ANTI-PROLIFERATIVE ACTIVITY OF CURCUMIN ANALOGS ON ACUTE PROMYELOCYTIC LEUKEMIA: SYNTHESIS AND MODE OF ACTION STUDIES TAN KHENG LIN (B. Sc. (Chemistry) (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirely. I have duly acknowledged all the sources of information which have been used in the thesis. The thesis has also not been submitted for any degree in any university previously. Tan Kheng Lin 12 August 2013 Table of Contents Acknowledgements 11 Conferences and Publications 13 Summary 15 List of Tables .18 List of Figures 20 List of Schemes 24 List of Abbreviations .25 Chapter 1: Introduction . 26 1.1. Leukemia .26 1.2. Acute Promyelocytic Leukemia (APL) .26 1.2.1. The molecular pathogenesis of APL 27 1.2.2. The role of N-CoR in APL pathogenesis .27 1.2.3. Therapeutic Modulation of N-CoR misfolding 30 1.3. Endoplasmic reticulum (ER) stress and the Unfolded Protein Response (UPR) .33 1.3.1 ER stress mechanism 33 1.3.2. Activation of UPR-induced apoptosis .35 1.4. The Proteasome .39 1.4.1. Structure and function of the proteasome 40 1.4.2. Current FDA approved proteasome inhibitors .41 1.5. Curcumin .43 1.5.1. Overview of structure, stability and bioavailability of curcumin 43 1.5.2. Biological activities of curcumin .47 1.5.3. Structural modification of curcumin 49 1.6. Statement of Purpose .52 Chapter 2: Design and Synthesis of Target Compounds 54 2.1. Introduction .54 2.2. Rationale of compound design 55 2.2.1 Overview of design rationale 55 2.2.2. Series I .58 2.2.3. Series II and III 60 2.2.4. Series IV .61 2.2.5. Series V 61 2.2.6. Series VI .62 2.3. Syntheses of Series I-VI: Mechanistic considerations 62 2.3.1. Syntheses of symmetrical analogs of Series I, II and III, IV and V by base-catalyzed aldol condensation 62 2.3.2. Syntheses of oximes .64 2.3.3. Syntheses of symmetrical analogs of Series IV by acid catalyzed aldol condensation .65 2.3.4. Synthesis of asymmetrical analogs of series III using sequential acid-catalyzed aldol condensation .68 2.3.5. Synthesis of asymmetrical analogs of Series IV 68 2.3.6. Synthesis of piperidinone analogs of Series VI .71 2.3.7. Synthesis of analogs containing hydroxyl substituents on phenyl ring .71 2.4. Materials and methods 72 2.4.1. General details .72 2.4.2. General procedure for syntheses of – 3, – 9, 11-15, 18 – 19, 21, 24 – 26, 31 – 33, 201, 206, 30, 36 – 37, 207 – 214, 42 – 44, 47, 228, 229 by aldol condensation under basic conditions. 73 2.4.3. Syntheses of 4, 10 and 22 73 2.4.4. General procedure for syntheses of 16, 17, 20, 23, 34 – 35, 38, 215, 216, 230, 231, 45 – 46 by aldol condensation under acidic conditions .74 2.4.5. 5-Bis-(4-fluoro-phenyl)-penta-1,4-dien-3-one oxime (27) .75 2.4.6. Syntheses of O-methyloximes of 1,5-bis-(fluorophenyl)-penta-1,4dien-3-ones (28 and 29) .75 2.4.7. General procedure for syntheses of 40, 41, 221 – 227 .76 2.4.8. Synthesis of 2-(3-Fluoro-benzylidene)-cyclohexanone (48) .76 2.4.9. Synthesis of (3-[(3-fluorobenzylidene)dihydro-2H-thiopyran4(3H)-one) (49) 76 2.4.10. General procedure for syntheses of 202 – 205, 217 – 220 .77 2.5. Summary .77 Chapter 3: Effects of synthesized compounds on viability of leukemic and non-malignant cells . 