KR100322240B1 - Invention of a new α-Glucosidase inhibitor from a Fungus, Penicillium sp. F70614(KCTC 8918P) - Google Patents
Invention of a new α-Glucosidase inhibitor from a Fungus, Penicillium sp. F70614(KCTC 8918P) Download PDFInfo
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- KR100322240B1 KR100322240B1 KR1019990036927A KR19990036927A KR100322240B1 KR 100322240 B1 KR100322240 B1 KR 100322240B1 KR 1019990036927 A KR1019990036927 A KR 1019990036927A KR 19990036927 A KR19990036927 A KR 19990036927A KR 100322240 B1 KR100322240 B1 KR 100322240B1
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- penicillium
- kctc
- glucosidase
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Abstract
본 발명은 신균주 페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P)와 α-글루코시다제 저해작용을 갖는 다음 화학식 1로 표시되는 디케토피페라진 유도체에 관한 것이다.The present invention relates to a diketopiperazine derivative represented by the following Chemical Formula 1 having Penicillium sp. F70614 (KCTC 8918P) and α-glucosidase inhibitory activity.
Description
본 발명은 신균주 페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P)와 α-글루코시다제 저해작용을 갖는 다음 화학식 1로 표시되는 디케토피페라진 유도체에 관한 것이다.The present invention relates to a diketopiperazine derivative represented by the following Chemical Formula 1 having Penicillium sp. F70614 (KCTC 8918P) and α-glucosidase inhibitory activity.
화학식 1Formula 1
당(Sugar)의 아노머 히드록시기(anomeric hydroxy group)는 당과 가역 적으로 축합되어 α,β-글리코시드를 형성하게 되며, 이때의 아노머 탄소와 산소를 연결하는 결합을 글리코시드 결합(glycosidic bond)이라고 한다. 이러한 글리코시드 결합의 가수분해는 글리코시다제라는 효소에 의해 촉매 화된다.Sugar's anmeric hydroxy group is reversibly condensed with sugars to form α, β-glycosides, and glycosidic bonds are used to link the anomer carbon and oxygen. It is called). Hydrolysis of these glycosidic bonds is catalyzed by an enzyme called glycosidase.
여러 종류의 카보하이드레이트 절단효소 중에서도 특히, α-글루코시다제는 탄수화물의 소화과정 중 마지막 단계에서 참여하게 되는 중요 효소로서[Jun. W., Biosci. Biotech. Bioch., 61(1), 177∼178, 1997], 당뇨나 비만의 억제 및 치료를 위하여 의학적으로 이용되고 있다[D. J. A. Diabetes, 30, 951∼954, 1981].즉, α-글루코시다제는 세포 내에서 다당류내의 당과 당, 당과 지질, 또는 당과 단백질간의 글리코시드 결합을 절단하는 기능을 가지는 바, 글루코시다제 효소반응을 저해하는 경우 소장에서 당의 흡수가 지연되어 당뇨와 비만증 치료에 효과적이다.Among the various carbohydrate cleavage enzymes, α-glucosidase is an important enzyme involved in the final stage of carbohydrate digestion [ Jun. W., Biosci. Biotech. Bioch. , 61 (1), 177-178, 1997], which has been used medically for the prevention and treatment of diabetes and obesity [ DJA Diabetes , 30, 951-954, 1981]. Has a function of cleaving glycosidic bonds between sugars and sugars, sugars and lipids, or sugars and proteins in polysaccharides. Inhibiting glucosidase enzyme reactions delays the absorption of sugars in the small intestine and is effective in treating diabetes and obesity. .
한편, α-글루코시다제 저해제는 전분과 이 당류의 섭취에 인한 식후 혈당상승의 억제에 관여하는데 있어, α-아밀라제 보다 특이적이다. α-글루코시다제 저해제는 술포닐우레아제 계열의 화합물과는 달리 인슐린 분비를 감소시켜 주며, 당뇨병 환자에게 동맥경화증과 고혈압을 유발시키는 인슐린 과다분비증(hyperinsulinemia)을 유발하지 않는 장점이 있다. 또한, 글리코시다제의 결여로 발생되는 질병 예를 들면 만노시도시스(mannosidosis)의 경우, 만노시다제(mannosidase)가 결여되어 있어 결과적으로 만노스를 포함한 슈가 체인(sugar chain)들이 세포 내에 축적됨으로써 독성을 나타내게 되는 것이다. 기타 여러 질병에서도 글리코시다제의 양적 변화와 관련이 있는 것으로 되어 있는 바, 예컨대 암 환자의 혈청 또는 암 세포사이의 분비액에서는 높은 농도의 글리코시다제가 발견되기도 하였다. 그리고, β-N-아세틸 글루코스 아미니다제와 β-글루쿠로니다제의in vitro실험에서 많은 암세포들이 이 효소들을 세포 외 액으로 분비한다는 것을 발견하였다. 이 효소들은 제1 사이트(primary site)로부터 암세포가 분화되는 것을 가속시킬 수 있을 뿐만 아니라, 다른 제2 사이트(secondary site)로의 이동이나 공격을 도와줌으로써 세포표면에서 올리고사카라이드(oligosaccharide)의 구조와 생합성 그리고 암세포에서의 형태 변화에도 관여를 한다. 따라서, 글리코시다제 저해제들은 암의 진행이나 변이를막을 수도 있다. 그리고, 세포 표면에서 올리고사카라이드의 구조와 생합성 그리고 암세포에서의 형태변화에도 관여한다. 