KR20160004648A

KR20160004648A - Cyclic phosphopeptide compounds as allosteric plk1-pbd inhibitor

Info

Publication number: KR20160004648A
Application number: KR1020140083248A
Authority: KR
Inventors: 심태보; 함영진; 윤호종
Original assignee: 한국과학기술연구원
Priority date: 2014-07-03
Filing date: 2014-07-03
Publication date: 2016-01-13
Also published as: KR101672975B1

Abstract

The present invention relates to a novel cyclic phosphopeptide compound having allosteric PLK1-PBD inhibitory activities. The compound of the present invention has PLK1-PBD inhibitory activities, and is useful as an antitumor agent. The present invention provides a cyclic phophopeptide compound represented by chemical formula 1, a hydrate thereof, a solvate thereof, an isomer thereof, or a racemic body and pharmaceutically acceptable salt thereof.

Description

알로스테릭 ＰＬＫ1-ＰＢＤ 저해활성을 가지는 고리형 포스포펩티드 화합물 {CYCLIC PHOSPHOPEPTIDE COMPOUNDS AS ALLOSTERIC PLK1-PBD INHIBITOR} [0001] CYCLIC PHOSPHOPEPTIDE COMPOUNDS AS ALLOSTERIC PLK1-PBD INHIBITOR WITH ALLOSERIC PLK1-PBD INHIBITOR ACTIVITY [0002]

본 발명은 알로스테릭 PLK1-PBD 저해활성을 갖는 신규 고리형 포스포펩티드 화합물에 관한 것이다.The present invention relates to a novel cyclic phosphopeptide compound having an allosteric PLK1-PBD inhibitory activity.

유사 폴로 키나아제 (Polo-Like Kinase, PLK)는 세린/쓰레오닌 단백질 키나아제로서 PLK1, PLK2(SNK), PLK3(FNK 또는 PRK), PLK4 (also known as SAK)의 4개로 하위 구성원으로 분류되고, 분자량은 약 66 kD 정도이다. PLK 중에서도 PLK1의 특성이 가장 잘 규명되어 있다. PLK1(Polo-like kinase 1)은 유사분열(mitosis)의 중심 단계를 조절하여 세포 증식을 증진시키는 과정에 중요한 역할을 담당한다. PLK1 은 N-터미날 키나아제 도메인과 C-터미날 폴로 박스 도메인(PBD)으로 구성하며, 두 도메인을 연결하는 링커로서 폴로 박스 캡(Pc)이 존재한다. PLK1 은 각종 기질을 포스포릴화 함으로써 유사분열기(M phase) 도입, 중심체의 조절, 염색체의 분리 및 세포질분열의 각 단계에 참여하는 것으로 보고되어 있다. 또한, PLK1 은 식도암, 췌장암, 폐암, 침윤성 직장암, 유방암, 전이성 전립선암, 난소암, 흑색종과 같은 다양한 암세포에서 과발현되는 것으로 보고되어 있으며, PLK1의 과발현이 이러한 질환을 앓고 있는 환자의 예후와 관련된 것으로 보고되어 있다. [Oncogene, Vol. 14, 543, 1997; Cancer Research, Vol. 15, 2794, 1999] 이처럼 PLK1 은 표적 항종양제의 분자표적으로서 유용하며, 전임상 및 초기 임상단계에 대래서는 그 가치가 이미 확립되어 있다. The polo-like kinase (PLK) is classified into four members as PLK1, PLK2 (SNK), PLK3 (FNK or PRK) and PLK4 (also known as SAK) as serine / threonine protein kinases, The molecular weight is about 66 kD. Among the PLKs, the properties of PLK1 are best understood. PLK1 (Polo-like kinase 1) plays an important role in the process of promoting cell proliferation by regulating the central phase of mitosis. PLK1 is composed of an N-terminal kinase domain and a C-terminal pollock box domain (PBD), and a pollo box cap (Pc) exists as a linker connecting the two domains. PLK1 has been reported to phosphorylate various substrates to participate in each step of introducing mitogenesis (M phase), regulation of centrosome, separation of chromosome, and cytoplasmic division. It has also been reported that PLK1 is overexpressed in various cancer cells such as esophageal cancer, pancreatic cancer, lung cancer, invasive rectal cancer, breast cancer, metastatic prostate cancer, ovarian cancer and melanoma and overexpression of PLK1 is associated with the prognosis of patients suffering from such diseases . [Oncogene, Vol. 14, 543, 1997; Cancer Research, Vol. 15, 2794, 1999] Thus, PLK1 is useful as a molecular target for targeted antineoplastic agents, and its value has already been established for preclinical and early clinical stages.

이에 PLK1 억제활성을 근거로 항종양제로서 유용한 화합물이 다수 공개되어 있다. 예를 들면, 국제특허공개 WO 2007-120752호에는 4,5-디히드로-[1,2,4]트리아졸로[4,3-Ｆ]프테리딘 화합물; 국제특허공개 WO 2006-082107호에는 티아졸리딘계 화합물; 국제특허공개 WO 2008-081910호에는 아미노피리미딘계 화합물; 국제특허공개 WO 2008/113711호는 피리미도디아제핀 화합물이 각각 개시되어 있다.
Accordingly, many compounds useful as antitumor agents have been disclosed based on the activity of inhibiting PLK1. For example, International Patent Publication No. WO 2007-120752 discloses 4,5-dihydro- [1,2,4] triazolo [4,3-F] pyridine compounds; International Patent Publication No. WO 2006-082107 discloses a thiazolidine-based compound; International Patent Publication No. WO 2008-081910 discloses aminopyrimidine-based compounds; International Patent Publication No. WO 2008/113711 discloses pyrimidodiazepine compounds, respectively.

본 발명은 신규 구조의 고리형 포스포펩티드 화합물 제공을 목적으로 한다.The present invention aims at providing a cyclic phosphopeptide compound having a novel structure.

또한, 본 발명은 상기한 신규 화합물이 PLK1 에 대한 선택적 억제활성을 가지고 있으므로, 상기 화합물을 부작용이 경감된 표적항종양제로 사용하는 용도 제공을 다른 목적으로 한다.
It is another object of the present invention to provide a use of the compound as a target anti-tumor agent with reduced side effects because the novel compound has selective inhibitory activity against PLK1.

상기한 과제 해결을 위하여, 본 발명은 하기 화학식 1로 표시되는 고리형 포스포펩티드 화합물, 이의 수화물, 이의 용매화물, 이의 이성질체, 라세미체 또는 약학적으로 허용 가능한 염을 제공한다.In order to solve the above problems, the present invention provides a cyclic phosphopeptide compound represented by the following formula (1), a hydrate thereof, a solvate thereof, an isomer thereof, a racemate thereof or a pharmaceutically acceptable salt thereof.

[화학식 1][Chemical Formula 1]

상기 화학식 1에서, In Formula 1,

R₁은 수소원자; C₁∼C₆ 알킬기; C₁∼C₆ 알콕시기; C₁∼C₆ 알킬아미노기; 또는

를 나타내고,R ₁ is a hydrogen atom; A C _{1 to} C ₆ alkyl group; A C _{1 to} C ₆ alkoxy group; A C ₁ -C ₆ alkylamino group; or

Lt; / RTI >

R₂는

를 나타내고, R ₂ is

Lt; / RTI >

A는 C₁∼C₆ 알킬기; C₁∼C₆ 알킬, C₁∼C₆ 알콕시 및 C₁∼C₆ 할로알킬로 이루어진 군으로부터 선택된 1 내지 3개의 치환기로 치환 또는 비치환된 페닐기; 또는 N, O 및 S로부터 선택된 헤테로원자가 1 내지 3개 포함된 5각형 내지 7각형의 포화헤테로고리기를 나타내고,A is a C ₁ -C ₆ alkyl group; A phenyl group substituted or unsubstituted with 1 to 3 substituents selected from the group consisting of C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy and C ₁ -C ₆ haloalkyl; Or a saturated 5- to 7-membered heterocyclic group containing 1 to 3 hetero atoms selected from N, O and S,

R₃, R₄, R₅, 및 R₆은 서로 같거나 다른 것으로서 수소원자; C₁∼C₆ 알킬기; 또는 (C₁∼C₆ 알킬싸이오)C₁∼C₆ 알킬기를 나타내고,R ₃ , R ₄ , R ₅ , and R ₆ are the same or different from each other and are a hydrogen atom; A C _{1 to} C ₆ alkyl group; Or a (C ₁ -C ₆ alkylthio) C ₁ -C ₆ alkyl group,

R₇은 C₁∼C₆ 알킬기를 나타내고,R ₇ represents a C ₁ -C ₆ alkyl group,

n은 0, 1 또는 2를 나타낸다.
n represents 0, 1 or 2;

또한, 본 발명은 상기 화학식 1로 표시되는 고리형 포스포펩티드 화합물, 이의 수화물, 이의 용매화물, 이의 이성질체, 라세미체 또는 약학적으로 허용 가능한 염으로부터 선택된 화합물이 활성성분으로 포함된 PLK1 저해제를 제공한다.
The present invention also provides a PLK1 inhibitor comprising a cyclic phosphopeptide compound represented by the formula (1), a hydrate thereof, a solvate thereof, an isomer thereof, a racemate thereof or a pharmaceutically acceptable salt thereof as an active ingredient to provide.

또한, 본 발명은 상기 화학식 1로 표시되는 고리형 포스포펩티드 화합물, 이의 수화물, 이의 용매화물, 이의 이성질체, 라세미체 또는 약학적으로 허용 가능한 염으로부터 선택된 화합물이 활성성분으로 포함된 항종양제를 제공한다.
The present invention also relates to an antineoplastic agent comprising a cyclic phosphopeptide compound represented by Formula 1, a hydrate thereof, a solvate thereof, an isomer thereof, a racemate thereof or a pharmaceutically acceptable salt thereof as an active ingredient Lt; / RTI >

본 발명의 화합물은 PLK1 에 대한 특이적 저해활성을 가지고 있으므로, 부작용이 경감된 표적항종양제로 유용하다.Since the compound of the present invention has a specific inhibitory activity against PLK1, it is useful as a target antitumor agent in which side effects are alleviated.

즉, 본 발명의 화합물은 식도암, 췌장암, 폐암, 침윤성 직장암, 유방암, 전이성 전립선암, 난소암, 흑색종과 같은 다양한 종양질환의 치료, 예방 및 경감에 유용하다.
That is, the compounds of the present invention are useful for the treatment, prevention and alleviation of various tumor diseases such as esophageal cancer, pancreatic cancer, lung cancer, invasive rectal cancer, breast cancer, metastatic prostate cancer, ovarian cancer and melanoma.

도 1은 실시예 5의 화합물에 대한 PLK1-PBD 저해활성을 확인한 결과이다.
Fig. 1 shows the results of confirming the PLK1-PBD inhibitory activity against the compound of Example 5. Fig.

본 발명은 하기 화학식 1로 표시되는 고리형 포스포펩티드 화합물에 관한 것이다.The present invention relates to a cyclic phosphopeptide compound represented by the following general formula (1).

[화학식 1][Chemical Formula 1]

상기 화학식 1에서, In Formula 1,

Lt; / RTI >

R₂는

를 나타내고, R ₂ is

Lt; / RTI >

n은 0, 1 또는 2를 나타낸다.
n represents 0, 1 or 2;

본 발명에 따른 상기 화학식 1로 표시되는 화합물은 하나 또는 그 이상의 비대칭 중심을 가질 수 있고, 이러한 화합물의 경우 거울상 이성체 또는 부분입체이성체가 존재할 수 있다. 따라서, 본 발명은 각 이성체 화합물, 이들 이성체의 혼합물 또는 라세미체 화합물을 포함한다.The compound represented by Formula 1 according to the present invention may have one or more asymmetric centers, and in the case of such a compound, an enantiomer or diastereomer may exist. Accordingly, the present invention includes each isomeric compound, a mixture of these isomers or a racemic compound.

또한, 본 발명은 상기 화학식 1로 표시되는 화합물에 방사성 원소를 도입시킨 방사성 유도체를 포함하며, 이들 방사성 화합물은 영상화 기술을 통한 생체연구 분야에 유용하다.The present invention also includes a radioactive derivative in which a radioactive element is introduced into the compound represented by Formula 1, and these radioactive compounds are useful in the field of biology research through imaging techniques.

또한, 본 발명에 따른 상기 화학식 1로 표시되는 화합물은 당해 기술 분야에서 통상적인 방법에 의해 약학적으로 허용 가능한 염을 형성할 수 있다. 예를 들면, 염산, 브롬산, 술폰산, 아미도황산, 인산, 질산과 같은 무독성의 무기산, 또는 프로피온산, 숙신산, 글리콜산, 스테아르산, 젖산, 타르타르산, 시트르산, 파라톨루엔설폰산, 메탄설폰산과 같은 무독성의 유기산과 함께 약학적으로 허용 가능한 이들의 산의 염을 형성할 수 있다.In addition, the compound represented by Formula 1 according to the present invention may form pharmaceutically acceptable salts by a conventional method in the art. Such as, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfonic acid, amidosulfuric acid, phosphoric acid, nitric acid, or organic acids such as propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, tartaric acid, citric acid, paratoluenesulfonic acid, Together with non-toxic organic acids, can form pharmaceutically acceptable salts of these acids.

또한, 본 발명에 따른 상기 화학식 1로 표시되는 화합물의 일부는 수성 및 유기 용매와 같은 용매로부터 결정화되거나 또는 재결정화될 수 있다. 그러한 경우, 용매화물이 형성될 수 있다. 동결건조와 같은 방법으로 제조 가능한 다양한 양의 물 함유 화합물 이외에 수화물을 비롯한 화학 양론적 용매화물도 본 발명의 범위에 속한다.In addition, some of the compounds represented by Formula 1 according to the present invention may be crystallized or recrystallized from a solvent such as aqueous and organic solvents. In such cases, solvates may be formed. In addition to various amounts of water-containing compounds that can be prepared by methods such as lyophilization, stoichiometric solvates, including hydrates, are also within the scope of the present invention.

본 발명에 따른 상기 화학식 1로 표시되는 화합물을 정의하기 위해 사용된 치환기를 좀 더 자세히 설명하면 다음과 같다. '알킬기'는 1 내지 6개의 탄소 원자를 가진 직쇄상, 분쇄상의 탄소사슬을 모두 포함하며, 선호하는 알킬기는 메틸, 에틸기, 노말프로필기, 이소프로필기, 노말부틸기, 이소부틸기, tert-부틸기 등이 있다. '알콕시기'는 산소에 연결된 탄소의 알킬기를 의미하는 것으로, 이때 알킬은 상기에서 정의한 바와 같다. '할로알킬기'는 플루오로, 클로로, 브로모, 아이오도와 같은 할로겐원자가 1 내지 13개 포함되고, 1 내지 6개의 탄소원자를 가진 직쇄상, 분쇄상의 탄소사슬을 모두 포함하며, 선호하는 할로알킬기는 플루오로메틸기, 트리플루오로메틸기, 1,2-디클로로에틸기, 1,1-디클로로에틸기, 펜타플루오로에틸기 등이 있다. '포화헤테로고리기'는 N, O, S로 구성된 1 내지 3개의 헤테로원자 및 탄소원자로 구성되는 싸이클로알킬기로서, 피페리디닐, 피페라지닐, 몰포리노, 피롤리디닐, 피라졸리디닐, 트리아졸리디닐 등이 있다.
The substituent used to define the compound represented by Formula 1 according to the present invention will be described in more detail as follows. "Alkyl group" is 1 to 6 straight-chain having a carbon atom, and include all of the carbon chain on the pulverization, preferred alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl group, isobutyl group, tert - Butyl group and the like. The term " alkoxy group " means an alkyl group of carbon linked to oxygen, wherein alkyl is as defined above. The term " haloalkyl group " includes all straight chain, branched chain carbon chains having 1 to 6 carbon atoms and 1 to 13 halogen atoms such as fluoro, chloro, bromo and iodo, A methyl group, a trifluoromethyl group, a 1,2-dichloroethyl group, a 1,1-dichloroethyl group, and a pentafluoroethyl group. The term " saturated heterocyclic group " means a cycloalkyl group composed of 1 to 3 heteroatoms and carbon atoms consisting of N, O, S, and includes piperidinyl, piperazinyl, morpholino, pyrrolidinyl, pyrazolidinyl, Decyl and the like.

본 발명에 따른 상기 화학식 1로 표시되는 고리형 포스포펩티드 화합물에 있어서, 바람직하기로는 상기 R₁은 C₁∼C₆ 알콕시기; C₁∼C₆ 알킬아미노기; 또는

를 나타내고, 상기 R₂는

를 나타내고, 상기 A는 C₁∼C₆ 알킬기; C₁∼C₆ 알콕시 및 C₁∼C₆ 할로알킬로 이루어진 군으로부터 선택된 1 내지 3개의 치환기로 치환 또는 비치환된 페닐기; 또는 피롤리디닐, 피라졸리디닐 및 트리아졸리디닐로 이루어진 군으로부터 선택된 포화헤테로고리기를 나타내고, 상기 R₃, R₄, R₅, 및 R₆은 서로 같거나 다른 것으로서 수소원자; C₁∼C₆ 알킬기; 또는 (C₁∼C₆ 알킬싸이오)C₁∼C₆ 알킬기를 나타내고, 상기 R₇은 C₁∼C₆ 알킬기를 나타내고, 상기 n은 0, 1 또는 2를 나타내는 화합물이다.
In the cyclic phosphopeptide compound represented by Formula 1 according to the present invention, preferably, R ₁ is a C ₁ -C ₆ alkoxy group; A C ₁ -C ₆ alkylamino group; or

, And R < ₂ >

, A represents a C ₁ -C ₆ alkyl group; A phenyl group substituted or unsubstituted with 1 to 3 substituents selected from the group consisting of C ₁ -C ₆ alkoxy and C ₁ -C ₆ haloalkyl; Or a saturated heterocyclic group selected from the group consisting of pyrrolidinyl, pyrazolidinyl and triazolidinyl, and R ₃ , R ₄ , R ₅ , and R ₆ , which are the same or different from each other, represent a hydrogen atom; A C _{1 to} C ₆ alkyl group; Or a (C ₁ -C ₆ alkylthio) C ₁ -C ₆ alkyl group, R ₇ represents a C ₁ -C ₆ alkyl group, and n represents 0, 1 or 2.

본 발명에 따른 상기 화학식 1로 표시되는 고리형 포스포펩티드 화합물에 있어서, 보다 바람직하기로는 상기 R₁은 메톡시기, 에톡시기, 노말프로폭시기, 이소프로폭시기, 노말부톡시기, 이소부톡시기, 메틸아미노기, 에틸아미노기, 노말프로필아미노기, 이소프로필아미노기, 또는 -NH-CH(이소부틸)-C(O)OCH₃를 나타내고, 상기 R₂는

를 나타내고, 상기 A는 메틸기, 에틸기, 노말프로필기, 이소프로필기, 노말부틸기, 이소부틸기, 3-메톡시페닐기, 4-메톡시페닐기, 3,5-다이메톡시페닐기, 3,5-다이(이소부톡시)페닐기, 3-(트리플루오로메틸)페닐기, 또는 피롤리딘-2-일기를 나타내고, 상기 R₃및 R₄는 서로 같거나 다른 것으로서 수소원자, 메틸기, 에틸기, 노말프로필기, 이소프로필기, 노말부틸기, 이소부틸기, 메틸싸이오메틸기, 또는 메틸싸이오에틸기를 나타내고, 상기 n은 0, 1 또는 2를 나타내는 나타내는 화합물이다.
In the cyclic phosphopeptide compound represented by Formula 1 according to the present invention, more preferably, R ₁ is a methoxy group, an ethoxy group, a normal propoxy group, an isopropoxy group, a normal butoxy group, an isobutoxy group , methylamino group, ethylamino group, normal propylamino group, an isopropyl group, or -NH-CH (isobutyl) -C (O) represents the OCH _3, wherein R ₂ is

Wherein A represents a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a normal butyl group, an isobutyl group, a 3- methoxyphenyl group, a 4- methoxyphenyl group, (Trifluoromethyl) phenyl group, or a pyrrolidin-2-yl group, and R ₃ and R ₄ are the same or different from each other and each represents a hydrogen atom, a methyl group, an ethyl group, a normal propyl group, An isopropyl group, a n-butyl group, an isobutyl group, a methylthiomethyl group, or a methylthioethyl group, and n is 0, 1 or 2.

