Development of Potent TPP-conjugated mtROS Inhibitors Targeting Mitochondria for Treatment of Rheumatoid Arthritis

다국어 초록 (Multilingual Abstract)

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease related to
oxidative stress. Rubicon interacts with the p22phox subunit of NOX (NADPH oxidase),
which is necessary for increased ROS-mediated RA pathogenesis. On the basis of a
p22phox-derived peptide mimetic compound, tetrahydroindazolylphenoxy-N-
(thiadiazolyl)propanamide (TIPTP) was synthesized, which showed high inhibitory
activity of the p22phox-Rubicon interaction. The previous study demonstrated that the
interaction of Rubicon with p22phox increases cellular ROS levels. However, the
correlation between Rubicon and mitochondrial ROS (mtROS) is poorly understood.
Rubicon-p22phox interaction also occurred in the mitochondrial membrane in
macrophages and mouse with ulcerative colitis.
In this study, we designed and synthesized TIPTP derivatives as therapeutic agents for
treatment of colitis through recovering mitochondrial function. Triphenyl-phosphonium
cation (TPP+) is widely used as mitochondrial targeting moiety because of negative
membrane potential of the mitochondrial inner membrane. Thus, new mitochondrial
targeting antioxidant Mito-TIPTPs, consisting of TPP alkyl chain linker and functionalized
TIPTP, were synthesized.
Mito-TIPTPs were biologically evaluated in in vitro and in vivo assays. The compound
possessing alkyl TPP group on the thiadiazole ring showed good inhibitory activity against
acute and chronic DSS-induced colitis by interfering interaction of p22phox-Rubicon in
mitochondria. Other mitochondrial targeting TIPTPs were also designed and synthesized,
and their biological evaluation will be performed.

국문 초록 (Abstract)

류마티스 관절염 (RA)은 산화 스트레스와 연관이 있는 만성적인 자가면역
염증질환이다. Rubicon 은 NOX (NADPH oxidase)의 구성요소인 p22phox 와
상호작용하며, 이로 인해 증가되는 ROS 는 RA 발병을 유발하는 매개체이다.
p22phox 로부터 유래된 펩타이드 모방 물질을 기반으로 하여 p22phox-Rubicon
상호작용에 대한 높은 억제력을 나타내는 tetrahydroindazolylphenoxy-N-
(thiadiazolyl)propanamide (TIPTP)를 합성하였다. 선행된 연구에서는 Rubicon 과
p22phox 의 상호작용에 의해 cellular ROS 의 생성이 증대됨을 입증하였다. 그러나
Rubicon 과 mitochondrial ROS (mtROS) 사이의 인과관계에 대한 연구는 충분히
이루어지지 않았으며, 본 연구를 통해 Rubicon-p22phox 상호작용이 대식세포와
궤양성 대장염이 유도된 쥐 내에서의 미토콘드리아 막에서도 일어남을 확인하였다.
본 연구에서는 미토콘드리아 기능의 회복을 통한 대장염 치료제로서 TIPTP
유도체를 디자인 및 합성하였다. 미토콘드리아의 내부막은 음의 전위를 가지고 있기
때문에 Triphenyl-phosphonium 양이온 (TPP+)은 미토콘드리아 표적 성분으로
광범위하게 사용되고 있다. 따라서, TIPTP 에 TPP alkyl chain linker 가 결합된 형태의
새로운 미토콘드리아 표적 항산화물질 Mito-TIPTP 를 합성하였다.
본 연구에서는 Mito-TIPTP 의 in vitro, in vivo 분석을 통한 생물학적 평가가 이루어졌다.
Mito-TIPTP 는 TIPTP 의 thiadiazole ring 에 alkyl TPP 그룹이 결합된
형태로, p22phox-Rubicon 상호작용의 억제를 통하여 DSS 에 의해 유도된 급성, 만성
대장염에 대한 좋은 억제 활성을 나타냈다. 또한, 다른 형태의 미토콘드리아 표적
TIPTP 들을 디자인 및 합성하여 추가적인 생물학적 활성 평가가 수행될 예정이다.

목차 (Table of Contents)

• List of Schemes ................................................................................................................................. ii
• List of Figures .................................................................................................................................. iii
• Abstract ............................................................................................................................................ iv
• 1. Introduction .............................................................................................................................. 1
• 1.1 Rheumatoid arthritis (RA) ...................................................................................................... 1
• 1.2 Pathology of RA ..................................................................................................................... 2
• 1.3 TIPTP, as the potent inhibitor of RA ...................................................................................... 3
• 1.4 Structure modification of TIPTP for targeting Mitochondria ................................................. 5
• 2. Result and Discussion .............................................................................................................. 8
• 2.1 Synthesis of Mito-TIPTP-1 .................................................................................................... 8
• 2.2 Effects of Mito-TIPTP (In vitro test) .................................................................................... 13
• 2.3 Effects of Mito-TIPTP (In vivo test) ..................................................................................... 17
• 2.4 Synthesis of Mito-TIPTP-2 .................................................................................................. 22
• 2.5 Synthesis of Mito-TIPTP-3 .................................................................................................. 24
• 2.6 Synthesis of Mito-TIPTP-4 .................................................................................................. 29
• 2.7 Synthesis of Mito-TIPTP-5 .................................................................................................. 32
• 2.8 Comparison of inhibition effects of Mito-TIPTP-1~5 .......................................................... 33
• 3. Conclusion ............................................................................................................................. 35
• 4. Experimental Section .................................................................................................................. 36
• 4.1 Equipment and Reagents ...................................................................................................... 36
• 4.2 Synthesis ............................................................................................................................... 37
• 5. References ................................................................................................................................... 67
• Spectra Data (1H and 13C NMR) ..................................................................................................... 69
• 국문요지 ........................................................................................................................................ 98