Phosphoinositide specific phospholipase C (PI-PLC) plays a pivotal role in the intracellular signal transduction induced by a variety of extracellular stimuli. In the course of screening programs for PLC inhibitors from natural sources such as fungal isolates and medicinal plants, eleven compounds were isolated.
Compound 1, 2 and 3 were isolated by the bioassay-guided purification from an unidentified fungus MT51005. By the various NMR techniques such as $^1H$-NMR, $^{13}C$-NMR, DEPT, $^{13}C-^1H$ COSY, NOESY, and HMBC, these compounds were determined as anguillosporal (1) (2,4-dihydroxy-3-ethyl-6-(1'-methylpentyl)-benzaldehyde), CRM-51005 (2) (2, 4-dihydroxy-3-ethyl-6-(1'-methyl-1'-pentenyl)-benzaldehyde) and CRM-51006 (3) (1-(3'-ethyl-2', 4-dihydroxyphenyl)-2-methyl-1-hexanone). Among these compounds, CRM-51005 (2) and CRM-51006 (3) were identified as new compounds.
When these compounds were evaluated for the ability to inhibit PLCγ1 activity in vitro, all of compounds inhibited PLCγ1 activity in dose-dependent manner. The compound 3 showed the most potent activity with an $IC_{50}$ of 2 ㎍/ml (8 μM), whereas 1 and 2 showed the similar inhibitory activity with the $IC_{50}$ of 4.7 ㎍/ml (18.8 μM). When the effects of these compounds on the PDGF-induced accumulation of inositol phosphates in PLCγ1-overexpressing NIH3T3 fibroblasts (NIH3T3γ1) cells were examined, 1 and 3 showed the similar inhibitory activity with the $IC_{50}$ of 0.8 ㎍/ml (3.2 μM). But, 2 showed slightly less activity with an $IC_{50}$ of 3.2 ㎍/ml (12.8 μM). The compound 2 showed the potent activity against T47D (breast cancer), PC-3 (prostate cancer) and OVCAR-4 (ovary cancer) among the examined human cancer cell lines. Especially, 2 showed the most potent growth inhibition (GI) against OVCAR-4 with the $GI_{50}$ values of 0.359 ㎍/ml.
Eight isoflavonoids were isolated from ${CH_2Cl_2}$ layer of Erythrina senegalensis (Leguminosae). By the various NMR techniques and physico-chemical properties, these compounds were determined as 8-prenylleutone (4), auriculatin (5), erysenegalensein O (6), erysenegalensein D (7), erysenegalensein N (8), derrone (9), alpinumisoflavone (10), and 6,8-diprenylgenistein (11). Among them, erysenegalensein O (5, 2', 4'-trihydroxy-6-(γ, {γ$-dimethylallyl})-3'''-hydroxy-2''',2'''-dimethyldihydropyrano [5''', 6''']isoflavone and erysenegalensein N (5, 7, 2', 4'-tetrahydroxy-6-(2"-hydroxy-3"-methylbut-3"-enyl)-8-(γ, {γ-dimethylallyl})isoflavone) were confirmed to be new compounds.
When the isolated compounds were evaluated for the ability to inhibit PLCγ1 activity in vitro, 8 showed the most potent activity with an $IC_{50}$ of 1 ㎍/ml (2.3 μM). The study on relationships of structure and activity indicates that the number of prenylated groups and the hydroxylation of prenyl group might play an important role at PI-PLC inhibitory activity. The compounds having two prenyl groups were more active than those having one prenyl group (4 ＜ 5, 7 or 8 ＜ 6). Furthermore, the compounds possessing a hydroxy group at C-2" or C-2''' of prenyl group were more effective than those which didn't contain a hydroxy group (7 or 8 ＞ 4). The compound 8 containing a hydroxy group at C-2" of prenyl group were more potent activity than that containing hydroxy group at C-2''' of prenyl group. Especially, 7 and 8 containing hydroxy group at C-2" or C-2''' of prenyl group inhibited more selectively in vitro PLCγ1 activity comparing with PLCβ1 activity (differences of 6 - 8 times in inhibitory activity against PLCγ1/ PLCβ1 were shown).
Compound 4, 6, 7, 8, and 9 reduced dose-dependently PDGF-induced $IP_t$ accumulation in NIH3T3γ1 cells. The compound 8 also showed the most potent inhibitory activities with an $IC_{50}$ of 10 ㎍/ml (23 μM). In the relationships of structure and activities, the compounds having the hydroxylated prenyl groups were generally more active than those having prenyl groups (7 and 8 ＞ 4 and 11). The compounds 4 and 5 were examined for the cytotoxic effects against several human cancer cell lines such as PC-3 (prostate), NCI-H226 (lung), CRL1579 (melanoma). These compounds have a moderate cytotoxicity ($GI_{50}$ of 9.0 - 20 μM).
Phosphoinositide specific phospholipase C (PI-PLC)는 외부 자극에 의한 세포내의 신호전달과정에서 중요한 역할을 한다. 곰팡이 분리주나 약용식물과 같은 천연자원으로부터 PLC 저해물질을 탐색하는 과정에서 11종의 화합물을 분리하였다. 화합물 1, 2, 3은 미 동정된 곰팡이 MT51005로부터 생물활성 지표에 따른 정제방법에 의하여 분리되었다.
