PART 1: TROGLITAZONE ANALOGUES AS CYCLIN D1 ABLATIVE AGENTS: THE POTENTIAL DRUGS FOR BREAST CANCER THERAPY
PART 2: VITAMIN E AND ITS ANALOGUES INDUCE APOPTOSIS IN PROSTATE CANCER CELLS IN PART THROUGH INHIBITION OF BCL-2/BCL-XL FUNCTIONS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By Jui-Wen Huang, M.S. *****
The Ohio State University 2005
Dissertation Committee Professor Ching-Shih Chen, Advisor Professor Robert W. Brueggemeier Approved by Professor Charles L. Shapiro Professor Tatiana M. Oberyszyn Advisor College of Pharmacy
ABSTRACT
Part 1: Troglitazone analogues as cyclin D1 ablative agents: the potential drugs for
breast cancer therapy
Cyclin D1 overexpression has been implicated in oncogene-induced mammary tumorigenesis as it is detected in over 50% of primary breast carcinomas and is correlated with poor prognosis. It has been reported that troglitazone (TG), a peroxisome proliferator-activated receptor-γ (PPARγ) agonist, can induce degradation of cyclin D1 as part of its mechanism for causing cell cycle arrest and growth inhibition in breast cancer cells. In this study, we obtained evidence that the ability of high doses of TG to repress cyclin D1 is independent of PPARγ activation. First, a PPARγ-inactive TG analogue
(Δ2-TG) causes cyclin D1 ablation with potency similar to that of TG in MCF-7 cells.
Secondly, MDA-MB-231 breast cancer cells, which exhibit higher PPARγ expression, are less sensitive to this TG-induced cyclin D1 down- regulation than MCF-7 cells. In addition, our data also indicate that TG- and Δ2-TG-induced cyclin D1 repression is mediated via proteasome-facilitated proteolysis as it can be inhibited by multiple proteasome inhibitors, including MG132, lactacystin, and epoxomicin, and is preceded by increased ubiquitination. The dissociation of these two pharmacological activities, i.e., PPARγ activation and cyclin D1 ablation, provides a molecular basis to use Δ2-TG as a scaffold to develop a novel class of cyclin D1-ablative agents. Accordingly, a small library of Δ2-TG ii derivatives has been synthesized. Among derivatives in this library, Δ2-TG-28 represents a
structurally optimized agent with potency an-order-of-magnitude higher than that of
Δ2-TG in cyclin D1 repression and MCF-7 cell growth inhibition.
Part 2: Vitamin E and its analogues induce apoptosis in prostate cancer cells in part
through inhibition of Bcl-2/Bcl-xL functions
Vitamin E and its analogues, such as a-tocopheryl succinate (α-TOS), have been shown to be proapoptotic agents in cancer cells, but the precise mechanism of their antineoplastic activity is not fully elucidated. To investigate the mechanism and the relationships between the structures and apoptosis-inducing activities of vitamin E and
α-TOS, a series of vitamin E analogues were synthesized. Among these analogues, including α-tocopherol and α-TOS, compound VEA-7 which has a two-unit isopranyled side chain and an ether linkage at the phenol position exhibits the most potent proapoptotic activity. Fluorescence polarization analysis and immunoprecipitation data confirmed that α-TOS and most apoptosis-inducing vitamin E analogues inhibit the proliferation of PC-3 and LNCaP prostate cancer cells in part by repressing the heterodimerization of Bcl-2/Bcl-XL and Bax. VEA-7, a more potent apoptotic inducer
than α-TOS, also displays stronger Bcl-XL binding affinity. In addition, α-TOS and these
vitamin E analogues selectively induce apoptosis in malignant prostate cancer cells but
not in normal prostate epithelial cells. The synthetic vitamin E analogue, VEA-7,
represents a novel apoptogenic agent that may have clinical value in chemotherapeutic
strategies for prostate cancer in the future.
iii
Dedicated to my parents
my sister, Rachel in the heaven and especially, my husband, Chung-Wai, for his support and everything
iv
ACKNOWLEDGMENTS
I would like to acknowledge Dr. Chen for his guidance, constant encouragement,
support, and for providing excellent working environment for his students.
I also would like to express my sincere appreciation for my defense committee Dr.
Brueggemeier, Dr. Shapiro, and Dr. Oberyszyn for their advice of my dissertation and
research. Thank Dr. Samuel K. Kulp for his patience in correcting my dissertation errors.
I would like to thank Dr. DashengWang who synthesized all vitamin E analogues
for my second project and Chung-Wai who synthesized most troglitazone and ciglitazone
analogues for my cyclin D1 project. Chung-Wai also performed PPARγ binding assay,
DNA fragmentation, and competitive fluorescence polarization analysis for this study.
Ya-Ting helped me to analyze cell cycle data. Ho-Pi and Yu-Chieh taught me how to do
RT-PCR. Jim and Chang-Shi provided a lot of ideas for the two projects and assisted me to complete some part of experiment. Kuen-Feng donated his hypostasis to my both projects. Nicole aided for immunohistochemitry experiment.
Kathy Brooks and Kelli Ballouz also provide a lot of assist to make graduate studies
going smoothly.