상기 화학식 1로 표시되는 고리형 포스포펩티드 화합물을 구체적으로 예시하면 다음과 같다:Specific examples of the cyclic phosphopeptide compound represented by the above formula (1) are as follows:

메틸 (3S, 6S, 9S, 17R, 19S)-9-아세트아미도-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이사이클로[15.2.1]이코산-19-카복실레이트;Methyl (3S, 6S, 9S, 17R, 19S) -9-acetamido-6- (hydroxymethyl) -2,5,8,12-tetraoxo- Yl) ethyl) -16-oxa-1,4,7,13-tetraaza- bicyclo [15.2.1] iso-19-carboxylate;

메틸 (3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-9-((S)-4-(메틸싸이오)-2-((S)-피롤리딘-2-카복스아미도)부탄아미도)-2,5,8,12-테트라옥소-3-( (R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카복실레이트;Methyl (3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -9 - ((S) -4- (methylthio) -2- (R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetra Azabicyclo [15.2.1] octanoic-19-carboxylate;

메틸 ((3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-9-((S)-4-(메틸싸이오)-2-((S)-피롤리딘-2-카복스아미도)부탄아미도)-2,5,8,12-테트라옥소-3-( (R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트;Methyl ((3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -9 - ((S) -4- (methylthio) -2- Carboxamido) butanamido) -2,5,8,12-tetraoxo-3- ((R) -1- (Phosphoroxy) ethyl) -16- Tetraaza bicyclo [15.2.1] iso-19-carbonyl) -L-leucinate;

메틸 ((3S, 6S, 9S, 17R, 19S)-9-((S)-2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트;Methyl ((3S, 6S, 9S, 17R, 19S) -9 - ((S) -2- 6- (hydroxymethyl) -2,5,8,12-tetraoxo-3 - ((R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetraaza Bicyclo [15.2.1] octanoic-19-carbonyl) -L-leucinate;

메틸 ((3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-9-(3-(트리플루오로메틸)벤자미도)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트;Methyl ((3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -2,5,8,12-tetraoxo-3 - ((R) -1- (Phosphoroxy) 16-oxa-1,4,7,13-tetraaza bicyclo [15.2.1] iso-19-carbonyl) -L-leucine Nate;

메틸 ((3S, 6S, 9S, 17R, 19S)-9-(3,5-다이이소부톡시벤자미도)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-카보닐)-L-로이시네이트; 또는Methyl ((3S, 6S, 9S, 17R, 19S) -9- (3,5-diisobutoxybenzamido) -6- (hydroxymethyl) -2,5,8,12-tetraoxo- ((R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetraaza bicyclo [15.2.1] iso-carbonyl) -L-isocyanate; or

메틸 (3S, 6S, 9S, 17R, 19S)-9-(3,5-다이이소부톡시벤자미도)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카복실레이트.
Methyl (3S, 6S, 9S, 17R, 19S) -9- (3,5-diisobutoxybenzamido) -6- (hydroxymethyl) -2,5,8,12-tetraoxo- (R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetraaza bicyclo [15.2.1] isoic acid-19-carboxylate.

한편, 본 발명에 따른 상기 화학식 1로 표시되는 고리형 포스포펩티드 화합물은 당해 기술분야에서 통상적으로 알려진 유기합성 경로를 통해 합성될 수 있다. 이에 본 발명은 제조방법에 특별한 제한을 두고 있지 않으며, 본 발명의 화합물을 합성하기 위한 몇 가지 경로를 제시하면 하기 반응식 1 내지 8과 같다.Meanwhile, the cyclic phosphopeptide compound represented by Formula 1 according to the present invention can be synthesized through an organic synthesis route commonly known in the art. Accordingly, the present invention does not impose any particular limitation on the preparation method, and some routes for synthesizing the compound of the present invention are shown in the following Reaction Schemes 1 to 8.

[반응식 1][Reaction Scheme 1]

[반응식 2][Reaction Scheme 2]

[반응식 3][Reaction Scheme 3]

[반응식 4][Reaction Scheme 4]

[반응식 5][Reaction Scheme 5]

[반응식 6][Reaction Scheme 6]

[반응식 7][Reaction Scheme 7]

[반응식 8][Reaction Scheme 8]

본 발명에 따른 제조방법에서 사용되는 반응 용매는 당 분야에서 사용되어 온 통상의 유기용매는 모두 사용될 수 있으며, 구체적으로는 디클로로메탄, 디클로로에탄 등의 할로겐화탄화수소류, 에틸 아세테이트 (EtOAc) 등의 아세테이트류, 아세토니트릴 등의 니트릴류, 메탄올, 에탄올 등의 알콜류; 헥산, 톨루엔 등의 탄화수소류, 테트라하이드로퓨란 (THF), 디에틸 에테르, 이소프로필 에테르 등의 에테르류, 디메틸포름아미드 (DMF) 등의 아미드류 등이 사용될 수 있다.
The reaction solvent used in the production process according to the present invention may be any conventional organic solvent used in the art. Specific examples thereof include halogenated hydrocarbons such as dichloromethane and dichloroethane, acetates such as ethyl acetate (EtOAc) Nitriles such as acetonitrile, etc .; alcohols such as methanol and ethanol; Hydrocarbons such as hexane and toluene, ethers such as tetrahydrofuran (THF), diethyl ether and isopropyl ether, and amides such as dimethylformamide (DMF).

한편, 본 발명에 따른 상기 화학식 1로 표시되는 고리형 포스포펩티드 화합물, 이의 수화물, 이의 용매화물, 이의 이성질체, 라세미체 또는 약학적으로 허용 가능한 염은 PLK1에 대한 선택적 억제활성을 나타낸다. 따라서 본 발명의 화합물은 표적항종양제의 활성성분으로서 유효하다. 이에 본 발명은 상기 화학식 1로 표시되는 고리형 포스포펩티드 화합물, 이의 수화물, 이의 용매화물, 이의 이성질체, 라세미체 또는 약학적으로 허용 가능한 염이 유효성분으로 함유하고, 여기에 통상의 부형제, 담체를 포함시킨 종양의 치료, 예방 또는 경감용 약학 조성물을 권리범위로 포함한다. 본 발명의 화합물에 의해 치료, 예방 또는 경감될 수 있는 종양은 식도암, 췌장암, 폐암, 침윤성 직장암, 유방암, 전이성 전립선암, 난소암, 흑색종 등이 포함될 수 있다.Meanwhile, the cyclic phosphopeptide compound represented by the formula 1, the hydrate thereof, the solvate thereof, the isomer thereof, the racemate thereof or the pharmaceutically acceptable salt thereof according to the present invention exhibit a selective inhibitory activity against PLK1. Accordingly, the compounds of the present invention are effective as active ingredients of a target anti-tumor agent. Accordingly, the present invention provides a pharmaceutical composition comprising a cyclic phosphopeptide compound represented by the general formula (1), a hydrate thereof, a solvate thereof, an isomer thereof, a racemate or a pharmaceutically acceptable salt thereof as an active ingredient, As well as pharmaceutical compositions for the treatment, prevention or alleviation of tumors containing the carrier. Tumors that can be treated, prevented or alleviated by the compounds of the present invention may include esophageal cancer, pancreatic cancer, lung cancer, invasive rectal cancer, breast cancer, metastatic prostate cancer, ovarian cancer, melanoma and the like.

본 발명의 약학 조성물은 상기 화학식 1로 표시되는 고리형 포스포펩티드 화합물, 이의 수화물, 이의 용매화물, 이의 이성질체, 라세미체 또는 약학적으로 허용 가능한 염에 통상의 무독성 약학적으로 허용 가능한 담체, 보강제 및 부형제 등을 첨가하여 약학적 분야에서 통상적인 제제 예를 들면 정제, 캅셀제, 트로키제, 액제, 현탁제 등의 경구투여용 제형 또는 비경구투여용 제형으로 제제화할 수 있다.The pharmaceutical composition of the present invention is a pharmaceutical composition comprising a cyclic phosphopeptide compound represented by the general formula (1), a hydrate thereof, a solvate thereof, an isomer thereof, a racemate thereof or a pharmaceutically acceptable salt thereof in the presence of a conventional non-toxic pharmaceutically acceptable carrier, An adjuvant, an excipient, and the like may be added to formulate a formulation for oral administration or a formulation for parenteral administration such as tablets, capsules, troches, liquids, and suspensions, which are customary in the pharmaceutical field.

상기 경구 투여용 제형으로는 예를 들면 정제, 환제, 경/연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제, 엘릭시르제 등이 포함될 수 있다. 이들 제형은 유효성분 이외에 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및/ 또는 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/또는 폴리에틸렌 글리콜)를 함유하고 있다. 상기 정제는 또한 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및/또는 폴리비닐피롤리딘과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염과 같은 붕해제 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제, 및 감미제를 함유할 수 있다.Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and the like. These formulations may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, a lubricant such as silica, talc, stearic acid and its magnesium or calcium salt and / Or polyethylene glycol). The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt The same disintegrating or boiling mixture and / or absorbing agent, coloring agent, flavoring agent, and sweetening agent.

상기 비경구 투여용 제형으로는 예를 들면 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사를 주입하는 방법에 의한다. 이때, 비경구 투여용 제형으로 제제화하기 위하여 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용되는 염을 안정제 또는 완충제와 함께 물에 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알 단위 투여형으로 제조할 수 있다. Examples of the formulations for parenteral administration include injections such as subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. In this case, in order to formulate the formulation for parenteral administration, the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof may be mixed with water or a stabilizer or a buffer to prepare a solution or suspension, .

상기 조성물은 필요에 따라 멸균할 수 있고, 또는 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 및/또는 완충제 등의 보조제, 및 기타 치료적으로 유용한 물질을 함유할 수 있으며, 통상적인 방법인 혼합, 과립화 또는 코팅 방법에 따라 제제화할 수 있다.The composition may be sterilized as needed or may contain other therapeutically useful substances such as preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for controlling osmotic pressure, and other therapeutically useful substances, Granulation, or coating method.

또한, 상기 화학식 1로 표시되는 화합물의 인체에 대한 투여용량은 환자의 나이, 몸무게, 성별, 투여형태, 건강상태 및 질환 정도에 따라 달라질 수 있다. 몸무게가 70 ㎏인 성인 환자를 기준으로 할 때, 일반적으로 0.01 ∼ 1,000 ㎎/일이며, 바람직하게는 1 ∼ 500 ㎎/일이며, 의사 또는 약사의 판단에 따라 일정시간 간격으로 1일 1회 내지 수회로 분할 투여할 수도 있다.
The administration dose of the compound represented by Formula 1 to the human body may be varied depending on the patient's age, weight, sex, dosage form, health condition, and disease severity. It is generally 0.01 to 1,000 mg / day, preferably 1 to 500 mg / day on the basis of an adult patient weighing 70 kg, and may be administered once a day, It may be divided into several doses.

이상에서 설명한 바와 같은 본 발명은 하기의 실시예 및 실험예를 통해 보다 구체적으로 설명하기로 한다. 다만, 하기의 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기의 실시예 및 실험예에 의해 한정되는 것은 아니다.
The present invention will now be described in more detail with reference to the following examples and experimental examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

[실시예][Example]

실시예 1: 메틸 (3S, 6S, 9S, 17R, 19S)-9-아세트아미도-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이사이클로[15.2.1]이코산-19-카복실레이트Example 1: Synthesis of methyl (3S, 6S, 9S, 17R, 19S) -9-acetamido-6- (hydroxymethyl) -2,5,8,12-tetraoxo- - (phosphonoxy) ethyl) -16-oxa-1,4,7,13-tetraaza- bicyclo [15.2.1]

하기와 같이 12단계의 합성과정을 통해 실시예 1의 화합물을 제조하였다.
The compound of Example 1 was prepared through the following 12 steps of synthesis.

계 1: (2S, 4R)-1-tert-부틸 2-메틸 4-(알릴록시)피롤리딘-1,2-디카복실레이트System 1: (2S, 4R) -l-tert-butyl 2-methyl 4- (allyloxy) pyrrolidine- 1,2-dicarboxylate

N-Boc-트랜스-4-하이드록시-L-프롤린 메틸 에스테르 (2.38 g, 9.70 mmol)를 DMF (40 mL)에 녹인 용액에, 0 ℃에서 NaH (582 mg, 60% in mineral oil, 14.5 mmol)를 가하였다. 20분 후, 알릴 브로마이드 (1.68 mL, 19.4 mmol)를 첨가하고 상온에서 8시간 동안 교반한 후 농축하였다. 잔사에 에틸아세테이트와 물을 가한 후, 수층을 여러 번 에틸아세테이트로 추출하였다. 유기층을 소금물로 씻어준 후, 마그네슘 설페이트로 건조하고 여과 및 농축하였다. 잔사를 컬럼 크로마토그래피 (실리카 겔, EtOAc : Hexane = 2:1)하여 무색 오일의 표제화합물 (1.80 g, 6.31 mmol, 수율 65%)을 얻었다. To a solution of N-Boc-trans-4-hydroxy-L-proline methyl ester (2.38 g, 9.70 mmol) in DMF (40 mL) was added NaH (582 mg, 60% in mineral oil, 14.5 mmol ) Was added. After 20 minutes, allyl bromide (1.68 mL, 19.4 mmol) was added and stirred at room temperature for 8 hours and then concentrated. Ethyl acetate and water were added to the residue, and the aqueous layer was extracted several times with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, filtered, and concentrated. The residue was subjected to column chromatography (silica gel, EtOAc: Hexane = 2: 1) to give the title compound (1.80 g, 6.31 mmol, yield 65%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 5.88(m, 1H), 5.28(d, 1H), 5.19(d, 1H), 4.34(t, 1H), 4.13-3.92(m, 2H), 3.97(m, 1H), 3.73(s, 3H), 3.74-3.60(m, 2H), 2.33-2.05(m, 2H), 1.41(s, 9H).
^{1 H NMR (400 MHz, CDCl} 3) δ 5.88 (m, 1H), 5.28 (d, 1H), 5.19 (d, 1H), 4.34 (t, 1H), 4.13-3.92 (m, 2H), 3.97 ( (s, 3H), 3.73 (s, 3H), 2.33-2.05 (m, 2H), 1.41 (s, 9H).

단계 2: (2S, 4R)-1-tert-부틸 2-메틸 4-(하이드록시에톡시)피롤리딘-1,2-디카복실레이트Step 2: (2S, 4R) -l-tert-Butyl 2-methyl 4- (hydroxyethoxy) pyrrolidine- 1,2-dicarboxylate

(2S, 4R)-1-tert-부틸 2-메틸 4-(알릴록시)피롤리딘-1,2-디카복실레이트(1.80 g, 6.31 mmol)와 N-메틸몰폴린-N-옥시드 (NMO; 813 mg, 6.94 mmol)에 THF/물 (28 mL, 3:1)을 가한 후, 2.5 중량% 사산화 오스뮴/tert-부탄올 용액 (3.26 mL, 0.252 mmol)을 상온에서 가하였다. 2시간 30분 후, 아황산나트륨 포화용액 (10 mL)을 가하였다. 반응액을 농축한 후 에틸아세테이트를 이용하여 추출하였다. 추출된 유기층을 소금물로 씻어준 후, 마그네슘 설페이트로 건조하고 여과 및 농축하여 무색의 디올 화합물을 얻었다. 상기에서 얻은 디올 화합물에 THF/물 (2:1, 30 mL)를 가한 후, 소듐 페리오데이트 (2.70 g, 12.62 mmol)를 0 ℃에서 가하였다. 2시간 동안 교반한 후, 에틸아세테이트를 이용하여 추출하였다. 추출된 유기층을 소금물로 씻어준 후, 마그네슘 설페이트로 건조하고 여과 및 농축하여 무색의 알데하이드 화합물을 얻었다. 상기에서 얻은 알데하이드 화합물을 메탄올 (40 mL)에 녹인 후, 소듐 보로하이드라이드 (408 mg, 10.78 mmol)를 0 ℃에서 가하였다. 30분 후, 반응물에 물을 가하여 반응을 종결시킨 후 농축시켰다. 얻어진 잔사에 에틸아세테이트와 물을 가한 뒤 여러 번 추출하였다. 추출된 유기층을 소금물로 씻어준 후, 마그네슘 설페이트로 건조하고 여과 및 농축하여 무색 오일의 표제화합물 (1.40 g, 4.84 mmol, 3단계 총수율 77%)을 얻었다. (1.80 g, 6.31 mmol) and N-methylmorpholine-N-oxide (prepared in Example 1) were added to a solution of (2S, Was added 2.5% by weight osmium tetroxide / tert-butanol solution (3.26 mL, 0.252 mmol) at room temperature, followed by addition of THF / water (28 mL, 3: 1). After 2 h 30 min, saturated sodium sulfite solution (10 mL) was added. The reaction mixture was concentrated and extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over magnesium sulfate, filtered and concentrated to obtain a colorless diol compound. THF / water (2: 1, 30 mL) was added to the diol compound obtained above, and sodium persodate (2.70 g, 12.62 mmol) was added at 0 ° C. After stirring for 2 hours, the mixture was extracted with ethyl acetate. The extracted organic layer was washed with brine, dried over magnesium sulfate, filtered, and concentrated to give a colorless aldehyde compound. The aldehyde compound obtained above was dissolved in methanol (40 mL), and then sodium borohydride (408 mg, 10.78 mmol) was added at 0 ° C. After 30 minutes, water was added to the reaction mixture to terminate the reaction, and the mixture was concentrated. Ethyl acetate and water were added to the obtained residue, followed by extraction several times. The extracted organic layer was washed with brine, dried over magnesium sulfate, filtered and concentrated to give the title compound (1.40 g, 4.84 mmol, three step total yield: 77%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 4.32(t, 1H), 4.11(m, 1H), 3.73(s, 3H), 3.62(m, 2H), 3.58(m, 2H), 3.49(m, 2H), 2.40-2.24(m, 1H), 2.09-2.04(m, 1H), 1.41(s, 9H).
^{1 H NMR (400 MHz, CDCl} 3) δ 4.32 (t, 1H), 4.11 (m, 1H), 3.73 (s, 3H), 3.62 (m, 2H), 3.58 (m, 2H), 3.49 (m, 2H), 2.40-2.24 (m, 1H), 2.09-2.04 (m, 1H), 1.41 (s, 9H).

단계 3: (2S, 4R)-1-tert-부틸 2-메틸 4-(2-아지도에톡시)피롤리딘-1,2-디카복실레이트Step 3: (2S, 4R) -l-tert-Butyl 2-methyl 4- (2-azidethoxy) pyrrolidine- 1,2-dicarboxylate

(2S,4R)-1-tert-부틸 2-메틸 4-(2-하이드록시에톡시)피롤리딘-1,2-다이카복실레이트 (1.40 g, 4.84 mmol)와 트리에틸아민 (TEA; 6.70 mL, 48.4 mmol)를 디클로로메탄 (30 mL)에 녹인 후, 0 ℃에서 메탄설포닐 클로라이드 (1.88 mL, 24.2 mmol)를 가하였다. 1 시간 후, 물을 가하여 반응을 종결하고 에틸아세테이트로 추출하였다. 유기층을 소금물로 씻어주고 마그네슘 설페이트로 건조한 후 농축하여 무색 오일의 메실레이트 화합물을 얻었다. 상기에서 얻은 메실레이트 화합물에 DMF (20 mL)를 가한 후, 소듐 아자이드 (1.89 g, 29.04 mmol)를 가하고 85 ℃에서 2시간 30 분동안 교반하였다. 반응물을 농축한 후 에틸아세테이트 (200 mL)를 가하였다. 수차례 물을 가하여 씻어준 후 (50 mL each, 5 times) 마그네슘 설페이트를 이용하여 건조하고 농축하였다. 잔사를 컬럼 크로마토그래피 (실리카 겔, EtOAc : Hexane = 1 : 1) 하여 무색 오일의 표제화합물 (1.29 g, 4.10 mmol, 2단계 총수율 85%)을 얻었다.Dicarboxylate (1.40 g, 4.84 mmol) and triethylamine (TEA; 6.70 g, 4.84 mmol) were added to a solution of (2S, mL, 48.4 mmol) was dissolved in dichloromethane (30 mL), and methanesulfonyl chloride (1.88 mL, 24.2 mmol) was added at 0 ° C. After 1 hour, water was added to terminate the reaction and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, and then concentrated to obtain a mesylate compound of a colorless oil. DMF (20 mL) was added to the mesylate compound obtained above, sodium azide (1.89 g, 29.04 mmol) was added, and the mixture was stirred at 85 ° C for 2 hours and 30 minutes. The reaction was concentrated and ethyl acetate (200 mL) was added. After washing several times with water (50 mL each, 5 times), it was dried with magnesium sulfate and concentrated. The residue was subjected to column chromatography (silica gel, EtOAc: Hexane = 1: 1) to give the title compound (1.29 g, 4.10 mmol, two step total yield 85%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 4.34(t, 1H), 4.12(m, 1H), 3.73(s, 3H), 3.66(m, 2H), 3.60(m, 2H), 3.35(m, 2H), 2.43-2.28(m, 1H), 2.11-2.02(m, 1H), 1.40(s, 9H).
^{1 H NMR (400 MHz, CDCl} 3) δ 4.34 (t, 1H), 4.12 (m, 1H), 3.73 (s, 3H), 3.66 (m, 2H), 3.60 (m, 2H), 3.35 (m, 2H), 2.43-2.28 (m, 1 H), 2.11-2.02 (m, 1 H), 1.40 (s, 9H).

