Synthesis and Structure-Activity Relationship Studies of HIF-1α Inhibitors, Chemical Probes for HIF-1α and P-glycoprotein Inhibitors

다국어 초록 (Multilingual Abstract)

Tumor hypoxia has been recognized as a common feature of solid tumor and a negative prognostic factor for response to treatment and survival of cancer patients. Hypoxia inducible factor (HIF)-1 is a key transcription factor that functions as a mast regulator in the response of growing tumor to hypoxia. In normoxia, the subunit HIF-1α becomes hydroxylated at several proline residues and this leads to ubiquitination and proteasomal degradation. Under hypoxic conditions, stabilized HIF-1α dimerizes with HIF-1β. The HIF-1 heterodimer binds to hypoxia response elements (HRE) in gene promoters along with coactivators and induces the expression of target genes involved in angiogenesis, metabolic reprogramming, cell proliferation, and drug resistance to apoptosis. Therefore, HIF-1α is considered as an important target for the development of novel cancer therapeutics. At present, a number of HIF-1α inhibitors including natural products are reported as anticancer drugs. However, most of presently available inhibitors are in early stage of drug development. Thus, in an attempt to develop novel small molecule inhibitors targeting HIF-1α pathway in solid tumor, we conducted phenotype-based structure-activity relationship (SAR) study to develop novel series of small molecule HIF-1α inhibitors. Our group previously identified an aryloxyacetylamino benzoic acid analogue, LW6, which potently inhibited HIF-1α accumulation by degrading HIF-1α without affecting the HIF-1α mRNA levels during hypoxia. LW6 has been used in various studies as an HIF-1α inhibitor.
To identify more potent and efficient HIF-1α inhibitors, we performed structural modifications of LW6 that include replacement of the oxyacetylamide linker portion with a more conformationally constrained oxyacrylic amide linker. Accordingly, we synthesized a series of (E)-phenoxyacrylic amide derivatives and evaluated as HIF-1α inhibitors using HRE dependent Luc assay. Among the synthesized analogues, a compound with morpholinoethyl containing ester was found to be the most potent inhibitor against HIF-1α under hypoxic conditions in HCT116 cells. Its effect on HIF-1α pathway was demonstrated by suppression of hypoxia-induced HIF-1α accumulation and target gene expression in dose dependent manner.
Of note, chemical biology approaches using multifunctional chemical probes are useful for identification of direct target of HIF-1α inhibitors, such as LW6. Photoaffinity labeling, click conjugation, and biotinylation, are very useful tools for detecting target proteins of biologically active molecules. On the basis of this technique, we designed and synthesized a series of LW6-derived chemical probes by installing a clickable tag and a photoactivatable moiety. We observed the mitochondrial localization of LW6 and identified malate dehydrogenase 2(MDH2) as a target protein. The intracellular localization of LW6 was visualized through click chemistry with probe containing an acetylene group, in colon cancer HCT116 cells. Labile trifluromethyl diazirine photoreactive group was introduced into the phenyl ring of LW6 analogue were used for photoafiinity labeling, which bound to MDH2 in the mitochondrial TCA cycle. Chemical probes installed with biotin derivatives were also synthesized to confirm the direct target by pull-down assay. Significantly, the structure-activity relationship of this series in HRE dependent Luc assay was consistent with that in MDH2 enzyme assay, suggesting that MDH2 is the direct target protein of LW6 and chemical biology techniques are the most reliable for target identification in drug discovery.
Furthermore, one of major problems in chemotherapy is multidrug resistance (MDR) against anticancer drugs. ATP-binding cassette (ABC) transporters, such as ABCB1 (P-gp), ABCC1-7 (multidrug resistant related protein 1-7, MRP1-7) and ABCG2 (breast cancer resistance protein, BCRP), are a family of proteins that mediate MDR via ATP-dependent drug efflux pumps. P-gp is the first found human ABC transporter and considered to be clinically significant drug target. Intensive efforts have been directed on developing P-gp modulators to overcome MDR. Many inhibitors of MDR transporters have been identified and some are undergoing clinical trials, but clinically useful drugs have yet to make it to market.
