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ISSN 0377-9556 (PRINT) ISSN 2383-9457 (ONLINE) 약학회지제 61 권제 5 호 260~266 (2017) Yakhak Hoeji Vol. 61, No. 5 DOI 10.17480/psk.2017.61.5.260 종설 Short Report 새로운메틸 3,4- 디알킬카보닐옥시 -5- 메톡시벤조에이트유도체의합성및항산화와세포증식억제작용 김채원 신다혜 정하숙 * 박명숙 # 덕성여자대학교약학대학, * 덕성여자대학교자연과학대학식품영양학과 (Received August 22, 2017; Revised October 11, 2017; Accepted October 12, 2017) Antioxidant, Antiproliferative Activity and Synthesis of Novel Methyl 3,4- Dialkylcarbonyloxy-5-methoxybenzoate Derivatives Chaewon Kim, Da Hae Shin, Hasook Jung*, and Myung-Sook Park # College of Pharmacy, Duksung Women s University, Seoul 01369, Korea * Department of Food and Nutrition, College of Natural Sciences, Duksung Women s University, Seoul 01369, Korea Abstract The antiproliferative and antioxidative properties of novel polyphenolic acid derivatives, structurally related with the natural model gallic acids, have been tested in cancer cell lines (MCF-7, Hep3B, RKO) and lipid peroxidation. A new series of methyl 3,4-dialkylcarbonyloxy-5-methoxybenzoate 4a~4e was synthesized by acylation from O-methyl gallicin 3 for search of bioactive compounds. The structures of the synthetic compounds were characterized using 1 H, 13 C NMR and HRMS spectroscopy. Two target compounds (4c, 4d) showed higher potency than propyl gallicin or drumstick-tree (Moringa oleifera Lam.) extracts for inhibiting the growth of cancer cell lines (MCF-7, Hep3B, RKO). The antioxidative properties of 4b among synthetic compounds was similar to the antioxidant propyl gallate and α-tocopherol. Keywords Methyl gallicins, Methyl 3,4-dialkylcarbonyloxy-5-methoxybenzoates, Acylation, Antioxidant, Antiproliferative activity 갈산또는몰식자산으로알려진 gallic acid는오배자, 옻나무, 차엽등여러가지식물에함유되어있는일종의유기산성분으로잉크, 염료, 제약산업의원료물질등으로이용되어왔다. 1) Gallic acid의 alkyl ester 유도체는주로약리적인관점에서연구자들의많은관심을끌어왔는데, 특히항암제로서의연구가활발하게보고되었다. 2) 그밖에 ethyl gallate의항혈전작용, 3) propyl gallate 의세포독성작용과 4) 항산화작용, 5) octyl gallates의항암작용등이연구되었다. 6) 본연구에서는기존항암제의부작용으로인한항암치료의문제점을개선하고자독성을감소시킨새로운항암물질의창출이 # Corresponding Author Myung-Sook Park College of Pharmacy, Duksung Women s University, Seoul 01369, Korea Tel.: 02-901-8395 Fax.: 02-901-8386 E-mail: mspark@duksung.ac.kr 시급하다는것에착안점을두었다. 이를위하여다양한식물에서발견되어여러가지생리활성작용을나타내고있는 gallic acid 유도체를주목하게되었다. 본연구에서는항산화제로알려져있는 propyl gallate의구조를변형하여새로운화합물의창출과그의활성을탐색하려고한다. 최근까지항암성 gallic acid 유도체를찾기위한연구들이보고되었지만보다획기적으로구조가변화된항암성유도체들에대한것은없다. 본연구에서는 Fig. 1에서보는바와같이 propyl gallate의 ester기인 propyl기대신에 methyl기를갖는 ester인 gallicin 구조를기본골격으로설계하였다. 본연구에서 methyl기구조를기본골격으로삼은것은의약시장에서간장질환치료제로사용되고있는닛셀 (Nissel @, 파마킹 ) 의성분인 biphenyl dimethyl dicarboxylates의구조를응용하였기때문이다. 그리고세개의페놀성 hydroxyl group에변화를주고자우선벤젠고리의 5-위치의 hydroxyl group를선택적인 monomethylation반응으로 O-methyl기를도입하고, 다음으로 3, 4-위치의 hydroxyl 260