Colleagues and friends: Julie, Qiang, Joe, Dr. Hung, Leo, Ping-Hui, Yeng-Jeng and
his family, Shih-Jiuan, Wen and Li-Shu, Jian, Dennis, Erica, Po-Hsien, Yukao, and Ma
are all nice people and gives a lot of help in the daily life.
v
VITA
September 1992- June 1996 B. S. Chemistry
National Chung-Hsin University, Tai-Chung, Taiwan
September 1996- June 1998 M. S. Chemistry (Organic)
National Taiwan University, Taipei, Taiwan
September 2000-May 2001 Graduate student for Ph.D program, Chemistry
State University of New York at Stony Brook, Stony
Brook, NY
August 2001-present Graduate Teaching and Research Assistant, Pharmacy
The Ohio State University, Columbus, OH
PUBLICATIONS
1. J. -W. Huang, C. -W. Shiau, Y. -T. Yang, S. K. Kulp, K.- F. Chen, R. W.
Brueggemeier, C. L. Shapiro, and C. -S Chen (2005) “Peroxisome
Proliferator-Activated Receptor γ-Independent Ablation of Cyclin D1 by
Thiazolidinediones and Their Derivatives in Breast Cancer Cells.” Molecular
Pharmacology, 67, 1342-1348.
vi 2. C. -W. Shiau, C. –C. Yang, S. K. Kulp, K.- F. Chen, C. –S. Chen, J. -W. Huang,
and C. -S Chen (2005) “Thiazolidenediones Mediate Apoptosis in Prostate
Cancer Cells, in part, through the Inhibition of Bcl-xL/Bcl-2 Functions
Independently of PPAR gamma.” Cancer Research, 65, 1561-1569
3. J. –X. Zhu, J. –W. Huang, P. –H. Tseng, Y. –T. Yang, J. Fowble, C. –W. Shiau,
Y. –J. Shaw, S. K. Kulp, C –S. Chen (2004) “From the cyclooxygenase-2
inhibitor celecoxib to a novel class of 3-pliosphoinositide-dependent protein
kinase-1 inhibitors.” Cancer Research, 64 , 4309-4318.
4. J. -W. Huang, C. –D. Chen, M. –K. Leung (1999) “Magnesium Bromide
promoted Barbier Type Intramolecular Cyclization of Halo-Substituted Acetals,
Ketals and Orthoesters.” Tetrahedron letters, 40, 8647-8650.
5. C. –D. Chen, J. -W. Huang, M. –K. Leung, H. –H. Li (1998) “S,S-dimethyl
dithiocarbonate: A Novel Carbonyl Dication Synthon in the synthesis of
ketones.” Tetrahedron, 54, 9067-9078.
FIELDS OF STUDY
Major Field: Pharmacy
Specification: Medicinal Chemistry
vii
TABLE OF CONTENTS
Page
Abstract……………………………………………………………………………………ii
Dedication ………………………………………………………………………………..iv
Acknowledgements ……………………………………………………………………….v
Vita …………………………………………………………...…………………………..vi
List of tables ………………………………………………………….……………..….xii
List of figures………………………………………………………………………...…xiii
List of schemes ………………………………………………………………………..xvii
Abbreviations…………………………………………………………………………xviii
Chapters:
1. Introduction……………………………………………………………………………1
1.1 Overview of cyclin D1…………………………………………………………….1
1.2 Cyclin D1 and cancer……………………………………………………………...2
1.3 Peroxisome proliferator-activated receptor γ and thiazolidinediones……………..6
1.4 TZDs as Anti-tumor Regents……………………………………………………...9
2. Discovery and synthesis of thiazolidinedione derivatives without PPARγ activities
………………………………………………………………………………………..19
2.1 The discovery of TZD derivatives without PPARγ activation…………………..19
2.2 Synthesis of TZD compounds and their derivatives……………………………..20
viii 3. Thiazolidinedione and derivatives down-regulate cyclin D1………………………..28
3.1 Effect of TZDs on cyclin D1 down-regulation is independent of PPARγ...... 28
3.2 TG and Δ2-TG facilitate proteasome-mediated proteolysis of cyclin D1……….30
3.3 Investigation of the mechanism of TG and Δ2-TG-mediated cyclin D1
degradation……………………………………………………………………..31
4. Development of novel Δ2-TG-derived cyclin D1-ablative agents………………..…47
4.1 Modification of Δ2-TG……………………………………………………….….47
4.2 Structure-activity relationship (SAR) study of Δ2-TG analogues……………….48
4.3 Bioactivities of STG-28………………………………………………………….50
5. Conclusions and future directions …………………………………………………64
5.1 Down-regulation of cyclin D1 by TG and Its Analogues Is PPARγ-
Independent……..……………………………….……………………………….64
5.2 Development of cyclin D1 ablative agents………………………………………65
5.3 Future directions………………………………...……………………………….66
6. Experimental methods and material for part 1 ………………………………………69
6.1 Reagents…...……………………………………………………………………..69
6.2 Cell culture……………………………………………………………………….70
6.3 Cell viability analysis………………………………………………………….....70
6.4 Analysis of PPARγ activation……………………………………………………70
6.5 Western blot analysis…………………………………………………………….71
6.6 Coimmunoprecipitation/western blot…………………………………………….72
6.7 Reverse transcriptase (RT)-PCR analysis of mRNA transcripts of cyclin D1 gene
ix ………………………………………………………………………………………..73
6.8 Cell cycle analysis………………………………………………………………..73
6.9 siRNA transfection procedure……………………………………………………74
6.10 Determination of IC50 values………………………………………………….75
6.11 General information on chemical methods…………………...……………….75
6.12 Synthetic procedures of Δ2-TG, Δ2-TG analogues, and Δ2-CG…………...…76
1 6.13 Nomenclatures, H NMR (proton nuclear magnetic resonance), and HRMS
(high resolution mass spectrometry) characterizations of Δ2-TG, Δ2-CG, and Δ2-TG
analogues ………………………………………………………………..…………79
Title page for part 2 ……………………………………………………………………..90
7. Introduction…………………………………………………………………………..91
7.1 Vitamin E and cancer…………………………………………………………….91
7.2 Bcl-2 family and its signaling pathways…………………………………………93
7.3 Vitamin E analogues target the Bcl-2 proteins for chemotherapy……………….95
8. Project design……………………………………………………………………….108
8.1 Aims and project design………………...………………………………………108