예를 들면 최근에 보고된 바에 의하면, 강력한 α-글루코시다제의 저해제인 노지리마이신(nojirimycin)은 T세포의 HIV 감염을 억제한다고 알려졌다[R. A. Gruters, Nature, 330, 74∼77, 1987].On the other hand, the α-glucosidase inhibitor is more specific than the α-amylase in being involved in the suppression of postprandial blood sugar elevation due to the ingestion of starch and saccharides. α-glucosidase inhibitors, unlike sulfonylurease-based compounds reduce insulin secretion, and has the advantage of not causing hyperinsulinemia, which causes atherosclerosis and hypertension in diabetic patients. In addition, diseases caused by lack of glycosidase, such as mannosidosis, lack mannosidase, resulting in the accumulation of sugar chains containing mannose in cells. It will be toxic. Many other diseases have been associated with quantitative changes in glycosidase. For example, high concentrations of glycosidase have been found in serum from cancer patients or secretions between cancer cells. In vitro experiments with β-N-acetyl glucose aminidase and β-glucuronidase found that many cancer cells secrete these enzymes into extracellular fluid. These enzymes not only accelerate the differentiation of cancer cells from the primary site, but also aid in the movement or attack of other secondary sites, and thus the structure of the oligosaccharides on the cell surface. It is also involved in biosynthesis and morphological changes in cancer cells. Thus, glycosidase inhibitors may prevent the progression or mutation of cancer. It is also involved in the structure and biosynthesis of oligosaccharides on the cell surface and in morphological changes in cancer cells. For example, recently reported nojirimycin, a potent α-glucosidase inhibitor, is known to inhibit T cell HIV infection ( RA Gruters, Nature , 330, 74-77, 1987).
본 발명은 토양 곰팡이류로부터 신균주 페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P)를 분리하였고, 이러한 신균주로부터 생산하거나 또는 화학적 방법으로 합성하여 수득된 상기 화학식 1로 표시되는 디케토피페라진 유도체가 α-글루코시다제 저해활성을 가지고 있음을 알게됨으로써 본 발명을 완성하였다.The present invention is isolated from the fungal strain Penicillium sp. Penicillium sp. F70614 (KCTC 8918P), diketopiperazine represented by the formula (1) produced from these new strains or obtained by synthesis by a chemical method The present invention has been completed by knowing that the derivative has α-glucosidase inhibitory activity.
도 1은 페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P)의 현미경 사진이다.1 is a micrograph of Penicillium sp. F70614 (KCTC 8918P).
도 2는 페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P) 균주 배양기간에 따른 세포성장 및 α-글루코시다제 저해율을 나타낸 그래프이다.Figure 2 is a graph showing cell growth and α-glucosidase inhibition rate according to Penicillium sp. F70614 (KCTC 8918P) strain culture period.
도 3은 화학식 1로 표시되는 디케토피페라진 유도체의 FAB 질량 분석 스펙트럼이다.3 is a FAB mass spectrometry spectrum of a diketopiperazine derivative represented by Chemical Formula 1. FIG.
도 4는 CD3OD 용액에서 화학식 1로 표시되는 디케토피페라진 유도체의 300MHz 수소핵자기공명 스펙트럼이다.FIG. 4 is a 300 MHz hydrogen nuclear magnetic resonance spectrum of a diketopiperazine derivative represented by Chemical Formula 1 in a CD 3 OD solution. FIG.
도 5는 CD3OD 용액에서 화학식 1로 표시되는 디케토피페라진 유도체의 300MHz 탄소핵자기공명 스펙트럼이다.FIG. 5 is a 300 MHz carbon nuclear magnetic resonance spectrum of a diketopiperazine derivative represented by Chemical Formula 1 in a CD 3 OD solution. FIG.
도 6은 화학식 1로 표시되는 디케토피페라진 유도체 투여량에 따른 돼지소장 α-글루코시다제 저해 활성을 나타낸 그래프이다.6 is a graph showing the activity of pig small intestine α-glucosidase according to the dose of diketopiperazine derivatives represented by the formula (1).
본 발명은 α-글루코시다제 저해물질을 생산하는 페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P)를 그 특징으로 한다.The invention is characterized by Penicillium sp. F70614 (KCTC 8918P), which produces α-glucosidase inhibitors.
또한, 본 발명은 α-글루코시다제 저해활성을 가지는 다음 화학식 1로 표시되는 디케토피페라진 유도체 및 이의 제조방법을 포함한다.In addition, the present invention includes a diketopiperazine derivative represented by the following formula (1) having an α-glucosidase inhibitory activity, and a preparation method thereof.
화학식 1Formula 1
또한, 본 발명은 상기 화학식 1로 표시되는 디케토피페라진 유도체가 포함되어 있는 α-글루코시다제 저해제를 포함한다.In addition, the present invention includes an α-glucosidase inhibitor containing a diketopiperazine derivative represented by the formula (1).
이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다.Referring to the present invention in more detail as follows.