78 3.1. Introduction .78 3.2. Materials and methods 79 3.2.1. Cell lines and culture medium .79 3.2.2. Medium and reagents for cell culture 80 3.2.3. Cell viability assay .80 3.3. Results .81 3.3.1. Anti-proliferative activity of curcumin and analogs on leukemic APL cell lines .81 3.3.2. Anti-proliferative activity of curcumin and analogs on leukemic non-APL cell lines .91 3.3.3. Anti-proliferative activity of curcumin and analogs on human nonmalignant cell lines 96 3.4. Discussion .99 3.5. Conclusions .102 Chapter 4: Curcumin and analogs upregulate misfolded N-CoR in APL . 104 4.1. Introduction .104 4.2. Materials and Methods 105 4.2.1. Reagents .105 4.2.2. Cell treatment and lysis using SDS sample buffer .106 4.2.3. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDSPAGE) 107 4.2.4. Western blotting .107 4.2.5. Coomassie blue staining 108 4.2.6. Immunofluoescence .108 4.2.7. Thermal shift assay 109 4.2.8. Surface plasmon resonance (SPR) assay .109 4.2.9. Determination of 20S proteasomal inhibition 110 4.2.10. Inhibition of 26S proteasome activity in NB4 cells .110 4.2.11. High content screening of NF-κB translocation in NB4 cells .111 4.3. Results .112 4.3.1. Determination of N-CoR levels in NB4 cells exposed to APL selective and APL non-selective compounds 112 4.3.2. N-CoR accumulated is in the misfolded state 116 4.3.3. Investigations on the interaction of curcumin and APL selective compounds 11, 22 and 41 with O-Sialoglycoprotein endopeptidase (OSGEP). .118 4.4. Investigations on the inhibition of 20S proteasome by curcumin and APL selective compounds 11, 22 and 41 .122 4.5. Investigations on the inhibition of 26S proteasome in NB4 cells by APL selective compounds (41, 206, 219, 220, 221, 227) and curcumin .124 4.6. Curcuminoids that are proteasome inhibitors similarly inhibit TNFαinduced activation of NF-κB 127 4.7. Discussion .130 4.8. Conclusions .132 Chapter 5: Mode of action studies on the APL selective compound 41. 134 5.1. Introduction .134 5.2. Materials and Methods 135 5.2.1. Microarray analysis 135 5.2.2. NB4 cell treatment and harvest 135 5.2.3. Isolation and purification of mRNA 135 5.2.4. Determination of mRNA integrity .135 5.2.5. Microarray chip hybridization and pathway analysis 136 5.2.6. Validation by real-time PCR 136 5.2.7. Effect on UPR pathways 137 5.2.7.1. Reagents 137 5.2.7.2. Western blot 138 5.2.7.3. Detection of apoptosis through flow cytometry 138 5.3. Results .138 5.3.1. Gene expression profile of NB4 cells treated with compound 41 and curcumin 138 5.3.2. Validation of selected genes identified in microarray analysis .144 5.3.3. Effects of 41, 219 and 227 on ER stress markers in NB4 cells .146 5.3.4. Effects of 41, 219 and 227 on the ER stress sensors PERK and IRE2 .148 5.3.5. Effects of 41, 219 and 227 on apoptotic markers caspases 3, and PARP 151 5.4. Discussion .153 5.5. Conclusion 155 Chapter 6: Evaluation of the hydrolytic stability and Michael acceptor reactivity of selected Series V thiopyranones and Series V thiopyranone dioxides . 156 6.1. Introduction .156 6.2. Materials and methods 156 6.2.1. Determination of hydrolytic stability of 41, 219, 227 and curcumin 156 6.2.2. Determination of Michael acceptor reactivity by NMR 157 6.3. Results .157 6.3.1. Hydrolytic stabilities of 41, 219 and 227 .157 6.3.2. Michael acceptor reactivities of 41 and 227. .159 6.4. Discussion .162 6.5. Conclusion 166 Chapter 7: Conclusions and Future work 167 Bibliography . 