8.2 Synthesis of vitamin E analogues………………………………………………110
9. Vitamin E analogues induce apoptosis through inhibiting Bcl-2/Bcl-XL function
………………………...…………………………………………………………….117
9.1 Vitamin E analogues induce apoptosis in prostate cancer cells……………….117
9.2 Vitamin E analogues induce apoptosis through inhibiting Bcl- XL function…..119
9.3 Effect of α-TOS and VEA-7 on intracellular Bcl-2 and Bcl-XL binding to Bak
x ……………………………………………………………………………………....121
9.4 Vitamin E analogues induce apoptosis selectively in prostate cancer cells but not
in normal prostate epithelial cells (PrEC)………………………………………122
10. Conclusions and future directions………………………………………………….134
10.1 Vitamin E analogues induce apoptosis in part by inhibiting Bcl-2/Bcl-XL
function in prostate cancer cells……………………………………………….134
10.2 Development Bcl-2/Bcl-XL inhibitors based on vitamin E scaffold………..136
10.3 Future directions……………………………………………………………..138
11. Experimental methods and material for part 2………..……………………………143
11.1 Reagents……………………………………………………………………..143
11.2 Cell culture…………………………………………………………………...143
11.3 Cell viability analysis………………………………………………………...144
11.4 Apoptosis detection by ELISA………………………………………………145
11.5 Western blot analysis of cytochrome c release into the cytoplasm………….145
11.6 Immunoblotting………………………………………………………………146
11.7 Competitive fluorescence polarization assay………………………………...147
11.8 Determination of IC50 values…………………………………………………147
11.9 Co-immunoprecipitation……………………………………………………..147
11.10 General information on chemical methods………………….……………….148
11.11 Synthetic procedures of vitamin E analogues (scheme 8.1)…………………149
1 11.12 Nomenclatures, H NMR, and HRMS characterizations of vitamin E
analogues………………………………………………………………….….150
Bibliography……………………………………………………………………….153 xi
LIST OF TABLES
Table Page
4.1 A series of compounds with dimethyl allyl protecting groups at the phenol
position………...……………………………………………………….…………….54
4.2 A series of compounds with allyl protecting groups at the phenol position
……………………………………………………………………………………………55
4.3 Three compounds with allyl or dimethyl allyl protecting groups at the phenol position
and replacement of the benzene group with naphthalene.………………..………….56
4.4 A series of compounds with different protecting groups at the phenol position……57
4.5 Compounds with different functional groups at the TZD position ………….………58
4.6 Anti-proliferative and cyclin D1-ablative activities of the S- or R-forms of TG-28...59
7.1 Summary of some proposed proapoptotic mechanisms of vitamin E analogues in
vitro……..…………………………………………………………………….…100
8.1 Structures of vitamin E analogues which are used in this study…………………....115
9.1 Structures and anti-proliferative activities in PC-3 and LNCaP cells of the ten vitamin
E analogues used in this study.……………………………………………………..124
9.2 Ectopic Bcl-XL protects LNCaP cells from vitamin E analogue-induced suppression
of proliferation..………………………………….…………..………………….….130
xii
LIST OF FIGURES
Figure page
1.1 A Schematic representation of pRb phosphorylation by G1 phase cyclins. B
Summary of biochemical functions of cyclin D1………………………………...…13
1.2 Schematic representation of the interacting domains of cyclin D1……………..…..14
1.3 MAPK signaling pathway which regulates cyclin D………………………………..15
1.4 PPARs function as heterodimers with their obligate partner, retinoid X receptor
………………………………………………………………………………………16
1.5 The chemical structures of some naturally occurring PPARγ ligand……………….17
1.6 Chemical structures of synthetic PPARγ agonists. (a) TZD class. (b) some non-TZD
compounds………………………………………………………………………... 18
2.1 A, Chemical structures of Rosiglitazone (or BRL49653) and its precursor compound
66 [( (Z )-5-[[4-[2-(methyl-2-pyridinylamino)ethoxy]phenyl]- methylenel
-2,4-thiazolidinedione)], which was renamed as Δ2-RG. B, Binding of
[3H]BRL49653 to PPARγ is specifically displaced by an excess of unlabeled
BRL49653 but not compound 66…………………………………………………….22
2.2 Chemical structures of troglitazone, ciglitazone, rosiglitazone, pioglitzone and their
analogues with double bond between the phenyl group and the TZD rings, Δ2-TG,
Δ2-CG, Δ2-RG, and Δ2-PG………………………………………………………..23
xiii 2.3 A, Δ-2-TZD derivatives lack activity in PPARγ activation. B, Similar result was
shown in MCF-7 cells…………………………………………….……………….…24
3.1 A, Differential expression levels of PPAR γ in MDA-MB-231 and MCF-7 cells. B,
dose-dependent effect of TG on cyclin D1 repression in MDA-MB-231 and MCF-7
cells…………………………………………………….…………………………….34
3.2 Dose-dependent effect of TG and CG on cyclin D1 in MCF-7 cells. B, Lack of effect
of RG on cyclin D1 in MCF-7 cells………………………….……………………....35
3.3 High doses of the PPARγ antagonist GW9662 have no effect on cyclin D1 expression
(top) or troglitazone-mediated cyclin D1 ablation (bottom) in MCF-7 cells………...36
3.4 Dose-dependent effect of TG, Δ2-TG, CG, and Δ2-CG on cyclin D1 and ERα
expression in MCF-7 cells…………………………………………….……………..37
3.5 Time-dependent effect of 40 μM TG and 30 μM Δ 2-TG on cyclin D1 expression in
MCF-7 cells………………………………………………………………………….38
3.6 RT-PCR analysis of cyclin D1 mRNA in MCF-7 cells after exposure to 40 μM TG or
30 μM Δ2-TG for 24 h…………………………..…………………………………...39
3.7 Dose-dependent effects of the proteasome inhibitors MG132, lactacystin, and