단계 4: (2S, 4R)-1-tert-부틸 2-메틸 4-(2-아미노에톡시)피롤리딘-1,2-디카복실레이트Step 4: (2S, 4R) -l-tert-Butyl 2-methyl 4- (2-aminoethoxy) pyrrolidine- 1,2-dicarboxylate

(2S,4R)-1-tert-부틸 2-메틸 4-(2-아지도에톡시)피롤리딘-1,2-다이카복실레이트 (2.04 g, 7.64 mmol)를 THF/물 (31 mL, 20:1)에 녹인 후, 트리페닐포스핀 (4.0 g, 15.3 mmol)를 가하였다. 50 ℃에서 2시간 동안 교반한 후, 반응물을 에틸아세테이트를 이용하여 추출하고 소금물로 씻어준 후 마그네슘 설페이트를 이용하여 건조한 후 농축하였다. 잔사를 컬럼 크로마토그래피 (실리카 겔, 디클로로메탄 : MeOH = 10 : 1)하여 무색 오일의 표제화합물 (1.95 g, 6.76 mmol, 수율 88%)을 얻었다. (2.04 g, 7.64 mmol) was dissolved in THF / water (31 mL, 1.44 mmol) 20: 1), triphenylphosphine (4.0 g, 15.3 mmol) was added. After stirring at 50 ° C for 2 hours, the reaction product was extracted with ethyl acetate, washed with brine, dried over magnesium sulfate, and then concentrated. The residue was subjected to column chromatography (silica gel, dichloromethane: MeOH = 10: 1) to give the title compound (1.95 g, 6.76 mmol, yield 88%) as a colorless oil.

¹H NMR (400 MHz, CDCl₃) δ 4.32(t, 1H), 4.09(m, 1H), 3.72(s, 3H), 3.64-3.44(m, 2H), 3.46(m, 2H), 2.85(m, 2H), 2.38-2.29(m, 1H), 2.09-2.02(m, 1H), 1.41(s, 9H, rotamer); ¹³C NMR (125 MHz, CD₃OD, rotamer) δ 174.3, 154.5, 80.3, 77.1, 69.2, 58.2 51.6, 48.7, 40.9, 36.0, 27.3.
^{1 H NMR (400 MHz, CDCl} 3) δ 4.32 (t, 1H), 4.09 (m, 1H), 3.72 (s, 3H), 3.64-3.44 (m, 2H), 3.46 (m, 2H), 2.85 ( m, 2H), 2.38-2.29 (m, 1H), 2.09-2.02 (m, 1H), 1.41 (s, 9H, rotamer); ¹³ C NMR (125 MHz, CD ₃ OD, rotamer)? 174.3, 154.5, 80.3, 77.1, 69.2, 58.2 51.6, 48.7, 40.9, 36.0, 27.3.

단계 5: (2S, 3R)-알릴 2-(tert-부톡시카보닐아미노)-3-하이드록시부타노에이트Step 5: (2S, 3R) -Allyl 2- (tert-butoxycarbonylamino) -3-hydroxybutanoate

Boc-L-쓰레오닌 (4.0 g, 18.2 mmol)과 다이이소프로필에틸아민 (DIPEA; 3.17 mL, 18.2 mmol)를 DMF (40 mL)에 녹인 후, 알릴 브로마이드 (2.20 g, 18.2 mmol)를 가하였다. 상온에서 15시간 교반 후, DMF를 증발시키고 에틸아세테이트를 가하였다. 유기층을 물로 여러 번 씻어준 후 마그네슘 설페이트로 건조한 후 농축하였고, 얻어진 잔사를 컬럼 크로마토그래피 (실리카 겔, EtOAc:Hexane=1:1)를 통해 정제하여 표제화합물 (2.84 g, 11.0 mmol, 수율 60%)을 얻었다. (2.20 g, 18.2 mmol) was dissolved in DMF (40 mL), followed by the addition of diisopropylethylamine (DIPEA, 3.17 mL, 18.2 mmol) and Boc-L- threonine Respectively. After stirring at room temperature for 15 hours, DMF was evaporated and ethyl acetate was added. The organic layer was washed with water several times, dried over magnesium sulfate and concentrated. The obtained residue was purified by column chromatography (silica gel, EtOAc: Hexane = 1: 1) to give the title compound (2.84 g, 11.0 mmol, ).

¹H NMR (400 MHz, CD₃OD) δ 5.79(m, 1H), 5.38(d, 1H), 5.23(d, 1H), 4.66(d, 2H), 4.26(m, 1H), 4.14(d, 1H), 1.46(s, 9H), 1.20(d, 3H).
¹ H NMR (400 MHz, CD ₃ OD)? 5.79 (m, IH), 5.38 (d, IH), 5.23 (d, , 1.46 (s, 9H), 1.20 (d, 3H).

단계 6: (2S, 3R)-알릴 2-((S)-3-(벤질옥시)-2-(tert-부톡시카보닐아미노)프로판아미도)-3-하이드록시부타노에이트Step 6: (2S, 3R) -Allyl 2 - ((S) -3- (benzyloxy) -2- (tert- butoxycarbonylamino) propanamido) -3-hydroxybutanoate

4 N HCl/다이옥산 용액 (2.5 mL)을 (2S, 3R)-알릴 2-(tert-부톡시카보닐아미노)-3-하이드록시부타노에이트 (555 mg, 2.14 mmol)에 가하였다. 상온에서 30분간 교반 후 농축하였고, 얻어진 잔사를 이소프로필에테르/헥산을 이용하여 고체화하여 백색의 고체 화합물을 얻었다. 상기에서 얻은 백색의 고체 화합물을 THF (10 mL)에 녹인 후 DIPEA (1.12 mL, 6.42 mmol)와 Boc-Ser(Bn)-OSu ((S)-2,5-다이옥소피롤리딘-1-일 3-(벤질옥시)-2-(tert-부톡시카보닐아미노)프로파노에이트, 840 mg, 2.14 mmol)를 가하고 상온에서 교반하였다. 1시간 후, 물을 가하여 반응을 종결시키고 에틸아세테이트로 추출하였다. 유기층을 소금물로 씻어준 후 마그네슘 설페이트를 가하여 건조하고 농축하였다. 얻은 잔사를 컬럼 크로마토그래피 (실리카 겔, EtOAc:Hexane=1:1) 하여 표제화합물 (940 mg, 2.11 mmol, 98% yield)을 얻었다. 4 N HCl / dioxane solution (2.5 mL) was added to (2S, 3R) -allyl 2- (tert-butoxycarbonylamino) -3-hydroxybutanoate (555 mg, 2.14 mmol). After stirring at room temperature for 30 minutes, the mixture was concentrated, and the obtained residue was solidified using isopropyl ether / hexane to obtain a white solid compound. The solid white compound obtained above was dissolved in THF (10 mL), and then DIPEA (1.12 mL, 6.42 mmol) and Boc-Ser (Bn) -OSu ((S) -2,5-dioxopyrrolidin- 3- (benzyloxy) -2- (tert-butoxycarbonylamino) propanoate (840 mg, 2.14 mmol) was added and stirred at room temperature. After 1 hour, water was added to terminate the reaction and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, and concentrated. The resulting residue was subjected to column chromatography (silica gel, EtOAc: Hexane = 1: 1) to give the title compound (940 mg, 2.11 mmol, 98% yield).

HPLC Retention time (condition A): 3.66min; ¹H NMR (400 MHz, CD₃OD) δ 7.37-7.27(m, 5H), 5.98-5.89(m, 1H), 5.34(d, 1H), 5.21(d, 1H), 4.64(m, 2H), 4.55(d, 2H), 4.51(d, 1H), 4.38(m, 1H), 4.31(m, 1H), 3.73(dd, 2H), 1.46(s, 9H), 1.60(d, 3H); ¹³C NMR (125 MHz, CD₃OD δ 172.0, 170.1, 156.5, 138.0, 132.1, 128.2, 127.7, 127.5, 117.4, 79.8, 73.1, 69.8, 67.4, 65.7, 58.0, 54.9, 27.7, 19.1; LRMS [M+1] 437.62(found), 437.22(calc.).
HPLC Retention time (condition A): 3.66 min; ^{1 H NMR (400 MHz, CD} 3 OD) δ 7.37-7.27 (m, 5H), 5.98-5.89 (m, 1H), 5.34 (d, 1H), 5.21 (d, 1H), 4.64 (m, 2H) 2H), 4.55 (d, 2H), 4.51 (d, 1H), 4.38 (m, 1H), 4.31 (m, 1H), 3.73 (dd, 2H), 1.46 ^{13 C NMR (125 MHz, CD} 3 OD δ 172.0, 170.1, 156.5, 138.0, 132.1, 128.2, 127.7, 127.5, 117.4, 79.8, 73.1, 69.8, 67.4, 65.7, 58.0, 54.9, 27.7, 19.1; LRMS [M +1] 437.62 (found), 437.22 (calc.).

단계 7: (5S, 8S, 11S)-알릴 8-(벤질옥시메틸)-5-(3-tert-부톡시-3-옥소프로필)-1-(9H-플루오렌-9-일)-11-((R)-1-하이드록시에틸)-3,6,9-트리옥소-2-옥사-4,7,10-트리아자도데칸-12-오에이트Step 7: (5S, 8S, llS) -Allyl 8- (benzyloxymethyl) -5- (3- tert -butoxy-3-oxopropyl) -1- (9H-fluoren- - ((R) -1-hydroxyethyl) -3,6,9-trioxo-2-oxa-4,7,10-triazododecan-12-

(2S, 3R)-알릴 2-((S)-3-(벤질옥시)-2-(tert-부톡시카보닐아미노)프로판아미도)-3-하이드록시부타노에이트 (3.00 g, 6.87 mmol)에 4 N HCl/다이옥산 용액 (10 mL)을 가하였다. 상온에서 30분간 교반한 후 농축하였다. 이소프로필에테르/헥산을 이용하여 고체화한 후 농축하여 (2S,3R)-알릴 2-((S)-2-아미노-3-(벤질옥시)프로판아미도)-3-하이드록시부타노에이트 염화수소를 백색 고체로 얻었다. [HPLC Retention time: 2.17 min, [M+1] 337.56(found), 337.17(calc.)] 상기에서 얻은 백색 고체 화합물과 DIPEA(3.57 mL, 20.52 mmol)를 THF (30 mL)에 녹인 다음, Fmoc-Glu(t-Bu)-OSu ((S)-5-tert-부틸 1-(2,5-다이옥소피롤리딘-1-일) 2-(((9H-플루오렌-9-일)메톡시)카보닐아미노)펜탄다이오에이트, 3.57 g, 6.84 mmol)를 상온에서 가하였다. 1시간 동안 교반한 후 물을 넣어서 반응을 종결시키고 에틸아세테이트를 이용하여 추출하였다. 유기층은 소금물을 이용하여 씻어준 후, 마그네슘 설페이트를 이용하여 건조하고 농축하였다. 잔사를 아세톤/이소프로필에테르에서 결정화하여 표제화합물 (4.64 g, 6.24 mmol, 수율 91%)을 얻었다. (3S, 3R) -allyl 2 - ((S) -3- (benzyloxy) -2- (tert- butoxycarbonylamino) propanamido) -3-hydroxybutanoate ) Was added 4 N HCl / dioxane solution (10 mL). The mixture was stirred at room temperature for 30 minutes and then concentrated. Solidified using isopropyl ether / hexane and then concentrated to give (2S, 3R) -allyl 2 - ((S) -2-amino-3- (benzyloxy) propanamido) -3- &Lt; / RTI > as a white solid. The white solid compound obtained above and DIPEA (3.57 mL, 20.52 mmol) were dissolved in THF (30 mL), and then Fmoc (0.25 g, -Glu (t-Bu) -OSu ((S) -5-tert-butyl 1- (2,5-dioxopyrrolidin- Carbonylamino) pentane diioate, 3.57 g, 6.84 mmol) at room temperature. After stirring for 1 hour, the reaction was terminated by adding water and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, and concentrated. The residue was crystallized from acetone / isopropyl ether to give the title compound (4.64 g, 6.24 mmol, 91% yield).

HPLC Retention time (condition A): 4.67min; ¹H NMR (400 MHz, CD₃OD) δ 7.78(d, 2H), 7.64(t, 2H), 7.38(t, 2H), 7.31-7.21(m, 7H), 5.98-5.89(m, 1H), 5.34(d, 1H), 5.21(d, 1H), 4.69(t, 1H), 4.62(d, 2H), 4.52(d, 2H), 4.51(m, 1H), 4.35(t, 2H), 4.31(m, 1H), 4.20(m, 2H), 3.73(m, 2H), 2.34(t, 2H), 2.18-1.86(m, 2H), 1.44(s, 9H), 1.12(d, 3H); ¹³C NMR (125 MHz, CD₃OD) δ 173.0, 172.8, 171.1, 170.2, 157.3, 144.1, 143.9, 141.4, 137.9, 132.1, 128.1, 127.7, 127.7, 127.5, 127.0, 125.1, 119.7, 117.4, 80.6, 73.1, 69.5, 68.9, 67.3, 66.9, 65.7, 62.7, 58.2, 54.4, 53.4, 47.2, 33.5, 31.4, 27.2, 25.5, 21.9, 19.2; LRMS [M+1] 744.86(found), 744.34(calc.)
HPLC Retention time (condition A): 4.67 min; ^{1 H NMR (400 MHz, CD} 3 OD) δ 7.78 (d, 2H), 7.64 (t, 2H), 7.38 (t, 2H), 7.31-7.21 (m, 7H), 5.98-5.89 (m, 1H) 2H), 4.52 (d, 2H), 4.51 (m, 1H), 4.35 (t, 2H) 2H), 1.24 (m, 2H), 2.32 (m, 2H), 2.31 (m, ; ^{13 C NMR (125 MHz, CD} 3 OD) δ 173.0, 172.8, 171.1, 170.2, 157.3, 144.1, 143.9, 141.4, 137.9, 132.1, 128.1, 127.7, 127.7, 127.5, 127.0, 125.1, 119.7, 117.4, 80.6, 73.1, 69.5, 68.9, 67.3, 66.9, 65.7, 62.7, 58.2, 54.4, 53.4, 47.2, 33.5, 31.4, 27.2, 25.5, 21.9, 19.2; LRMS [M + 1] 744.86 (found), 744.34 (calc.)

단계 8: (2S, 4R)-1-tert-부틸 2-메틸 4-((7S, 10S, 13S)-7-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-10-(벤질옥시메틸)-13-((R)-1-하이드록시에틸)-4,8,11,14-테트라옥소-15-옥사-3,9,12-트리아자옥타덱-17-에닐록시)피롤리딘-1,2-디카복실레이트Step 8: (2S, 4R) -l-tert-Butyl 2-methyl 4 - ((7S, 10S, 13S) -7 - ((9H- fluoren-9- yl) methoxy) carbonylamino) 13 - ((R) -1-hydroxyethyl) -4,8,11,14-tetraoxo-15-oxa- Ethynyloxy) pyrrolidine-1, 2-dicarboxylate

(5S, 8S, 11S)-알릴 8-(벤질옥시메틸)-5-(3-tert-부톡시-3-옥소프로필)-1-(9H-플루오렌-9-일)-11-((R)-1-하이드록시에틸)-3,6,9-트리옥소-2-옥사-4,7,10-트리아자도데칸-12-오에이트 (1.57 g, 2.11 mmol)에, 20% 트리플루오로아세틱 에시드/디클로로메탄 용액(15 mL)을 가하였다. 상온에서 1시간 동안 교반한 후 농축하였다. 잔사를 톨루엔/헥산으로 다시 한번 농축시켜 (S)-4-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-5-((S)-1-((2S,3R)-1-(알릴옥시)-3-하이드록시-1-옥소부탄-2-일아미노)-3-(벤질옥시)-1-옥소프로판-2-일아미노)-5-옥소펜타노익 에시드 (1.41 g, 2.05 mmol)을 얻었다. [HPLC Retention time (condition A): 3.90 min, [M+1] 688.75(found), 688.28(calc.)] 상기에서 얻은 (S)-4-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-5-((S)-1-((2S,3R)-1-(알릴옥시)-3-하이드록시-1-옥소부탄-2-일아미노)-3-(벤질옥시)-1-옥소프로판-2-일아미노)-5-옥소펜타노익 에시드 (1.41 g, 2.05 mmol)를 THF (15 mL)에 녹인 후 DIPEA (0.71 mL, 4.10 mmol)와 (2S,4R)-1-tert-부틸 2-메틸 4-(2-아미노에톡시)피롤리딘-1,2-다이카복실레이트 (591 mg, 2.05 mmol), 1-[비스(다이메틸아미노)메틸렌]-1H-1,2,3-트리아졸로[4,5-b]피리디늄 3-옥사이드 헥사플루오로포스페이트 (HATU; 1.56 g, 4.10 mmol)를 상온에서 가하였다. 1시간 동안 교반한 후 물을 가하여 반응을 종결시키고 에틸아세테이트로 추출하였다. 유기층을 소금물을 이용하여 씻어주고 마그네슘 설페이트를 이용하여 건조 후 농축하였다. 잔사를 컬럼 크로마토그래피 (실리카 겔, 디클로로메탄 : MeOH = 10:1)를 통해 정제하여 표제화합물 (1.95 g, 2.04 mmol, 수율 99%)을 얻었다. (5S, 8S, 11S) -allyl 8- (benzyloxymethyl) -5- (3-tert-butoxy-3-oxopropyl) -1- (9H-fluoren- (1.57 g, 2.11 mmol) was added to a solution of 20% trifluoroacetic acid in toluene A solution of ro acetic acid / dichloromethane (15 mL) was added. The mixture was stirred at room temperature for 1 hour and then concentrated. The residue was again concentrated with toluene / hexane to give (S) -4 - (((9H-fluoren-9-yl) methoxy) carbonylamino) -5 - ) -1- (allyloxy) -3-hydroxy-1-oxobutan-2-ylamino) -3- (benzyloxy) -1-oxopropan-2- ylamino) -5-oxopentanoic acid 1.41 g, 2.05 mmol). (S) -4 - ((9H-Fluoren-9-yl) methane obtained in the above-mentioned manner) [HPLC Retention time (condition A): 3.90 min, [M + 1] 688.75 (found), 688.28 3-hydroxy-1-oxobutan-2-ylamino) -3- (benzyl) (1.41 g, 2.05 mmol) was dissolved in THF (15 mL), and DIPEA (0.71 mL, 4.10 mmol) and (2S, 4R) -1-tert-butyl 2-methyl 4- (2-aminoethoxy) pyrrolidine-1,2-dicarboxylate (591 mg, 2.05 mmol), 1- [bis (dimethylamino) methylene] -1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate (HATU; 1.56 g, 4.10 mmol) was added at room temperature. After stirring for 1 hour, water was added to terminate the reaction and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and concentrated. The residue was purified by column chromatography (silica gel, dichloromethane: MeOH = 10: 1) to give the title compound (1.95 g, 2.04 mmol, yield 99%).

HPLC Retention time (condition A): 4.30 min; ¹H NMR (400 MHz, CD₃OD) δ 7.80(d, J=7.5 Hz, 2H; Fmoc-CH), 7.67(t, J=7.5 Hz, 2H; Fmoc-CH), 7.39(t, J=7.5 Hz, 2H; Fmoc-CH), 7.33-7.21(m, 7H; 벤질 and Fmoc-CH), 5.98-5.88(m, 1H; 알릴-CH-), 5.33(dd, J=17.2, 1.3 Hz, 1H; 알릴-CH), 5.21(d, J=10.5 Hz, 1H; 알릴-CH'), 4.69(m, 1H; Ser-Ca-H), 4.63(d, J=5.5 Hz, 2H; 알릴-CH₂-), 4.54(d, J=4.5 Hz, 2H; 벤질-CH₂-), 4.51(m, 1H; Thr-Ca-H), 4.37-4.28(m, 5H; Fmoc-CH₂-O, Glu-Ca-H, Thr-Ca-H, Pro-Cd-H), 4.23-4.18(m, 2H; Thr-Cb-H, Fmoc-CH-), 4.10(m, 1H; Pro-Cc-H), 3.80(m, 2H; Ser-Cb-H,H'), 3.69(s, 3H; OMe), 3.50-3.35(m, 5H; X-Cb-H,H', X-Ca-H,H', Pro-Cd-H'), 2.37-2.25(m, 3H; Glu-Cc-H,H', Pro-Cb-H), 2.11-1.95(m, 3H; Pro-Cb-H', Glu-Cb-H,H'), 1.45-1.38(4s, 9H; Boc,rotamer), 1.17(d, J=6.2 Hz, 3H; Thr-Cc-H); LRMS [M+1] 958.90(found), 958.44(calc.).
HPLC Retention time (condition A): 4.30 min; ^{1 H NMR (400 MHz, CD} 3 OD) δ 7.80 (d, J = 7.5 Hz, 2H; Fmoc-CH), 7.67 (t, J = 7.5 Hz, 2H; Fmoc-CH), 7.39 (t, J = J = 17.2, 1.3 Hz, 2H), 7.33-7.21 (m, 7H, benzyl and Fmoc-CH), 5.98-5.88 (m, 1H, allyl-CH), 5.21 (d, J = 10.5 Hz, 1H; allyl-CH '), 4.69 _{CH 2 -), 4.54 (d} , J = 4.5 Hz, 2H; benzyl _{-CH 2 -), 4.51 (m} , 1H; Thr-Ca-H), 4.37-4.28 (m, 5H; Fmoc-CH 2 -O , 4.10 (m, 1H, Pro-Cb-H, Thr-Ca-H, Thr-Ca-H, Pro-Cd-H), 4.23-4.18 H), X-Ca-H (3H, O), 3.80 (m, 2H, Ser-Cb-H, H, Pro-Cd-H '), 2.37-2.25 (m, 3H; Glu- , Glu-Cb-H, H '), 1.45-1.38 (4s, 9H; Boc, rotamer), 1.17 (d, J = 6.2 Hz, 3H; Thr-Cg-H); LRMS [M + 1] 958.90 (found), 958.44 (calc.).