1. 신균주의 동정1. Identification of Mycobacteria
대한민국 전국의 토양으로부터 분리된 곰팡이 균주를 포테이토-덱스트로스 아가, 차벡크 아가(Czapek's agar) 및 맥아 추출 아가에 접종한 후에 관찰한 결과는 다음 표 1에 나타내었다.The results observed after inoculation of fungal strains isolated from soils in Korea were inoculated in potato-dextrose agar, Czapek's agar and malt extract agar.
생육은 매우 양호하였으며 25℃에서 7일간 배양시 콜로니 직경이 30 ∼ 40㎜에 이르렀다. 표면 색깔은 노란색을 띤 녹색이었고, 뒷면 색깔은 짙은 녹색을 보였다.Growth was very good and colony diameter reached 30-40 mm at 7 ° C. for 7 days. The surface color was yellowish green and the backside color was dark green.
형태적인 관찰을 위해 포테이토-덱스트로스 아가 배지에서 광학현미경과 전자현미경을 이용하여 관찰한 결과[도 1 참조]에 의하면, 분생자 자루(conidiophore)는 기저 균사에서 시작되었으며, 페니실리아는 단윤생(monoverticillate)으로 보였다. 피아리드(Phialides)의 크기는 2 ∼ 3 ×7.5 ㎛이고, 분생자(conidia)의 형태는 구형(globose)에서 반구형(subglobose)로 변했으며, 직경은 2.0 ∼ 2.5 ㎛ 이었다.According to the results of observation using optical and electron microscopy in potato-dextrose agar medium for morphological observation [see FIG. 1], the conidiophore started from the basal hyphae, and penicillia was the only monoverticillate). Phialides range in size from 2 to 3 × 7.5 Μm, the shape of conidia changed from globose to subglobose, and the diameter was 2.0-2.5 μm.
상기한 특성으로 미루어보아, 이 생산균주는 페니실리움 속(Penicilliumsp.)에 속하는 것으로 밝혀졌다. 이에, 본 발명에서는 신균주를 페니실리움 속(Penicilliumsp.) F70614로 명명하고, 1998년 12월 16일자로 생명공학연구소 내 유전자원센터에 기탁하여 KCTC 8918P의 수탁 번호를 부여받았다.In view of the above characteristics, the production strains are in the genus Penicillium (Penicilliumsp.). Thus, in the present invention, the new strain is penicillium (Penicilliumsp.) Designated as F70614 and deposited on 16 December 1998 at the Genetic Resources Center in the Institute of Biotechnology, KCTC 8918P. You have been assigned an accession number.
2. 신균주의 배양2. Cultivation of Mycobacteria
본 발명에 있어서의 균주 배양은 통상의 미생물이 사용할 수 있는 영양 원을 함유하는 배지에서 배양한다. 예를 들어 탄소원은 글루코스를 사용하고, 질소원으로 효모추출액, 폴리펩톤 등을 사용한다. 그리고 황산 마그네슘, 다이포타슘 포스페이트 등의 무기염류를 첨가하여 배양한다. 배양방법으로는 호기적 조건에서 진탕 배양 혹은 통기교반 배양하는 것이 좋다. 배양온도는 각 조건에 따라서 약간씩 상이하기는 하나 25℃ ∼ 30℃에서 배양하는 것이 적당하였으나, 28℃에서 배양하였다. 또한, 배양기간이나 진탕 배양의 경우 통상 5 ∼ 7일 배양함으로써 상기 화학식 1로 표시되는 신규 화합물의 생산이 최고에 달하였다[도 2참조].Strain culture in the present invention is cultured in a medium containing a nutrient source that can be used by ordinary microorganisms. For example, glucose is used as the carbon source, and yeast extract and polypeptone are used as the nitrogen source. And inorganic salts, such as magnesium sulfate and dipotassium phosphate, are added and cultured. As a culture method, shaking culture or aeration stirring culture in aerobic conditions is preferable. The culture temperature was slightly different depending on the conditions, but it was appropriate to incubate at 25 ℃ ~ 30 ℃, it was incubated at 28 ℃. In addition, in the case of incubation period or shaking culture, the production of the new compound represented by Chemical Formula 1 reached the highest by culturing for 5 to 7 days [see FIG. 2].
