylamino) -2-deoxy-4,6- O, O- (2-butynyl Den) -β -D-glucosyl] -4'- O -demethyl-epipodophyllotoxin (Compound 1f), 4- O- [2- ( N, N -dimethylamino) -2-deoxy-4 , 6- O, O- (3-hour Claw-hexenyl methylidene) - β -D- glucosyl] -4'- O-de-methyl-epi-podophyllotoxin (compound 1g), 4- O - [2- (N, N - dimethylamino) -2- Deoxy-4,6- O, O- (trans-cinnamylidene) -β -D-glucosyl] -4'- 0 -demethyl- epipodophyllotoxin (compound 1 h).

이와 같은 결과를 살펴볼 때 본 화학식 1의 화합물은 비교화합물보다 세포독성 및 효소저해능이 매우 우수함을 알 수 있었다.Looking at the results, it was found that the compound of Chemical Formula 1 is much better in cytotoxicity and enzyme inhibition than the comparative compound.

시험예 3: 생체내 항암활성 측정Test Example 3: In vivo anticancer activity measurement

6주령 BDF1 생쥐 8마리를 1군으로 하여 7군의 대조군 및 11개의 실험군으로 나누었다. DBA/2 생쥐에서 계대배양된 루케미아(leukemia) L1210 세포 1 ×10⁵세포/0.1 ㎖ 씩을 각각 생쥐의 복강내로 이식하였다. 루케미아 세포를 투여한지 1,2, 3, 4 및 5 일째에 5개의 실험 화합물, 즉, 에토포사이드(etoposide), NK-611 및 본 발명의 화합물 1a, 1b 및 1g를 하기 표 2에 나타낸 각 농도로 각 군의 생쥐의 복강내로 투여하였다.Eight 6-week-old BDF1 mice were divided into seven groups as controls and eleven experimental groups. 1 × 10 ⁵ cells / 0.1 ml of leukemia L1210 cells passaged in DBA / 2 mice were implanted intraperitoneally of the mice, respectively. Five experimental compounds, namely etoposide, NK-611 and compounds 1a, 1b and 1g of the present invention, at 1, 2, 3, 4 and 5 days after the administration of leuchemia cells, are shown in Table 2 below. Concentration was administered intraperitoneally of the mice of each group.

생쥐를 매일 관찰하면서 생존기간을 측정하고 하기 수학식 3과 같이 I.R.(inversion recovery)을 계산함으로써 항암활성을 측정하였다. 이 결과는 하기 표 2에 나타내었다.Survival was measured by observing mice daily, and anticancer activity was measured by calculating I.R. (inversion recovery) as shown in Equation 3 below. The results are shown in Table 2 below.

상기에서, At는 실험군 생쥐의 평균 생존일수이고, Ac는 대조군 생쥐의 평균 생존일수이다.In the above, At is the average survival days of the experimental mice, Ac is the average survival days of the control mice.

투여량(mg/kg)Dose (mg / kg) I.R.(%)I.R. (%) 60일이상 생존 마리수Survival over 60 days 에토포사이드Etoposide 2010520105 508.0323.1251.9508.0323.1251.9 521521 NK-611 HClNK-611 HCl 1052.511052.51 독성발현545.1198.6159.1Toxicity expression 545.1198.6159.1 06000600 화합물 1aHClCompound 1aHCl 1052.511052.51 독성발현450.9158.3151.4Toxicity Expression 02000200 화합물 1bHClCompound 1bHCl 1052.511052.51 독성발현450.9520.7170.8Toxic expression 450.9520.7170.8 02500250 화합물 1gHClCompound 1gHCl 2010520105 685.7462.9385.2685.7462.9385.2 842842

상기 결과에서 보듯이, 본 발명의 화합물들의 루케미아 세포에 대한 항암활성은 에토포사이드 및 NK-611의 경우에 비하여 우수함을 알 수 있다.As shown in the above results, it can be seen that the anticancer activity of the compounds of the present invention against leuchemia cells is superior to that of etoposide and NK-611.

시험예 4: 급성독성 시험Test Example 4: Acute Toxicity Test

4주령 ICR 계통의 특정병원체 부재 마우스(암수 각각 15마리) 3마리를 1군으로 하여 암수 각각 5개의 군으로 나누었다.Three mice without specific pathogens (15 males and females) of 4-week-old ICR strains were divided into five male and female groups.

증류수에 에토포사이드(etoposide), NK-611 HCl 및 화합물 1a HCl, 1b HCl 및 1g HCl 각각을 녹인 용액을 5주령이 된 생쥐의 복강에 68.3, 88.8 및 115.4 mg/kg의 양으로 투여하였다. 1회 투여후 7일동안 생쥐의 일반 증상 및 투여된 화합물의 양에 따른 체중변화를 관찰하였다. LD₅₀(mg/kg) 값을 구하기 위해 독성 시험용 프로그램인 Labcatmodule(Innovative Origramming Associates Inc., New Jersey, USA)에서 제공되는 Probit법을 이용하여 구하였다. 또한, 하기 수학식 4에 의해 치료지수(therapeutic index)를 계산함으로써 항암활성을 측정하였다. 이 결과는 하기 표 3에 나타내었다.A solution of etoposide, NK-611 HCl and Compound 1a HCl, 1b HCl, and 1g HCl in distilled water was administered in the amount of 68.3, 88.8 and 115.4 mg / kg to the abdominal cavity of 5 week old mice. After 7 days of administration, body weights were observed according to the general symptoms of the mice and the amount of the compound administered. LD ₅₀ (mg / kg) values were obtained using the Probit method provided by Labcatmodule (Innovative Origramming Associates Inc., New Jersey, USA). In addition, anticancer activity was measured by calculating the therapeutic index by the following equation (4). The results are shown in Table 3 below.

