신경염증에 의해 매개되는 미세아교세포의 활성화는 뇌 손상의 특징으로, 알츠 하이머병, 파킨슨병, 다발성 경화증과 같은 신경 퇴행성 질환의 발병 기전에서 중요한 역할을 한다. 미세아교세포는 외부 스트레스나 LPS와 같은 병원체에 의해 미세아교세포가 과도하게 활성화되게 되면 TNF-α, IL-1β, IL-6 등과 같은 염증성 사이토카인이나 ROS, NO, O2- 등을 과도하게 분비하여 퇴행성 질환의 발 달에 기여하게 된다. 그러므로 비정상적으로 활성화된 미세아교세포로부터 생성 되는 다량의 염증 매개물질의 활성 및 생성을 조절하는 것이 퇴행성 질환의 치료 표적으로 인식되고 있다. 본 연구에서는 새로운 항염증제를 찾기 위해, 탈리도마이드와 퀘르세틴과 같이 항염증, 면역조절, 항암효과 등의 다양한 생물학적 효과가 있는 것으로 보고되어 있는 두 화합물의 구조적 변형이 LPS로 활성화된 BV2 세 포에서 항염증 활성을 향상시킬 수 있는지를 조사하였다. 이에 따라 프탈리마이드의 벤젠 고리를 두 개의 디페닐 고리로 융합하고 글루타리미드 구조를 다양한 아민으로 대체한 탈리도마이드 유사체 41개를 합성하여 약 리 효과를 확인하였다. 합성된 화합물의 대부분은 BV2 세포에서 LPS에 의한 NO 의 생성을 더 잘 억제하는 것으로 나타났다. 특히 말레이미드에 두 개의 페닐 고리 를 융합하고 글루타리미드 대신 디메틸아닐린으로 대체된 유사체 19u (IC50 = 7.1 μM)는 탈리도마이드 (100 μM 까지 효과 없음)에 비해 항염증 효과가 크게 향상되는 것으로 나타났다. 또한 유사체 19u는 세포 독성 없이 용량 의존적으로 NO 생성을 억제하는 것이 확인되었으며, 기전적으로 NF-κB 및 p38 MAPK 경로를 억제하고 염증성 사이토카인 생산과 iNOS 및 COX-2의 발현을 억제하였다. 퀘르세틴의 B-고리 치환체의 수산기 위치와 개수를 변경한 30개의 퀘르세틴 유사체를 합성하여 BV2 세포에서 이들의 NO 생성 억제 활성을 테스트 하였다. 합성된 대부분의 유사체는 퀘르세틴보다 약한 활성을 보였지만, 3, 4번 위치의 수산 기가 메톡시기로 전환된 유사체 14io는 20 μM 농도에서 82.5 ± 2.0 %로 퀘르세 틴 (65.7 ± 1.1 %)보다 뛰어난 NO 억제 효과를 나타냈다. 그러나 B-고리의 3, 4 위치에 수소 결합 능력을 제거한 메틸 유사체 14im (25.1 ± 1.8 %)의 경우 항염증 활성이 감소하는 것을 확인하였다. 이러한 결과는 퀘르세틴 구조에서 3번과 4 번 위치의 수산기의 수소 결합 가능성이 항염증 활성을 유지하는 데 중요하다는 것을 시사한다. 이러한 탈리도마이드와 퀘르세틴 유사체의 구조-활성 결과는 미세아교세포 매개 신경염증 질환의 새로운 치료제 개발에 기초자료로 활용될 수 있다.|Activation of microglia mediated by neuroinflammation is a hallmark of brain injury and plays an important role in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. When microglia are over-activated by external stress or pathogens such as LPS, they secrete inflammatory cytokines, including TNF-α and IL-1β, as well as ROS, NO and O2-, which contribute to the development of neurodegenerative diseases. Therefore, the modulation of the activity and production of large amounts of inflammatory mediators by abnormally activated microglia is recognized as a therapeutic target for degenerative diseases. In the present study, for the purpose of finding new anti-inflammatory agents, we investigated whether structural modifications of thalidomide and quercetin could enhance their anti-inflammatory activity in BV2 cells because both compounds have various biological effects, including anti-inflammatory, immunomodulatory, and anticancer activities. Accordingly, forty-one analogues of thalidomide, in which the benzene ring of phthalimide in the structure was fused with two diphenyl rings and the glutarimide moiety was replaced by various amines, were synthesized and tested for their pharmacological effects. Most of the synthesized compounds exhibited better inhibition of LPS-induced NO production in BV2 cells than thalidomide. Especially, analogue 19u (IC50 = 7.1 μM), which contains two phenyl rings fused to maleimide and a dimethylaniline moiety in replace of glutarimide, showed significantly improved inhibition compared to thalidomide (no effect up to 100 μM). The analogue 19u also showed dose-dependent inhibition of NO production without cytotoxicity. Mechanistically, analogue 19u inhibited the NF-κB and p38 MAPK pathways, resulting in the suppression of proinflammatory cytokine production and expression of iNOS and COX-2._x000D_