3. 신규 화합물의 분리 및 정제3. Isolation and Purification of New Compounds
상기한 바와 같은 배양 조건하에서 페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P) 균주를 배양하여 상기 화학식 1로 표시되는 신규한 디케토피페라진 유도체를 얻는다. 상기 화학식 1로 표시되는 디케토피페라진 유도체는 균주 배양액 뿐 아니라 균체 부분에서도 존재하므로 배양액을 원심 분리하여 배양 상등 액과 균체를 분리한 후 다음에서 기술하는 방법에 의하여 효율적으로 추출 정제를 실시한다. 즉, 상기 화학식 1로 표시되는 디케토피페라진 유도체는 지용성 물질이므로 배양액을 에틸아세테이트, 클로로포름 등의 유기용매를 가하여 추출한다. 한편 균체 부분은 아세톤 등의 유기용매를 가하여 교반하여 균체로 부터도 유효성분을 추출한 후 아세톤 등의 용매는 증발시키고 다시 에틸아세테이트, 클로로포름 등으로 추출한 후 배양액과 합친다. 얻어진 유효성분이 포함된 혼합액으로부터 에틸아세테이트, 클로로포름 등의 용매를 증발 건조시킨 후 클로로포름/메탄올 = 100/1 ∼ 10/1(V/V)의 용매를 사용하여 실리카겔 컬럼크로마토그라피를 실시한다. 그리고, 얻어진 활성부위를 다시 세파덱스 LH-20 컬럼 크로마토그라피를 클로로포름/메탄올 = 1/1(V/V)로 재차 실시한다. 그런 다음, HPLC에 의해서 순수한 유효 화합물을 얻을 수 있다. 상기한 바와 같은 분리 정제방법을 단독 또는 복합적으로 반복하면 더욱 손쉽게 순수한 정제 품을 얻을 수 있다. Penicillium sp. F70614 (KCTC 8918P) strains were cultivated under the culture conditions as described above to obtain a novel diketopiperazine derivative represented by Chemical Formula 1. Since the diketopiperazine derivative represented by Chemical Formula 1 is present in the bacterial culture as well as the strain culture solution, the culture solution is centrifuged to separate the culture supernatant and the bacteria and then efficiently extracted and purified by the method described below. That is, since the diketopiperazine derivative represented by Chemical Formula 1 is a fat-soluble substance, the culture solution is extracted by adding an organic solvent such as ethyl acetate or chloroform. On the other hand, the cell part is agitated by adding an organic solvent such as acetone, and extracting the active ingredient from the cell, the solvent such as acetone is evaporated and extracted again with ethyl acetate, chloroform and then combined with the culture solution. After solvents such as ethyl acetate and chloroform are evaporated to dryness from the mixture containing the obtained active ingredient, silica gel column chromatography is performed using a solvent of chloroform / methanol = 100/1 to 10/1 (V / V). Then, the obtained active site was subjected to Sephadex LH-20 column chromatography again with chloroform / methanol = 1/1 (V / V). The pure effective compound can then be obtained by HPLC. By repeating the separation and purification method as described above alone or in combination, it is possible to obtain a pure purified product more easily.
구조분석을 위한 NMR 분석은 Varian UNITY 300을 이용하였으며, 용매는CD3OD를 이용하여 측정하였다. 분자량 결정은 ESIMS(electrospray ionization mass spectroscopy)로 측정하였으며, UV spectrum(Shimazu UV-260)은 메탄올을 용매로 측정하였다.NMR analysis for structural analysis was carried out using Varian UNITY 300, the solvent was measured using CD 3 OD. The molecular weight was determined by electrospray ionization mass spectroscopy (ESIMS), and the UV spectrum (Shimazu UV-260) was measured by methanol as a solvent.
페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P) 균주가 생산하는 활성물질은 백색분말로 UV λmax(MeOH) 208 ㎚에서 최대흡수 피크를 보였으며, FAB, ESIMS 분석결과 분자량이 182로 확인되었으며 HR-mode 상에서는 계산치 182.1055, 실측치 182.1056 으로 나타났으며, 그 결과 분자식은 C9H14N2O2로 결정되었으며,1H,13C, HMBC(heteronuclear multiple bond coherence) 등에 의해 구조를 확인하였다.The active substance produced by Penicillium sp. F70614 (KCTC 8918P) strain was white powder and showed a maximum absorption peak at UV λ max (MeOH) 208 nm, and the molecular weight was 182 as determined by FAB, ESIMS analysis. On the HR-mode, the calculated value was 182.1055 and found 182.1056. As a result, the molecular formula was determined as C 9 H 14 N 2 O 2 , and the structure was confirmed by 1 H, 13 C, and heteronuclear multiple bond coherence (HMBC). .
그 밖의 활성물질에 대한 이화학적 특징은 다음과 같다:The physicochemical characteristics of the other active substances are as follows:
1) 물질의 성상 : 백색분말1) Property: White powder
2) 분자량: 1822) Molecular Weight: 182
3) 분자식: C9H14N2O2 3) Molecular Formula: C 9 H 14 N 2 O 2
4) 질량분석(M+H) : 182[도 3 참조]4) Mass spectrometry (M + H): 182 [See FIG. 3]
5) 자외선 흡수 스펙트럼(UV λmax(MeOH)) : 208, 350 ㎚5) UV absorption spectrum (UV λ max (MeOH)): 208, 350 nm
6) 적외선 흡수 스펙트럼(KBr)㎝-1: 3447, 2928, 1678, 1637, 1458, 13376) Infrared absorption spectrum (KBr) cm -1 : 3447, 2928, 1678, 1637, 1458, 1337
7) 핵자기공명(NMR) 흡수스펙트럼 : CD3OD를 용매로 하고 TMS(Si(CH3)4)를 표준물질로 하여 수소핵자기공명(1H-NMR) 스펙트럼과 탄소핵자기공명(13C-NMR) 스펙트럼을 조사하여 도 4 및 도 5에 각각 나타내었다.7) nuclear magnetic resonance (NMR) absorption spectrum: a CD 3 OD as solvent and TMS (Si (CH 3) 4), hydrogen nuclear magnetic resonance (1 H-NMR) spectra and carbon nuclear magnetic resonance (13 to a standard C-NMR) spectra were examined and shown in FIGS. 4 and 5, respectively.