단계 9: (3S,6S,9S,17R,19S)-메틸 9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복실레이트Step 9: (3S, 6S, 9S, 17R, 19S) -methyl 9 - (((9H-fluoren- R) -1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo [15.2.1]

(5S,8S,11S)-알릴 8-(벤질옥시메틸)-5-(3-tert-부톡시-3-옥소프로필)-1-(9H-플루오렌-9-일)-11-((R)-1-하이드록시에틸)-3,6,9-트리옥소-2-옥사-4,7,10-트리아자도데칸-12-오에이트 (1.57 g, 2.11 mmol)에, 20% 트리플루오로아세틱 에시드/디클로로메탄 용액 (15 mL)을 가하였다. 상온에서 1시간 동안 교반한 후 농축하였다. 잔사를 톨루엔/헥산으로 다시 한번 농축시켜 (S)-4-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-5-((S)-1-((2S,3R)-1-(알릴옥시)-3-하이드록시-1-옥소부탄-2-일아미노)-3-(벤질옥시)-1-옥소프로판-2-일아미노)-5-옥소펜타노익 에시드 (1.41 g, 2.05 mmol)을 얻었다. [HPLC Retention time (condition A): 3.90 min, [M+1] 688.75(found), 688.28(calc.)] 얻어진 (S)-4-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-5-((S)-1-((2S,3R)-1-(알릴옥시)-3-하이드록시-1-옥소부탄-2-일아미노)-3-(벤질옥시)-1-옥소프로판-2-일아미노)-5-옥소펜타노익 에시드 (1.41 g, 2.05 mmol)를 THF (15 mL)에 녹인 후 DIPEA (0.71 mL, 4.10 mmol)와 (2S,4R)-1-tert-부틸 2-메틸 4-(2-아미노에톡시)피롤리딘-1,2-다이카복실레이트 (591 mg, 2.05 mmol), HATU (1.56 g, 4.10 mmol)를 상온에서 가하였다. 1시간 동안 교반한 후 물을 가하여 반응을 종결시키고 에틸아세테이트로 추출하였다. 유기층을 소금물을 이용하여 씻어주고 마그네슘 설페이트를 이용하여 건조 후 농축하였다. 잔사를 컬럼 크로마토그래피 (실리카 겔, 디클로로메탄 : MeOH = 10:1)를 통해 정제하여 표제화합물 (1.95 g, 2.04 mmol, 수율 99%)을 얻었다. (5S, 8S, 11S) -allyl 8- (benzyloxymethyl) -5- (3-tert-butoxy-3-oxopropyl) -1- (9H-fluoren- (1.57 g, 2.11 mmol) was added to a solution of 20% trifluoroacetic acid in toluene A solution of ro acetic acid / dichloromethane (15 mL) was added. The mixture was stirred at room temperature for 1 hour and then concentrated. The residue was again concentrated with toluene / hexane to give (S) -4 - (((9H-fluoren-9-yl) methoxy) carbonylamino) -5 - ) -1- (allyloxy) -3-hydroxy-1-oxobutan-2-ylamino) -3- (benzyloxy) -1-oxopropan-2- ylamino) -5-oxopentanoic acid 1.41 g, 2.05 mmol). The obtained (S) -4 - ((9H-fluoren-9-yl) methoxy) 3- (benzyloxy) -3-hydroxy-1-oxobutan-2-ylamino) (1.41 g, 2.05 mmol) was dissolved in THF (15 mL), and then DIPEA (0.71 mL, 4.10 mmol) and (2S, 4R) - 1 -oxopropane-2-ylamino) -5-oxopentanoic acid dicarboxylate (591 mg, 2.05 mmol) and HATU (1.56 g, 4.10 mmol) were added at room temperature. After stirring for 1 hour, water was added to terminate the reaction and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and concentrated. The residue was purified by column chromatography (silica gel, dichloromethane: MeOH = 10: 1) to give the title compound (1.95 g, 2.04 mmol, yield 99%).

단계 10: (3S,6S,9S,17R,19S)-메틸 9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴록시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복실레이트Step 10: (3S, 6S, 9S, 17R, 19S) -methyl 9 - (((9H-fluoren-9- yl) methoxy) carbonylamino) -6- (benzyloxymethyl) -3- R) -1- (Bis (benzyloxy) phospholyloxy) ethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo [ ] Isoic acid-19-carboxylate

(3S,6S,9S,17R,19S)-메틸 9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복실레이트 (200 mg, 0.25 mmol)와 1H-테트라졸 (53 mg, 0.75 mmol)을 디클로로메탄 (3 mL)에 녹인 다음, 다이벤질 N,N-다이이소프로필포스포아미다이트 (90% tech. grade, 259 mg, 0.75 mmol)를 가하였다. 1시간 동안 교반한 후 물을 가하여 반응을 종결하고 디클로로메탄으로 추출하였다. 유기층을 소금물로 씻어준 후 마그네슘 설페이트를 가하여 건조하고 농축하였다. 얻어진 잔사에 디클로로메탄 (3 mL)을 가하고 0 ℃로 냉각한 후 m-클로로퍼벤조익 에시드 (129 mg, 0.75 mmol)를 가하고 1시간 동안 교반하였다. 물을 가하여 반응을 종결시킨 후 혼합물을 디클로로메탄으로 추출하였다. 유기층을 소금물로 씻어준 후 마그네슘 설페이트를 가하여 건조하고 농축한 후 컬럼 크로마토그래피 (실리카 겔, EtOAc:Hexane:MeOH = 8:1:1→16:8:1)를 이용해 정제하여 표제화합물 (87 mg, 0.082 mmol, 2단계 총수율 33%)을 얻었다. (3S, 6S, 9S, 17R, 19S) -methyl 9 - ((9H-fluoren-9-yl) methoxy) carbonylamino) -6- (benzyloxymethyl) -3- 1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo [15.2.1] isoic acid- 19-carboxylate , 0.25 mmol) and 1H-tetrazole (53 mg, 0.75 mmol) were dissolved in dichloromethane (3 mL) and then dibenzyl N, N- diisopropylphosphoramidite (90% , 0.75 mmol). After stirring for 1 hour, water was added to terminate the reaction and extracted with dichloromethane. The organic layer was washed with brine, dried over magnesium sulfate, and concentrated. Dichloromethane (3 mL) was added to the resulting residue, and the mixture was cooled to 0 ° C, m-chloroperbenzoic acid (129 mg, 0.75 mmol) was added, and the mixture was stirred for 1 hour. Water was added to terminate the reaction and the mixture was extracted with dichloromethane. The organic layer was washed with brine, dried over magnesium sulfate, concentrated, and then purified by column chromatography (silica gel, EtOAc: Hexane: MeOH = 8: 1: 1 to 16: 8: 1) to obtain the title compound , 0.082 mmol, two step total yield 33%).

HPLC Retention time (condition A): 4.51 min, LRMS [M+1] 1061.08(found), 1060.40(calc.)HPLC Retention time (condition A): 4.51 min, LRMS [M + 1] 1061.08 (found), 1060.40 (calc.

단계 11: (3S,6S,9S,17R,19S)-메틸 9-아세트아미도-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴록시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복실레이트Step 11: (3S, 6S, 9S, 17R, 19S) -methyl 9-acetamido-6- (benzyloxymethyl) -3 - ((R) -1- (bis (benzyloxy) phospholyloxy) ethyl ) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza-bicyclo [15.2.1] isoic acid-

(3S,6S,9S,17R,19S)-메틸 9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴옥시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복실레이트 (87 mg, 0.082 mmol)에 1% 피페리딘/THF 용액 (0.7 mL)을 가한 후 상온에서 15시간 동안 교반하였다. 그 후 농축하고 톨루엔을 이용하여 재농축하여 (3S,6S,9S,17R,19S)-메틸 9-아미노-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴옥시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복실레이트를 얻었다. [HPLC Retention time (condition A): 2.94 min, LRMS [M+1] 838.99(found), 838.34(calc.)] 별도의 정제 없이 다음 반응에 이용하였다. 상기에서 얻은 (3S,6S,9S,17R,19S)-메틸 9-아미노-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴옥시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복실레이트를 디클로로메탄/물 용액 (0.65 mL, 10:3)에 녹인 후, 상온에서 소듐 카보네이트 (9.6 mg, 0.902 mmol)를 가하였다. 반응물에 아세틸 클로라이드 (9.7 mg, 0.123 mmol)를 가한 후 35 ℃로 가온하였다. 1시간 동안 교반한 후 디클로로메탄과 물을 가한 후 추출하였다. 유기층을 소금물로 씻어준 후 마그네슘 설페이트를 이용하여 건조, 농축하였다. 잔사를 컬럼 크로마토그래피 (실리카 겔, EtOAc:Hexane:MeOH = 8:1:1)응 이용해 정제하여 표제화합물 (35 mg, 0.040 mmol, 2단계 총수율 49%)을 얻었다. (3S, 6S, 9S, 17R, 19S) -methyl 9 - ((9H-fluoren-9-yl) methoxy) carbonylamino) -6- (benzyloxymethyl) -3- (Bis (benzyloxy) phospholyloxy) ethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo [15.2.1] -1-carboxylate (87 mg, 0.082 mmol) was added 1% piperidine / THF solution (0.7 mL), and the mixture was stirred at room temperature for 15 hours. The mixture was concentrated and re-concentrated using toluene to obtain (3S, 6S, 9S, 17R, 19S) -methyl 9-amino-6- (benzyloxymethyl) -3 - ((R) ) Phosphoryloxy) ethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo [15.2.1] icoc acid- 19-carboxylate . [HPLC Retention time (condition A): 2.94 min, LRMS [M + 1] 838.99 (found), 838.34 (calc.)] To a solution of (3S, 6S, 9S, 17R, 19S) -methyl 9-amino-6- (benzyloxymethyl) -3 - ((R) -1- (bis (benzyloxy) Oxa-1,4,7,13-tetraaza-bicyclo [15.2.1] isoic acid-19-carboxylate was dissolved in a dichloromethane / water solution (0.65 mL, 10: 3), and sodium carbonate (9.6 mg, 0.902 mmol) was added thereto at room temperature. Acetyl chloride (9.7 mg, 0.123 mmol) was added to the reaction, followed by heating to 35 占 폚. After stirring for 1 hour, dichloromethane and water were added and extracted. The organic layer was washed with brine, dried over magnesium sulfate, and concentrated. The residue was purified by column chromatography (silica gel, EtOAc: Hexane: MeOH = 8: 1: 1) to give the title compound (35 mg, 0.040 mmol, 49% overall yield in two steps).

HPLC Retention time (condition A): 3.45 min, LRMS [M+1] 880.72(found), 880.35(calc.)
HPLC Retention time (condition A): 3.45 min, LRMS [M + 1] 880.72 (found), 880.35 (calc.

단계 12: (3S,6S,9S,17R,19S)-메틸 9-아세트아미도-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복실레이트 (실시예 1 화합물)Step 12: (3S, 6S, 9S, 17R, 19S) -methyl 9-acetamido-6- (hydroxymethyl) -2,5,8,12-tetraoxo- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetraaza-bicyclo [15.2.1]

(3S,6S,9S,17R,19S)-메틸 9-아세트아미도-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴옥시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복실레이트 (35 mg, 0.040 mmol)을 에탄올/물 용액(1.5 mL, 2:1)에 녹인 후, Pd/C (10 중량%, 7 mg)을 가하였다. 혼합물을 수소가스 (1 atm)하에서 12시간 동안 교반한 후, Pd/C (40 mg)을 더 가하고 4시간 동안 교반하였다. 셀라이트 패드를 통해 여과한 후 에탄올/물 (2/1) 용액으로 여러 번 씻어준 후 농축하고, 얻어진 잔사를 preparatory HPLC로 정제하여 표제화합물 (14.7 mg, 0.024 mmol, 60% yield)을 얻었다. (3S, 6S, 9S, 17R, 19S) -methyl 9-acetamido-6- (benzyloxymethyl) -3 - ((R) -1- (bis (benzyloxy) (35 mg, 0.040 mmol) was dissolved in a mixture of ethanol / water (10 ml) and water (1.5 mL, 2: 1), and Pd / C (10 wt%, 7 mg) was added thereto. The mixture was stirred under hydrogen gas (1 atm) for 12 hours, then further Pd / C (40 mg) was added and stirred for 4 hours. The filtrate was filtered through a pad of celite, washed several times with an ethanol / water (2/1) solution and then concentrated. The resulting residue was purified by preparatory HPLC to obtain the title compound (14.7 mg, 0.024 mmol, 60% yield).

HPLC Retention time (condition A): 1.56 min; ¹H NMR (900 MHz, DMSO-d₆) δ 9.20(br, 1H; Thr-NH), 8.18(d, J=7.2 Hz, 1H; Ser-NH), 7.91(d, J=8.1 Hz, 1H; Glu-NH), 7.05(m, 1H; X-NH), 4.35(m, 1H; Glu-Ca-H), 4.32(m, 1H; Ser-Ca-H), 4.24(m,3H; Pro-Ca-H, Thr-Ca-H, Thr-Cb-H), 4.19(d, J=10.8 Hz, 1H; Pro-Cd-H'), 4.09(m, 1H; Pro-Cc-H), 3.70(m, 1H; Ser-Cb-H'), 3.60(s, 3H; -OMe), 3.55(m, 2H; X-Cb-H', Ser-Cb-H), 3.43(m, 1H; X-Ca-H'), 3.40(t, J=7.2 Hz, 1H; X-Cb-H), 2.57(m, 1H; Glu-Cc-H'), 2.97(t, J=11.7 Hz, 1H; X-Ca-H), 2.24(m, 1H; Pro-Cb-H'), 2.01(m, 1H; Glu-Cc-H), 1.93(m, 1H; Pro-Cb-H), 1.87(m, 1H; Glu-Cb-H'), 1.82(s, 3H; -COMe), 1.62(m, 1H; Glu-Cb-H), 1.26(m, 3H; Thr-Cc-H); ¹³C NMR (125 MHz, CD₃OD)δ 174.3, 172.7, 172.6, 171.7, 171.1, 169.8, 78.3, 73.0, 68.4, 61.6, 58.6, 58.2, 55.5, 54.0, 52.6, 51.5, 39.3, 35.6, 31.4, 29.2, 21.1, 17.1; [M+1] 610.70(found), 610.20(calc.); 31P NMR (200MHz, DMSO-d6) δ 6.134; HRMS [M+2Na-H] 654.2350 (found), 654.1759 (calc.); [α]D20= -0.009 (c=0.1, MeOH).
HPLC Retention time (condition A): 1.56 min; ^{1 H NMR (900 MHz, DMSO} -d 6) δ 9.20 (br, 1H; Thr-NH), 8.18 (d, J = 7.2 Hz, 1H; Ser-NH), 7.91 (d, J = 8.1 Hz, 1H H), 4.24 (m, 3H, Pro: H-Glu-NH), 7.05 (D, J = 10.8 Hz, 1H, Pro-Cd-H '), 4.09 (m, 1H, Pro-Cc-H), Thr- 3.70 (m, 1H), 3.70 (m, 1H, Ser-Cb-H '), 3.60 (s, 3H; J = 11.7 Hz, 1H), 3.40 (t, J = 7.2 Hz, 1H; X-Cb-H) H), 1.93 (m, 1H, Pro-Cb-H), 1.87 (m, m, 1H, Glu-Cb-H '), 1.82 (s, 3H, -COMe), 1.62 (m, 1H, Glu-Cb-H), 1.26 (m, 3H, Thr-C? ^{13 C NMR (125 MHz, CD} 3 OD) δ 174.3, 172.7, 172.6, 171.7, 171.1, 169.8, 78.3, 73.0, 68.4, 61.6, 58.6, 58.2, 55.5, 54.0, 52.6, 51.5, 39.3, 35.6, 31.4, 29.2, 21.1, 17.1; [M + 1] 610.70 (found), 610.20 (calc.); 31 P NMR (200 MHz, DMSO-d 6)? 6.134; HRMS [M + 2Na-H] 654.2350 (found), 654.1759 (calc.); [[alpha]] D < 20 > = -0.009 (c = 0.1, MeOH).

실시예 2: 메틸 (3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-9-((S)-4-(메틸싸이오)-2-((S)-피롤리딘-2-카복스아미도)부탄아미도)-2,5,8,12-테트라옥소-3-( (R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카복실레이트Example 2: Synthesis of methyl (3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -9 - ((S) -4- (methylthio) -2- (R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7 , 13-tetraaza bicyclo [15.2.1] isoic acid-19-carboxylate

하기와 같이 4단계의 합성과정을 통해 실시예 2의 화합물을 제조하였다.
The compound of Example 2 was prepared by the following four-step synthesis procedure.

단계 1: (S)-벤질 2-((S)-1-메톡시-4-(메틸싸이오)-1-옥소부탄-2-일카바모일)피롤리딘-1-카복실레이트Step 1: (S) -Benzyl 2- ((S) -1-methoxy-4- (methylthio) -1-oxobutan- 2- ylcarbamoyl) pyrrolidine-

L-메티오닌 메틸 에스터 염화수소 (0.80 g, 4.01 mmol)를 THF (15 mL)에 녹인 후, N-Cbz-L-프로린 (1.00 g, 4.01 mmol)과 DIPEA (1.4 mL, 8.02 mmol), HATU (2.29 g, 6.02 mmol)를 상온에서 가하였다. 2시간 동안 교반한 후 반응물을 물에 쏟고, 에틸아세테이트를 이용하여 여러 번 추출하였다. 유기층을 소금물을 이용하여 씻어준 후 마그네슘 설페이트로 건조하고 여과하여 표제화합물을 얻었다.L-proline (1.00 g, 4.01 mmol), DIPEA (1.4 mL, 8.02 mmol), HATU (2.29 g, 4.01 mmol) were dissolved in THF (15 mL) g, 6.02 mmol) at room temperature. After stirring for 2 hours, the reaction was poured into water and extracted several times with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and filtered to give the title compound.

HPLC Retention time (condition A): 3.30 min, LRMS [M+1] 395.45 (found), 395.16 (calc.).
HPLC Retention time (condition A): 3.30 min, LRMS [M + 1] 395.45 (found), 395.16 (calc.).

단계 2: (S)-2-((S)-1-(벤질옥시카보닐)피롤리딘-2-카복스아미도)-4-(메틸싸이오)부타노익 에시드Step 2: (S) -2 - ((S) -1- (Benzyloxycarbonyl) pyrrolidine-2-carboxamido) -4- (methylthio) butanoic acid

(S)-벤질 2-((S)-1-메톡시-4-(메틸싸이오)-1-옥소부탄-2-일카바모일)피롤리딘-1-카복실레이트 (1.07 g, 2.71 mmol)의 THF (10 mL)용액에, 0 ℃에서 1 M LiOH 용액 (3.4 mL, 3.4 mmol)을 가하였다. 3시간 동안 교반한 후, 1 N HCl 용액을 이용하여 pH 4로 맞추었다. 반응물을 에틸아세테이트로 추출한 후 유기층을 마그네슘 설페이트로 건조하고 여과한 후 농축하였다. 잔사를 아세톤/이소프로필에테르 용매 하에 재결정하여 백색 고체의 표제 화합물 (1.02 g, 2.68 mmol, 2단계 수율 67% 수율)을 얻었다. (S) -benzyl 2 - ((S) -1-methoxy-4- (methylthio) -1-oxobutan- 2- ylcarbamoyl) pyrrolidine- 1-carboxylate (1.07 g, 2.71 mmol ) In THF (10 mL) was added 1 M LiOH solution (3.4 mL, 3.4 mmol) at 0 ° C. After stirring for 3 hours, the pH was adjusted to 4 with 1 N HCl solution. After the reaction was extracted with ethyl acetate, the organic layer was dried over magnesium sulfate, filtered, and concentrated. The residue was recrystallized in an acetone / isopropyl ether solvent to give the title compound as a white solid (1.02 g, 2.68 mmol, 67% yield in two steps).

HPLC Retention time (condition A): 2.95 min; ¹H NMR (400 MHz, CD₃OD) δ 7.33(m, 5H; phenyl-CH), 5.12(s, 2H; phenyl-CH₂-), 4.40(m, 1H; Pro-Ca-H), 4.34(m, 1H; Met-Ca-H), 3.51(m, 1H; Pro-Cd-H), 3.48(m, 1H; Pro-Cd-H'), 2.60(m, 1H; Pro-Cb-H'), 2.50-2.10(m, 3H; Pro-Cb-H, Met-Cb-H,H'), 2.09-1.88(m, 4H; Pro-Cc-H,H', Met-Cc-H,H'), 2.01(s, 3H; Met-SMe); LRMS [M+1] 381.46 (found), 381.14 (calc.).
HPLC Retention time (condition A): 2.95 min; ^{1 H NMR (400 MHz, CD} 3 OD) δ 7.33 (m, 5H; phenyl-CH), 5.12 (s, 2H; phenyl-CH 2 -), 4.40 (m, 1H; Pro-Ca-H), 4.34 (m, 1H, Met-Ca-H), 3.51 (m, 1H, Pro-Cd-H), 3.48 H), Met-Cb-H, H), 2.09-1.88 (m, 4H, Pro-Cb- H '), 2.01 (s, 3H, Met-SMe); LRMS [M + 1] 381.46 (found), 381.14 (calc.).