171 Appendixes . 181 Table of Contents .181 Appendix 2-1. Spectroscopic data of synthesized compounds 181 Appendix 2-2. Purity data of final compounds as determined by reverse phase HPLC .181 Appendix 3-1. Cell Viability (%) of NB4-R1 cells treated with fixed concentrations (5 µM, 20 µM) of “Inactives” .181 Appendix 4-1. IC50 determination of curcumin and 41 on trypsin-, caspaseand chymotrypsin-like active sites of 20S proteasome 181 Appendix 4-2. Determination of substrates Km. Initial rate of reaction containing each substrate from µM to 200 µM with µg protein of NB4 lysate was measured and Km and Vmax was calculated using the MichaelisMenten equation .181 Appendix 5-1. qRT -PCR reaction conditions 181 Appendix 5-2. List of TaqMan® primers for real-time PCR .181 Appendix 5-3. List of genes involved in DNA replication, cell cycle, apoptosis and proteasomal structure that are significantly regulated by both curcumin and 41 .181 Appendix References .181 10 Compoun d Mobile Phase A Composition RT Area a (min) (%)d c 5e 10 11 12 17 18 20 21 22 38 46 201 e 202 203 e A1 A1 A1 A1 A1 A1 A1 A1 A1 A2 A1 A1 A1 A2 A1 A1 A2 5.0 2.7 3.1 22.0 6.6 7.7 12.1 7.5 5.0 2.8 5.7 4.8 2.5 4.0 5.02 6.46 7.65 204 A1 205 Mobile Phase B Compositionb/ RT Area c (min) (%)d c B1 B1 B2 B1 B1 B1 B1 B1 B1 B4 B1 B1 C1 B3 B1 B1 B2 3.1 3.3 2.8 4.2 3.1 2.4 5.5 3.1 4.0 2.8 2.8 1.7 2.3 4.2 3.68 4.54 3.52 4.56 99.5 98.5 95.5 97.1 99.3 96.9 98.7 98.3 95.4 97.9 100.0 100.0 99.0 98.8 94.75 95.98 91.02 100.0 B1 2.87 A1 4.88 95.76 B1 2.86 206 207 A1 A2 7.83 3.10 98.32 95.50 B1 B1 4.46 2.88 208 209 210 212 213 215 A2 A2 A1 A1 A1 A1 2.41 1.56 3.84 5.49 6.42 4.06 B2 B2 B1 B1 B1 B1 1.71 3.81 2.48 3.46 3.81 2.53 216 217 e 218 e 219 220 A2 A1 A2 A1 A1 3.35 5.55 6.96 4.06 4.18 99.11 95.07 96.59 97.86 98.90 98.12 100.0 94.82 91.56 98.12 96.23 B2 B1 B2 B1 B1 1.91 3.45 2.51 2.53 2.52 221 A2 4.52 B2 3.10 222 223 A2 A2 2.57 5.27 99.50 100.0 97.13 99.7 99.8 94.6 99.3 99.8 96.6 98.6 97.7 96.8 99.0 97.8 99.8 100.0 98.2 94.65 97.08 94.49 100.0 100.0 100.0 98.01 100.0 97.94 96.65 96.38 97.93 98.19 100.0 95.52 95.58 98.44 98.68 100.0 B2 B2 2.13 3.21 99.15 96.32 203 224 A1 2.18 95.71 B1 1.94 225 A1 1.57 B2 1.85 226 A1 1.63 99.33 100.0 B1 1.46 227 e A1 2.35 92.78 B1 2.02 100.0 100.0 100.0 100.0 a Composition of Mobile Phase A: Methanol and Water A1: 80% methanol A2: 70% methanol b Composition of Mobile Phase B: Acetonitrile and Water B1: 80% acetonitrile B2: 70% acetonitrile c Retention time of Major Peak in chromatogram. Chromatogram was run for at least 15 for the detection of the major peak d Area (%) of Major Peak = [Area of Major Peak / Total Area of All Peaks] x 100 e Did not comply with > 95% purity on two solvent systems. Appendix 3-1. Cell Viability (%) of NB4-R1 cells treated with fixed concentrations (5 µM, 20 µM) of “Inactives” Ref no. Cur 10 12 13 15 Compound Linker Keto-enol Substituents 3OCH3, 4OH H 4F H 2F 3F 4F 3OH, 4OCH3 H 2F 4F 204 % NB4-R1 cell viability ± SDa µM 20 µM 4.63 ± 2.5 0.85 ± 0.6 43.83 ± 32.7 1.25 ± 0.6 59.60 ± 8.5 0.99 ± 1.8 83.08 ± 4.6 36.48 ± 5.7 60.11 ± 10.7 47.12 ± 11.9 90.40 ± 10.0 71.60 ± 5.0 91.30 ± 11.2 87.81 ± 9.1 83.18 ± 4.7 73.84 ± 3.7 75.15 ± 12.1 0.52 ± 0.5 56.20 ± 27.9 1.70 ± 2.1 69.24 ± 11.4 1.36 ± 2.0 27 4F 93.83 ± 9.4 86.95 ± 6.5 28 3F 89.08 ± 10.1 69.05 ± 5.7 29 4F 92.11 ± 9.5 