8) 용해성8) Solubility
용해: 아세톤, 클로로포름, 에틸아세테이트, 메탄올Soluble: Acetone, Chloroform, Ethyl Acetate, Methanol
불용: 물Insoluble: water
9) 박층 크로마토 그라피 : Rf값 0.399) Thin layer chromatography: R f value 0.39
흡착제 : 머크사 실리카겔 60F 254Adsorbent: Merck Silica Gel 60F 254
전개용매 : CHCl3/MeOH = 10/1(V/V)Developing Solvent: CHCl 3 / MeOH = 10/1 (V / V)
10) 화학 구조식10) Chemical Structural Formula
4. 신규 화합물의 화학적 합성4. Chemical Synthesis of Novel Compounds
상기 화학식 1로 표시되는 디케토피페라진 유도체는 페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P) 균주 배양물로부터 얻을 수 있고, 또한 화학적 합성방법으로도 제조가 가능하다. 즉, 상기 화학식 1로 표시되는 디케토피페라진 유도체는 데하이드로알라닌과 루이신이 결합하고 있는 구조를 지니고 있는 바, 먼저 t-boc-세린과 루이신 메틸 에스테르를 고리화 반응시킨 후 tos-클로라이드를 이용하여 세린 잔기의 OH기를 제거하므로써 합성한다.The diketopiperazine derivative represented by Chemical Formula 1 may be obtained from Penicillium sp. F70614 (KCTC 8918P) strain culture, and may also be prepared by chemical synthesis. That is, the diketopiperazine derivative represented by Chemical Formula 1 has a structure in which dehydroalanine and leucine are bound. First, cyclization of t-boc-serine and leucine methyl ester is performed, followed by tos-chloride. It synthesize | combines by removing OH group of serine residue using
5. 신규 화합물에 대한 α-글루코시다제 저해활성 검정5. Assay of α-glucosidase inhibitory activity on novel compounds
상기 화학식 1로 표시되는 디케토피페라진 유도체에 대한 α-글루코시다제 저해활성은 검체 30 ㎕, 완충액(0.1M 인산완충액 pH 7.0) 250 ㎕ 및 기질로서 p-니트로페닐-α-D-글루코피라노사이드(5 mM, pH 7.0) 250 ㎕를 넣은 후, 효소용액(α-글루코시다제(4.75 unit/㎖, 시그마사) 10 ㎕를 첨가하였다. 37℃에서 30분간 반응시킨 후 0.4M 글리신-수산화나트륨용액(pH 10.4) 2 ㎖를 가해 반응을 정지시킨 후 410 ㎚에서 흡광도를 측정하였다. 그리고 천연기질인 말토스를 이용하여 효소활성을 측정할때는 효소작용에 의해서 유리된 글루코스의 양을 신양화학의 지엘자임 키트(GLzyme kit)를 사용하여 500 ㎚에서 측정하였다. 그후 결과를 다음 수학식 1에 대입하여 효소활성 저해율을 계산하였다.Α-glucosidase inhibitory activity of the diketopiperazine derivative represented by Formula 1 was 30 μl of the sample, 250 μl of buffer (0.1 M phosphate buffer pH 7.0) and p-nitrophenyl-α-D-glucose as a substrate. 250 μl of ranoside (5 mM, pH 7.0) was added, followed by 10 μl of enzyme solution (α-glucosidase (4.75 unit / ml, Sigma). 2 ml of sodium hydroxide solution (pH 10.4) was added to stop the reaction, and the absorbance was measured at 410 nm, and the amount of glucose liberated by enzymatic action was measured when enzyme activity was measured using maltose, a natural substrate. The enzyme was measured at 500 nm using the GLzyme kit, and then the results were substituted into the following equation to calculate the enzyme activity inhibition rate.
상기 수학식 1에서 : A는 저해제를 넣은 것의 반응전 흡광도이고; B는 저해제를 넣은 것의 반응후 흡광도이고; C는 저해제를 넣지 않은 것의 반응전 흡광도이고; D는 저해제를 넣지 않은 것의 반응후 흡광도이다.In Equation 1: A is the absorbance before the reaction of the inhibitor; B is the absorbance after the reaction with the inhibitor; C is the absorbance before the reaction without the inhibitor; D is the absorbance after the reaction with no inhibitor.
이에, 본 발명은 상기 화학식 1로 표시되는 디케토피페라진 유도체가 유효성분으로 함유되어 있는 α-글루코시다제 저해제를 포함한다. 본 발명에 따른 약제에는 상기 화학식 1로 표시되는 디케토피페라진 유도체 이외에도 해당 기술분야에서 통상의 지식을 가진 전문가라면 용이하게 알 수 있는 약학적으로 허용 가능한 담체 또는 부형제가 함께 함유되어 정제, 산제, 과립, 캅셀제, 현탁제, 유화액 또는 비경구 투여용의 단위투여용 또는 수회 투여용 제제로 제형화할 수 있다. 약제의 유효투입량은 환자의 나이, 신체적 조건, 몸무게 등에 의해 다양해질 수 있느며, 일반적으로 1 ∼ 10 ㎎/㎏(몸무게)/1일 범위내에서 투여된다. 그리고, 1일 유효투입량 범위내에서 하루에 한 번 또는 하루에 여러번 나누어 투입할 수 있다.Accordingly, the present invention includes an α-glucosidase inhibitor containing the diketopiperazine derivative represented by Formula 1 as an active ingredient. In addition to the diketopiperazine derivative represented by Chemical Formula 1, the medicament according to the present invention contains a pharmaceutically acceptable carrier or excipient which can be easily understood by those skilled in the art, and includes tablets, powders, It may be formulated into granules, capsules, suspensions, emulsions or formulations for parenteral or parenteral administration for parenteral administration. The effective dosage of the medicament may vary depending on the age, physical condition, weight, etc. of the patient, and is generally administered within the range of 1 to 10 mg / kg (weight) / day. In addition, within a daily effective dosage range, it can be injected once or several times a day.