단계 3: (3S,6S,9S,17R,19S)-메틸 9-((S)-2-((S)-1-(벤질옥시카보닐)피롤리딘-2-카복스아미도)-4-(메틸싸이오)부타나미도)-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴록시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복실레이트Step 3: (3S, 6S, 9S, 17R, 19S) -methyl 9 - ((S) -2 - ((S) -1- (benzyloxycarbonyl) pyrrolidine- 2- carboxamido) 4- (methylthio) butanamide) -6- (benzyloxymethyl) -3 - ((R) -1- (bis (benzyloxy) phospholyloxy) ethyl) Tetraoxo-16-oxa-1,4,7,13-tetraaza-bicyclo [15.2.1] isoic acid-l9-carboxylate

(3S,6S,9S,17R,19S)-메틸 9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴옥시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복실레이트 (57 mg, 0.054 mmol)에 1% 피페리딘/THF 용액 (0.7 mL)을 가한 후 상온에서 15시간 동안 교반하였다. 그 후 농축하고 얻어진 잔사를 THF (2 mL)에 녹인 후, DIPEA (7 mg, 0.054 mmol)와 (S)-2-((S)-1-(벤질옥시카보닐)피롤리딘-2-카복스아미도)-4-(메틸싸이오)부타노익 에시드 (21 mg, 0.054 mmol), HATU (41 mg, 0.108 mmol)를 가하였다. 1시간 동안 교반한 후 물을 가해 반응을 종결시키고 에틸아세테이트를 이용하여 추출하였다. 유기층을 소금물을 이용하여 씻어준 후 마그네슘 설페이트를 이용하여 건조한 후 농축하였다. 잔사를 컬럼 크로마토그래피 (실리카 겔, EA:Hexane:MeOH = 8:1:1)를 이용해 정제하여 표제화합물 (24 mg, 0.020 mmol, 2단계 총수율 34%)을 얻었다. (3S, 6S, 9S, 17R, 19S) -methyl 9 - ((9H-fluoren-9-yl) methoxy) carbonylamino) -6- (benzyloxymethyl) -3- (Bis (benzyloxy) phospholyloxy) ethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo [15.2.1] -1-carboxylate (57 mg, 0.054 mmol) was added 1% piperidine / THF solution (0.7 mL), and the mixture was stirred at room temperature for 15 hours. The resulting residue was dissolved in THF (2 mL), and then DIPEA (7 mg, 0.054 mmol) and (S) -2 - ((S) -1- (benzyloxycarbonyl) pyrrolidin- (Methylthio) butanoic acid (21 mg, 0.054 mmol) and HATU (41 mg, 0.108 mmol). After stirring for 1 hour, water was added and the reaction was terminated and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, and then concentrated. The residue was purified by column chromatography (silica gel, EA: Hexane: MeOH = 8: 1: 1) to give the title compound (24 mg, 0.020 mmol, 34% overall yield in two steps).

HPLC Retention time (condition A): 4.11 min, LRMS [M+1] 1200.90(found), 1200.47(calc.)
HPLC Retention time (condition A): 4.11 min, LRMS [M + 1] 1200.90 (found), 1200.47 (calc.)

단계 4: 메틸 (3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-9-((S)-4-(메틸싸이오)-2-((S)-피롤리딘-2-카복스아미도)부탄아미도)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카복실레이트 (실시예 2의 화합물)Step 4: Methyl (3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -9 - ((S) -4- (methylthio) -2- 2-carboxamido) butanamido) -2,5,8,12-tetraoxo-3 - ((R) -1- (Phosphoroxy) ethyl) 13a-tetraaza-bicyclo [15.2.1] isoic acid-19-carboxylate (compound of Example 2)

(3S,6S,9S,17R,19S)-메틸 9-((S)-2-((S)-1-(벤질옥시카보닐)피롤리딘-2-카복스아미도)-4-(메틸싸이오)부탄아미도)-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴옥시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복실레이트 (67 mg, 0.056 mmol)을 에탄올/물 (3 mL, 2:1) 용액에 녹인 후, Pd/C (20 중량%, 130 mg)을 가하였다. 수소가스(1 atm)하에서 혼합물을 8시간 동안 교반한 후, 셀라이트 패드를 통해 여과하고 에탄올/물 (2:1) 로 씻어준 후 농축하였다. 잔사를 preparatory HPLC를 이용하여 정제하여 표제화합물 (10.3 mg, 0.013 mmol, 수율 23%)을 얻었다. ((S) -1- (benzyloxycarbonyl) pyrrolidine-2-carboxamido) -4- ( Methylthio) butanamido) -6- (benzyloxymethyl) -3- ((R) -1- (bis (benzyloxy) phosphoryloxy) ethyl) -2,5,8,12-tetraoxo- (67 mg, 0.056 mmol) was dissolved in a solution of ethanol / water (3 mL, 2: 1) in tetrahydrofuran After dissolving, Pd / C (20 wt%, 130 mg) was added. The mixture was stirred under hydrogen gas (1 atm) for 8 hours, then filtered through a pad of celite, washed with ethanol / water (2: 1) and then concentrated. The residue was purified by preparative HPLC to give the title compound (10.3 mg, 0.013 mmol, yield 23%).

HPLC Retention time (condition A): 2.74 min; ¹H NMR (900 MHz, DMSO-d₆) δ 10.33(br, 1H; Thr-NH), 8.72(d, J=5.4 Hz, 1H; Met-NH), 8.21(d, J=7.2 Hz, 1H; Ser-NH), 7.84(d, J=6.3 Hz, 1H; Glu-NH), 7.04(m, 1H; X-NH), 4.41(m, 1H; Met-Ca-H), 4.36(d, J=3.6 Hz, 1H; Glu-Ca-H), 4.32(m, 1H; Ser-Ca-H), 4.23(t, J=9.0 Hz, 1H; Pro2-Ca-H), 4.17(d, J=10.8 Hz, 1H; Pro2-Cd-H'), 4.14(m, 2H; Pro1-Ca-H, Thr-Cb-H), 4.09(m, 1H; Pro2-Cc-H), 4.04(d, J=9.0 Hz, 1H; Thr-Ca-H), 3.74(m, 1H; Ser-Cb-H'), 3.62(m, 1H; X-Cb-H'), 3.59(s, 3H; -OMe), 3.54(m, 2H; Pro2-Cd-H, Ser-Cb-H), 3.44(m, 1H; X-Ca-H'), 3.41(t, J=9.9 Hz, 1H; X-Cb-H), 3.18-3.14(m, 2H; Pro1-Cd-H', Pro1-Cd-H), 2.95(t, J=10.8 Hz, 1H; X-Ca-H), 2.45(m, 2H; Met-Cc-H,H'), 2.43(m, 2H; Glu-Cc-H,H'), 2.28(m, 1H; Pro1-Cb-H'), 2.23(m, 1H; Pro2-Cb-H'), 2.02(s, 3H; Met-SCH3), 1.95-1.90(m, 3H; Glu-Cb-H', Met-Cb-H', Pro2-Cb-H), 1.86-1.84(m, 3H; Pro1-Cb-H, Pro1-Cc-H, H'), 1.82-1.79(m, 1H; Met-Cb-H), 1.68(m, 1H; Glu-Cb-H), 1.21(d, J=5.4 Hz, 3H; Thr-Cc-H); ¹³C NMR (125 MHz, CD₃OD) δ 174.9, 173.7, 172.5, 172.0, 170.9, 170.5, 169.3, 78.5, 71.5, 68.5, 61.6, 59.8, 58.9, 55.9, 54.2, 53.1, 52.6, 52.4, 50.3, 46.5, 39.5, 35.6, 31.3, 30.9, 30.0, 29.6, 29.1, 23.9, 17.5, 14.2; 31P NMR (200MHz, DMSO-d6) δ 6.438; HRMS [M+Na] 818.2773 (found), 818.2772 (calc.); LRMS [M+1] 796.69(found), 796.29(calc.).
HPLC Retention time (condition A): 2.74 min; ^{1 H NMR (900 MHz, DMSO} -d 6) δ 10.33 (br, 1H; Thr-NH), 8.72 (d, J = 5.4 Hz, 1H; Met-NH), 8.21 (d, J = 7.2 Hz, 1H H-NMR), 7.84 (d, J = 6.3Hz, 1H; Glu-NH), 7.04 J = 3.6 Hz, 1H; Glu-Ca-H), 4.32 (m, 1H, Ser-Ca-H), 4.23 (M, 2H, Pro1-Ca-H, Thr-Cb-H), 4.09 (m, 1H, Pro2-Cc-H), 4.04 H), 3.74 (m, 1H, Ser-Cb-H '), 3.62 ), 3.54 (m, 2H, Pro2-Cd-H, Ser-Cb-H), 3.44 (m, H), 2.45 (m, 2H, Met), 3.18-3.14 (m, 2H, Pro1-Cd-H ', Pro1-Cd- Cb-H '), 2.23 (m, 1H, Pro2-Cb-H), 2.43 (M, 3H), 2.02 (s, 3H, Met-SCH3), 1.95-1.90 H), 1.21 (d, J), 1.68-1.79 (m, 1H, Met-Cb-H) = 5.4 Hz, 3H; Thr-Cg-H); ^{13 C NMR (125 MHz, CD} 3 OD) δ 174.9, 173.7, 172.5, 172.0, 170.9, 170.5, 169.3, 78.5, 71.5, 68.5, 61.6, 59.8, 58.9, 55.9, 54.2, 53.1, 52.6, 52.4, 50.3, 46.5, 39.5, 35.6, 31.3, 30.9, 30.0, 29.6, 29.1, 23.9, 17.5, 14.2; 31 P NMR (200 MHz, DMSO-d 6)? 6.438; HRMS [M + Na] 818.2773 (found), 818.2772 (calc.); LRMS [M + 1] 796.69 (found), 796.29 (calc.).

실시예 3: 메틸 ((3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-9-((S)-4-(메틸싸이오)-2-((S)-피롤리딘-2-카복스아미도)부탄아미도)-2,5,8,12-테트라옥소-3-( (R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트
Example 3: Methyl ((3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -9 - ((S) -4- (methylthio) -2- 2-carboxamido) butanamido) -2,5,8,12-tetraoxo-3- ((R) -1- (Phosphoroxy) ethyl) 7,13-tetraaza bicyclo [15.2.1] iso-19-carbonyl) -L-leucinate

하기와 같이 4단계의 합성과정을 통해 실시예 3의 화합물을 제조하였다.
The compound of Example 3 was prepared through the following four steps of synthesis.

단계 1: (S)-메틸 2-((3S,6S,9S,17R,19S)-9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복스아미도)-4-메틸펜타노에이트Step 1: (S) -Methyl 2- ((3S, 6S, 9S, 17R, 19S) -9 - ((9H-Fluoren-9- yl) methoxy) carbonylamino) -6- (benzyloxy Methyl) -3 - ((R) -1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa- 1,4,7,13- tetraaza- bicyclo [ &Lt; RTI ID = 0.0 > octanoic-19-carboxamido) -4-methylpentanoate

(3S,6S,9S,17R,19S)-메틸 9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복실레이트 (1.0 g, 1.25 mmol)과 트리메틴 하이드록사이드(4.52 g, 25.0 mmol)을 1,2-다이클로로에탄 (10 mL)에 녹인 후 80℃로 가온하였다. 1시간 동안 교반한 후 트리메틴 하이드록사이드 (1.0 g, 5.53 mmol)를 더 가하고 1시간 동안 80 ℃에서 교반하였다. 혼합물을 상온으로 식힌 후 0.5 N HCl 수용액에 쏟은 후, 에틸아세테이트를 이용하여 세차례 추출하였다. 유기층을 마그네슘 설페이트를 이용하여 건조한 후 여과하여 흰색 잔사를 얻었다. 얻은 잔사를 THF (20 mL)에 녹인 후, L-로이신 메틸 에스터 염화수소 (342 mg, 1.88 mmol)와 HATU(950 mg, 2.5 mmol)를 상온에서 순차적으로 가하였다. 상온에서 2시간 동안 교반한 후 물에 혼합물을 붓고, 에틸아세테이트로 세 차례 추출하였다. 유기층을 소금물로 씻어주고 마그네슘 설페이트로 건조한 후 농축하였다. 얻어진 잔사를 컬럼 크로마토그래피 (실리카 겔, EtOAc:Hexane:MeOH = 8:1:1)를 이용해 정제하여 표제화합물 (190 mg, 0.208 mmol, 수율 17%)을 얻었다. (3S, 6S, 9S, 17R, 19S) -methyl 9 - ((9H-fluoren-9-yl) methoxy) carbonylamino) -6- (benzyloxymethyl) -3- 1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo [15.2.1] isoic acid- 19- carboxylate , 1.25 mmol) and trimethine hydroxide (4.52 g, 25.0 mmol) were dissolved in 1,2-dichloroethane (10 mL) and heated to 80 ° C. After stirring for 1 hour, trimethine hydroxide (1.0 g, 5.53 mmol) was further added and the mixture was stirred at 80 DEG C for 1 hour. The mixture was cooled to room temperature, poured into 0.5 N HCl aqueous solution, and extracted three times with ethyl acetate. The organic layer was dried over magnesium sulfate and filtered to give a white residue. The resulting residue was dissolved in THF (20 mL), L-leucine methyl ester hydrochloride (342 mg, 1.88 mmol) and HATU (950 mg, 2.5 mmol) were successively added at room temperature. After stirring at room temperature for 2 hours, the mixture was poured into water and extracted three times with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and concentrated. The obtained residue was purified by column chromatography (silica gel, EtOAc: Hexane: MeOH = 8: 1: 1) to give the title compound (190 mg, 0.208 mmol, yield: 17%).

HPLC Retention time (condition A): 3.78 min, LRMS [M+1] 913.98 (found), 913.43(calc.)
HPLC Retention time (condition A): 3.78 min, LRMS [M + 1] 913.98 (found), 913.43 (calc.

단계 2: (S)-메틸 2-((3S,6S,9S,17R,19S)-9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복스아미도)-4-메틸펜타노에이트Step 2: (S) -Methyl 2 - ((3S, 6S, 9S, 17R, 19S) -9 - ((9H- fluoren-9- yl) methoxy) carbonylamino) -6- (benzyloxy Methyl) -3 - ((R) -1- (bis (benzyloxy) phosphoryloxyethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- Bicyclo [15.2.1] octanoic-19-carboxamido) -4-methylpentanoate

(S)-메틸 2-((3S,6S,9S,17R,19S)-9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복스아미도)-4-메틸펜타노에이트 (370 mg, 0.41 mmol)과 1H-테트라졸 (128 mg, 1.82 mmol)를 디클로로메탄 (5 mL)에 녹인 후, 다이벤질 N,N-다이이소프로필포스포아미다이트(90% tech. grade, 628 mg, 1.82 mmol)를 상온에서 가한 후 1시간 동안 교반하였다. 물을 가하여 반응을 종결시키고 디클로로메탄으로 수차례 추출한 후 유기층을 소금물로 씻어주었다. 마그네슘 설페이트로 건조하고 농축한 후 얻은 잔사를 디클로로메탄 (5 mL)로 녹인 후 0 ℃로 냉각하였다. m-클로로퍼벤조익 에시드 (314 mg, 1.82 mmol)를 가하고 1시간 동안 교반한 후, 물을 가해 반응을 종결시키고 디클로로메탄으로 여러 번 추출하였다. 유기층을 소금물로 씻어준 후 마그네슘 설페이트 로 건조한 뒤 농축하였다. 얻은 잔사를 컬럼 크로마토그래피 (실리카 겔, EtOAc:MeOH = 100:1→100:3)를 이용해 정제하여 백색 고체의 표제화합물 (320 mg, 0.273 mmol, 수율 68%)을 얻었다. (S) -methyl 2 - ((3S, 6S, 9S, 17R, 19S) -9 - ((9H-fluoren-9- yl) methoxy) carbonylamino) -6- (benzyloxymethyl) 3 - ((R) -1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo [ (370 mg, 0.41 mmol) and 1H-tetrazole (128 mg, 1.82 mmol) were dissolved in dichloromethane (5 mL), and dibenzyl N, N-dimethylcarbamoyl chloride Diisopropylphosphoramidite (90% tech. Grade, 628 mg, 1.82 mmol) was added at room temperature and then stirred for 1 hour. The reaction was terminated by adding water, extracted several times with dichloromethane, and the organic layer was washed with brine. After drying with magnesium sulfate and concentration, the obtained residue was dissolved in dichloromethane (5 mL) and then cooled to 0 ° C. m-Chloroperbenzoic acid (314 mg, 1.82 mmol) was added and the mixture was stirred for 1 hour. Water was added to terminate the reaction and extracted several times with dichloromethane. The organic layer was washed with brine, dried over magnesium sulfate and concentrated. The obtained residue was purified by column chromatography (silica gel, EtOAc: MeOH = 100: 1 → 100: 3) to obtain the title compound (320 mg, 0.273 mmol, yield 68%) as a white solid.

HPLC Retention time (condition A): 4.70 min, LRMS [M+1] 1174.14 (found), 1173.49 (calc.)
HPLC Retention time (condition A): 4.70 min, LRMS [M + 1] 1174.14 (found), 1173.49 (calc.)

단계 3: (S)-벤질 2-(S)-1-((3S,6S,9S,17R,19S)-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴록시)에틸)-19-((S)-1-메톡시-4-메틸-1-옥소펜탄-2-일카바모일)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-일아미노)-4-(메틸싸이오)-1-옥소부탄-2-일카바모일)피롤리딘-1-카복실레이트Step 3: (S) -Benzyl 2- (S) -1 - ((3S, 6S, 9S, 17R, 19S) -6- (benzyloxymethyl) -3- Oxy) phospholyloxy) ethyl) -19- ((S) -1-methoxy-4-methyl-1-oxopentan- 2- ylcarbamoyl) -2,5,8,12-tetraoxo- Oxa-1,4,7,13-tetraaza-bicyclo [15.2.1] iso-19-ylamino) -4- (methylthio) -1-oxobutan- 2- ylcarbamoyl) pyrrole Di-l-carboxylate

(S)-메틸 2-((3S,6S,9S,17R,19S)-9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴옥시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복스아미도)-4-메틸펜타노에이트 (320 mg, 0.273 mmol)를 THF(2.7 mL)에 녹인 후, 상온에서 피페리딘 (27 uL, 0.273 mmol)을 넣고 18시간 동안 교반하였다. 반응 혼합물을 농축한 후 얻은 잔사를 THF (5 mL)에 녹인 후, (S)-2-((S)-1-(벤질옥시카보닐)피롤리딘-2-카복스아미도)-4-(메틸싸이오)부타노익 에시드 (156 mg, 0.410 mmol)와 DIPEA(95 uL, 0.546 mmol)를 순차적으로 넣었다. 혼합물에 HATU (156 mg, 0.410 mmol)를 가한 후 세 시간 동안 교반하고 물을 가해 반응을 종결한 후 에틸아세테이트로 추출하였다. 유기층을 소금물로 씻어준 후 마그네슘 설페이트를 가하여 건조한 후 여과하였다. 얻어진 잔사를 컬럼 크로마토그래피 (실리카 겔, EtOAc:Hexane:MeOH = 8:1:1)를 이용해 정제하여 표제화합물 (185 mg, 0.163 mmol, 수율 60%)을 얻었다. (S) -methyl 2 - ((3S, 6S, 9S, 17R, 19S) -9 - ((9H-fluoren-9- yl) methoxy) carbonylamino) -6- (benzyloxymethyl) 3 - ((R) -1- (Bis (benzyloxy) phospholyloxy) ethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo (32 mg, 0.273 mmol) was dissolved in THF (2.7 mL), and then piperidine (27 uL, 0.273 mmol) was added thereto at room temperature. ) And stirred for 18 hours. After the reaction mixture was concentrated, the residue was dissolved in THF (5 mL), and (S) -2 - ((S) -1- (benzyloxycarbonyl) pyrrolidine- - (methylthio) butanoic acid salt (156 mg, 0.410 mmol) and DIPEA (95 uL, 0.546 mmol) were added sequentially. HATU (156 mg, 0.410 mmol) was added to the mixture, stirred for 3 hours, and water was added thereto to terminate the reaction, followed by extraction with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, and filtered. The obtained residue was purified by column chromatography (silica gel, EtOAc: Hexane: MeOH = 8: 1: 1) to give the title compound (185 mg, 0.163 mmol, yield 60%).

HPLC Retention time (condition A): 4.33 min, LRMS [M+1] 1313.97 (found), 1313.55(calc.)
HPLC Retention time (condition A): 4.33 min, LRMS [M + 1] 1313.97 (found), 1313.55 (calc.