Antioxidant and Antiproliferative Methyl 3,4-Dialkylcarbonyloxy-5-methoxybenzoates 261 Fig. 1 Reported antioxidant propyl gallate, liver-protective biphenyl dimetnyl dicarboxylates, and target compounds 4a~4e. group을 acylation반응으로각각 O-alkylcarbonyl기로치환하기로하였다. 본연구에서는설계및합성한 acylated O-methyl gallicin유도체들의항산화효과와세포증식억제효과실험들을수행하였다. 또한, 항산화물질인 propyl gallate 뿐만아니라모링가추출물이세포증식억제효과가있다는최근의연구와 7, 13-16) 접목하였다. 새로운합성화합물들을모링가추출물등을이용하여항암성질을비교해보고새로운항암성및항산화성물질창출에대한유의한지견을얻었기에보고하고자한다. 실험방법 (Experimental Methods) 시약및분석기기시약은 Sigma-Aldrich, TCI 등에서구입했고, 융점측정은 Büchi 545 Melting Point apparatus를이용하였다. Bruker사의 300 MHz NMR spectrometer로 1 H 및 13 C NMR data를측정하였다. TMS를기준으로하여 ppm단위로 chemical shift 값을기록했다. Silica gel 60F 254로피막된 TLC plate를이용하여반응의진행을확인하였다. 전개용매로 n-hexanes: ethyl acetate (3/1, 2/1, 1/1 등 ) 를사용하였다. HRMS는 JMS-700 Mass spectrometer에서측정하였다. 흡광도측정을위한 ELISA reader 는 ASYS Biotech, Cambridge, UK를사용하였다. 모링가추출물제조본실험에사용한모링가는탄자니아 (2013년, 9월 ) Dar es Saalam에서채집하여부위별로추출하였다. 건조한모링가잎과열매의추출방법은 70% 에탄올에담구어 3시간동안실온에서 sonication 하였다. 추출액을여과하여 60 o C에서감압농축한다음건조하여 20 o C에보관하였다가 7) 본연구의 CCK-8 assay에사용하였다. 합성 General synthetic procedure for the methyl 3,4- dihydroxy-5-methoxybenzoate (3) Na 2 B 4 O 7 10H 2 O (17.2 g, 45 mmol) 을물 (160 ml) 에녹여 5% 수용액으로만들고, gallicin 2 (5.5 g, 30 mmol) 을용해시킨후실온에서 2시간동안교반하였다. 이용액에 dimethyl sulfate (7 ml, 75 mmol) 와 sodium hydroxide (3.0 g, 75 mmol) 의포화수용액을적가하였다. 반응액은 22시간동안교반하면서흑갈색으로변했다. 과량의 dimethyl sulfate를분해하기위하여 c- NH 4 OH (5 ml) 용액을가하여교반하고, 황산을가해산성용액 (ph 2~3) 으로만든다음, ethyl acetate (25 ml 4회 ) 로추출하였다. 유기층을모아물로세척하고무수 sodium sulfate로건조하여감압농축하였다. 용매를제거하고얻은수지상물질을 methanol로재결정하여미황색의결정 3 (mp: 118~120 o C) 을얻었다. 물질의 NMR data를이용하여새로운 O-methyl기의도입을확인하였고, 그결과문헌과일치하였다. 8) Method A. General synthetic procedure for the methyl 3,4-diethylcarbonyloxy-5-methoxybenzoate (4a, 4b) Methyl 3,4-dihydroxy-5-methoxybenzoate 3 (1.0 g, 5 mmol) 에 propionic anhydride 10 μl (78 mmol) 을가하여혼합한다음, 반응용액을 heating mantle에서 100~120 를유지하며약 1시간동안교반하면서반응시켰다. 반응용액의색깔은처음갈색 J. Pharm. Soc. Korea