epoxomicin on TG- and Δ2-TG-mediated cyclin D1 ablation……………………….40
3.8 Cyclin D1 ubiquitination in TG- and Δ2-TG-treated MCF-7 cells…………………..41
3.9 Dose-dependent effects of TG and Δ2-TG on viability of MCF-7 cells
…………………………..………………………………..…………………………42
3.10 Cell cycle analysis following treatment with DMSO vehicle, TG or Δ2-TG in
MCF-7 cells………………………………………………………………………….43
xiv 3.11 Dose-dependent effects of TG and Δ2-TG on the expression of cyclins and CDKs
..………………………………………………….…………………………………...44
3.12 Evidence that troglitazone and Δ2-TG-induced cyclin D1 downregulation is
independent of GSK-3 β activation…………………………………………………45
3.13 Western blot analysis of cyclin D1 and SKP2 in MCF-7 cells with or without SKP2
siRNA transfection…………………………………………………………………...46
4.1 Three parts of Δ2-TG are modified…………………………………………………..52
4.2 Dose-dependent effects of STG-28 on the expression of cyclins, CDKs, and CKIs
………………………………………………………………………………………..60
4.3 Dose-dependent effects of the proteasome inhibitors MG132 on STG-28-mediated
cyclin D1 ablation…………………………………………………………………..61
4.4 RT-PCR analysis of the mRNA levels of cyclin D1 in MCF-7 cells
…………………………………………………………………………...…………62
4.5 Evidence that STG-28-induced cyclin D1 downregulation is independent of GSK-3 β
activation………………………………………………………………………...….63
7.1 Chemical structures of tocopherols and tocotrienols……………………….………..99
7.2 Three subfamilies of Bcl-2-related proteins. “TM” refers to a hydrophobic region in
the carboxyl terminus……………..………………………………………………...104
7.3 The model for Bcl-2 survival activity in mammals………………………………...105
7.4 Caspases inhibitor model for Bcl-2 function……………………………………….106
7.5 A Structure of HA14-1, a small molecule that binds Bcl-2 protein. B Structural model
for the complex of HA14-1 with the Bcl-2 surface pocket as predicted by computer
xv docking calculation…………………………………………………………………107
8.1 Domains in vitamin E analogues…………………………………………………...112
8.2 Some vitamin E analogues………………………………………………………….113
8.3 A. Chemical structures of γ-T2H and γ-T3H. B. Structure of phytyl succinate……114
9.1 Formation of nucleosomal DNA in PC-3 cells that were treated with α-TOS or
VEA-7 at the indicated concentrations for 24 hours……...………………………125
9.2 Dose- and time-dependent effects of α-TOS and VEA-7 on cytochrome c release in
PC-3 cells…………………………………………………………………………...126
9.3 PARP cleavage by α-TOS and VEA-7 in PC-3 cells…………………………..…..127
9.4 Effect of α-TOS and VEA-7 on the expression level of Bcl-2 family members in
PC-3 cells………………………………………………………………………....128
9.5 Differential inhibition of BH3 domain-mediated protein interactions of Bak BH3
peptide with Bcl-XL by vitamin E analogues……………………………………….129
9.6 α-TOS and VEA-7 trigger apoptotic death in PC3 cells by inhibiting heterodimer
formation of Bcl-2 and Bcl-XL with Bak…………………………………………...131
9.7 Dose-dependent effect of α-TOS and VEA-7 on caspase-9 activation in PC-3 cells
…………………………………………………………………………………...….132
9.8 Normal prostate epithelial cells (PrEC) are resistant to vitamin E analogue-induced
suppression of proliferation.……………………………...………………………...133
xvi
LIST OF SCHEMES
Scheme page
2.1 The first synthetic strategy of troglitazone……………………….………………….25
2.2 Our modified synthetic strategy of TG and Δ2-TG………………………….………26
2.3 Synthesis of CG and Δ2-CG…………………………………………………………27
4.1 General synthetic procedure for Δ2-TG analogues…………………………………..53
8.1 Synthetic procedures of VEA-1 to VEA-8………………………………………....116
10.1 Designed synthetic procedures to construct the main structure of vitamin E, the
chromanol ring...……………………………………………………………………140
10.2 Designed synthetic procedures to construct saturated or unsaturated phytyl side
chains with different lengths……………………………………………………...141
10.3 Designed synthetic procedures to create different carboxylic groups at the phenol
position of tocopherols or tocotrienols……………………….…………………….142
xvii
ABBREVIATIONS
1H-NMR proton nuclear magnetic resonance
12,14 15d-PGJ2 15-deoxy-Δ -prostaglandin J2
Akt protein kinase B
ANT antennapedia
Apaf1 apoptotic protease-activating factor 1
APC adenomatous polyposis coli
α−TOS α−tocopheryl succinate
Bcl-2 B-cell lymphoma gene 2
BH Bcl homology
CDK cycline dependent kinase
CG ciglitazone
CKI cyclin dependent kinase inhibitors
CREB cAMP response element binding protein
DIBAL diisobutylaluminium
DMEM Dulbecco's modified eagle's Medium
DN Fas-dominant negative
EGFR epidermal growth factor receptor
Egr-1 early growth response protein-1
xviii eIF2 eukaryotic initiation factor 2
ELISA enzyme-linked immunosorbent assay
ERK extracellular signal-regulated kinases
FBS fetal bovine serum
FP fluorescence polarization
GADD45 growth arrest and DNA damage-inducible gene 45
GSK-3 glycogen synthase kinase-3
GTP guanosine triphosphate
HDAC histone deacetylase
HER2/neu human epidermal growth factor receptor 2
HR-MS high resolution mass spectrum
IAP inhibitor of apoptosis protein
IGF-1R Insulin-like Growth Factor-1 Receptor
JNK c-Jun amino-terminal kinase
MAPK Mitogen-Activated Protein Kinase
MEK MAPK/ ERK kinases
MOM methoxymethyl mTOR mammalian target of rapamycin
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
NF-κB nuclear factor-κB
NHR nuclear-hormone-receptor
xix ODN oligodeoxynucleotide p-Akt phospho-protein kinase B