<br>Thirty analogues of quercetin, in which the position and number of hydroxyl groups in the B-ring substituents were changed, were synthesized, and their NO-inhibitory activities were tested in BV2 cells. Although most of the synthesized analogues revealed weaker activity than quercetin, the methoxy analogue 14io, in which the hydroxyl groups at the 3 and 4 position were converted into a methoxy group, revealed better NO inhibition with 82.5 ± 2.0 % at a concentration of 20 μM, than that of quercetin (65.7 ± 1.1 %). However, when the hydrogen bonding capability at positions 3 and 4 of the B-ring was removed, the inhibitory activities of the corresponding methyl analogues decreased. This result indicates that hydrogen bonding potential of the hydroxyl group at positions 3 and 4 in the structure of quercetin is important for maintaining its anti-inflammatory activity. These structure-activity results of thalidomide and quercetin analogues could be used to develop new treatments for microglia-mediated neuroinflammatory diseases.
Alternative Abstract
Activation of microglia mediated by neuroinflammation is a hallmark of brain injury and plays an important role in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis. When microglia are over-activated by external stress or pathogens such as LPS, they secrete inflammatory cytokines, including TNF-α and IL-1β, as well as ROS, NO and O2-, which contribute to the development of neurodegenerative diseases. Therefore, the modulation of the activity and production of large amounts of inflammatory mediators by abnormally activated microglia is recognized as a therapeutic target for degenerative diseases. In the present study, for the purpose of finding new anti-inflammatory agents, we investigated whether structural modifications of thalidomide and quercetin could enhance their anti-inflammatory activity in BV2 cells because both compounds have various biological effects, including anti-inflammatory, immunomodulatory, and anticancer activities. Accordingly, forty-one analogues of thalidomide, in which the benzene ring of phthalimide in the structure was fused with two diphenyl rings and the glutarimide moiety was replaced by various amines, were synthesized and tested for their pharmacological effects. Most of the synthesized compounds exhibited better inhibition of LPS-induced NO production in BV2 cells than thalidomide. Especially, analogue 19u (IC50 = 7.1 μM), which contains two phenyl rings fused to maleimide and a dimethylaniline moiety in replace of glutarimide, showed significantly improved inhibition compared to thalidomide (no effect up to 100 μM). The analogue 19u also showed dose-dependent inhibition of NO production without cytotoxicity. Mechanistically, analogue 19u inhibited the NF-κB and p38 MAPK pathways, resulting in the suppression of proinflammatory cytokine production and expression of iNOS and COX-2._x000D_
<br>Thirty analogues of quercetin, in which the position and number of hydroxyl groups in the B-ring substituents were changed, were synthesized, and their NO-inhibitory activities were tested in BV2 cells. Although most of the synthesized analogues revealed weaker activity than quercetin, the methoxy analogue 14io, in which the hydroxyl groups at the 3 and 4 position were converted into a methoxy group, revealed better NO inhibition with 82.5 ± 2.0 % at a concentration of 20 μM, than that of quercetin (65.7 ± 1.1 %). However, when the hydrogen bonding capability at positions 3 and 4 of the B-ring was removed, the inhibitory activities of the corresponding methyl analogues decreased. This result indicates that hydrogen bonding potential of the hydroxyl group at positions 3 and 4 in the structure of quercetin is important for maintaining its anti-inflammatory activity. These structure-activity results of thalidomide and quercetin analogues could be used to develop new treatments for microglia-mediated neuroinflammatory diseases.