262 김채원 신다혜 정하숙 박명숙 에서점점노란색으로변화하였다. 반응종결후실온까지냉각시킨반응혼합액에 diethyl ether 50 ml를가하여녹이고, 5% Na 2 CO 3 수용액 20 ml로 2회추출한다. 계속하여, diethyl ether 층을정제수 15 ml로 2회세척한다. Ether 층을취해 magnesium sulfate를이용하여건조하였고, 여과후감압농축하여용매를제거하였다. 그결과연한노란색의 oil상물질을얻었다. 이잔류물을 column chromatography (silicagel, solvent; n-hexane: ethyl acetate = 1: 1) 로분리하여 4b를얻었다. Methyl 3,4-dimethylcarbonyloxy-5-methoxybenzoate (4a): Yield: 93%, mp.: 91-94 o C, TLC [n-hexane: ethyl acetate (1:1)] R f 0.50. 1 H NMR(CDCl 3 ) δ 7.54(s, 1H, phenyl), 7.49(s, 1H, phenyl), 3.90(s, 3H, OCH 3 ), 3.88(s, 3H, OCH 3 ), 2.31(s, 3H, CH 3 ), 2.29(s, 3H, CH 3 ). 13 C NMR(CDCl 3 ) δ 167.92, 167.16, 165.63(carbonyl), 152.40, 143.27, 135.94, 128.25(phenyl), 116.94, 110.84(acetyl), 56.45, 52.38(methoxy). HRMS(m/z) (FAB, M+Na) Calcd for 305.0637, found 305.0627. Methyl 3,4-diethylcarbonyloxy-5-methoxybenzoate (4b): Yield: 38%, oil, TLC [n-hexane: ethyl acetate (1:1)] R f 0.80. 1 H NMR(CDCl 3 ) δ 7.53(s, 1H, phenyl), 7.49(s, 1H, phenyl), 3.89(s, 3H, OCH 3 ), 3.87(s, 3H, OCH 3 ), 2.60(t, J=7.6 Hz, 2H, CH 2 ), 2.55(t, J=7.6 Hz, 2H, CH 2 ) 1.25(tt, J=7.5 Hz, 6H, CH 3 ). 13 C NMR(CDCl 3 ) δ 171.44, 170.70, 165.71(carbonyl), 152.44, 143.38, 136.05, 128.10(phenyl), 116.96, 110.75(methylene), 56.45, 52.34(methoxy), 27.38(ethyl). HRMS(m/z) (FAB, M+H) Calcd for 310.1053, found 310.1054. Method B. General synthetic procedure for the methyl 3,4-dipropylcarbonyloxy-5-methoxybenzoate (4c~4e) Methyl 3,4-dihydroxy-5-methoxybenzoate 3 (1.0 g, 5 mmol) 를 toluene 10 ml에가하고, butyric anhydride 2.5 ml (15 mmol) 을더해 1시간동안환류하면서반응하였다. Methyl 3,4-dihydroxy-5- methoxybenzoate 3은온도가올라가면서 toluene에잘녹아갈색용액이되었고, 반응이진행될수록반응용액의색깔이점점연해져서투명한주황색으로변화하였다. 반응이종결된후반응용매인 toluene 을감압농축하여제거하고, diethyl ether 50 ml 과 5% Na 2 CO 3 15 ml로총 2회추출하였다. 다시, ethyl ether 층을정제수 10 ml로 2회세척한다. Ether 층을취해 magnesium sulfate를이용하여건조하였고, 여과후감압농축하여유기용매를제거하였다. 그결과연한노란색의 oil상물질을얻었다. 이잔류물을 column chromatography (silica gel, solvent; n-hexane: ethyl acetate = 3: 1) 로분리하여 4c를얻었다. Methyl 3,4-di-n-propylcarbonyloxy-5-methoxybenzoate (4c): Yield: 31%, oil, TLC [n-hexane: ethyl acetate (1:1)] R f 0.80. 1 H NMR(CDCl 3 ) δ 7.55(s, 1H, phenyl), 7.49(s, 1H, phenyl), 3.91(s, 3H, OCH 3 ), 3.89(s, 3H, OCH 3 ), 2.56(t, J=7.2 Hz, 2H, CH 2 ), 2.53(t, J=7.2 Hz, 2H, CH 2 ), 1.83-1.74(m, 4H, CH 2 ), 1.04(tt, J=7.3 Hz, 6H, CH 3 ). 13 C NMR (CDCl 3 ) δ 170.63, 169.87, 165.72(carbonyl), 152.41, 143.37, 136.09, 128.08(phenyl), 117.00, 110.72(methylene), 56.41, 52.34(methoxy), 35.80, 35.59, 18.45, 13.49(propyl). HRMS(m/ z) (FAB, M+H) Calcd for 338.1366, found 338.1366. Methyl 3,4-di-n-butylcarbonyloxy-5-methoxybenzoate (4d): Yield: 11%, oil, TLC [n-hexane: ethyl acetate (1:1)] R f 0.65. 