77.19 ± 6.9 a Percent growth inhibition of NB4-R1, human acute promyelocytic leukemia (APL) cells resistant to retinoic acid. Activity data performed on three independent experiments, two concentrations (5 and 20 µM), each experiment performed in triplicate. Appendix 4-1. IC50 determination of curcumin and 41 on trypsin-, caspaseand chymotrypsin-like active sites of 20S proteasome. Inhibition of Proteasome T -like activity by Curcumin Inhibition of Proteasome Casp-like activity by Curcumin 100 y it iv t c a e k il T % 50 -50 0.0 0.5 1.0 1.5 150 150 y itv it 100 c a e 50 k il p s a C % y it 100 iv t c a 50 e k il T C % -50 0.0 2.0 0.5 Log [Cur uM] -50 0.0 2.0 50 25 .5 .0 Log [41  M] .5 .0 1.0 1.5 2.0 In h ib itio n o f P ro te a s o m e C T -lik e a c tiv ity b y 100 ty i v tic a e k il T C % 75 50 25 0 .0 0.5 Log [Cur uM] 100 y itv it c a e k i -l p s a C % 75 -2 .0 1.5 In h ib itio n o f P ro te a s o m e C a s p -lik e a c tiv ity b y 100 % 1.0 Log [Cur uM] In h ib itio n o f P ro te a s o m e T -lik e a c tiv ity b y y it iv t c a e k liT Inhibition of Proteasom e CT -like activity by Curcum in .5 .0 Log [41  M] 205 .5 .0 75 50 25 0 .0 .5 .0 Log [41  M] .5 .0 Appendix 4-2. Determination of substrates Km. Initial rate of reaction containing each substrate from µM to 200 µM with µg protein of NB4 lysate was measured and Km and Vmax was calculated using the MichaelisMenten equation. Km Determination T-like e g a e v a e l m c o s C a e M to A r f p o y e ta b R Casp-like CT-like 50 100 150 200 250 [peptide] uM Appendix 5-1. qRT -PCR reaction conditions. Total RNA was isolated as described in Section 5.2.3 and converted to cDNA as described in Section 5.2.6. Real-time PCR of selected genes was carried out using the TaqMan® Gene Expression Assay System (Applied Biosystems, CA, USA) and recorded using the ABI 7500 Fast PCR System (Applied Biosystems, CA, USA). The PCR reaction, conditions and list of TaqMan primers were as follows: Reagents for qRT-PCR reaction using TaqMan® Gene Volume used per Expression Assay System reaction (µL) TaqMan® Gene Expression Master Mix (2X) 10 TaqMan® Gene Expression Assay or ACTB control (20X) RNase-free H2O 206 cDNA template Total volume 20 qRT-PCR assay conditions Step Temperature (˚C) Time Number of Cycles UDG Incubation 50 Hold 95 10 Hold Denaturation 95 15 sec Anneal/Extention 60 AmpliTaq Gold, UP enzyme activation 40 cycles Appendix 5-2. List of TaqMan® primers for real-time PCR Gene Dye Assay number UBE2E1 FAM Hs00979831_m1 EIF2AK2 FAM Hs00169345_m1 SEC62 FAM Hs00162786_m1 LMAN1 FAM Hs01557242_m1 MAP3K7 FAM Hs01105682_m1 THAP5 FAM Hs00973505_g1 ACTB VIC Hs99999903_m1 Calculation of fold-change (with respect to vehicle control) using the comparative Ct method was described as follows: ΔCt =Ct (sample) - Ct (endogenous control) ΔΔCt = ΔCt (sample) - ΔCt (vehicle control) Fold-change = 2-ΔΔCt Where Ct refers to the cycle number at which the increase in fluorescence (i.e. amount of cDNA) crosses a preset threshold. Sample refers to individual gene investigated and endogenous control refers to the housekeeping gene ACTB. 207 Appendix 5-3. List of genes involved in DNA replication, cell cycle, apoptosis and proteasomal structure that are significantly regulated by both curcumin and 41. Class Symbol MCM6 DNA2 DNA replication RFC4 