Our efforts are focused on development of effective MDR reversal agents via inhibition of P-gp that result in an increased intracellular accumulation of anticancer drugs. In order to synthesize less toxic and more potent inhibitors of P-gp, screening process was proceeded from natural products and chemical library. Herein we described synthesis and biological evaluation of novel classes of adamantyl derivatives as P-gp inhibitors. The core structure modification of adamantyl based compounds was carried out and MDR reversal activities of the synthesized derivatives were assessed in P-gp overexpressed human cancer cell lines. These results indicated that some derivatives were potential MDR reversal agents and are promising for further development.

국문 초록 (Abstract)

종양 저산소증(Tumor hypoxia)은 주로 고형암에서 발견되며 환자의 나쁜 예후와 관련하여 낮은 감수성(susceptibility)과 생존율(rate of survival)을 보인다. HIF-1(Hypoxia Inducible Factor-1)은 저산소증(hypoxia)에서 유도되는 중요한 전사인자로써 암세포의 성장과 관련한다. 산소 존재 하에서 HIF-1α 서브 유니트(subunit)의 여러 프롤린 잔기들은 수산화 되고 이 잔기들은 유비퀴틴화(ubiquitination)와 단백질 분해를 초래한다. 저산소증에서 HIF-1α는 HIF-1β와 다이머(dimerization)를 구성한다. HIF-1 헤테로 다이머(heterodimer)는 공활성제 (coactivatior)와 함께 프로모터 유전자의 저산소 반응 요소(Hypoxia Response Elements, HRE)와 결합하여 혈관 형성, 대사적 재프로그래밍, 세포 확산, 아포토시스의 약제 내성 등의 유전자 발현을 유도한다. 그러므로 HIF-1α는 신규한 항암제 개발에 주요한 타깃으로 여겨진다. 현재까지 항암제로써 다수의 저분자 및 천연물 HIF-1α 저해제가 보고되어 있지만 개발 가능성이 있는 약물들 대부분이 임상 초기 단계에서 연구되고 있다. 따라서, 신규한 저분자 HIF-1α 저해제를 개발하기 위해 페노 타입(phenotype-based)을 기반으로 한 구조-활성 상관관계 (structure-activity relationship, SAR) 연구를 수행하였다. 지난 연구에서 저산소증에서 HIF-1α mRNA 수준에 영향을 주지 않으면서 HIF-1α를 분해해 축적을 저해하는 아릴옥소아세틸아미노 벤조익 액시드 유도체인 LW6를 규명하였다. LW6는 여러 연구에서 HIF-1α 저해제로 사용되고 있다.
더 높은 효과와 활성이 있는 HIF-1α 저해제를 발견하기 위하여, 본 연구에서는 LW6의 옥시아세틸아미드 링커 부분을 옥시아크릴릭 아미드 링커 부분으로 더 견고한 구조적 변형을 수행하였다. 이에 따라서, (E)-페녹시아크릴릭 아미드 유도체 시리즈를 합성하였고 HRE 의존적 Luc 어세이(HRE dependent Luc assay)를 통하여 HIF-1α 저해제의 활성을 확인하였다. 그 중에서 모르폴리노에틸기를 포함한 에스테르 화합물이 저산소 HCT116 cell 조건 하에서 가장 좋은 활성을 보였으며, HIF-1α 축적을 저해하는 활성을 용량 의존적으로 나타내었다.