PARP Poly- (ADP-ribose) polymerase
PG pioglitzone
PI3-kinase phosphatidylinositol 3-kinase
PKC protein kinase C
PPAR peroxisome proliferator-activated receptors
PPRE peroxisome-proliferator response element
PrEC normal prostate epithelial cells
Rb retinoblastoma
RG rosiglitazone or BRL49653
RT-PCR reverse transcriptase-polymerase chain reaction
RXR retinoid X receptor
SCF Skp1-cullin-F-box protein
STAT signal transducer and activator of transcriptions
TBS-Cl tert-butyldimethylsilyl chloride
Tf trifliate
TG troglitazone
TGF transforming growth factor
TNF tumor necrosis factor
TRAIL TNF-related apoptosis-inducing ligand
TZD thiazolidinedione
xx
CHAPTER 1
INTRODUCTION
1.1 Overview of Cyclin D1
Cyclin D1, one of three family members (D1, D2, D3), serves as a key sensor and
integrator of extracellular signals in early to mid-G1 phase in the cell cycle. It binds
preferentially to two cyclin dependent kinases, CDK4 and CDK6. The best studied target
of the cyclin D/CDK4 complex is the retinoblastoma protein (Rb), which releases the
E2F transcription factor after phosphorylation by the cyclin D/CDK4 complex,
permitting expression of many proliferation-related genes. phosphorylated Rb (pRb)
serves as a gatekeeper of the G1 phase, and passage through the restriction point leads to
DNA synthesis in S phase (Fig. 1.1 A) (1). It also associates with histone deacetylase
(HDAC) through the pRb pocket domain and recruits HDACs to E2Fs. Phosphorylation of pRb by cyclin D/CDK4 complex displaces HDAC, which leads to increased cyclin E expression, S phase progression, and repression of the cyclin A and cdc2 genes (2).
The functions of the cyclin D/CDK4 complex are regulated by levels of cyclin
dependent kinase inhibitors (CKI). Two general classes of CKIs are the ink4 and Cip/Kip
families. Members of the Ink4 family, consisting of p15, p16, p18, and p19, compete with 1 cyclin D1 for binding to the CDK. The Cip/Kip inhibitors, consisting of p21, p27 and p57, bind all cyclinD/CDK4 complexes to suppress the activity of the complex and may serve to titrate out ‘free’ Cip/Kip inhibitors, thereby facilitating cyclin E/CDK2 function (3).
In addition to its CDK-binding function, a body of evidence now indicates that
D-type cyclins have CDK-independent properties (4). Cyclin D1 forms physical associations with more than 30 transcription factors or transcriptional coregulators (1).
Cyclin D1 directly binds to several nuclear receptors, including the androgen receptor
(AR), estrogen receptor (ER) α, thyroid hormone receptor, and peroxisome proliferator-activated receptor (PPAR) γ, and regulates the basal and ligand-dependent transactivation of these nuclear receptors within cultured cells (1). The biological functions of cyclin D1 are summarized in Fig 1.1 B.
1.2 Cyclin D1 and Cancer
Cyclin D1 functions in a large complex network of interactions, some of which are independent of its role as a CDK partner. Many oncogenic signals induce cyclin D1 expression and transactivate the cyclin D1 promoter, including STAT (signal transducer and activator of transcription) (5), NF-κB (nuclear factor-κB) (6), Egr-1 (early growth response protein-1) (7), CREB (cAMP response element binding protein) (8), Src (8),
Ras (9), HER2/neu (human epidermal growth factor receptor 2) (10), and β-catenin
(11-12). Therefore, it is not surprising that cyclin D1 plays an essential role, not only in cell cycle progression but also in malignant transformation.
In breast carcinomas, cyclin D1 is overexpressed at the mRNA and protein levels in up to 50% of primary breast cancers correlating with poor prognosis (13-14).
Moreover, substantial evidence documents roles for cyclin D1 in ER action. Cyclin D1 2 regulates the estrogen response of breast epithelial cells (15). Cyclin D1, but not D2 or
D3, can activate ER-mediated transcription. Significantly, a mutant form of cyclin D1
that fails to bind CDKs potentiates ER activity to the same extent as wild-type cyclin D1.
When associated with CDK4, cyclin D1 has no effect on ER activity, probably because
the kinase titrates its partner away from the transcription factor. Evidence also indicates
that kinase activity of CDK4 is not involved in the effect of cyclin D1 on ER action
because p16INK4, a CDK4 inhibitor that competes with cyclin D1 for binding to the kinase,
did not inhibit the activation of ER-mediated transcription. Therefore, free cyclin D1
activates ER transcription in a cell cycle-independent manner. These findings suggest that
two different forms of cyclin D1, unbound and bound to CDK4, play tremendous roles
within signaling transduction networks (16). Other studies have reported that cyclin D1
can form a specific complex with ER, and further regulate ER-mediated transcription.
Since ER activity is induced in the absence of estrogen, but in the presence of cyclin D1, it is suggested that cyclin D1 can directly activate ER through its direct association with
ER. An important implication of this finding is that cyclin D1-overexpressing tumor cells
should be cable of estrogen-independent growth. Moreover, it also explains how cyclin
D1 overexpression confers resistance to antiestrogens in breast cancer cells and
represents a negative predictive factor for tamoxifen and ICI 182780 respponse (17-18).