POLD3 BUB1B BUB3 STAG2 CUL1 Cell cycle MCM6 RB1 NRAS YWHAB CHP1 Apoptosis PRKAR1A Function Log2FCa 41 Log2FCa Cur Req for DNA replication initiation and 2.806 2.909 elongation DNA2 is a conserved helicase/nuclease involved in the maintenance of 2.689 2.674 mitochondrial and nuclear DNA stability Replication factor-4 involved in the elongation of primed DNA templates by 2.549 2.482 DNA polymerase delta Required for optimal DNA polymerase 2.425 2.656 delta activity Required for normal mitosis progression. 3.241 3.425 Mitotic checkpoint protein to ensure 3.214 3.337 correct chromosome segregation Component of cohesin complex, a complex required for the cohesion of sister 2.963 3.092 chromatids after DNA replication. Mediate the ubiquitination of proteins involved in cell cycle progression, signal 3.107 3.274 transduction and transcription Req for DNA replication initiation and 2.806 2.909 elongation Active form of RB1 interacts with E2F1 and represses its transcription activity, leading 3.068 3.212 to cell cycle arrest. N-Ras membrane protein that bind GDP/GTP and possess intrinsic GTPase 2.415 2.829 activity 14-3-3 family, interact with RAF1 and CDC25 phosphatases, play a role in linking 2.109 2.350 mitogenic signaling and the cell cycle machinery Calcium-binding protein involved in regulation of vesicular trafficking, plasma 3.545 3.952 membrane Na(+)/H(+) exchanger and gene transcription A cAMP-dependent protein kinase is a component of the signal transduction 208 2.346 2.512 mechanism of certain GPCRs and transduces the signal through phosphorylation of different target proteins PIK3R1 Phosphoinositide 3-kinases capable of phosphorylating 3'OH of inositol ring of phosphoinositides. 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L.; Balog, M. r.; Selvendiran, K.; Rivera, B. K.; Kuppusamy, P.; Hideg, K. l. n. Synthesis of N-Substituted 3,5Bis(arylidene)-4-piperidones with High Antitumor and Antioxidant Activity. Journal of Medicinal Chemistry 2011, 54, 5414-5421. 218 [...]... Lymphoblastic leukemias are categorized into L1 – L3 subtypes base on cytological features such as size, nuclear shape and amount of cytoplasm Myeloid leukemias are categorized into six main types, M1 to M6, depending on the differentiation direction and degree of maturation 1.2 Acute Promyelocytic Leukemia (APL) Acute promyelocytic leukemia (APL) accounts for 10 – 15 % of acute myeloid leukemia (AML) and is... 2-10: Reaction mechanism of asymmetric thiopyranone analogs of series IV 70 Scheme 2-11: Synthesis of selected hydroxyl-substituted benzylidene analogs, 4, 10, 22 71 Scheme 2-12: Protection of alcohols using tetrahydropyran 72 24 List of Abbreviations 13 Carbon-13 nuclear magnetic resonance 1 Proton nuclear magnetic resonance AML Acute myelogenous leukemia Anal Combustion elemental... Acid-catalyzed aldol condensation reaction (X = CH2, S) 66 Scheme 2-6: Synthesis of thiopyranone dioxides from series IV .66 Scheme 2-7: Oxidation of thiopyranone analogs to thiopyranone dioxide in the presence of peracetic acid as oxidizing agent .67 Scheme 2-8: Synthesis of asymmetrical series III and IV analogues, 202 – 205 68 Scheme 2-9: Synthesis of asymmetrical series III and IV analogues,... The aim of this thesis was to test the hypothesis that curcuminoids induce potent growth inhibitory