$^1H$-NMR, $^{13}C$-NMR, DEPT, $^{13}C-^1H$ COSY, NOESY, HMBC등과 같은 NMR 분석에 의하여 이 화합물들은 anguillosporal (1), CRM-51005 (2) (2, 4-dihydroxy-3-ethyl-6-(1'-methyl-1'-pentenyl)-benzaldehyde), CRM-51006 (3) (1-(3'-ethyl-2', 4'-dihydroxyphenyl)-2-methyl-1-hexanone)으로 결정되었다. 이중 화합물 CRM-51005 (2) 와 CRM-51006 (3)은 신규 화합물이었다.
분리된 화합물의 PLCγ1 저해활성을 조사한 결과 모두 농도 의존적으로 PLCγ1을 저해하였다. 화합물 3이 가장 강한 저해활성 (2㎍/ml, 8㎛)을 나타냈고 1과 2는 비슷한 저해활성 (4.7㎍/ml, 18.8㎛)을 보여주었다. 세포 내에서의 PLC 저해활성을 알아보기 위하여 NIH3T3γ1 세포에서 혈소판유래성장인자 (PDGF)에 의해 유도되는 총 inositol phosphates ($IP_t$)의 생성에 대한 저해활성 측정시 1과 3은 비슷한 저해활성 (0.8㎍/ml, 3.2μM)을 보여주었으나 화합물 2의 활성은 1과 3에 비하여 상대적으로 약했다 (3.2㎍/ml, 12.8μM). 화합물 2는 여러 암세포주에 대한 성장저해 효과를 보였으며 그 중에서 T47D (breast cancer), PC-3 (prostate cancer), OVCAR-4 (ovary cancer)등에 강한 저해활성을 보여주었다. 특히, OVCAR-4에 대하여 가장 강력한 성장 저해활성을 보여주었다 ($GI_{50}$, 0.359㎍/ml).
8종의 이소풀라보노이드 화합물들이 아프리카산 식물 Erythrina senegalensis로부터 PLC 저해물질로 분리되었다. 여러가지 NMR 기법과 물리 화학적 성질에 의하여 이 화합물들은 8-prenylleutone (4), auriculatin (5), erysenegalensein O (6), erysenegalensein D (7), erysenegalensein N (8), derrone (9), alpinumisoflavone (10)과 6,8-diprenylgenistein (11)으로 결정되었다. 이중 erysenegalensein O (5, 2', 4'-trihydroxy-6-(γ, {γ-dimethylallyl})-3'''-hydroxy-2''',2'''-dimethyldihydropyrano[5''', 6''']isoflavone와 erysenegalensein N (5, 7, 2', 4'-tetrahydroxy-6-(2"-hydroxy-3"-methylbut-3"-enyl)-8-(γ, {γ-dimethylallyl})isoflavone)은 신규 화합물이었다.
분리된 화합물들의 PLCγ1 저해활성을 조사한 결과 화합물 8이 가장 강한 저해활성 (1㎍/ml, 2.3μM)을 보여주었다. 구조활성 관계를 조사한 결과 프레닐화된 기의 수와 프레닐기내의 수산기 존재가 중요한 역할을 하고 있음을 알았다. 두개의 프레닐기를 갖는 화합물이 한 개의 프레닐 기를 갖는 화합물보다 활성이 좋았다 (4 ＞ 5, 7 or 8 > 6). 또한, 프레닐기의 C-2"번이나 C-2'''에 수산기를 갖는 화합물들이 수산기를 포함하지 않는 화합물들보다 더 효과적이었다 (7 or 8 ＞ 4). 더욱이, 프레닐기의 C-2"번에 수산기를 갖는 화합물이 C-2'''에 수산기를 갖는 화합물보다 저해활성이 강하였다. 프레닐기의 C-2"번이나 C-2'''에 수산기를 갖는 화합물 7 과 8은 PLC의 isozyme인 PLCβ1에대한 저해활성에 비하여 PLCγ1에 대한 저해활성이 강하였다 PLCγ1과 PLCβ1의 저해활성은 6 - 8배 차이가 관찰되었다).
화합물 4, 6, 7, 8, 9는 농도 의존적으로 NIH3T3γ1 세포에서 PDGF 자극에대한 inositol phosphates생성을 저해하였다. 이중 화합물 8이 가장 강한 저해활성 (10㎍/ml, 23μM)을 보여주었다. 구조활성 관계로서 수산기가 치환된 프페닐기를 갖는 화합물이 수산기를 포함하지 않는 프레닐기를 갖는 화합물보다 활성이 강하였다 (7 과 8 ＞ 4 과 11). 화합물 4 와 5를 PC-3 (prostate), NCI-H226 (lung), CRL1579 (melanoma) 암세포에 대한 세포독성을 측정하였을때 이 화합물들은 9.0 - 20μM의 성장 저해활성을 보여주었다.