1 H NMR(CDCl 3 ) δ 7.55(s, 1H, phenyl), 7.49(s, 1H, phenyl), 3.91(s, 3H, OCH 3 ), 3.89(s, 3H, OCH 3 ), 2.58(t, J=7.5 Hz, 2H, CH 2 ), 2.53(t, J=7.5 Hz, 2H, CH 2 ), 1.80-1.69(m, 4H, CH 2 ), 1.52-1.35(m, 4H, CH 2 ), 0.97(tt, J=7.3 Hz, 6H, CH 3 ). 13 C NMR(CDCl 3 ) δ 170.79, 70.02, 165.72 (carbonyl), 152.43, 143.39, 136.11, 128.08(phenyl), 117.01, 110.72(methylene), 56.42, 52.33(methoxy), 33.68, 26.95, 22.18, 13.60(butyl). HRESIMS(m/z) (FAB, M+Na) Calcd for 389.1576, found 389.1564. Methyl 3,4-di-n-pentylcarbonyloxy-5-methoxybenzoate (4e): Yield: 35%, oil, TLC [n-hexane: ethyl acetate (3:1)] R f 0.51. 1 H NMR(CDCl 3 ) δ 7.52(s, 1H, phenyl), 7.47(s, 1H, phenyl), 3.89(s, 3H, OCH 3 ), 3.87(s, 3H, OCH 3 ), 2.56(t, J=7.5 Hz, 2H, CH 2 ), 2.53(t, J=7.5 Hz, 2H, CH 2 ), 1.78-1.59(m, 6H, CH 2 ), 1.48-1.34(m, 6H, CH 2 ), 0.95(tt, J=7.3 Hz, 6H, CH 3 ). 13 C NMR(CDCl 3 ) δ 170.80, 169.56, 165.72(carbonyl), 152.41, 143.38, 136.09, 128.07(phenyl), 117.00, 110.71(methylene), 56.41, 52.34(methoxy), 34.96, 33.68, 26.95, 22.11, 13.61 (pentyl). HRESIMS(m/z) (FAB, M+Na) Calcd for 417.1889, found 417.1891. 지질과산화억제 (Inhibition of lipid peroxidation) 실험지질과산화억제율을 Koleva등의 9, 10) β-carotene/linoleic acid system을응용하여측정하였다. β-carotene용액 (400 μg/ml in chloroform) 3 ml에 linoleic acid (20 mg), Tween 40 (200 mg) 을천천히첨가하여잘섞고 chloroform을 N 2 gas를이용하여완전히제거한다. 잔사에증류수 50 ml을천천히넣으면서격렬하게잘섞어준후희석용액을만든다. 합성화합물 4a~4e 을증류수로용해한용액이들어있는시험관 (ep tube) 에희석용액 (emulsion) 900 μl 를첨가하였다. 시험관 (ep tube) 의내용물 (200 μl) 을 96 Well 배양접시에옮기고 ELISA reader를이용하여 470 nm에서흡광도 (Ac) 를측정하였다. 배양접시를 50 o C incubator에서 2 시간반응한후다시흡광도 (As) 를구하였다. 합성화합물 4a~4e의 antioxidant activity(aa) (%) 은 [(control Vol. 61, No. 5, 2017

Antioxidant and Antiproliferative Methyl 3,4-Dialkylcarbonyloxy-5-methoxybenzoates 263 Ac control Ac) (sample As sample As)]/(control Ac control Ac) 100의식으로구하여비교하였다. CCK-8 (cell counting kit-8) assays 4a~4e에대한항암활성을측정하기위하여 CCK-8 Kit (Dojindo, Kumamoto, Kyushu, Japan) assay방법을이용하였다. 11) 본연구의활성검색에사용된 3종의암세포 (MCF-7, Hep3B, RKO 세포 ) 는 ATTC (Manassas, USA) 에서구입하였다. 96 Well 배양접시에각세포를 5 10 3 cells/well로분주하여 CO 2 incubator (37 o C, 5% CO 2 ) 에서 24 시간동안보관하였다. 24시간후에배지를갈아주고, 미리정한 0.1, 1, 10, 100, 1000 μg/ ml 의농도로화합물 4a~4e를처리하여각각 48시간동안배양하였다. 합성한화합물 4a~4e을처리한세포에 Kit-8 시약 10 μl를넣고 3시간동안반응시킨후 ELISA reader(asys Biotech, Cambridge, UK) 로 450 nm에서흡광도 (A 450 ) 를측정하였다. 세포생존율은다음과같이계산하였다 : Cell viability = (test group A 450 / control group A 450 ) 100%, IC 50 값은세번의독립적인실험값으로결정하였다. 결과및고찰 (Results and Discussion) 본연구에서는상업적으로이용가능한 gallic acid 10H 2 O을출발물질로사용하였다. 최종화합물은 esterification, methylation, 그리고 acylation 12) 의합성방법을이용하여제조하였다 (Scheme 1). 