이상에서 설명한 바와 같은 본 발명은 다음의 실시 예에 의거하여 더욱 상세히 설명하겠는 바, 본 발명이 다음의 실시예에 의해 한정되는 것은 아니다.The present invention as described above will be described in more detail based on the following examples, but the present invention is not limited by the following examples.
실시예 1 : 미생물 배양Example 1 Microbial Culture
종배지 및 생산배지로서 글루코스 2%, 효모추출액 0.2%, 폴리펩톤 0.5%, 황산마그네슘 0.05%, 포타슘 다이포스페이트 0.1%를 함유한 배지에 pH 5.6으로 조정한 후 사용하였다. 500 ㎖의 삼각프라스크 2개에 각각 100 ㎖씩 분주한 종배지를 121℃에서 20분간 살균하고, 이것에 페니실리움 속(Penicilliumsp.) F70614 균주의 사면배양 1∼2 백금이를 접종하고 28℃에서 3일간 진탕 배양하여 배양기의 종배양으로 사용하였다. 121℃에서 30분 살균한 3ℓ생산배지를 함유한 5ℓ퍼멘터(fermentor)에 종배양액 200 ㎖를 접종하여 28℃에서 7일간 통기 하에서 400 rpm의 교반속도로 배양하였다.It was used after adjusting to pH 5.6 in a medium containing 2% glucose, 0.2% yeast extract, 0.5% polypeptone, 0.05% magnesium sulfate, and 0.1% potassium diphosphate as seed and production medium. 100 ml of two 500 ml Erlenmeyer flasks were sterilized for 20 minutes at 121 ° C., and inoculated with 1 to 2 platinum seeds of Penicillium sp. F70614 strain. Shaking culture for 3 days at 28 ℃ was used as species culture of the incubator. 200 ml of the seed culture solution was inoculated into a 5 L fermentor containing 3 L production medium sterilized at 121 ° C. for 30 minutes, and incubated at 28 ° C. for 7 days under aeration at 400 rpm.
실시예 2 : 배양물질의 구조 결정Example 2 Determination of Structure of Culture Material
상기 실시예 1에서 얻은 배양여액 6ℓ를 12,000 ×g에서 15분간 원심 분리하여 배양 상등액과 균체를 분리하고, 균체는 다시 동량의 100% 아세톤으로 2회 반복 추출하고 감압 농축하여 아세톤을 제거한 후 배양 상등액과 합하여 다시 동량의 에틸아세테이트로 3회 반복 추출하였다. 에틸아세테이트 추출물을 농축한 후 클로로포름에 용해하여 실리카겔(70∼230 mesh, Merck) 크로마토그라피상에서 클로로포름/메탄올 = 100/1 →10/1(V/V)로 조성된 유기용매로 전개시켜 활성분획을 모았다. 이 활성분획을 농축한 후 클로로포름/메탄올 = 1/1(V/V)의 전개용매로 하여 세파덱스 LH-20(Pharmacia) 컬럼크로마토그라피상에서 전개하여 활성 분획을 모았다. 이 활성 분획을 농축한 후 고압액체 크로마토그라피(칼럼: YMC-pack ODS 250 ×100 ㎜, 5 ㎛, 유속 1 ㎖/분, 254 ㎚ 검출, 용출액 50% 메탄올)상에서 분리하여 체류시간 20분 근처에서 활성물질을 순수분리하고, 용매를 감압 건조기로 제거한 후 냉동건조 하여 백색의 분말 5 ㎎을 얻었다.The culture supernatant and the cells were separated by centrifuging 6 l of the culture filtrate obtained in Example 1 at 12,000 × g for 15 minutes, and the cells were again extracted twice with the same amount of 100% acetone and concentrated under reduced pressure to remove acetone. Combined and extracted again three times with the same amount of ethyl acetate. The ethyl acetate extract was concentrated, dissolved in chloroform, and developed on silica gel (70-230 mesh, Merck) chromatography with an organic solvent composed of chloroform / methanol = 100/1 → 10/1 (V / V) to obtain an active fraction. Collected. The active fractions were concentrated and developed on Sephadex LH-20 (Pharmacia) column chromatography using a developing solvent of chloroform / methanol = 1/1 (V / V) to collect the active fractions. The active fractions were concentrated and separated on high pressure liquid chromatography (column: YMC-pack ODS 250 × 100 mm, 5 µm, flow rate 1 ml / min, 254 nm detection, eluent 50% methanol) at a residence time of about 20 minutes. The active substance was purely separated, the solvent was removed with a reduced pressure drier, and lyophilized to obtain 5 mg of white powder.