While most evidence supports an important role for the interplay between cyclin
D1 and the estrogen pathway in the regulation of mammary epithelial cells, these results
are complicated by studies using a knock in model in which cyclin D1 was replaced by
cyclin E (19). In this model, cyclin E rescued mammary gland development that was
3 arrested by cyclin D1 deletion. Since cyclin E has no effect on ER transcriptional activity,
the finding suggests that proliferation of the mammary epithelium is not regulated by the
interaction between ER and cyclin D1. However, a second possibility is that the
transcriptional effects of cyclin D1 are specific to pathological situations. In support of
their idea, the overexpression of cyclin D1 in tumor cells, which mimicked cyclin D1
gene amplification, resulted in saturation of CDK4 and the regulation of ER activity by
free cyclin D1. Under conditions in which normal levels of cyclin D1 were expressed,
binding of CDK4 to cyclin D1 prevented its effects on ER action (20). These results
suggest that only the cyclin D1 oncogene has transcriptional functions.
Side-directed mutagenesis have further elucidated that mechanism of cyclin D1’s
effects on ER activity. These studies have confirmed earlier findings which showed that
cyclin D transcriptional activity generally does not depend on CDK4 or CDK6 kinase
activity. These findings are often based on the use of K112E mutated form of cyclin D1
that does not interact with CDKs, the use of p16Ink4a or dominant negative forms of CDKs, or by mutating the putative CDK phosphorylation sites of the target protein. Collectively, these data indicate that the regulatory functions of cyclin D1 do not rely on CDKs, and the cyclin box (amino acids 56–152) is generally not involved in transcriptional regulation. In contrast, the C-terminal part of cyclin D1 appears to be necessary for the modulation of ER transcriptional activities. This carboxyl terminus of cyclin D1 diverges from the other D-type cyclins, due to the presence of an acid region encompassing amino acids 272–280 and to the presence of a leucine-rich LLXXXL motif present at position
254–259 (Fig. 1.2). Mutation of leucine 254 and 255 abrogates the transcriptional effect
4 of cyclin D1 on the estrogen receptor. Therefore, some of the transcriptional effects of cyclin D1 might rely on the functional domain at the C-terminal part of the protein (16).
The mitogen-activated protein kinase (MAPK) signaling pathway is one of the
well known pathways which regulate proliferation of tumor cells. The expression of
cyclin D1 is also mediated by this signaling pathway. Mitogen-induction of cyclin D1
relies on the activation of a class of intracellular serine/threonine protein kinases, MAPKs
or extracellular signal-regulated kinases (ERKs) (21). This pathway sequentially involves
the small GTP-binding protein Ras and the Raf-1 protein kinase. Raf-1 phosphorylates
MAPK/ ERK kinases (MEKs) and in turn activate ERKs to induce their nuclear
translocation and gene activation. Inactivation of Ras causes a decline in cyclin D1 levels
(22) and it has been shown that this signaling pathway is implicated in cyclin D1
regulation via at least two mechanisms (Fig. 1.3) (23). Mitogen-induced Ras signaling
and ERK activation promote transcription of the cyclin D1 gene. Cyclin D1 promoter
activity is stimulated by overexpression of MAPKs or their targets, c-ets 2 or AP-1, and
transcriptional activation by EGF is accordingly reduced by inhibitors of MAPKs and ets
proteins (24). Cyclin D1 accumulation is generally modest because of ubiquitination and
rapid turnover, with an approximate half-life of 20 min in fibroblasts. Following
synthesis, cyclin D associates with Cul-1, a component of the SCF (Skp1p-cullin-F-box
protein) ubiquitin E3 ligase complex, and is then degraded by the 26S proteasome (25).
Secondly, following its association with CDK4, cyclin D1 is phosphorylated on threonine
286 by the glycogen synthetase 3β (GSK-3β), which represents another mechanism by
which cyclin D1 is translocated out of the nucleus to induce its proteasomal degradation
(26). However, this GSK-3β-dependent turnover is Ras-dependent since the activity of 5 the kinase is inhibited following phosphorylation by Akt (27). In fibroblasts, the
pharmacological inactivation of the PI-3 kinase/Akt signaling pathway extends cyclin D
half-life two to threefold, as does the transfection of a constitutively active form of Akt.
Interestingly, free cyclin D1 is ubiquitinated by a different mechanism that does not
require GSK-3β or the phosphorylation of Thr286 (28). This finding could suggest the
existence of two different pools of cyclin D1, free or bound to CDK4, that are regulated
by different mechanisms and that could have different functions. Altogether, these
observations indicate that intracellular signaling pathways inducing cyclin D expression
are probably coordinated to transactivate the promoter and to prevent degradation of the
protein at the same time.
Cyclin D1 also can be considered as an oncogene. Cyclin D1 was shown to
cooperate with various oncogenes, such as Myc, in the induction of tumors in transgenic
mice (29-30), or Ras in the transformation of cultured cells (23, 31). In addition,
antisense cyclin D1 cDNA inhibits the growth of colon cancer cells in nude mice (32).
Importantly, cyclin D1-/- mice are resistant to breast cancers induced by the Neu and Ras oncogenes (33), but are still sensitive to oncogenic pathways driven by the Myc or Wnt1
oncogenes. This suggests that some oncogenic pathways, such as Neu/Ras, may strictly rely on cyclin D1, whereas others, such as Myc or Wnt, may use different cell cycle proteins.
1.3 Peroxisome Proliferator-activated Receptor γ and Thiazolidinediones
The peroxisome proliferator-activated receptors (PPARs) are a group of three nuclear receptor isoforms, PPARα, PPARβ/δ, and PPARγ, encoded by different genes.