출발물질을황산촉매하에서메탄올과 esterification반응으로 methyl ester화합물 2로전환시켰다. 다음으로온화한반 Scheme 1. Synthetic route of target O-acylated O-methyl gallicins; methyl 3,4-dialkylcarbonyloxy-5-methoxybenzoates 4a~4e. Fig. 2 Antioxidant activities of five synthetic compounds, propyl gallate, and α-tochopherol (β-carotene-linoleic acid test). Values of each curve are means±sd (n=2). 응조건하에서세개의 hydroxyl group를갖고있는 methyl gallate 2의구조에서선택적으로한개의 hydroxyl group만 methyl ether 3로바꾸었다. 즉, borax 150 mol% 를사용하여 saltation 을완성하고, methyl화제로서 dimethyl sulfate (250 mol%) 와 NaOH수용액을실온에서 22시간동안반응시켰다. 벤젠고리의 3, 4-위의 hydroxyl기에아실기도입을위한시약으로는 carboxylic acid anhydride 를이용하였다. Alkylcarbonyl기의도입을위해이용된 alkyl기는 methyl, ethyl, n-propyl, n-butyl, n-pentyl 기의 5종이었고, acetic anhydride, propionic anhydride, butyric anhydride, valeric anhydride, hexanoic anhydride와같은 acyclic acid anhydride 시약을구입하여이용하였다. 화합물 4a와 4b는용매없이과량의 anhydride 를사용하는 method A 방법으로합성하였으나, alkyl 기의길이가길어지면서반응의수득율이아주떨어지는결과를얻게되었다. 아실화반응의효율성을위하여화합물 4c~4e는 anhydride (300 mol%) 와 toluene을용매로사용하는 method B 방법으로제조하였다. O-acylation 반응에서는 anhydride의 carbonyl기에 3,4-위의페놀성 hydroxyl기가첨가되면서 zwitterionic 중간체가형성되었다가, 다음으로분해가일어나 alkylcarbonyloxy 화합물 4a~4e 과 carboxylic acid로전환되었다. 최종화합물의구조는 1 H NMR, 13 C NMR과 HRMS의 data로확인하였다. 지질과산화현상은자유라디칼에의해서세포막의손상으로발생한다. 항산화효과는지질과산화억제의정도로측정할수있다. 본연구에서의합성화합물들은항산화제인 propyl gallate와구조적으로유사하다. 항산화제로서의개발가능성을타진해보기위해서합성화합물의지질과산화억제실험을수행하였다. 합성화합물 4a~4e의지질과산화반응의억제 ( 항산화 ) 율은 Fig. 2에서보는바와같이항산화제로사용되고있는 α-tocopherol, propyl gallate의항산화율과비교하였다. 4b는농도의존적으로 α-tocopherol 값과유사한항산화억제효과를보였다. 또한, 합성화합물과구조적으로유사한산화제인 propyl gallate와 4b 의 J. Pharm. Soc. Korea

264 김채원 신다혜 정하숙 박명숙 Fig. 3 Antiproliferative activities of five synthetic compounds, propyl gallate, and Moringa oleifera extracts (ML, MF) in RKO human colon carcinoma cells (CCK-8 assays). Values of each curve are means±sd (n=3). Fig. 4 Antiproliferative activities of five synthetic compounds, propyl gallate, and Moringa oleifera extracts (ML, MF) in MCF-7 human breast cancer cells (CCK-8 assays). Values of each curve are means±sd (n=3). Fig. 5 Antiproliferative activities of five synthetic compounds, propyl gallate, and Moringa oleifera extracts (ML, MF) in Hep3B hepatocellular carcinoma cells (CCK-8 assays). Values of each curve are means±sd (n=3). 활성을비교하였을때, 저농도 (10, 20 μg/ml) 에서비슷한억제율을나타내어항산화제로의가능성을보였다. 4d, 4e 에서도 α- tocopherol과비슷한추세선을보이고있어서약한항산화작용을가지고있는것으로생각된다. 최근열대지방에널리분포되어있는식의약용나무인모링가 (drumstick-tree) 에대한관심이높아져그성분에대한연구가활발하다. 암세포증식억제실험의비교물질로서사용한모링가 (Moringa oleifera Lam.) 잎의추출물은암세포의 apoptosis와 antiproliferation 효과가있다고보고되었다. 13) 모링가잎추출물은항산화효과가크고, 동물모델의실험에서안전성이높은 Vol. 61, No. 5, 2017