구조분석을 위한 NMR 분석은 Varian UNITY 300을 이용하였으며, 용매는 CD3OD를 이용하여 측정하였다. 분자량 결정은 FAB(fast atom bombardment), ESIMS(electrospray ionization mass spectroscopy)로 측정하였으며, UV spectrum(Shimazu UV-260)은 메탄올을 용매로 측정하였다.NMR analysis for structural analysis was carried out using Varian UNITY 300, the solvent was measured using CD 3 OD. Molecular weight was determined by fast atom bombardment (FAB) and electrospray ionization mass spectroscopy (ESIMS), and UV spectrum (Shimazu UV-260) was measured by methanol as a solvent.
그 결과, 페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P) 균주가 생산하는 활성물질의 이화학적 특징은 다음 표 1과 같다. 즉, 활성물질은 백색분말로 UV λmax(MeOH) 208 ㎚에서 최대흡수 피크를 보였으며, FAB, ESIMS 분석결과 분자량이 182로 확인되었다. 또한, NMR에 의한 구조분석 결과 분자식은 C9H14N2O2로 결정되었으며, HMBC(heteronuclear multiple bond coherence) 등에 의해 구조를 확인하였다.As a result, the physicochemical characteristics of the active material produced by Penicillium sp. F70614 (KCTC 8918P) strain are shown in Table 1 below. That is, the active material showed a maximum absorption peak at a UV λ max (MeOH) 208 nm as a white powder, the molecular weight was found to be 182 by FAB, ESIMS analysis. In addition, as a result of structural analysis by NMR, the molecular formula was determined as C 9 H 14 N 2 O 2 , and the structure was confirmed by heteronuclear multiple bond coherence (HMBC).
실시예 3 : 화학식 1로 표시되는 디케토피페라진 유도체의 화학적 합성Example 3 Chemical Synthesis of Diketopiperazine Derivatives of Formula 1
t-Boc-세린(205 ㎎, 1 mmol) 205 ㎎을 염화메틸렌(10 ㎖)에 가하고, 얼음 중탕 하에서 루이신 메틸 에스테르(181 ㎎, 1 mmol), 트리에틸아민(140 ㎕, 1 mmol) 및 N-에틸-N'-(3-디메틸아미노프로필)카르보디이미드 염산염(192 ㎎, 1 mmol)을 투입하였다. 반응용액은 얼음 중탕 하에서 하룻동안 반응시켰다. 염화메틸렌(10 ㎖)을 가하고 증류수(40 ㎖)로 세척한 다음, 유기 층은 1N 시트르산(40 ㎖)으로 세척하고 1N 탄산수소나트륨(40 ㎖)으로 계속해서 세척하고 건조시켜 액상(240 ㎎)을 얻었다. 포름산(20 ㎖)을 첨가한 후에 상온에서 2시간 30분 동안 유지시키고, sec-부탄올(20 ㎖) 및 톨루엔(10 ㎖)을 가하고 2시간 30분 동안 환류하였다. 에테르(20 ㎖)를 첨가하고 메탄올으로 세척한 후에 건조하여 노란 액상의 싸이클로 L-ser-L-leu를 얻었다. 상기에서 합성한 싸이클로 L-ser-L-leu에 0℃의 p-톨루엔술포닐 클로라이드/피리딘 용액을 2시간동안 첨가하고, 0℃에서 12시간동안 유지시킨 후 건조시켰다. 에테르와 석유에테르의 혼합용액(1:1)을 사용하여 세척한 결과, 싸이클로 L-leu-ser-Tos를 얻었다. 디메틸포름아마이드(DMF)에 상기에서합성한 싸이클로 L-leu-ser-Tos를 가하고 용해시킨 다음, 디에틸포름아마이드(10.45 ㎖)를 가하고 상온에서 5시간동안 유지시킨 후 건조하여 상기 화학식 1로 표시되는 디케토피페라진 유도체를 합성하였다.205 mg of t-Boc-serine (205 mg, 1 mmol) was added to methylene chloride (10 ml), leucine methyl ester (181 mg, 1 mmol), triethylamine (140 μl, 1 mmol) and N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride (192 mg, 1 mmol) was added. The reaction solution was reacted under ice bath for one day. Methylene chloride (10 mL) was added and washed with distilled water (40 mL), then the organic layer was washed with 1N citric acid (40 mL), washed with 1N sodium bicarbonate (40 mL) and dried to give a liquid (240 mg). Got. Formic acid (20 mL) was added and then maintained at room temperature for 2 hours 30 minutes, sec-butanol (20 mL) and toluene (10 mL) were added and refluxed for 2 hours 30 minutes. Ether (20 mL) was added, washed with methanol and dried to obtain L-ser-L-leu as a yellow liquid cycle. The above synthesized cycle was added to the L-ser-L-leu at 0 ℃ p-toluenesulfonyl chloride / pyridine solution for 2 hours, maintained at 0 ℃ for 12 hours and dried. The resultant was washed with a mixed solution of ether and petroleum ether (1: 1) to obtain L-leu-ser-Tos as a cycle. L-leu-ser-Tos was added to dimethylformamide (DMF) and dissolved in the above synthesized cycle. Then, diethylformamide (10.45 ml) was added thereto, maintained at room temperature for 5 hours, and then dried to show the formula (1). Diketopiperazine derivatives were synthesized.