activity on acute promyelocytic leukemia (APL) via the misfolded N-CoR pathway Six series of curcumin analogs comprising of 78 compounds were synthesized and evaluated on two APL cell lines (NB4 and NB4-R1), two leukemic non-APL cell lines (HL60 and K562) and two nonmalignant cell lines (IMR90 and MCF10A)... Keto-enol tautomers of curcumin 45 Figure 1-7: Michael acceptor motifs in curcumin 45 Figure 1-8: Curcumin analogs reported to inhibit ubiquitin isopeptidase activity 46 Figure 1-9: Examples of curcumin analogs 49 Figure 1-10: Monocarbonyl cross-conjugated dienone analogs of curcumin 50 Figure 1-11: Selected examples of C5-Bridged analogs bearing piperidinone ring ... conformational change in PML-RARα that disrupted the physical association of N-CoR and PML-RARα This would destabilize the fusion protein and cause N-CoR to refold to its native conformation Like ATRA, genistein intercepts the association between N-CoR and PML-RARα through conformational effects and in this way circumvents the transcriptional outcomes (repression of RA target genes, derepression of. .. 5th April 2011) – poster presentation  Poster presentation: Synthesis and Evaluation of Curcumin analogs targeting Nuclear Receptor Co-repressor 4 1st PharmSci@Singapore Symposium, 7th American Association for Pharmaceutical Science – Student Chapter (National University of Singapore, 6th – 7th June 2012)  Poster presentation: Synthesis and Evaluation of Curcumin analogs targeting Nuclear Receptor... who has been my source of motivation, my pillar of strength and my confidante in this journey Their continuous encouragement gave me the inspiration and strength to persevere on 12 Conferences and Publications International conferences 1 21st International Symposium for Medicinal Chemistry, EFMC/ISMC (Brussels, Belgium, 5th – 9th Sept 2010)  Poster presentation: Investigation of agents targeting Nuclear... Oral presentation - Manipulation of Nuclear Receptor Co-Repressor by Curcumin analogs in Acute Promyelocytic Leukemia  Best oral presentation award 3 RIKEN Institute Summer Camp 2011 (Tokyo and Hakone, Japan, 26th – 29th Aug 2011)  Poster presentation: Development of Small Molecule Therapeutics targeting Nuclear Receptor Co-Repressor in Acute Promyelocytic Leukemia 4 GPEN, Globalization of Pharmaceutics... increase in local concentrations of repression complexes at RA target promoters lead to transcriptional repression of RA target genes and ultimately, differentiation arrest of promyelocytic cells The role of N-CoR in APL pathogenesis is of particular relevance in this thesis and is elaborated in the following section 1.2.2 The role of N-CoR in APL pathogenesis N-CoR is a component of the generic corepressor . condensation under acidic conditions 74 2.4.5. 5-Bis-(4-fluoro-phenyl)-penta-1,4-dien-3-one oxime (27) 75 2.4.6. Syntheses of O-methyloximes of 1,5-bis-(fluorophenyl)-penta-1, 4- dien-3-ones (28 and. lines 81 3.3.2. Anti-proliferative activity of curcumin and analogs on leukemic non-APL cell lines 91 3.3.3. Anti-proliferative activity of curcumin and analogs on human non- malignant cell lines. for syntheses of 40, 41, 221 – 227 76 2.4.8. Synthesis of 2-( 3-Fluoro-benzylidene)-cyclohexanone (48) 76 2.4.9. Synthesis of ( 3-[ (3-fluorobenzylidene)dihydro-2H-thiopyran- 4(3H)-one) (49) 76 2.4.10.