단계 4: 메틸 ((3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-9-((S)-4-(메틸싸이오)-2-((S)-피롤리딘-2-카복스아미도)부탄아미도)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트 (실시예 3의 화합물)Step 4: Preparation of methyl ((3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -9 - ((S) -4- (methylthio) -2- (R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7 , 13-tetraaza bicyclo [15.2.1] iso-19-carbonyl) -L-leucinate (the compound of Example 3)

(S)-벤질-2-((S)-1-((3S,6S,9S,17R,19S)-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴옥시)에틸)-19-((S)-1-메톡시-4-메틸-1-옥소펜탄-2-일카바모일)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-9-일아미노)-4-(메틸싸이오)-1-옥소부탄-2-일카바모일)피롤리딘-1-카복실레이트 (185 mg, 0.163 mmol)를 에탄올/물 (10 mL, 7:3)에 녹인 후, Pd/C (20 중량%, 400 mg)를 가하였다. 수소가스 (1 atm) 하에서 15시간 동안 교반한 후 셀라이트 패드를 통해 여과한 후 에탄올/물 (7:3)으로 여러 번 씻어준 후 농축하였다. 얻어진 잔사를 에탄올/물 (10 mL, 7:3)에 녹인 후 1,2-에탄다이싸이올 (41 uL, 0.49 mmol)를 가하였다. 2시간 동안 교반한 후 혼합물을 셀라이트 패드를 통해 여과한 후 preparatory HPLC를 이용하여 정제하여, 백색 고체의 표제화합물 (34 mg, 0.037 mmol, 수율 23%)을 얻었다. (S) -benzyl-2 - ((S) -1 - ((3S, 6S, 9S, 17R, 19S) -6- (benzyloxymethyl) -3- ) Phosphoyloxy) ethyl) -19 - ((S) -1-methoxy-4-methyl- 1 -oxopentan- 2- ylcarbamoyl) -2,5,8,12-tetraoxo-16- -1,4,7,13-tetraaza-bicyclo [15.2.1] iso-9-ylamino) -4- (methylthio) -1-oxobutan- 2- ylcarbamoyl) pyrrolidine -1-carboxylate (185 mg, 0.163 mmol) was dissolved in ethanol / water (10 mL, 7: 3) and Pd / C (20 wt%, 400 mg) was added. The mixture was stirred under hydrogen gas (1 atm) for 15 hours, filtered through a celite pad, washed several times with ethanol / water (7: 3) and concentrated. The resulting residue was dissolved in ethanol / water (10 mL, 7: 3) and 1,2-ethanedithiol (41 uL, 0.49 mmol) was added. After stirring for 2 hours, the mixture was filtered through a pad of celite and purified by preparative HPLC to give the title compound as a white solid (34 mg, 0.037 mmol, yield 23%).

HPLC Retention time (condition A): 4.60 min; ¹H NMR (900 MHz, DMSO-d₆) δ 9.25(br, 1H; Thr-NH), 8.70(d, J=7.2 Hz, 1H; Met-NH), 8.27(d, J=6.3 Hz, 1H; Leu-NH), 8.21(d, J=7.2 Hz, 1H; Ser-NH), 7.91(d, J=6.3 Hz, 1H; Glu-NH), 7.03(s, 1H; X-NH), 4.43(q, J=8.1 Hz, 1H; Met-Ca-H), 4.37(t, J=9.0 Hz, 1H; Pro2-Ca-H), 4.35(q, J=7.2 Hz, 1H; Glu-Ca-H), 4.32(q, J=7.2 Hz, 1H; Ser-Ca-H), 4.20-4.18(m, 3H; Thr-Ca-H, Thr-Cb-H, Leu-Ca-H), 4.16(d, J=11.7 Hz, 1H; Pro2-Cd-H') 4.05(m, 1H; Pro2-Cc-H), 3.70(d, J=7.2 Hz, 1H; Ser-Cb-H), 3.61(m, 1H; X-Cb-H), 3.58(s, 3H; -OMe), 3.57(d, J=7.2 Hz, 1H; Ser-Cb-H'), 3.48(m, 2H; Pro2-Cd-H, X-Ca-H'), 3.40(t, J=9.0 Hz, 1H; X-Cb-H), 3.22-3.16(m, 2H; Pro1-Cd-H), 2.97(t, J=12.6 Hz, 1H; X-Ca-H), 2.51-2.43(m, 3H; Met-Cc-H,H', Glu-Cc-H), 2.30(m, 1H; Pro1-Cb-H), 2.14(m, 1H; Pro2-Cb-H), 2.01(s, 3H; Met-SMe), 2.01(m, 1H; Glu-Cc-H). 1.94-1.91(m, 2H; Glu-Cb-H, Met-Cb-H), 1.90-1.83(m, 4H; Pro1-Cb-H, Pro1-Cc-H,H', Pro2-Cb-H), 1.78(m, 1H; Met-Cb-H), 1.68-1.66(m, 2H; Glu-Cb-H, Leu-Cc-H), 1.52-1.44(m, 2H; Leu-Cb-H), 1.27(s, 3H; Thr-Cc-H), 0.88(d, J=6.3 Hz, 3H; Leu-Cd-H), 0.83(d, J=6.3 Hz, 3H; L-Cd-H); ¹³C NMR (125 MHz, CD₃OD) δ 174.5, 173.7, 173.0, 172.2, 171.6, 171.0, 169.7, 168.8, 78.5, 72.5, 68.5, 61.6, 59.8, 59.4, 58.5, 55.8, 54.5, 52.8, 52.4, 51.7, 51.2, 46.2, 40.1, 39.4, 35.9, 31.8, 31.5, 29.9, 29.8, 29.7, 24.6, 23.9, 22.1, 20.7, 17.4, 14.1; 31P NMR (200MHz, MeOD) δ 6.551; HRMS [M+Na] 931.3611(found), 931.3611(calc.); LRMS [M+1] 909.80 (found), 909.37 (calc.); [α]D20= -0.037 (c=0.1, MeOH).
HPLC Retention time (condition A): 4.60 min; ^{1 H NMR (900 MHz, DMSO} -d 6) δ 9.25 (br, 1H; Thr-NH), 8.70 (d, J = 7.2 Hz, 1H; Met-NH), 8.27 (d, J = 6.3 Hz, 1H H-NMR), 8.21 (d, J = 7.2 Hz, 1H, Ser-NH), 7.91 (d, J = 6.3 Hz, (q, J = 8.1 Hz, 1H; Met-Ca-H), 4.37 (t, J = 9.0 Hz, 1H, Pro2- H), 4.32 (q, J = 7.2 Hz, 1H, Ser-Ca-H), 4.20-4.18 (m, 3H; Thr-Ca-H, Thr- H), 3.61 (d, J = 11.7 Hz, 1H; Pro2-Cd-H ') 4.05 , 3.58 (d, J = 7.2 Hz, 1H, Ser-Cb-H '), 3.48 (m, 2H, Pro2-Cd-H , 3.40 (t, J = 9.0 Hz, 1H, X-Cb-H), 3.22-3.16 (m, 2H, Pro1-Cd- , H-X-Ca-H), 2.51-2.43 (m, 3H, Met-C-H, H ', Glu- , 1H, Pro2-Cb-H), 2.01 (s, 3H, Met-SMe), 2.01 (m, 1H; H-Pro-Cb-H), 1.94-1.91 (m, 2H, Glu-Cb-H, Met- , 1.78 (m, 1H, Met-Cb-H), 1.68-1.66 (m, 2H, Glu-Cb-H, Leu- 1.27 (s, 3H, Thr-C? -H), 0.88 (d, J = 6.3 Hz, 3H; Leu-Cd-H), 0.83 (d, J = 6.3 Hz, 3H; ^{13 C NMR (125 MHz, CD} 3 OD) δ 174.5, 173.7, 173.0, 172.2, 171.6, 171.0, 169.7, 168.8, 78.5, 72.5, 68.5, 61.6, 59.8, 59.4, 58.5, 55.8, 54.5, 52.8, 52.4, 51.7, 51.2, 46.2, 40.1, 39.4, 35.9, 31.8, 31.5, 29.9, 29.8, 29.7, 24.6, 23.9, 22.1, 20.7, 17.4, 14.1; 31 P NMR (200 MHz, MeOD)? 6.551; HRMS [M + Na] 931.3611 (found), 931.3611 (calc.); LRMS [M + 1] 909.80 (found), 909.37 (calc.); [[alpha]] D < 20 > = -0.037 (c = 0.1, MeOH).

실시예 4: 메틸 ((3S, 6S, 9S, 17R, 19S)-9-((S)-2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트
Example 4: Synthesis of methyl ((3S, 6S, 9S, 17R, 19S) -9 - ((S) -2 - ((S) -2-acetamido-4-methylpentanamido) Amido) -6- (hydroxymethyl) -2,5,8,12-tetraoxo-3 - ((R) -1- (Phosphoroxy) ethyl) 13-tetraaza bicyclo [15.2.1] iso-19-carbonyl) -L-leucinate

하기와 같이 7단계의 합성과정을 통해 실시예 4의 화합물을 제조하였다.
The compound of Example 4 was prepared through the following seven steps of synthesis.

단계 1: (S)-메틸 2-((S)-2-아세트아미도-4-메틸펜타미도)-4-메틸펜타노에이트Step 1: (S) -Methyl 2- ((S) -2-acetamido-4-methylpentimido) -4-methylpentanoate

N-아세틸-로이신(0.50 g, 2.89 mmol), L-로이신 메틸 에스터 염화수소 (0.524 mg, 2.89 mmol), DIPEA (1.26 mL, 7.23 mmol)를 THF (10 mL)에 녹인 후, HATU (1.36 g, 3.57 mmol)를 가하였다. 상온에서 3시간 동안 교반한 후 탄산수소나트륨 포화용액에 반응물을 붓고 에틸아세테이트를 이용하여 추출하였다. 유기층을 소금물로 씻고 마그네슘 설페이트를 이용하여 건조한 후 농축하였다. 얻은 잔사를 컬럼 크로마토그래피(실리카 겔, EA:Hexane=2:1)롤 이용해 정제하여 (S)-메틸 2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜타노에이트 (840 mg, 2.80 mmol, 수율 97%)을 얻었다. L-leucine methyl ester hydrochloride (0.524 mg, 2.89 mmol) and DIPEA (1.26 mL, 7.23 mmol) were dissolved in THF (10 mL), HATU (1.36 g, 3.57 mmol). After stirring at room temperature for 3 hours, the reaction mixture was poured into saturated sodium hydrogencarbonate solution and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate, and concentrated. The resulting residue was purified by column chromatography (silica gel, EA: Hexane = 2: 1) to give (S) -methyl 2 - ((S) -2-acetamido- Methylpentanoate (840 mg, 2.80 mmol, yield 97%).

HPLC Retention time (condition B): 2.54 min; ¹H NMR (400 MHz, CD₃OD) δ 8.10(d, J=7.6 Hz, 1H), 7.86(d, J=7.4 Hz, 1H), 4.45(m, 2H), 3.66(s, 3H), 1.95(s, 3H), 1.67-1.56(m, 6H), 0.95-0.88(m, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 173.8, 173.3, 171.9, 52.0, 51.6, 50.8, 40.6, 39.9, 24.4, 22.2, 22.1, 21.4, 20.9, 20.7; LRMS [M+1] 301.24 (found), 301.20 (calc.).
HPLC Retention time (condition B): 2.54 min; ^{1 H NMR (400 MHz, CD} 3 OD) δ 8.10 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 7.4 Hz, 1H), 4.45 (m, 2H), 3.66 (s, 3H), 1.95 (s, 3H), 1.67-1.56 (m, 6H), 0.95-0.88 (m, 12H); ¹³ C NMR (100 MHz, CDCl ₃ ) 隆 173.8, 173.3, 171.9, 52.0, 51.6, 50.8, 40.6, 39.9, 24.4, 22.2, 22.1, 21.4, 20.9, 20.7; LRMS [M + 1] 301.24 (found), 301.20 (calc.).

단계 2: (S)-메틸 2-((S)-2-아세트아미도-4-메틸펜타미도)-4-메틸펜타노익산Step 2: (S) -Methyl 2- ((S) -2-acetamido-4-methylpentimido) -4-methylpentanoic acid

(S)-메틸 2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜타노에이트 (0.21 g, 0.70 mmol)을 THF (5 mL)에 녹인 후, 0 ℃에서 1 M LiOH 용액 (1.05 mL, 1.05 mmol)을 가하였다. 3시간 동안 교반한 후 6N HCl (100 uL, 0.60 mmol)을 이용하여 pH를 5로 맞추었다. 마그네슘 설페이트를 가한 후, 메탄올을 가하였다. 혼합물을 셀라이트 패드를 통해 정제하고 THF/MeOH로 수차례 씻어준 후 농축하여 표제화합물 (190 mg, 0.67 mmol, 95% yield)을 얻었다. 얻은 화합물은 더 이상 정제하지 않고 다음 반응에 이용하였다. Methylpentanoate (0.21 g, 0.70 mmol) was dissolved in THF (5 mL), and a solution of O (S) -methyl 2- 1 M LiOH solution (1.05 mL, 1.05 mmol) was added. After stirring for 3 hours, the pH was adjusted to 5 with 6N HCl (100 uL, 0.60 mmol). Magnesium sulfate was added, and then methanol was added. The mixture was purified through a pad of celite, washed several times with THF / MeOH and concentrated to give the title compound (190 mg, 0.67 mmol, 95% yield). The resulting compound was used in the next reaction without further purification.

¹H NMR (400 MHz, CD₃OD) δ 4.75(m, 1H), 4.66(m, 1H), 2.35(s, 3h), 2.00-1.93(m, 6H), 1.33-1.26(m, 12H). ¹ H NMR (400 MHz, CD ₃ OD)? 4.75 (m, 1H), 4.66 (m, 1H), 2.35 .

단계 3: (3S,6S,9S,17R,19S)-메틸 9-((S)-2-((S)-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복실레이트Step 3: (3S, 6S, 9S, 17R, 19S) -methyl 9 - ((S) -2 - ((S) -acetamido-4-methylpentanamido) - (benzyloxymethyl) -3 - ((R) -1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa- 1,4,7,13- tetraaza- bicyclo [ 15.2.1] octanoic-19-carboxylate

(3S,6S,9S,17R,19S)-메틸 9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복실레이트 (60% purity, 500 mg, 0.63 mmol) 을 THF(6.8 mL) 에 녹인 후, 상온에서 피페리딘 (68 uL, 0.693 mmol)을 가하였다. 15시간 동안 교반한 후, 혼합물을 농축하였다. 얻은 잔사를 THF (5 mL)에 다시 녹인 후, (S)-2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜타노익 에시드 (200 mg, 0.70 mmol)를 THF (2 mL)에 녹인 용액과 DIPEA (165 uL, 0.95 mmol), HATU (360 mg, 0.95 mmol)를 상온에서 순차적으로 가하였다. 15시간 동안 교반한 후, 탄산수소나트륨 포화용액을 이용하여 반응 종결하고 에틸아세테이트로 수차례 추출하였다. 유기층을 소금물로 씻어준 후 건조하고 컬럼 크로마토그래피 (실리카 겔, EtOAc only → EtOAc:MeOH = 9:1→8:1)를 이용해 정제하여 표제화합물 (145 mg, 0.17 mmol, 27% 수율of 2 steps)을 얻었다. (3S, 6S, 9S, 17R, 19S) -methyl 9 - ((9H-fluoren-9-yl) methoxy) carbonylamino) -6- (benzyloxymethyl) -3- Oxo-1,4,7,13-tetraaza-bicyclo [15.2.1] isoic acid-19-carboxylate (60% purity, 500 mg, 0.63 mmol) was dissolved in THF (6.8 mL), and then piperidine (68 uL, 0.693 mmol) was added at room temperature. After stirring for 15 hours, the mixture was concentrated. The resulting residue was redissolved in THF (5 mL), and then 200 mg (0.70 mmol) of (S) -2 - ((S) -2-acetamido-4-methylpentanamido) -4-methylpentanoic acid ) Was dissolved in THF (2 mL), DIPEA (165 uL, 0.95 mmol) and HATU (360 mg, 0.95 mmol) were successively added at room temperature. After stirring for 15 hours, the reaction was terminated using a saturated solution of sodium hydrogencarbonate and extracted several times with ethyl acetate. The organic layer was washed with brine, dried and purified by column chromatography (silica gel, EtOAc only → EtOAc: MeOH = 9: 1 → 8: 1) to obtain the title compound (145 mg, 0.17 mmol, 27% ).

HPLC Retention time (condition B): 4.11min, LRMS [M+1] 846.57 (found), 846.45(calc.)
HPLC Retention time (condition B): 4.11 min, LRMS [M + 1] 846.57 (found), 846.45 (calc.

단계 4: (3S,6S,9S,17R,19S)-메틸 9-((S)-2-((S)-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복실산Step 4: (3S, 6S, 9S, 17R, 19S) -methyl 9 - ((S) -2 - ((S) -acetamido-4-methylpentanamido) - (benzyloxymethyl) -3 - ((R) -1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa- 1,4,7,13- tetraaza- bicyclo [ 15.2.1] octanoic acid-9-carboxylic acid

(3S,6S,9S,17R,19S)-메틸 9-((S)-2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도)-3-((R)-1-하이드록시에틸)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복실레이트 (130 mg, 0.154 mmol)를 THF (2 mL)에 녹인 후, 0 ℃에서 1M LiOH 용액 (230 uL, 0.230 mmol)을 가하였다. 같은 온도에서 3시간 동안 교반한 후, 디클로로메탄 과 1 N HCl 용액 (230 uL)를 가하여 pH 4로 맞추어 백색의 고체를 얻었다. 얻은 고체를 여과하여 백색 고체의 표제화합물 (50 mg, 0.060 mmol, 수율 39%)을 얻었다. (S) -2 - ((S) -2-acetamido-4-methylpentanamido) -4-methylpentanamido) -3 - ((R) -1-hydroxyethyl) -6- (hydroxymethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13- tetraaza- bicyclo [ 15.2.1] iodic acid-19-carboxylate (130 mg, 0.154 mmol) was dissolved in THF (2 mL), and then 1M LiOH solution (230 uL, 0.230 mmol) was added at 0 ° C. After stirring at the same temperature for 3 hours, dichloromethane and 1 N HCl solution (230 uL) were added to adjust pH to 4 to obtain a white solid. The obtained solid was filtered to obtain the title compound (50 mg, 0.060 mmol, yield 39%) as a white solid.

HPLC Retention time (condition B): 3.83 min, LRMS [M+1] 832.38 (found), 832.44 (calc.)
HPLC Retention time (condition B): 3.83 min, LRMS [M + 1] 832.38 (found), 832.44 (calc.)

단계 5: (S)-메틸 2-((3S,6S,9S,17R,19S)-9-((S)-2-((S)-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복스아미도)-4-메틸펜타노에이트Step 5: (S) -Methyl 2- ((3S, 6S, 9S, 17R, 19S) -9 - ((S) -2- -Methylpentanamido-6- (benzyloxymethyl) -3 - ((R) -1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa- -Tetraaza-bicyclo [15.2.1] octanoic-19-carboxamido) -4-methylpentanoate

(3S,6S,9S,17R,19S)-9-((S)-2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도)-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복시산 (50 mg, 0.060 mmol), L-로이신 메틸 에스터 염화수소(14.2 mg, 0.078 mmol), 및 DIPEA(26 uL, 0.15 mmol)를 THF(5 mL)에 녹인 후, HATU(23 mg, 0.09 mmol)를 가하고 4시간 동안 상온에서 교반한 후, 탄산수소나트륨 포화용액에 첨가하였다. 수층을 에틸아세테이트로 수차례 추출한 후 유기층을 감압건조 및 여과하였다. 얻은 잔사를 컬럼 크로마토그래피 (실리카 겔, EA:MeOH = 8:1)로 정제하여 표제화합물 (34 mg, 0.035 mmol, 59% yield)을 얻었다. (S) -2 - ((S) -2-acetamido-4-methylpentanamido) -4-methylpentanamido) -6- (Benzyloxymethyl) -3 - ((R) -1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa- 1,4,7,13- tetraaza- bicyclo [ (14.2 mg, 0.078 mmol) and DIPEA (26 uL, 0.15 mmol) were dissolved in THF (5 mL), followed by the addition of diisopropylethylamine , HATU (23 mg, 0.09 mmol) was added, and the mixture was stirred at room temperature for 4 hours, and then added to a saturated solution of sodium hydrogencarbonate. The aqueous layer was extracted several times with ethyl acetate, and the organic layer was dried under reduced pressure and filtered. The obtained residue was purified by column chromatography (silica gel, EA: MeOH = 8: 1) to obtain the title compound (34 mg, 0.035 mmol, 59% yield).

HPLC Retention time (condition B): 4.81 min, LRMS [M+1] 959.50 (found), 959.54(calc.) HPLC Retention time (condition B): 4.81 min, LRMS [M + 1] 959.50 (found), 959.54 (calc.)