실시예 4 : α-글루코시다제 저해활성 측정Example 4: Measurement of α-glucosidase inhibitory activity
α-글루코시다제에 대한 상기 화학식 1로 표시되는 디케토피페라진 유도체의 저해활성 정도를 조사하였다. 디케토피페라진 유도체 투여량에 따른 효소 저해 율은 상기 수학식 1과 같이 계산하여 도 6에 나타내었으며, IC50값은 35 ㎍/㎖(효모 효소), 50 ㎍/㎖(돼지 소장) 이었다.The degree of inhibitory activity of the diketopiperazine derivatives represented by the formula (1) on α-glucosidase was investigated. Enzyme inhibition rate according to the diketopiperazine derivative dosage was calculated as shown in Equation 1 shown in Figure 6, the IC 50 value was 35 ㎍ / ㎖ (yeast enzyme), 50 ㎍ / ㎖ (pork small intestine).
또한, 다른 글루코시다제에 대한 상기 화학식 1로 표시되는 디케토피페라진 유도체의 활성 비교는 다음 표 2에 나타내었다.In addition, a comparison of the activity of the diketopiperazine derivative represented by Formula 1 with respect to other glucosidase is shown in Table 2 below.
실시예 6 : α-글루코시다제 저해제의 급성독성 조사Example 6 Acute Toxicity Study of α-Glucosidase Inhibitors
실험동물은 생명공학연구소에서 유지 관리되고 있는 SPF(Specific Pathogens Free; 특정 병원체 부재) 상태의 6주령 암컷 ICR 마우스를 사용하였다. 실험동물은 온도 22 ±1℃, 습도 55 ±5%, 조명 12L/12D의 동물실내에서 유지되었다. 마우스는 실험에 사용하기 전에 순화시켰다. 시료의 조제는 30 ㎎/㎏의 용량으로 조제하였다. 투여방법은 먼저 그룹당 마우스를 3 마리로 나눈 뒤 마우스 10 g-몸무게를 기준으로 하여 0.1 ㎖ 씩 1회 경구 투여하였다. 약물 투여 후 10일 동안 마우스 몸무게 변화 및 사망여부를 관찰하였다. 투여당일은 투여 후 1시간, 4시간, 8시간, 12시간 뒤에, 투여 익일부터 10일째까지는 매일 오전, 오후 1회 이상씩 일반증상의 변화 및 사망동물의 유무를 관찰하였다. 또한 투여 당일부터 3일 간격으로 체중의 변화를 측정하여 시료에 의한 동물의 체중변화를 관찰하였다. 그 결과 급성 경구독성 실험결과 본 시료를 100 ㎎/㎏의 용량으로 경구 투여한 후 10일간 치사동물은 관찰되지 않았다. 그리고 부검시 특별한 병변 육안 소견이 없었으며, 투여 익일부터 10일간 어떠한 체중의 감소 없이 정상적인 체중의 증가가 관찰되었다. 결론적으로 본 시료는 경구 투여시 독성이 관찰되지 않았다[표 3 및 표 4 참조].The experimental animals used 6-week-old female ICR mice with SPF (Specific Pathogens Free) maintained by the Biotechnology Institute. The experimental animals were maintained in an animal room with a temperature of 22 ± 1 ° C., a humidity of 55 ± 5% and illumination of 12L / 12D. Mice were purified prior to use in experiments. The sample was prepared at a dose of 30 mg / kg. The administration method was first divided into three mice per group and then orally administered once by 0.1 ml based on the 10 g-weight of the mouse. Mouse weight change and death were observed for 10 days after drug administration. On the day of dosing, 1 hour, 4 hours, 8 hours, and 12 hours after the administration, the change of general symptoms and the presence of dead animals were observed at least once in the morning and afternoon every day from the next day to the 10th day of administration. In addition, the change in body weight was measured at intervals of 3 days from the day of administration to observe the weight change of the animal by the sample. As a result of the acute oral toxicity test, no mortality was observed for 10 days after oral administration of this sample at a dose of 100 mg / kg. At necropsy, there were no gross lesions, and normal body weight gain was observed without any weight loss for 10 days from the day after administration. In conclusion, no toxicity was observed in this sample upon oral administration (see Tables 3 and 4).
이상에서 설명한 바와 같이, 본 발명에 따른 신균주 페니실리움 속(Penicilliumsp.) F70614(KCTC 8918P)로부터 생산 또는 화학적인 합성 방법으로 합성된 상기 화학식 1로 표시되는 디케토피페라진 유도체는 α-글루코시다제에 대한 저해활성이 우수하므로 당뇨, 비만증 등의 치료 및 예방을 위한 유효약물로 유용하다.As described above, the diketopiperazine derivative represented by Chemical Formula 1 synthesized by Penicillium sp. F70614 (KCTC 8918P) or synthesized by a chemical synthesis method according to the present invention is α- Because of its excellent inhibitory activity against glucosidase, it is useful as an effective drug for the treatment and prevention of diabetes and obesity.
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