흥미롭게도, 다기능적인 화학적 탐침(multifunctional chemical probe)을 이용한 화학 생물적 접근은 LW6 같은 HIF-1α 저해제의 타깃을 규명하는데 유용하다. 광 친화성 레이블링, 클릭 컨쥬게이션, 바이오티닐레이션은 생물학적으로 활성이 있는 분자의 타깃 단백질을 감지하는데 유용하게 쓰인다. 이 기술을 바탕으로, 본 연구에서는 LW6에 화학적 탐침을 이용한 유도체들을 고안하고 합성하였다. 또한 LW6가 세포 내 미토콘드리아에 위치함(mitochondrial localization)을 관찰하였고 타깃 단백질로서 말레이트 디하이드로지네이즈 2(MDH2)를 규명하였다. 결장암 HCT116 cells (colon cancer HCT116 cells) 내 LW6의 로컬라이제이션은 아세틸렌 그룹을 포함한 탐침인 클릭 반응응 통해 가시화 하였다. 반응성이 큰 트라이플루오르메틸 다이아지린 광활성 그룹(labile trifluoromethyl diazirine photoreactive group)은 LW6의 페닐 고리에 도입하여 미토콘드리아의 TCA 사이클(mitochondrial TCA cycle)에서 MDH2와 결합하는데 이용하였다. 바이오틴 유도체 또한 새로 합성하여 풀-다운 어세이(pull-down assay)를 통해 타깃 단백질과의 결합을 재확인하는데 활용하였다. 특히, HRE 의존적 Luc 어세이 및 MDH2 어세이를 활용한 구조-활성 상관관계 연구를 통해 LW6의 타깃 단백질이 MDH2라는 것을 일관되게 나타낸 것으로 미루어 보아 화학 생물학적 기술은 신약 개발에서 타깃을 규명하는데 매우 유용하다고 판단 된다.
한편, 화학요법의 주요 문제 중 하나는 항암제에 대한 다중 약물 내성 (multidrug resistance, MDR)이다. ABCB1 (P-gp), ABCC1-7 (multidrug resistant related protein 1-7, MRP1-7), ABCG2(breast cancer resistance protein, BCRP)와 같은 ATP-binding cassette (ABC) transporter 단백질을 통하여 MDR은 ATP 의존적 약물 유출 펌프작용을 나타낸다. P-gp는 임상적으로 중요하게 여겨지는 타깃으로 처음으로 발견된 인간 ABC transporter이다. 또한 P-gp의 조절게 개발에 집중하여 MDR을 극복하고자 한다. 다양한 ABC transporter 저해제가 개발되었으며, 그 중 몇몇의 저해제는 임상 실험 단계를 진행하고 있으나 아직 약물로써 사용되는 사례는 없다.
본 연구에서는 P-gp의 활성을 저해함으로써 효과적인 MDR 가역제(reversal agent)를 개발함으로써 세포 내 항암제의 축적 증가 효과를 높이고자 하였다. 독성이 적고 활성이 높은 P-gp 저해제를 합성하기 위해, 스크리닝 기법을 이용하여 천연물과 화홥물 라이브러리를 검색하였다. 이러한 연구를 바탕으로 신규한 P-gp 저해제로써 아다맨틸 유도체(adamantyl derivatives)를 합성하고 생물학적 활성을 측정하였다. P-gp가 과발현된 인간 암 cell line에서 MDR reversal activity를 나타내는 신규한 화합물들을 아다맨틸 구조의 변형 등을 통하여 발굴하였다. 본 연구는 MDR 가역제로써 높은 잠재성과 개발 가능성을 제안하고 있다.