PPARs are ligand-regulated transcription factors that belong to the 6 nuclear-hormone-receptor (NHR) family. They bind as heterodimers with a retinoid X
receptor (RXR) and recruit a cofactor complex either co-activators or co-repressors that
modulate its transcriptional activity (Fig.1.4) (34). After forming heterodimers with RXR
and being activated by their ligands, PPARs interact with peroxisome-proliferator
response elements (PPREs) in the promoter of their target gene. Binding PPAR-RXR to
the PPRE is a mechanism by which organisms can modulate lipid levels by altering transcription. The pattern of expression and the main functions of three PPAR isoforms are diverse. The main role of PPARα is the regulation of energy homeostasis. It also
activates fatty-acid catabolism (β- and ω– oxidations), stimulates gluconeogenesis and
ketone-body synthesis and is involved in the control of lipoprotein assembly. PPARα
attenuates inflammatory responses and participates in the control of amino-acid
metabolism and urea synthesis. PPARβ/δ is required for placental development and is
involved in the control of lipid metabolism. It is also important in the regulation of cell
proliferation, differentiation and survival, especially in keratinocytes, the main cell type
of the epidermis. PPARγ has a pivotal role in adipocyte differentiation, lipid storage in
the white adipose tissue and energy dissipation in the brown adipose tissue. In addition, it
mediates inflammatory reactions and is involved in glucose metabolism through
12,14 improvement of insulin sensitivity (34). The 15-deoxy-Δ -prostaglandin J2 (15d-PGJ2)
is a naturally occurring PPARγ ligand. This eicosanoid can activate PPARγ at
micromolar concentrations. Some unsaturated fatty acids (Fig.1.5) also are natural
ligands of the receptor. Synthetic PPARγ ligands, known as thiazolidinediones (TZDs),
include rosiglitazone (Avandia), pioglitazone (Actos), and troglitazone (Rezulin).
7 Troglitazone (TG) causes a severe idiosyncratic liver toxicity and thus has been
withdrawn from the market. The TZDs are a breakthrough in the therapy of type II
diabetes mellitis because they decrease insulin resistance. TZDs enhance insulin action
by increasing glucose uptake in the peripheral tissues, as well as reducing the hepatic
glucose output. A strong correlation between the ability of the ligand to activate PPARγ
and the antidiabetic potency of the PPARγ ligand has been established (35).
PPARγ is thought to have broad anti-carcinogenic effects in many different cell
types, due to its anti-proliferative, pro-differentiation, and pro-apoptotic properties.
Nevertheless, the involvement of PPARγ in the development of tumours in various
tissues is still debated. In many cell types, PPARγ activation has anti-tumorigenic effects.
For instance, both ER-positive (MCF-7) and -negative (MDA-MB-231) breast cancer cell
lines undergo cell cycle arrest when treated with 15d-PGJ2 or TG. In addition, 15d-PGJ2 induced an irreversible apoptosis in an MDA-MB-231 nude mouse model (36-37).
However, conflicting results have been obtained from some other experimental settings or in vivo models. For instance, two independent groups reported that treatment of
APCmin+/ mice with PPARγ activators for 5–8 weeks results in an increase of tumors or polyps in the colon (38, 39). Due to the numerous different models that are being utilized, defining roles for PPARγ is complicated.
The synthetic TZDs were the first class of compounds to be identified as PPARγ
ligands (Fig. 1.6a). It has been demonstrated that the insulin-sensitizer, rosiglitazone (RG)
is the most potent and selective PPARγ agonist in this series of compounds (40). The
relevance of PPARγ activation to the insulin-sensitizing properties of the TZDs is
8 underscored by the high correlation between PPARγ agonist potency and antidiabetic
efficacy. Furthermore, selective non-TZD agonists of PPARγ, such as GW 1929, have
been developed that also improve insulin sensitivity in animal models (Fig. 1.6b) (41,
42).
1.4 TZDs as Anti-tumor Reagents
Studies have demonstrated that the some compounds in the TZD class of PPARγ
ligands not only induce differentiation of PPARγ-expressing preadipoctytes (43) and
primary human liposarcoma cells (44), but also exhibit antitumor effects in some cancer
cell lines including lung (45), breast (46), colon (47), prostate (48), hematiopoietic (49),
gastric (50), and pancreatic (51) cells. Although it was suggested that the TZD
compounds inhibit the proliferation of cancer cells through increasing the activity of
PPARγ (45−47, 51−52), some data indicated that there is no strong association between
PPARγ−activating function of the TZDs and their antitumor effects. Halperin and
coworkers showed that the inhibition of cell proliferation by TZDs is independent of
PPARγ by using PPARγ−/− and PPARγ+/+ mouse embryonic stem cells. The study
demonstrated that TG and ciglitazone (CG) block G1-S transition in both PPARγ−/− and
PPARγ+/+ mouse embryonic stem cells by inhibiting translation initiation. Inhibition of
translation initiation was the consequence of partial depletion of intracellular calcium
stores and the resulting activation of protein kinase R which phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2), thus rendering eIF2 inactive (53). Song and coworkers latter showed that TG but not RG, induces G1 cell cycle arrest and apoptosis in human and rat hepatoma cell lines. In addition, these results showed that only TG, but
9 not RG, increased the levels of p53, p27, and p21, but it reduced the levels of cyclin D1
and pRb in a time-dependent manner. Theretofore, TZD-mediated growth inhibition
appears to be independent of PPARγ activation in hepatoma cells (54). These
contradictory conclusions might result from the use of different cancer cell lines or
different experimental conditions or models. Different TZD compounds might also affect
specific signaling pathways in diverse cancer cells. Since TZDs are claimed to be
potential anticancer drugs, it is worthwhile to determine whether the antitumor effects of
each TZD compound are dependent upon their PPARγ activities.