Antioxidant and Antiproliferative Methyl 3,4-Dialkylcarbonyloxy-5-methoxybenzoates 265 Table I IC 50 values of synthesized compounds 4a~4e, propylgallate, and α-tochopherol against cancer cell lines for 48 h and β- carotene-linoleic acid test. IC 50 (µm) Comp. No RKO a MCF-7 a Hep3B β-carotene-linoleic a acid b 4a 2478.3 233.3 213.1 >1000 4b 2243.0 202.4 197.5 <10 4c 685.8 156.3 143.3 627.4 4d 904.2 110.1 118.9 42.3 4e >3000 98.9 708.9 45.4 propyl gallate >3000 >3000 >3000 <10 α-tochopherol - - - <10 a CCK-8 assay b β-carotene-linoleic acid test 것으로평가되어왔다. 14) 모링가뿌리추출물은암세포침투및전이과정의주요한인자인 MMP-9에대한억제효과를보고되어있다. 15) 모링가의잎뿐만아니라나무껍질의추출물에서도유방암과대장암에항암효과를보였다는연구결과가보고된바있다. 16) 본연구에서는 gallicin 유도체의구조를기반으로한저분자화합물들을합성하였고, 그의세포증식억제효과를모링가의잎과열매의추출물과함께비교하였다. 세포증식억제효과는약물처리후 48시간배양하여그결과를얻었다. Fig. 3에서보는바와같이대장암세포인 RKO cell에서의 CCK- 8 실험에서는 5종의합성화합물 4a~4e과비교물질로항산화제 propyl gallate 그리고두종류의모링가추출물 ( 잎, 열매 ) 의세포증식억제작용을비교하였다. 4a~4e는미리정한 0.1, 1, 10, 100, 1000 μg/ml의농도에서농도의존적으로높은억제율을나타내었다. 이합성화합물들중특히 4c가가장높은억제율을보였다. 유방암세포인 MCF-7 cell에서의 CCK-8 실험에서는 4d와 4e가높은억제율을나타내었다 (Fig. 4). 간암세포인 Hep3B cell에서는 4c와 4d가우수한세포증식억제율을보였다 (Fig. 5). Table I에서보는바와같이항산화효과실험에서는 4b가 α- tochopherol 및 propyl gallate와비슷한정도로우수한활성을가졌다. 암세포증식억제효과실험에서는 5종의합성화합물 4a~4e 모두가암세포 (MCF-7, Hep3B, RKO cells) 에서모링가추출물 (MF, ML) 과 propyl gallate 보다좋은활성을나타내었다. 특히, 4c와 4d 에서우수한세포증식억제작용을보였다. 결론 (Conclusion) 본실험에서는치환된 O-methyl gallicin 유도체 4a~4e 5종을합성하였고, 목표화합물을얻기위한합성경로로 esterification, methylation과 acylation을이용하였다. 합성화합물중 4b의항산 화효과가항산화제인 α-tochopherol 및 propyl gallate 와유사하게나타났다. 화합물 4c, 4d은 3종 (MCF-7, Hep3B, RKO cells) 암세포에서세포증식억제작용을보이기는했으나 IC 50 상으로는그효과가크지는않았고, 비교물질모링가추출물 (MF, ML) 보다는좋은활성을나타내었다. 구조활성관계를기반으로한생리활성이높은유도체탐색에대한연구는추후보고할예정이다. References 1) Ow, Y. Y. and Stupans, I. : Gallic acid and gallic acid derivatives: effects on drug metabolizing enzymes. Curr. Drug Metab. 4, 241 (2003). 2) Locatelli, C., Filippin-Monteiro, F. B. and Creczynski-Pasa, T. B. : Alkyl esters of gallic acid as anticancer agents: A review. Eur. J. Med. Chem. 60, 233 (2013). 3) Yun, H. S., Kang, S. S., Kim, M. H. and Jung, K. H. : Antithrombotic effects of analogs of procatechuic acid and gallic acid. Yakhak Hoeji 37, 453 (1993). 4) Jacobi, H., Eicke, B. and Witte, I. : DNA strand break induction and enhanced cytotoxicity of propyl gallate in the presence of copper(ii). Free Radic. Biol. Med. 24, 972 (1998). 