상기에서, C는 항암활성을 나타내는 화합물의 최저농도이다.In the above, C is the minimum concentration of the compound showing anticancer activity.

LD₅₀ LD ₅₀ 치료지수Treatment index 일반증상General symptoms 체중증감Weight loss 에토포사이드Etoposide 수컷cock 200200 1010 특이 증상 없음No specific symptoms 투여후 1일째 체중감소 투여후 7일째 회복Weight loss 1 day after administration Recovery 7 days after administration 암컷female 200200 1010 NK-611 HClNK-611 HCl 수컷cock 24.524.5 4.94.9 운동성 감소, 설사, 하복부 오염, 및 안검하수Decreased mobility, diarrhea, lower abdominal contamination, and ptosis 투여후 1일부터체중감소Weight loss from 1 day after administration 암컷female 67.267.2 13.413.4 화합물 1a HClCompound 1a HCl 수컷cock 101.2101.2 2020 용량상관성 운동성 감소 및 유루Dose correlates with reduced mobility 용량 상관성체중 감소Dose Correlation 암컷female 110.5110.5 2020 화합물 1b HClCompound 1b HCl 수컷cock <68.3<68.3 2727 운동성 감소, 설사 및 혼수Decreased mobility, diarrhea and lethargy 용량 상관성체중 감소Dose Correlation 암컷female <68.3<68.3 2727 화합물 1g HClCompound 1g HCl 수컷cock 131.5131.5 6.56.5 용량상관성 운동성 감소 및 유루Dose correlates with reduced mobility 용량 상관성체중 감소Dose Correlation 암컷female 152.6152.6 7.67.6

상기의 급성독성의 결과에서 알 수 있는 바와 같이, 본 발명의 세 가지 화합물 들은 모두 기준 화합물인 에토포사이드 및 NK-611보다 치료지수가 높음을 알 수 있다. 따라서, 이들 화합물들은 암치료제로서 사용될 수 있다.As can be seen from the results of the acute toxicity, all three compounds of the present invention can be seen that the treatment index is higher than the reference compounds etoposide and NK-611. Therefore, these compounds can be used as cancer treatments.

본 발명의 화학식 1의 새로운 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(알케닐리덴 또는 알키닐리덴)-β-D-글루코실]-4'-O-데메틸-에피-포도필로톡신 화합물은 인간 유전자 위상이성질화 II 효소에 대한 활성 및 암세포 독성 및 생체내 항암효과 시험에서 종래의 에토포사이드 보다 우수한 활성 및 낮은 독성을 갖고 있으며, 수용성이 개선되어 매우 우수한 생체내 항암효과를 나타낸다.4- O new of the general formula (I) of the present invention - [2- (N, N - dimethylamino) -2-deoxy -4,6- O, O - (alkenyl or alkynyl alkylpiperidinyl alkylpiperidinyl) - β -D- Glucosyl] -4'- O -demethyl-epi-podophyllotoxin compounds have superior activity and lower toxicity than conventional etoposides in the activity and cancer cell toxicity and in vivo anticancer effects test for human gene topology isomerization II enzyme. It has a good water solubility and shows an excellent in vivo anticancer effect.

Claims (4)