단계 6: (S)-메틸 2-((3S,6S,9S,17R,19S)-9-((S)-2-((S)-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴록시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복스아미도)-4-메틸펜타노에이트Step 6: (S) -Methyl 2- ((3S, 6S, 9S, 17R, 19S) -9 - ((S) -2- -Methylpentanamido-6- (benzyloxymethyl) -3- ((R) -1- (bis (benzyloxy) phospholyloxy) ethyl) -2,5,8,12-tetraoxo-16- -1,4,7,13-tetraaza-bicyclo [15.2.1] iso-19-carboxamido) -4-methylpentanoate

2-((3S,6S,9S,17R,19S)-9-((S)-2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도)-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복스아미도)-4-메틸펜타노에이트 (28 mg, 0.0292 mmol, 반응 전에 톨루엔을 가해서 농축 2회)와 1H-테트라졸 (10.2 mg, 0.146 mmol)를 디클로로메탄 (3 mL)에 녹인 후, 다이벤질 N,N-다이이소프로필포스포아미다이트 (90% tech. grade, 50 mg, 0.146 mmol)를 상온에서 가하고 1시간 동안 교반하였다. 반응 혼합물을 -78 ℃로 냉각한 후, m-클로로퍼벤조익 에시드 (70% purity, 36 mg, 0.0.146 mmol)을 디클로로메탄 (1 mL)에 녹인 용액을 가하였다. 차차 상온으로 온도를 올린 후 1시간 동안 더 교반하고 물을 가해 반응을 종결시킨 후 수 차례 추출하였다. 얻어진 유기층을 소금물로 씻어준 후 건조하고, 컬럼 크로마토그래피 (실리카 겔, EtOAc only→ EtOAc:MeOH = 20:1→9:1)를 이용해 정제하여 백색 고체의 표제화합물 (15 mg, 0.0123 mmol, 42% yield)을 얻었다. (S) -2 - ((S) -2-acetamido-4-methylpentanamido) -4-methylpentanamido) -2 - {(3S, 6S, 9S, -6- (benzyloxymethyl) -3 - ((R) -1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- (28 mg, 0.0292 mmol, twice concentrated by addition of toluene before the reaction) and 1 H-tetrazole (10.2 mg, 0.146 mmol) were added to a solution of 2-amino- ) Was dissolved in dichloromethane (3 mL), dibenzyl N, N-diisopropylphosphoamidite (90% tech. Grade, 50 mg, 0.146 mmol) was added at room temperature and stirred for 1 hour. The reaction mixture was cooled to -78 ° C, and a solution of m-chloroperbenzoic acid (70% purity, 36 mg, 0.0.146 mmol) in dichloromethane (1 mL) was added. After increasing the temperature to room temperature, the reaction mixture was further stirred for 1 hour, and water was added thereto to terminate the reaction and then extracted several times. The obtained organic layer was washed with brine, dried and purified by column chromatography (silica gel, EtOAc only → EtOAc: MeOH = 20: 1 → 9: 1) to obtain the title compound (15 mg, 0.0123 mmol, % yield).

HPLC Retention time (condition B): 5.40 min, LRMS [M+1] 1219.48 (found), 1219.60 (calc.)
HPLC Retention time (condition B): 5.40 min, LRMS [M + 1] 1219.48 (found), 1219.60 (calc.

단계 7: 메틸 ((3S, 6S, 9S, 17R, 19S)-9-((S)-2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트 (실시예 4의 화합물)Step 7: A solution of methyl ((3S, 6S, 9S, 17R, 19S) -9 - ((S) -2 - ((S) -2-acetamido-4-methylpentanamido) (Hydroxymethyl) -2,5,8,12-tetraoxo-3 - ((R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13 -Tetraaza-bicyclo [15.2.1] iso-19-carbonyl) -L-leucinate (the compound of Example 4)

(S)-메틸 2-((3S,6S,9S,17R,19S)-9-((S)-2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도)-6-(벤질옥시메틸)-3-((R)-1-(비스(벤질옥시)포스포릴옥시)에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이사이클로[15.2.1]이코산-19-카복스아미도)-4-메틸펜타노에이트 (15 mg, 0.0158 mmol)를 에탄올/물 (5 mL, 7:3)에 녹인 후, Pd/C (10 중량%, 15 mg)을 가하였다. 수소기체 (1 atm)하에서 30 시간 동안 교반한 후 여과하고 수차례 씻어주어 백색 고체의 표제화합물 (13 mg, 0.0137 mmol, 87% yield)을 얻었다. (S) -2 - ((S) -2-acetamido-4-methylpentanamido) -4- Methylpentanamido) -6- (benzyloxymethyl) -3- ((R) -1- (bis (benzyloxy) phosphoryloxy) ethyl) -2,5,8,12-tetraoxo-16- -1,4,7,13-tetraaza- bicyclo [15.2.1] iso-19-carboxamido) -4-methylpentanoate (15 mg, 0.0158 mmol) was dissolved in ethanol / water , 7: 3), and Pd / C (10% by weight, 15 mg) was added thereto. The reaction mixture was stirred under hydrogen gas (1 atm) for 30 hours, filtered and washed several times to obtain the title compound (13 mg, 0.0137 mmol, 87% yield) as a white solid.

HPLC Retention time (condition B): 4.88 min; ¹H NMR (900 MHz, DMSO-d₆) δ 10.40(br, 1H; Thr-NH), 8.26(d, J=6.3 Hz, 1H; Leu-NH), 8.12(d, J=7.2 Hz, 1H; Ser-NH), 7.98(d, J-7.2 Hz, 1H; Leu1-NH), 7.97(d, J=6.3 Hz, 1H; Leu2-NH), 7.65(d, J=8.1 Hz, 1H; Glu-NH), 6.99(m, 1H; X-NH), 4.37(t, J=9.0 Hz, 1H; Pro-Ca-H), 4.32(m, 2H; Glu-Ca-H, Ser-Ca-H), 4.27(m, 2H; Leu1-Ca-H, Leu2-Ca-H), 4.19(m, 1H; Leu-Ca-H), 4.16(d, J=11.7 Hz, 1H; Pro-Cd-H), 4.10(m, 1H; Thr-Ca-H), 4.04(m, 1H; Pro-Cc-H), 4.00(d, J=9.0 Hz, 1H; Thr-Cb-H), 3.75(d, J=9.0 Hz, 1H; Ser-Cb-H), 3.58(m, 1H; X-Cb-H'), 3.55(m, 1H; Ser-Cb-H), 3.42(m, 3H; Pro-Cd-H, X-Ha', X-Cb-H), 2.98(t, J=11.7 Hz, 1H; X-Ca-H), 2.50(m, 1H; Glu-Cc-H'), 2.12(m, 1H; Pro-Cb-H'), 2.00(m, 1H; Glu-Cc-H), 1.88(m, 1H; Glu-Cb-H'), 1.82(m, 1H; Pro-Cb-H), 1.68(m, 1H; Leu-Cc-H), 1.60(m, 1H; Glu-Cb-H), 1.58-1.54(m, 2H; Leu1-Cc-H, Leu2-Cc-H), 1.50(m, 1H; Leu-Cb-H'), 1.46-1.40(m, 5H; Leu-Cb-H, Leu1-Cb-H, H', Leu2-Cb-H,H'), 1.18(d, J=6.3 Hz, 3H; Thr-Cc-H), 0.88(d, J=6.3 Hz, 3H; Leu-Cd-H'), 0.86(d, J=6.3 Hz, 3H; Leu-Cd-H), 0.85-0.80(d X4, J=6.3 Hz, 12H; Leu1-Cd-H,H', Leu2-Cd-H,H'); ¹³C NMR (100 MHz, CD₃OD) δ 174.2, 173.6, 173.2, 172.8, 172.5, 171.9, 171.4, 170.4, 170.2, 78.2, 74.4, 70.9, 67.7, 61.9, 59.1, 55.3, 54.0, 52.1, 51.8, 51.4, 51.1, 50.8, 40.4, 40.3, 40.0, 39.2, 35.7, 31.2, 29.4, 24.5, 24.3, 22.1, 22.0, 21.9, 20.9, 20.5, 20.4, 17.5; 31P NMR (200MHz, DMSO-d6) δ 6.326; HRMS [M+Na] (found), (calc.); LRMS [M+1] 949.40 (found), 949.46 (calc.); [α]D20= -0.063 (c=0.1, MeOH).
HPLC Retention time (condition B): 4.88 min; ^{1 H NMR (900 MHz, DMSO} -d 6) δ 10.40 (br, 1H; Thr-NH), 8.26 (d, J = 6.3 Hz, 1H; Leu-NH), 8.12 (d, J = 7.2 Hz, 1H H-Leu2-NH), 7.98 (d, J = 7.2 Hz, 1H, Leu1-NH), 7.97 (d, J = 6.3 Hz, H), 4.32 (m, 2H), 6.99 (m, 1H, X-NH) ), 4.27 (m, 2H, Leu1-Ca-H, Leu2-Ca-H), 4.19 (D, J = 9.0 Hz, 1H, Thr-Cb-H), 3.75 (d, H), 3.42 (m, 3H, Pro-Cd-H), 3.58 (m, (M, 1H, Glu-Cc-H), 2.12 (m, H-X- H-Pro-Cb-H '), 2.00 (m, 1H, Glu-Cc-H), 1.88 , 1.50-1.54 (m, 2H, Leu1-Cc-H, Leu2-Cc-H), 1.50 (m, H, Leu-Cb-H), 1.18 (d, J), 1.46-1.40 (m, 5H, Leu-Cb-H, Leu-Cb- Leu-Cd-H), 0.86 (d, J = 6.3 Hz, 3H, Leu-Cd-H) 0.85-0.80 (d X4, J = 6.3 Hz, 12H; Leu1-Cd-H, H ', Leu2-Cd-H, H'); ^{13 C NMR (100 MHz, CD} 3 OD) δ 174.2, 173.6, 173.2, 172.8, 172.5, 171.9, 171.4, 170.4, 170.2, 78.2, 74.4, 70.9, 67.7, 61.9, 59.1, 55.3, 54.0, 52.1, 51.8, 51.4, 51.1, 50.8, 40.4, 40.3, 40.0, 39.2, 35.7, 31.2, 29.4, 24.5, 24.3, 22.1, 22.0, 21.9, 20.9, 20.5, 20.4, 17.5; 31 P NMR (200 MHz, DMSO-d 6)? 6.326; HRMS [M + Na] (found), (calc.); LRMS [M + 1] 949.40 (found), 949.46 (calc.); [[alpha]] D < 20 > = -0.063 (c = 0.1, MeOH).

실시예 5: 메틸 ((3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-9-(3-(트리플루오로메틸)벤자미도)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트Example 5: Preparation of methyl ((3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -2,5,8,12-tetraoxo- ) Ethyl) -9- (3- (trifluoromethyl) benzamido) -16-oxa-1,4,7,13-tetraaza bicyclo [15.2.1] L-leucinate

상기 실시예 1의 단계 9에서 얻은 화합물인 (3S,6S,9S,17R,19S)-메틸 9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복실레이트와 3-트리플루오로메틸 벤조산을 이용하여, 상기 실시예 4와 같은 방법으로 합성을 수행하여 상기 표제화합물을 얻었다.(9S-fluoren-9-yl) methoxy) carbonylamino) -6- (benzyl Oxethyl) -3 - ((R) -1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo [ ] The title compound was obtained by the same procedure as in Example 4 using iodic acid-19-carboxylate and 3-trifluoromethylbenzoic acid.

HPLC Retention time (condition B): 3.91 min; ¹H NMR (900 MHz, DMSO-d₆) δ 8.67(d, J=7.2 Hz, 1H; Glu-NH), 8.27(d, J=6.3 Hz, 1H; Leu-NH), 8.24(d, J=7.2 Hz, 1H; Ser-NH), 8.20(s, 1H; phenyl-C2-H), 8.15(d, J=7.2 Hz, 1H; phenyl-C6-H), 7.03(br, 1H; X-NH), 7.88(d, J=8.1 Hz, 1H; phenyl-C4-H), 7.70(t, J=7.2 Hz, 1H; phenyl-C5-H), 4.57(m, 1H; Glu-Ca-H), 4.39(t, J=9.0 Hz, 1H; Pro-Ca-H), 4.36(m, 1H; Ser-Ca-H), 4.19(m, 2H; Pro-Cd-H', Leu-Ca-H), 4.14(m, 1H; Thr-Ca-H), 4.05(m, 2H; Pro-Cc-H, Thr-Cb-H), 3.77(m, 1H; Ser-Cb-H'), 3.60(m, 2H; Ser-Cb-H, X-Cb-H'), 3.58(s, 3H; -OMe), 3.47-3.38(m, 3H, Pro-Cd-H, X-Cb-H, X-Ca-H), 3.00(t, J=12.6 Hz, 1H; X-Ca-H), 2.80(t, J=11.7 Hz, 1H; Glu-Cc-H'), 2.14(m, 1H; Pro-Cb-H'), 2.09(m, 1H; Glu-Cc-H), 2.03(m, 1H; Glu-Cb-H), 1.84(m, 1H; Pro-Cb-H), 1.80(m, 1H; Glu-Cb-H), 1.69(m, 1H; Leu-Cc-H), 1.53-1.44(m, 2H; Leu-Cb-H,H'), 1.21(m, 3H; Thr-Cc-Me), 0.89(d, J=6.3 Hz, 3H; Leu-Cd-Me), 0.84(d, J=6.3 Hz, 3H; Leu-Cd-Me'); ¹³C NMR (100 MHz, CDCl₃) δ 174.8, 174.6, 173.1, 172.9, 170.8, 169.7, 168.2, 133.7, 130.7, 129.4, 128.5, 124.4, 78.4, 71.4, 68.0, 62.4, 61.0, 59.1, 58.3, 55.5, 54.2, 53.3, 52.4, 51.2, 48.6, 39.2, 35.5, 31.0, 28.0, 24.2, 21.9, 20.4, 17.2; ³¹P NMR (200MHz, DMSO-d₆) δ 5.894; HRMS [M+2Na-H] 897.2434 (found), 897.2635 (calc.); LRMS [M+1] 853.19 (found), 853.29 (calc.).
HPLC Retention time (condition B): 3.91 min; ^{1 H NMR (900 MHz, DMSO} -d 6) δ 8.67 (d, J = 7.2 Hz, 1H; Glu-NH), 8.27 (d, J = 6.3 Hz, 1H; Leu-NH), 8.24 (d, J = 7.2 Hz, 1 H-Ser-NH), 8.20 (s, 1H; phenyl-C2-H), 8.15 (d, J = 7.2 Hz, H), 7.88 (d, J = 8.1 Hz, 1H; phenyl-C4-H), 7.70 (t, J = 7.2 Hz, ), 4.39 (t, J = 9.0 Hz, 1H; Pro-Ca-H), 4.36 H), 3.14 (m, 1H, Thr-Ca-H), 4.05 (m, 2H, Pro-Cc-H, Thr- (m, 2H, Ser-Cb-H, X-Cb-H '), 3.58 (s, 3H; -OMe), 3.47-3.38 (T, J = 11.7 Hz, 1H, Glu-Cc-H), 2.14 (M, 1H, Pro-Cb-H), 2.09 (m, Leu-Cb-H), 1.21 (m, 3H, Thr-Cb-H), 1.69 (m, Me), 0.89 (d, J = 6.3 Hz, 3H; Leu-Cd-Me), 0.84 (d, J = 6.3 Hz, 3H; Leu-Cd-Me '); ^{13 C NMR (100 MHz, CDCl} 3) δ 174.8, 174.6, 173.1, 172.9, 170.8, 169.7, 168.2, 133.7, 130.7, 129.4, 128.5, 124.4, 78.4, 71.4, 68.0, 62.4, 61.0, 59.1, 58.3, 55.5 , 54.2, 53.3, 52.4, 51.2, 48.6, 39.2, 35.5, 31.0, 28.0, 24.2, 21.9, 20.4, 17.2; ^{31 P NMR (200MHz, DMSO-} d 6) δ 5.894; HRMS [M + 2Na-H] 897.2434 (found), 897.2635 (calc.); LRMS [M + 1] 853.19 (found), 853.29 (calc.).

실시예 6: 메틸 ((3S, 6S, 9S, 17R, 19S)-9-(3,5-다이이소부톡시벤자미도)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-카보닐)-L-로이시네이트Example 6: Synthesis of methyl ((3S, 6S, 9S, 17R, 19S) -9- (3,5-diisobutoxybenzamido) -6- (hydroxymethyl) -2,5,8,12-tetra (R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetraaza bicyclo [15.2.1] iso-carbonyl) -L- Sinate

상기 실시예 1의 단계 9에서 얻은 화합물인 (3S,6S,9S,17R,19S)-메틸 9-(((9H-플루오렌-9-일)메톡시)카보닐아미노)-6-(벤질옥시메틸)-3-((R)-1-하이드록시에틸)-2,5,8,12-테트라옥소-16-옥사-1,4,7,13-테트라아자-바이시클로[15.2.1]이코산-19-카복실레이트와 3,5-다이이소부톡시 벤조산을 이용하여, 상기 실시예 4와 같은 방법으로 합성을 수행하여 상기 표제화합물을 얻었다.(9S-fluoren-9-yl) methoxy) carbonylamino) -6- (benzyl Oxethyl) -3 - ((R) -1-hydroxyethyl) -2,5,8,12-tetraoxo-16-oxa-1,4,7,13-tetraaza- bicyclo [ ] Icosan-19-carboxylate and 3,5-diisobutoxybenzoic acid in the same manner as in Example 4, the title compound was obtained.

HPLC Retention time (condition B): 5.38 min; ¹H NMR (900 MHz, DMSO-d₆) δ 10.25(br, 1H; Thr-NH), 8.27(d, J=7.2 Hz, 1H; Leu-NH), 8.25(d, J=7.2 Hz, 1H; Glu-NH), 8.20(d, J=7.2 Hz, 1H; Ser-NH), 7.02(br, 1H; X-NH), 6.97(s, 2H; phenyl-C2,6-H), 6.59(s, 1H; phenyl-C4-H), 4.51(m, 1H; Glu-Ca-H), 4.39(t, J=7.2 Hz, 1H; Pro-Ca-H), 4.34(m, 1H; Ser-Ca-H), 4.20-4.18(m, 2H; Leu-Ca-H, Pro-Cd-H'), 4.13(m, 1H; Thr-Cd-H), 4.05(m, 1H; Pro-Cc-H), 4.03(m, 1H; Thr-Cb-H), 3.77(m, 1H; Ser-Cb-H'), 3.75(d, J=6.3 Hz, 4H; i-BuO-Ca-H), 3.61-3.59(m, 2H; Ser-Cb-H', X-Cb-H'), 3.58(s, 3H; -OMe) 3.47-3.43(m, 3H; Pro-Cd-H, X-Cb-H, X-Ca-H'), 3.01(t, J=10.8 Hz, 1H; X-Ca-H), 2.78(t, J=11.7 Hz, 1H; Glu-Cc-H'), 2.13(m, 1H; Pro-Cb-H'), 2.07(m, 1H; Glu-Cc-H), 2.05(m, 1H; Glu-Cb-H'), 1.98(m, 2H; i-BuO-Cb-H), 1.84(m, 1H; Pro-Cb-H), 1.78(m, 1H; Glu-Cb-H), 1.69(m, 1H; Leu-Cc-H), 1.53-1.44(m, 2H; Leu-Cb-H,H'), 1.20(d, J=5.4 Hz, 1H; Thr-Cc-H), 0.96(d, J=6.3 Hz, 12H; i-BuO-Cc-Me), 0.88(d, J=6.3 Hz, 3H; Leu-Cd-Me), 0.84(d, J=6.3 Hz, 3H; Leu-Cd-Me'); ¹³C NMR (100 MHz, CD₃OD) δ 174.2, 173.2, 172.8, 172.2, 170.4, 168.0, 165.9, 160.5, 105.4, 104.3, 78.3, 74.3, 67.8, 59.2, 59.1, 54.4, 54.0, 53.0, 51.2, 50.8, 50.2, 42.3, 40.0, 39.3, 29.3, 28.1, 24.4, 21.9, 20.4, 18.1, 16.6; ³¹P NMR (200MHz, DMSO-d₆) δ 6.038; HRMS [M+2Na-H] 973.5461(found), 973.3912 (calc.); LRMS [M+1] 929.61 (found), 929.42 (calc.); [α]_D ²⁰= -0.004 (c=0.1, MeOH).
HPLC Retention time (condition B): 5.38 min; ^{1 H NMR (900 MHz, DMSO} -d 6) δ 10.25 (br, 1H; Thr-NH), 8.27 (d, J = 7.2 Hz, 1H; Leu-NH), 8.25 (d, J = 7.2 Hz, 1H 2H, phenyl-C2, 6-H), 6.59 (d, J = J = 7.2 Hz, 1H-Pro-Ca-H), 4.34 (m, 1H, Ser- (M, 1H, Pro-Cc-H), 4.20-4.18 (m, 2H, Leu- H), 3.75 (d, J = 6.3 Hz, 4H, i-BuO-Ca-H) (M, 3H, Pro-Cd-H, X-Cb-H ', X- H, X-Ca-H '), 3.01 (t, J = 10.8 Hz, H-Bu-Cb-H '), 2.07 (m, 1H, Glu-Cg-H), 2.05 H), 1.84 (m, 1H, Pro-Cb-H), 1.78 (m, 1H, Glu-Cb-H), 1.69 (m, 1H, Leu-Cc-H), 1.53-1.44 (m, 2H; (D, J = 6.3 Hz, 12H, i-BuO-Cc-Me), 0.88 (d, J = 5.4 Hz, (d, J = 6.3 Hz, 3H; Leu-Cd-Me), 0.84 (d, J = 6.3 Hz, 3H; Leu-Cd-Me '); ^{13 C NMR (100 MHz, CD} 3 OD) δ 174.2, 173.2, 172.8, 172.2, 170.4, 168.0, 165.9, 160.5, 105.4, 104.3, 78.3, 74.3, 67.8, 59.2, 59.1, 54.4, 54.0, 53.0, 51.2, 50.8, 50.2, 42.3, 40.0, 39.3, 29.3, 28.1, 24.4, 21.9, 20.4, 18.1, 16.6; ³¹ P NMR (200 MHz, DMSO-d ₆ )? 6.038; HRMS [M + 2Na-H] 973.5461 (found), 973.3912 (calc.); LRMS [M + 1] 929.61 (found), 929.42 (calc.); [?] _D ²⁰ = -0.004 (c = 0.1, MeOH).

