Development of Novel Compounds for Melanin Inhibition Using Biochemical, Chemoinformatics and Pharmacological Approaches

국문 초록 (Abstract)

현재 논문에서, 다른N-(substituted-phenyl)-4-{(4-[(E)-3-
phenyl-2-propenyl]-1-piperazinyl}butanamides가 반응식 1에 기재된 프로토콜에 따라 합성되었다. 수성 염기성 매질에서 다양하게 치환 된anilines (1a-e)과4-chlorobutanoyl chloride (2)를 반응시켜 다양한 친전자체4-chloro-N-(substitutedphenyl)butanamides (3a-e)를 생성함으로써 합성을 개시하였다. 이어서, 극성 비양성자성 용매에서 이들 친전자체를 1-[(E)-3- phenyl-2-propenyl]piperazine(4)과 결합하여 목표한 화합물 N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl} butanamides(5a-e)를 얻었다. 모든 유도체의 구조는 양성자 핵자기공명 (1H-NMR), 탄소 핵자기공명 (13C-NMR), Infra-Red (IR) 스펙트럼 데이터. 및 CHN 분석을 통해 동정되었고 특성이 조사되었다. 이들 부탄 아미드의 버섯 티로시나제에 대한 시험관내 억제능이 평가되었으며 , 모든 화합물이 생물학 활성을 가진 것이 밝혀졌다 . 이들 중 5b는 IC50 값이 0.013 ± 0.001 μM로 가장 높은 억제능을 나타냈다. 동일한 화합물 5b를 또한 생체내실험을 통해 분석하였고, 그것이 제브라 피쉬에서 피부색소를 상당히 감소시키는 것이 밝혀졌다. 또한 컴퓨터 분석결과는 전술한 결과와 일치했다. 또한, 용혈 활성을 통해 이들 분자의 세포 독성이 조사되었으며, 5e를 제외하고 다른 모든 화합물은 최소 독성을 나타내는 것이 밝혀졌다. 화합물 5a는 또한 유사한 결과를 나타냈다. 따라서, 이들 화합물 중 일부는 부작용이 적은 탈색소 약물의 제형 및 개발에 적합한 후보가 될 수있다. (1 장).
새로운N-(substituted-phenyl)-4-(4-phenyl1-piperazinyl) butanamides (4a-c) 의 합성이 뛰어난 2 단계 전략을 통해서 수행되었다. 이들 화합물의 구조는 CHN 분석 데이터와 함께 IR, EI-MS, 1H-NMR, 13C-NMR 스펙트럼에 의해 확증되었다. 버섯 타이로시나제에 대한 실험실 억제의 결과는 모든 화합물이 이 효소를 잘 억제하였으며 그 중에서 4b는 표준 (16.841 ± 1.146 μM)에 비해 IC50 값이 0.168 ± 0.057 μM 이었으며 가장 억제능이 높은 화합물로 확인되었다. 이 분자의 동력학적 분석 (Ki = 0.22 μM)을 통해 티로시나아제 효소를 경쟁적으로 억제하지 않는 것이 밝혀졌다. 또한 제브라 피쉬 배아에서 연구했을 때 생체내 실험에서 피부 색소를 현저하게 감소시켰다 (75.373 %, P <0.05). 또한, 이들 부탄 아미드의 세포 독성도 조사되었으며, 이들 분자들은 매우 낮은 세포 독성을 갖는 것이 밝혀졌다. 따라서, 본 연구로부터 이들 화합물은 피부 관련 질환의 개선을 위해보다 적은 세포 독성 치료제로서 사용될 수 있다는 결론을 내렸다. (제 2 장).
3 장의 연구는 자외선 차단을 위해 필수적인 필수 단백질 (멜라닌)의 보호작용을 연구했다. 본 연구에서 3 가지 다른 (시험 관내, 생체 내 및 컴퓨터 분석) 방법을 사용하여 새로운 약물 Acemetacin (ACE)의 멜라닌 억제능력을 검사하였다. ACE는 표준 코지산 (IC50 = 16.841 ± 1.146 µM)과 비해서 현저한 억제능을 (IC50 = 0.353 ± 0.003 μM) 보였고, 티로시나아제에 대한 억제 메커니즘 분석 결과 ACE는 경쟁적 억제를 나타냈다. 생체 내 연구에서 어린 제브라 피쉬를 5, 10, 15 및 20 μM의 ACE 및 양성 대조군 용량으로 노출하였다. 72 시간 처리에서, ACE는 코지산 (39.64 %)에 비해 20 μM의 농도에서 색소 침착 수준을 표현 적으로 (P <0.05) 감소시켰다(62.89 %). ACE의 결합 양상이 분자 도킹에 의해 확인되었고 도킹된 복합체의 안정성이 MD 시뮬레이션에 의해 확인되었다. 이런 결과에 기초하여, ACE가 티로시나제에 대해서 멜라닌 생성에 대해 우수한 치료 가능성을 보유한 것으로 결론 내렸다. (3 장).