하기 화학식 1의 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(알케닐리덴 또는 알키닐리덴)-β-D-글루코실]-4'-O-데메틸-에피-포도필로톡신 화합물 또는 그의 약학적으로 허용가능한 염:4- O to the formula 1 - [2- (N, N - dimethylamino) -2-deoxy -4,6- O, O - (alkenyl or alkynyl alkylpiperidinyl alkylpiperidinyl) - β -D- glucosyl; -4'- O -demethyl-epi-podophyllotoxin compound or a pharmaceutically acceptable salt thereof: 화학식 1Formula 1 상기식에서,In the above formula, R¹및 R²는 각각 서로 독립적으로 수소, C₁-C₁₀알킬 또는 C₅-C₁₀시클로알킬이거나; 또는 상호 연결되어 C₄-C₁₀의 질소원자 함유 고리화합물을 형성하고;R ¹ and R ² are each independently of each other hydrogen, C ₁ -C ₁₀ alkyl or C ₅ -C ₁₀ cycloalkyl; Or are interconnected to form a C ₄ -C ₁₀ nitrogen atom-containing cyclic compound; R³은 치환되거나 치환되지 않은 비닐, 에티닐, 알릴 또는 C₅-C₁₀시클로알케닐이며,이때 치환체는 분쇄상 또는 직쇄상 C₁-C₈알킬, 알콕시, 알콕시알킬 또는 아릴이다.R ³ is substituted or unsubstituted vinyl, ethynyl, allyl or C ₅ -C ₁₀ cycloalkenyl, wherein the substituents are ground or straight chain C ₁ -C ₈ alkyl, alkoxy, alkoxyalkyl or aryl. 제 1 항에 있어서,The method of claim 1, 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(프로페닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1a), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(3-부테닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1b), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(2-부테닐리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1d), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(시클로프로필메틸리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1e), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(2-부티닐리덴)-β-D-글루코 실]-4'-O-데메틸-에피포도필로톡신(화합물 1f), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(3-시클로헥세닐메틸리덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1g), 4-O-[2-(N,N-디메틸아미노)-2-데옥시-4,6-O,O-(트랜스-신나밀리 덴)-β-D-글루코실]-4'-O-데메틸-에피포도필로톡신(화합물 1h)으로 이루어진 군 중에서 선택된 화합물 또는 그의 약학적으로 허용 가능한 염.4- O- [2- ( N, N -dimethylamino) -2-deoxy-4,6- O, O- (propenylidene) -β -D-glucosyl] -4'- 0 -demethyl - epi podophyllotoxin (compound 1a), 4- O - [2- (N, N - dimethylamino) -2-deoxy -4,6- O, O - (3- butenyl alkylpiperidinyl) - β -D -Glucosyl] -4'- O -demethyl-epipodophyllotoxin (Compound 1b), 4- O- [2- ( N, N -dimethylamino) -2-deoxy-4,6- O, O - (2-butenyl alkylpiperidinyl) - β -D- glucosyl] -4'- O-de-methyl-epi-podophyllotoxin (compound 1d), 4- O - [2- (N, N - dimethylamino) - 2-deoxy-4,6- O, O- (cyclopropylmethylidene) -β -D-glucosyl] -4'- 0 -demethyl-epipodophyllotoxin (Compound 1e), 4- O- [ 2- (N, N-dimethylamino) -2-deoxy -4,6- O, O - (2- butynyl alkylpiperidinyl) - β -D- glucosyl] -4'- O-de-methyl-epi grapes Philo toxin (compound 1f), 4- O - [2- (N, N - dimethylamino) -2-deoxy -4,6- O, O - (3- cyclohexenyl methylidene) - β -D- Glucosyl] -4'- O -Demethyl-Epipodophyllotoxin (Compound 1g), 4- O- [2- ( N, N -dimethylamino ) -2-deoxy-4,6- O, O- (trans-cinnamylidene) -β -D-glucosyl] -4'- O -demethyl-epipodophyllotoxin (compound 1h) Or a pharmaceutically acceptable salt thereof. ⅰ) 하기 화학식 2의 4'-O-데메틸-에피-포도필로톡신-4-O-[2-아미노-2-(테트라클로로프탈이미도일)-2-데옥시-β-D-글루코시드]와 하기 화학식 3의 알데히드 또는 화학식 4의 아세탈과 산 촉매하에 반응시켜 하기 화학식 5의 화합물을 얻는 단계; ⅱ) 화학식 5의 아미노 보호기를 탈보호시켜 하기 화학식 6의 화합물을 얻는 단계; ⅲ) 화학식 6의 화합물을 환원제의 존재하에 알킬화반응시키는 단계를 포함하는 제1 항에 따른 화학식 1 화합물의 제조방법.Ⅰ) to the 4'- O- methyl having the formula 2-epi-podophyllotoxin -4- O - [2- amino-2- (tetrachloride GRAUFTHAL yimido yl) -2-deoxy-β -D- glucoside Seed] is reacted with an aldehyde of formula (3) or acetal of formula (4) under an acid catalyst to obtain a compound of formula (5); Ii) deprotecting the amino protecting group of formula 5 to obtain a compound of formula 6; V) preparing a compound of formula 1 according to claim 1 comprising alkylating the compound of formula 6 in the presence of a reducing agent. 화학식 2Formula 2 화학식 3Formula 3 화학식 4Formula 4 화학식 5Formula 5 화학식 6Formula 6 상기식에서, R¹, R²및 R³은 제 1 항에서 정의한 바와 같다.Wherein R ¹ , R ² and R ³ are as defined in claim 1. 제 1 항에 따른 화학식 1의 4-O-[2-(N,N-디알킬아미노)-2-데옥시-4,6-O,O-(알케닐리덴 또는 알키닐리덴)-β-D-글루코실]-4'-O-데메틸-에피-포도필로톡신 화합물 또는 그 산성 염, 항암 효과량 및 약제학적으로 허용 가능한 담체를 포함하는 항암제 조성물.Agent of formula (I) according to claim 1 4- O - [2- (N, N - di-alkylamino) -2-deoxy -4,6- O, O - (alkenyl or alkynyl alkylpiperidinyl alkylpiperidinyl) - β - An anticancer composition comprising a D-glucosyl] -4′- O -demethyl-epi-podophyllotoxin compound or an acid salt thereof, an anticancer effective amount and a pharmaceutically acceptable carrier.