5) Massoni, M., Clavijo, T., ColinapVegas, L., Villarreal, W., Dias, J. S. M., Silva, G. A. F., Ionta, M., Soares, M., Ellena, J., Dorigueto, A., Barbosa, M. and Batista, A. A. : Propyl gallate metal complexes: circular dichroism, BSA-binding, antioxidant and cytotoxic activity. Polyhedron 129, 214 (2017). 6) Fiuza, S. M., Gomes, C., Teixeira, J., Girao-Cruz, M. T., Cordeiro, M. N. D. S., Milhazes, N., Borges, F. and Marques, M. P. M. : Phenolic acid derivatives with potential anticancer properties a structure activity relationship study. Part 1: methyl, propyl and octyl esters of caffeic and gallic acids. Bioorg. Med. Chem. 12, 3581 (2004). 7) Guon, T. E. and Chung, H. S. : Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells. Oncol. Lett. 14, 1703, (2017). 8) Park, M. S. and Kwon, S. K. : Selective methylation of phenolic hydroxyl group; optimal condition for synthesis of methyl 3,4- dihydroxy-5-methoxy benzoate. Duk-Sung Bull Natural Sci. 3, 29 (1997). 9) Koleva, I. I., Beek, T. A., Linssen, J. P. H., Groot, A. and Evstatieva, L. N. : Screening of plant extracts for antioxidant activity: a comparative study on tree testing methods. Phytochem. Anal. 13, 8 (2002). 10) Wu, N., Fu, K., Fu, Y. J., Zu, Y. G., Chang, F. R., Chen, Y. H., Liu, X. L., Kong, Y., Liu, W. and Gu, C. B. : Antioxidant activities of extracts and main components of pigeonpea [Cajanus cajan (L.) Millsp.] leaves. Molecules 14, 1032 (2009). J. Pharm. Soc. Korea

266 김채원 신다혜 정하숙 박명숙 11) Kim, C., Kim, S. B., Jung J. and Park, M. S. : Design, synthesis and biological evaluation of selenium-incorporated aminopyridazines as anticancer agents. Bull. Korean Chem. Soc. 36, 1669 (2015). 12) Park, E. H. and Park, M. S. : Acylation of pyridazinylamines by acyclic anhydrides: synthesis of N-substituted 3-amino-6- chloropyridazines. Yakhak Hoeji 49, 56 (2005). 13) Sreelatha, S., Jeyachitra, A. and Padma, P. R. : Antiproliferation and induction of apoptosis by Moringa oleifera leaf extract on human cancer cells. Food Chem. Toxicol. 49, 1270 (2011). 14) Stohs, S. and Hartman, M. J. : Review of the safety and efficacy of Moringa oleifera. Phytother. Res. DOI: 10.1002/ptr.5325 (2015). 15) Cho, H. J. and Chang, Y. C. : Extract of Moringa root inhibits PMA-induced invasion of breast cancer cells. J. Lif. Sci. 24, 8 (2014). 16) Al-Asmari, A. K., Albalawi, S. M., Athar, M. T., Khan, A. Q., Al- Shahrani, H. and Islam, M. : Moringa oleifera as an anti-cancer agent against breast and colorectal cancer cell lines. PLOS ONE 10, DOI:10.1371/pone.0135814 (2015). Vol. 61, No. 5, 2017