다국어 초록 (Multilingual Abstract)

In the current thesis work, different N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamides have been synthesized according to the protocol described in scheme 1. The synthesis was initiated by reacting various substituted anilines (1a-e) with 4-chlorobutanoyl chloride (2) in aqueous basic medium to give various electrophiles, 4-chloro-N-(substituted-phenyl)butanamides (3a-e). These electrophiles were then coupled with 1-[(E)-3-phenyl-2-propenyl]piperazine (4) in polar aprotic medium to attain the targeted N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamides (5a-e). The structures of all derivatives were identified and characterized by proton-nuclear magnetic resonance (1H-NMR), carbon-nuclear magnetic resonance (13C-NMR) and Infra-Red (IR) spectral data along with CHN analysis. The in vitro inhibitory potentials of these butanamides were evaluated against mushroom tyrosinase, whereby all compounds were found to be biologically active. Among them, 5b exhibited highest inhibitory potential with IC50 value of 0.013 ± 0.001 µM. The same compound 5b was also assayed through in vivo approach, and it was explored that it significantly reduced the pigments in zebrafish. The in silico studies were also in agreement with aforesaid results. Moreover, these molecules were profiled for their cytotoxicity through hemolytic activity, and it was found that except 5e, all other compounds showed minimal toxicity. The compound 5a also exhibited comparable results. Hence, some of these compounds might be worthy candidates for the formulation and development of depigmentation drugs with minimum side effects. (Chapter 1).
The syntheses of some new N-(substituted-phenyl)-4-(4-phenyl1-piperazinyl)butanamides (4a-c) were carried out through a facile bi-step strategy. The structures of these compounds were corroborated by their IR, EI-MS, 1H-NMR, 13C-NMR spectra along with CHN analysis data. The results of mushroom tyrosinase in vitro inhibition revealed that all compounds were superb inhibitors of this enzyme and among them 4b was identified as the most active compound having IC50 value of 0.168 ± 0.057 µM, relative to the standard (16.841 ± 1.146 µM). The kinetic analysis (Ki = 0.22 µM) of this molecule revealed that it does not competitively inhibit the tyrosinase enzyme. It also significantly reduced (P<0.05) the enormous amount of pigments to about 75.373% in an in vivo protocol, when studied on the zebrafish embryos. Moreover, the cytotoxicity of these butanamides was also profiled and it was an inferred that these molecules possess very mild cytotoxicity. So, it was consummated from the present investigation that these compounds might be utilized as less cytotoxic therapeutic agents for the betterment of skin related ailments. (Chapter 2).
The present study describes that the protection. Essential protein (melanin) that is vital for the skin for defense from UV rays. In present research, emerging drug Acemetacin (ACE) was examined for their melanin inhibition using three different (in vitro, in vivo and computational) methods. ACE showed potentially remarkable potency (IC50 = 0.353 ± 0.003 µM) against tyrosinase in the comparison of standard, kojic acid (IC50 = 16.841 ± 1.146 µM) and enzyme inhibition mechanism analysis, it was exposed ACE exhibited competitive inhibition. In the in vivo study zebrafish seeds were exposed with 5, 10, 15 and 20 µM of ACE and positive control doses. At 72 h treatment, ACE expressively (P<0.05) reduced the level of pigmentation (62.89%) at a concentration of 20 µM, relative to that of kojic acid (39.64%). The binding profile of ACE was confirmed by molecular docking and stability of the docked complexes was justified by MD simulation. Based on our results, it was concluded as ACE possessed good therapeutic potential against melanogenesis by targeting the tyrosinase. (Chapter 3).