실시예 7: 메틸 (3S, 6S, 9S, 17R, 19S)-9-(3,5-다이이소부톡시벤자미도)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카복실레이트Example 7: Synthesis of methyl (3S, 6S, 9S, 17R, 19S) -9- (3,5- diisobutoxybenzamido) -6- (hydroxymethyl) -2,5,8,12-tetraoxo -3 - ((R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetraaza bicyclo [15.2.1]

HPLC Retention time (condition B): 5.14 min; ¹H NMR (900 MHz, DMSO-d₆) δ 8.28(br, 1H; Ser-NH), 8.24(d, J=7.2 Hz, 1H; Glu-NH), 7.07(br, 1H; X-NH), 6.97(s, 2H; phenyl-C2,6-H), 6.60(s, 1H; phenyl-C4-H), 4.486(m, 1H; Glu-Ca-H), 4.33(m, 1H; Ser-Ca-H), 4.26(m, 3H; Pro-Ca-H, Thr-Ca-H, Thr-Cb-H), 4.21(d, J=11.7 Hz, 1H; Pro-Cd-H), 4.10(m, 1H; Pro-Cc-H), 3.75(d, J=8.1, 4H; i-BuO-Ca-H), 3.71(m, 1H; Ser-Cb-H'), 3.60(m, 1H; Ser-Cb-H), 3.60(s, 3H; -OMe), 3.55(m, 2H; X-Cb-H', Pro-Cd-H), 3.42(m, 2H; X-Ca-H', X-Cb-H), 3.00(t, J=12.6 Hz, 1H; X-Ca-H), 2.72(m, 1H; Glu-Cc-H'), 2.25(m, 1H; Pro-Cb-H'), 2.08(m, 1H; Glu-Cc-H), 1.99(m, 1H; Glu-Cb-H'), 1.98(m, 2H; i-BuO-Cb-H), 1.93(m, 1H; Pro-Cb-H), 1.82(m, 1H; Glu-Cb-H), 1.27(m, 3H; Thr-Cc-Me), 0.97(d, J=6.3 Hz, 12H; i-BuO-Cc-Me); ¹³C NMR (100 MHz, CD₃OD) δ 174.1, 172.6, 172.3, 169.6, 168.4, 168.1, 132.6, 130.5, 105.4, 104.3, 78.1, 74.3, 73.4, 68.1, 58.4, 58.0, 55.3, 53.6, 53.1, 51.2, 37.4, 35.3, 31.2, 28.6, 28.1, 18.1, 16.9;³¹P NMR (200MHz, DMSO-d₆) δ 5.995; HRMS [M+2Na-H] 860.3883 (found), 860.3071 (calc.); LRMS [M+1] 816.48 (found), 816.34 (calc.); [α]_D ²⁰= -0.035 (c=0.1, MeOH).
HPLC Retention time (condition B): 5.14 min; ^{1 H NMR (900 MHz, DMSO} -d 6) δ 8.28 (br, 1H; Ser-NH), 8.24 (d, J = 7.2 Hz, 1H; Glu-NH), 7.07 (br, 1H; X-NH) H-phenyl-C2-H), 6.60 (s, 1H, phenyl-C4-H), 4.486 (m, C-H), 4.26 (m, 3H, Pro-Ca-H, Thr-Ca-H, Thr- (m, 1H, Pro-Cc-H), 3.75 (d, J = 8.1, 4H; i-BuO-Ca-H). H), 3.42 (m, 2H, X-Ca-H ', 3 ' H-Pro-Cb-H), 3.00 (t, J = 12.6 Hz, H), 1.98 (m, 2H, i-BuO-Cb-H), 1.93 (m, 1H ; Pro-Cb-H), 1.82 (m, 1H; Glu-Cb-H), 1.27 (m, 3H; Thr- -Me); ^{13 C NMR (100 MHz, CD} 3 OD) δ 174.1, 172.6, 172.3, 169.6, 168.4, 168.1, 132.6, 130.5, 105.4, 104.3, 78.1, 74.3, 73.4, 68.1, 58.4, 58.0, 55.3, 53.6, 53.1, 51.2, 37.4, 35.3, 31.2, 28.6, 28.1, 18.1, 16.9; ^{31 P NMR (200MHz, DMSO-} d 6) δ 5.995; HRMS [M + 2Na-H] 860.3883 (found), 860.3071 (calc.); LRMS [M + 1] 816.48 (found), 816.34 (calc.); [?] _D ²⁰ = -0.035 (c = 0.1, MeOH).

한편, 본 발명에 따른 상기 화학식 1로 표시되는 신규 화합물은 목적에 따라 여러 형태로 제제화가 가능하다. 다음은 본 발명에 따른 상기 화학식 1로 표시되는 화합물을 활성성분으로 함유시킨 몇몇 제제화 방법을 예시한 것으로 본 발명이 이에 한정되는 것은 아니다.Meanwhile, the novel compounds represented by Formula 1 according to the present invention can be formulated into various forms according to the purpose. The following is a description of some formulations containing the compound of Formula 1 according to the present invention as an active ingredient, but the present invention is not limited thereto.

[제제예]
[Formulation Example]

제제 1 : 정제(직접 가압)Formulation 1: Tablets (direct pressurization)

활성성분 5.0 ㎎을 체로 친 후, 락토스 14.1 ㎎, 크로스포비돈 USNF 0.8 ㎎ 및 마그네슘 스테아레이트 0.1 ㎎을 혼합하고 가압하여 정제로 만들었다.
After 5.0 mg of the active ingredient was sieved, 14.1 mg of lactose, 0.8 mg of crospovidone USNF and 0.1 mg of magnesium stearate were mixed and pressurized to make tablets.

제제 2 : 정제(습식 조립)Formulation 2: Tablet (wet assembly)

활성성분 5.0 ㎎을 체로 친 후, 락토스 16.0 ㎎과 녹말 4.0 ㎎을 섞었다. 폴리솔베이트 80 0.3 ㎎을 순수한 물에 녹인 후 이 용액의 적당량을 첨가한 다음, 미립화하였다. 건조 후에 미립을 체질한 후 콜로이달 실리콘 디옥사이드 2.7 ㎎ 및 마그네슘 스테아레이트 2.0 ㎎과 섞었다. 미립을 가압하여 정제로 만들었다.
After 5.0 mg of the active ingredient was sieved, 16.0 mg of lactose and 4.0 mg of starch were mixed. 0.3 mg of Polysorbate 80 was dissolved in pure water, and an appropriate amount of this solution was added, followed by atomization. After drying, the granules were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The granules were pressurized to make tablets.

제제 3 : 분말과 캡슐제Formulation 3: Powder and Capsule

활성성분 5.0 ㎎을 체로 친 후에, 락토스 14.8 ㎎, 폴리비닐 피롤리돈 10.0 ㎎, 마그네슘 스테아레이트 0.2 ㎎와 함께 섞었다. 혼합물을 적당한 장치를 사용하여 단단한 No. 5 젤라틴 캡슐에 채웠다.
5.0 mg of the active ingredient was sieved and mixed with 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone and 0.2 mg of magnesium stearate. The mixture was filtered through a hard No. 5 gelatin capsules.

제제 4 : 주사제Formulation 4: Injection

활성성분으로서 100 mg을 함유시키고, 그 밖에도 만니톨 180 mg, Na₂HPO₄·12H₂O 26 mg 및 증류수 2974 mg를 함유시켜 주사제를 제조하였다.
100 mg as an active ingredient, 180 mg of mannitol, 26 mg of Na ₂ HPO ₄ .12H ₂ O and 2974 mg of distilled water were added to prepare an injection.

[실험예] 생리활성 평가
[Experimental Example] Evaluation of physiological activity

실험예 1. in vitro PLK-PBD 저해활성Experimental Example 1: In vitro PLK-PBD inhibitory activity

형광단이 부착된 PBD-결합 펩타이드로서 FITC-펩타이드와 본 발명의 화합물들이 PLK-PBD 결합부위에서 직접 경쟁에 기인한 형광 편광(fp)의 변화를 측정하였다. 본 발명의 화합물의 PLK-PBD 저해활성을 측정한 결과는 하기 표 1에 나타내었다.As a PBD-binding peptide with a fluorescent moiety, the FITC-peptide and the compounds of the present invention were measured for the change in fluorescence polarization (fp) due to direct competition at the PLK-PBD binding site. The results of measuring the PLK-PBD inhibitory activity of the compounds of the present invention are shown in Table 1 below.

실험화합물Experimental compound PLK-PBD 저해활성 (IC₅₀, uM)PLK-PBD inhibitory activity (IC ₅₀ , uM) 실시예 3Example 3 <3<3 실시예 4Example 4 <3<3 실시예 5Example 5 <3<3 실시예 6Example 6 <3<3

실험예 2. in vivo 유사분열중단(mitotic arrest) 효능Experimental Example 2: In vivo mitotic arrest efficacy

본 발명의 실시예 5 화합물 12.24 ng 및 24.48 ng을 각각 제브라피시(zebrafish) 배아에 미세주사(microinjection)한 후에, PLK1-PBD 저해활성을 확인한 결과를 도 1의 (A) 및 (B)에 나타내었다. 12.24 ng and 24.48 ng of the compound of Example 5 of the present invention were microinjected into zebrafish embryos respectively and the results of confirming PLK1-PBD inhibitory activity were shown in Figs. 1 (A) and 1 (B) .

도 1의 (A)에 의하면, phospho-H3-positive 세포수는 실시예 5 화합물의 용량에 비례하게 증가함을 확인할 수 있다.1 (A), it can be seen that the number of phospho-H3-positive cells increases in proportion to the dose of the compound of Example 5. [

또한, 도 1의 (B)는 웨스턴 블롯 분석한 결과로서 실시예 5 화합물의 용량에 비례하게 증가함을 확인할 수 있다. In addition, FIG. 1 (B) shows that as a result of western blot analysis, it increases in proportion to the capacity of the compound of Example 5.

따라서 본 발명의 화합물은 in vivo 유사분열중단(mitotic arrest) 효능을 가지고 있으므로, PLK1-PBD 저해에 기인하여 항종양제로 유용함을 알 수 있다.
Thus the compounds of the present invention can be prepared by reacting in vivo Since it has a mitotic arrest effect, it is useful as an antitumor agent due to inhibition of PLK1-PBD.

Claims

하기 화학식 1로 표시되는 고리형 포스포펩티드 화합물, 이의 수화물, 이의 용매화물, 이의 이성질체, 라세미체 및 약학적으로 허용 가능한 이의 염 :
[화학식 1]

상기 화학식 1에서,
R₁은 수소원자; C₁∼C₆ 알킬기; C₁∼C₆ 알콕시기; C₁∼C₆ 알킬아미노기; 또는

를 나타내고,
R₂는

를 나타내고,
A는 C₁∼C₆ 알킬기; C₁∼C₆ 알킬, C₁∼C₆ 알콕시 및 C₁∼C₆ 할로알킬로 이루어진 군으로부터 선택된 1 내지 3개의 치환기로 치환 또는 비치환된 페닐기; 또는 N, O 및 S로부터 선택된 헤테로원자가 1 내지 3개 포함된 5각형 내지 7각형의 포화헤테로고리기를 나타내고,
R₃, R₄, R₅, 및 R₆은 서로 같거나 다른 것으로서 수소원자; C₁∼C₆ 알킬기; 또는 (C₁∼C₆ 알킬싸이오)C₁∼C₆ 알킬기를 나타내고,
R₇은 C₁∼C₆ 알킬기를 나타내고,
n은 0, 1 또는 2를 나타낸다.
CLAIMS 1. A cyclic phosphopeptide compound represented by the following formula (1), a hydrate thereof, a solvate thereof, an isomer thereof, a racemate thereof and a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

In Formula 1,
R ₁ is a hydrogen atom; A C _{1 to} C ₆ alkyl group; A C _{1 to} C ₆ alkoxy group; A C ₁ -C ₆ alkylamino group; or

Lt; / RTI >
R ₂ is

Lt; / RTI >
A is a C ₁ -C ₆ alkyl group; A phenyl group substituted or unsubstituted with 1 to 3 substituents selected from the group consisting of C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy and C ₁ -C ₆ haloalkyl; Or a saturated 5- to 7-membered heterocyclic group containing 1 to 3 hetero atoms selected from N, O and S,
R ₃ , R ₄ , R ₅ , and R ₆ are the same or different from each other and are a hydrogen atom; A C _{1 to} C ₆ alkyl group; Or a (C ₁ -C ₆ alkylthio) C ₁ -C ₆ alkyl group,
R ₇ represents a C ₁ -C ₆ alkyl group,
n represents 0, 1 or 2;

제 1 항에 있어서,
R₁은 C₁∼C₆ 알콕시기; C₁∼C₆ 알킬아미노기; 또는

를 나타내고,
R₂는

를 나타내고,
A는 C₁∼C₆ 알킬기; C₁∼C₆ 알콕시 및 C₁∼C₆ 할로알킬로 이루어진 군으로부터 선택된 1 내지 3개의 치환기로 치환 또는 비치환된 페닐기; 또는 피롤리디닐, 피라졸리디닐 및 트리아졸리디닐로 이루어진 군으로부터 선택된 포화헤테로고리기를 나타내고,
R₃, R₄, R₅, 및 R₆은 서로 같거나 다른 것으로서 수소원자; C₁∼C₆ 알킬기; 또는 (C₁∼C₆ 알킬싸이오)C₁∼C₆ 알킬기를 나타내고,
R₇은 C₁∼C₆ 알킬기를 나타내고,
n은 0, 1 또는 2를 나타내는 것을 특징으로 하는 화합물.
The method according to claim 1,
R ₁ is a C ₁ -C ₆ alkoxy group; A C ₁ -C ₆ alkylamino group; or

Lt; / RTI >
R ₂ is

Lt; / RTI >
A is a C ₁ -C ₆ alkyl group; A phenyl group substituted or unsubstituted with 1 to 3 substituents selected from the group consisting of C ₁ -C ₆ alkoxy and C ₁ -C ₆ haloalkyl; Or a saturated heterocyclic group selected from the group consisting of pyrrolidinyl, pyrazolidinyl and triazolidinyl,
R ₃ , R ₄ , R ₅ , and R ₆ are the same or different from each other and are a hydrogen atom; A C _{1 to} C ₆ alkyl group; Or a (C ₁ -C ₆ alkylthio) C ₁ -C ₆ alkyl group,
R ₇ represents a C ₁ -C ₆ alkyl group,
and n represents 0, 1 or 2.

제 1 항에 있어서,
R₁은 메톡시기, 에톡시기, 노말프로폭시기, 이소프로폭시기, 노말부톡시기, 이소부톡시기, 메틸아미노기, 에틸아미노기, 노말프로필아미노기, 이소프로필아미노기, 또는 -NH-CH(이소부틸)-C(O)OCH₃를 나타내고,
R₂는

를 나타내고,
A는 메틸기, 에틸기, 노말프로필기, 이소프로필기, 노말부틸기, 이소부틸기, 3-메톡시페닐기, 4-메톡시페닐기, 3,5-다이메톡시페닐기, 3,5-다이(이소부톡시)페닐기, 3-(트리플루오로메틸)페닐기, 또는 피롤리딘-2-일기를 나타내고,
R₃, 및 R₄는 서로 같거나 다른 것으로서 수소원자, 메틸기, 에틸기, 노말프로필기, 이소프로필기, 노말부틸기, 이소부틸기, 메틸싸이오메틸기, 또는 메틸싸이오에틸기를 나타내고,
n은 0, 1 또는 2를 나타내는 것을 특징으로 하는 화합물.
The method according to claim 1,
R ₁ is a group selected from a methoxy group, an ethoxy group, a normal propoxy group, an isopropoxy group, a normal butoxy group, an isobutoxy group, a methylamino group, an ethylamino group, a normal propylamino group, an isopropylamino group or -NH- -C (O) represents the OCH _3,
R ₂ is

Lt; / RTI >
A is a group selected from the group consisting of a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a normal butyl group, an isobutyl group, a 3- methoxyphenyl group, a 4- methoxyphenyl group, (Trifluoromethyl) phenyl group, or a pyrrolidin-2-yl group,
R ₃ and R ₄ are the same or different and each represents a hydrogen atom, a methyl group, an ethyl group, a normal propyl group, an isopropyl group, a normal butyl group, an isobutyl group, a methylthiomethyl group, or a methylthioethyl group,
and n represents 0, 1 or 2.

제 1 항에 있어서,
메틸 (3S, 6S, 9S, 17R, 19S)-9-아세트아미도-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이사이클로[15.2.1]이코산-19-카복실레이트;
메틸 (3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-9-((S)-4-(메틸싸이오)-2-((S)-피롤리딘-2-카복스아미도)부탄아미도)-2,5,8,12-테트라옥소-3-( (R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카복실레이트;
메틸 ((3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-9-((S)-4-(메틸싸이오)-2-((S)-피롤리딘-2-카복스아미도)부탄아미도)-2,5,8,12-테트라옥소-3-( (R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트;
메틸 ((3S, 6S, 9S, 17R, 19S)-9-((S)-2-((S)-2-아세트아미도-4-메틸펜탄아미도)-4-메틸펜탄아미도)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트;
메틸 ((3S, 6S, 9S, 17R, 19S)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-9-(3-(트리플루오로메틸)벤자미도)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카보닐)-L-로이시네이트;
메틸 ((3S, 6S, 9S, 17R, 19S)-9-(3,5-다이이소부톡시벤자미도)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-카보닐)-L-로이시네이트; 및
메틸 (3S, 6S, 9S, 17R, 19S)-9-(3,5-다이이소부톡시벤자미도)-6-(하이드록시메틸)-2,5,8,12-테트라옥소-3-((R)-1-(포스포녹시)에틸)-16-옥사-1,4,7,13-테트라아자바이시클로[15.2.1]이코산-19-카복실레이트; 로부터 선택되는 것을 특징으로 하는 화합물.
The method according to claim 1,
Methyl (3S, 6S, 9S, 17R, 19S) -9-acetamido-6- (hydroxymethyl) -2,5,8,12-tetraoxo- Yl) ethyl) -16-oxa-1,4,7,13-tetraaza- bicyclo [15.2.1] iso-19-carboxylate;
Methyl (3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -9 - ((S) -4- (methylthio) -2- (R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetra Azabicyclo [15.2.1] octanoic-19-carboxylate;
Methyl ((3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -9 - ((S) -4- (methylthio) -2- Carboxamido) butanamido) -2,5,8,12-tetraoxo-3- ((R) -1- (Phosphoroxy) ethyl) -16- Tetraaza bicyclo [15.2.1] iso-19-carbonyl) -L-leucinate;
Methyl ((3S, 6S, 9S, 17R, 19S) -9 - ((S) -2- 6- (hydroxymethyl) -2,5,8,12-tetraoxo-3 - ((R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetraaza Bicyclo [15.2.1] octanoic-19-carbonyl) -L-leucinate;
Methyl ((3S, 6S, 9S, 17R, 19S) -6- (hydroxymethyl) -2,5,8,12-tetraoxo-3 - ((R) -1- (Phosphoroxy) 16-oxa-1,4,7,13-tetraaza bicyclo [15.2.1] iso-19-carbonyl) -L-leucine Nate;
Methyl ((3S, 6S, 9S, 17R, 19S) -9- (3,5-diisobutoxybenzamido) -6- (hydroxymethyl) -2,5,8,12-tetraoxo- ((R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetraaza bicyclo [15.2.1] iso-carbonyl) -L-isocyanate; And
Methyl (3S, 6S, 9S, 17R, 19S) -9- (3,5-diisobutoxybenzamido) -6- (hydroxymethyl) -2,5,8,12-tetraoxo- (R) -1- (Phosphoroxy) ethyl) -16-oxa-1,4,7,13-tetraaza-bicyclo [15.2.1] isoic acid-19-carboxylate; &Lt; / RTI >

청구항 제 1 항 내지 제 4 항의 화합물이 포함된 항종양제.
An antitumor agent comprising the compound of any one of claims 1 to 4.

제 5 항에 있어서,
식도암, 췌장암, 폐암, 침윤성 직장암, 유방암, 전이성 전립선암, 난소암, 또는 흑색종의 치료, 예방 또는 개선을 위하여 사용되는 항종양제.
6. The method of claim 5,
An antineoplastic agent for the treatment, prevention or amelioration of cancer of the esophagus, pancreatic cancer, lung cancer, invasive rectal cancer, breast cancer, metastatic prostate cancer, ovarian cancer, or melanoma.