목차 (Table of Contents)

• Acknowledgement = i
• 국문 초록 = iv
• Abstract of the Thesis = viii
• Definition of Abbreviations = xiii
• List of Figures = xvii
• List of Schemes = xix
• List of Tables = xxi
• Part-I: Synthesis and biological evaluation of (E)-phenoxyacrylic amide derivatives as potent HIF-1α inhibitors
• 1. Introduction = 2
• 1.1 The cellular responses to hypoxia = 5
• 1.2 HIF-1 signaling pathway in hypoxia = 10
• 1.3 HIF-1α as a valid target for cancer therapy = 20
• 1.4 Small molecule HIF-1 inhibitors = 23
• 1.5 Potent HIF-1α inhibitors derived from LW6 = 35
• 2. Results and Discussion = 39
• 2.1 Synthesis of the key intermediates 9a-f = 39
• 2.2 Synthesis of (E)-phenoxyacrylic amide derivatives 4a-g and 4j-k= = 40
• 2.3 Synthesis of (E)-phenoxyacrylic amide derivatives 4i and 4n-r= = 41
• 2.4 Synthesis of (E)-phenoxyacrylic amide derivatives 4h, 4l-m, and 4s-v = 42
• 2.5 Structure-activity relationship studies = 47
• 3. Materials and Methods= 59
• 3.1 Experimental section= = 59
• 3.1.1 Chemistry = 59
• 3.1.1.1 General procedures = 59
• 3.1.1.2 Synthetic procedures = 61
• 3.1.2 Biology = 96
• 3.1.2.1 Cell-based HRE reporter assay = 96
• 4. Conclusions = 98
• 5. References = 100
• Part-II: Synthesis and structure-activity relationship studies of chemical probes as potent HIF (hypoxia induced factor)-1α inhibitors
• 1. Introduction = 129
• 1.1 Anatomy of chemical probes = 133
• 1.1.1 Reactive group = 133
• 1.1.2 Linker region = 134
• 1.1.3 Reporter tag = 134
• 1.2 The search for ideal photoaffinity probes = 136
• 1.2.1 Fluorinated aryl azide = 137
• 1.2.2 Benzophenone group= 138
• 1.2.3 Diaazirine group = 138
• 1.3 Approaches to target identificatio n = 140
• 1.3.1 Affinity based target identification = 140
• 1.3.2 Two-dimensional gel electrophoresis = 148
• 1.3.3 Fluorescence difference in two-dimensional gel electrophoresis = 149
• 1.4 Target confirmation and validation = 150
• 1.5 Chemical probes used for target identification = 152
• 2. Results and Discussion = 155
• 2.1 Chemistry = 155
• 2.1.1 Synthesis of click-conjgated chemical probes 4c and 4d = 156
• 2.1.2 Synthesis of benzophenone chemical probes 4e and 4f = 157
• 2.1.3 Synthesis of key intermediate = 158
• 2.1.4 Synthesis of photoaffinity trimodular chemical probes 4g and 4h = 159
• 2.1.5 Synthesis of photoaffinity trimodular chemical praobes 4a and 4b = 160
• 2.2 Biological Evaluation = 167
• 3. Materials and Methods = 176
• 3.1 Experimental section = 176
• 3.1.1 Chemistry = 176
• 3.1.1.1 General procedures = 176
• 3.1.1.2 Synthetic procedures = 178
• 3.1.2 Biolog y= 217
• 3.1.2.1 HRE-Luciferase reporter assay = 217
• 3.1.2.2 MDH2 activity assay = 217
• 4. Conclusions = 219
• 5. References= = 221
• Part-III: Synthesis and bioactivity of novel adamantyl derivatives as potent MDR reversal agent
• 1. Introduction = 237
• 1.1 ABC transporters = 239
• 1.2 Structure and function of P-gp = 245
• 1.3 Development of MDR reversal agents = 248
• 1.4 P-gp inhibitor derived from adamantyl derivatives = 257
• 2. Results and Discussion = 259
• 2.1 Synthesis of adamantyl derivatives = 259
• 2.2 Reversal activities of synthesized compounds = 264
• 3. Materials and Methods = 268
• 3.1 Experimental section = 268
• 3.1.1 Chemistry = 268
• 3.1.1.1 General procedures = 268
• 3.1.1.2 Synthetic procedures = 270
• 3.1.2 Biology = 296
• 3.1.2.1 MDR reversal assay = 296
• 4. Conclusions = 297
• 5. References = 298