목차 (Table of Contents)

• VI) Abstract 1
• 1. Introduction 5
• 2. Material and Methods 14
• 2.1. Chemistry (Chapter 1) 14
• 2.1.1. Procedure for the synthesis of 4-Chloro-N-(substituted-phenyl)butanamides (3a-e) 14
• 2.1.2. Procedure for the synthesis of N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamides (5a-e) 15
• 2.2. Structural characterization 15
• 2.2.1. N-(2,3-Dimethylphenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamide (5a) 15
• 2.2.2. N-(2,4-Dimethylphenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamide (5b) 16
• 2.2.3. N-(3-Methylphenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamide (5c) 17
• 2.2.4. N-(4-Methylphenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamide (5d) 18
• 2.2.5. N-(4-Ethylphenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamide (5e) 19
• 2.3. Chemistry (Chapter 2) 20
• 2.3.1. General Procedure for the synthesis of 4-Chloro-N-(substituted-phenyl)butanamides (3a-c) 20
• 2.4. General procedure for the synthesis of N-(Substituted-phenyl)-4-(4-phenyl-1-piperazinyl)butanamides (4a-c) 20
• 2.4.1. N-(4-Methylphenyl)-4-(4-phenyl-1-piperazinyl)butanamide (4a) 21
• 2.4.2. N-(2,5-Dimethylphenyl)-4-(4-phenyl-1-piperazinyl)butanamide (4b) 21
• 2.4.3. N-(3,4-Dimethylphenyl)-4-(4-phenyl-1-piperazinyl)butanamide (4c) 22
• 2.5. In vitro methodology 26
• 2.5.1. Tyrosinase assay 26
• 2.5.2. Kinetic analysis 27
• 2.5.3. Hemolytic activity 27
• 2.6. In vivo methodology 29
• 2.6.1. Determination of pigmentation reducing capacity of compound in embryos of Zebrafish 29
• 2.6.2. Zebrafish Farming 29
• 2.6.3. Compound treatment and depigmentation examination 29
• 2.6.4. Quantification of melanin 30
• 2.7. Statistical Analysis 31
• 2.8. In silico Methodology 32
• 2.8.1. Grid generation and molecular docking 32
• 2.8.2. Molecular Dynamics 33
• 3. Results and Discussion 34
• 3.1. Chemistry (Chapter 1 34
• 3.2. Biology 38
• 3.2.1. Enzyme inhibition and structure-activity relationship 38
• 3.2.2. Kinetic analysis of 5b 39
• 3.2.3. Cytotoxicity 42
• 3.2.4. In vivo depigmentation zebrafish assay 42
• 3.3. Molecular docking analysis 45
• 3.3.1. Binding energy evaluation of synthesized compounds 45
• 3.3.2. Binding analysis-against tyrosinase 48
• 3.4. Molecular dynamic simulations 51
• 3.4.1. Root mean square deviation and fluctuation (RMSD/RMSF) analysis 51
• 3.5. Chemistry (Chapter 2) 53
• 3.6. Biology 56
• 3.6.1. Enzyme inhibition and structure-activity relationship 56
• 3.6.2. Kinetic analysis of 4b 57
• 3.6.3. Hemolytic activity 60
• 3.6.4. In vivozebrafish analysis 60
• 3.7. Molecular docking analysis 63
• 3.7.1. Binding energy evaluation of synthesized compounds 63
• 3.7.2. Binding analysis of ligands against tyrosinase 65
• 3.8. Molecular dynamic simulations 67
• 3.8.1. RMSD and RMSF analysis 67
• 3.9. Biology 69
• 3.9.1. Enzyme Inhibition of ACE 69
• 3.9.2. Kinetic analysis of ACE 71
• 3.9.3. Depigmentation zebrafish assay of ACE 73
• 3.9.4. Melanin quantification of ACE 75
• 3.10. Molecular docking analysis 77
• 3.10.1. Binding energy calculation of ACE 77
• 3.10.2. Ligand-Binding analysis of tyrosinase docked complexes 77
• 3.11. Molecular dynamic simulations 80
• 3.11.1. Root mean square deviation and fluctuation (RMSD/RMSF) analysis 80
• 3.11.2. Radius - protein stability 80
• 4. Conclusion 83
• Abstract in Korean 85
• References 90
• Acknowledgement 105
• List of Publications 108
• Conferences and Poster Presentations 120