In breast cancer research, plentiful studies using PPARγ ligands, such as TZDs, to
induce cell cycle arrest or apoptosis in vitro or in vivo have been reported (55). For
example, Safe and coworkers reported that CG and 15d-PGJ2, inhibit the growth of
ER-positive MCF-7 breast cancer cells, through actions on the cell cycle arrest machinery.
The data showed that CG or 15d-PGJ2 treatment of MCF-7 cells resulted in a
concentration- and time-dependent decrease of cyclin D1 and ERα proteins, which also
accompanied by decreased cell proliferation and G0-G1 →S-phase progression.
Downregulation of cyclin D1 and ERα by these two PPARγ agonists was inhibited in
cells co-treated with the proteasome inhibitors, MG132 and PSII, but not in cells
co-treated with the protease inhibitors, calpain II and calpeptin. Moreover, after treatment
of MCF-7 cells with 15d-PGJ2 and immunoprecipitation with cyclin D1 or ERα
antibodies, enhanced levels of ubiquitinated cyclin D1 and ERα were observed (56).
Another study demonstrated that TG inhibited MCF-7 cell proliferation by blocking
events critical for G1 → S progression. Flow cytometry results showed that TG at 20
10 μM increased the percentage of cells in G1 phase from 51 to 69% after 24 h. This
phenomenon also was accompanied by an attenuation of Rb protein phosphorylation
associated with decreased CDK4 and CDK2 activities. Inhibition of CDK activity by TG
correlated with decreased protein levels for several G1 regulators of Rb phosphorylation
(cyclin D1, and CDKs 2, 4, and 6). Overexpression of cyclin D1 partially rescued MCF-7
cells from TG-mediated G1 arrest (57). The details of the mechanism of how TG or CG
downregulates the expression of cyclin D1 and induces cell cycle arrest or apoptosis is
not completely elucidated. One report described the screening of a limited DNA array
containing 23 genes involved in regulating either the cell cycle and/or apoptosis to
investigate the mechanism of TG’s antiproliferative and pro-apoptotic effects. Among
these 23 genes, four of them exhibited regulation by TG, with growth arrest and DNA
damage-inducible gene 45 (GADD45) being the most strongly upregulated. TG induced
GADD45 mRNA expression in a time- and dose-dependent manner. Depletion of
GADD45 by siRNA abrogated TG-induced apoptosis in MCF-7 cells demonstrating the physiological relevance of GADD45 upregulation. Signaling pathways mediating
TG-induced GADD45 expression were investigated in this study. Several MAPK
pathways were involved in the induction of GADD45 by TG. Inhibition of the c-jun
N-terminal kinase MAPK pathway by SP600125 partially abolished TG-induced
GADD45 mRNA and protein expression, and apoptosis. In contrast, inhibition of the p38
MAPK pathway by SB203580, or through overexpression of a dominant-negative mutant
of p38 MAPK, augmented GADD45 mRNA induction and GADD45 promoter activation,
as well as cell apoptosis by TG. Blockade of the extracellular signal-regulated kinase
11 MAPK pathway by PD98059 also enhanced the effects on GADD45 and apoptosis by
TG. However, two other PPARγ agonists, pioglitazone (PG) and RG, did not induce
GADD45 expression (58). According to these research data, some PPARγ agonists showed potential value
for treatment of various cancers. However, numerous questions remain to be clarified.
First, it is still debated whether the induction of apoptosis by these PPARγ ligands in
certain cancer cell lines and animal models is related to their PPARγ activities. The
detailed mechanisms of the antiproliferative actions of each TZD in different carcinoma
types are still not clear. Moreover, the toxicity of the drugs requires further study since
hepatoxicity of both TG and CG has been reported (59). Finally, the role of PPARγ as an
important regulator of lipid and glucose metabolism suggests possible side effects related
to the PPARγ activities of TZDs when used in cancer therapy. Therefore, the development of novel agents based on the TZD structure that retain antitumor effects, but are devoid of PPARγ activities is a worthwhile endeavor that could avoid these potential toxicities.
12
Fig 1.1 A Schematic representation of pRb phosphorylation by G1 phase cyclins. B
Summary of biochemical functions of cyclin D1 (1)
13
Fig. 1.2 Schematic representation of the interacting domains of cyclin D1 (16).
14
Fig. 1.3 MAPK signaling pathway which regulates cyclin D (23).
15
Fig.1.4 PPARs function as heterodimers with their obligate partner, retinoid X receptor
(RXR). The dimer probably interacts with co-regulators, either co-activators (CoAct) or
co-repressors (CoRep). The peroxisome proliferator response element (PPRE) is located
in the promoters of target genes of PPARs (34).
16
Fig. 1.5 The chemical structures of some naturally occurring PPARγ ligand
17
Fig. 1.6 Chemical structures of synthetic PPARγ agonists. (a) TZD class, (b) some non-TZD compounds
18
CHAPTER 2
DISCOVERY AND SYNTHESIS OF THIAZOLIDINEDIONE
DERIVATIVES WITHOUT PPARγ ACTIVITIES
2.1 The Discovery of TZD Derivatives without PPARγ Activation
Evidence that the anti-proliferative activity of TZD is unrelated to PPARγ
activiation has been obtained through the use of PPARγ−/− and PPARγ +/+ mouse embryonic stem cells (53) and different TZD compounds (54). These approaches however have been restricted to specific cancer cell lines. Therefore, other strategies are needed to thoroughly investigate the relationship between these two activities of TZD compounds.
A literature search revealed an interesting study of the structure-activity relationship of
RG in which a series of TZD compounds were synthesized and their antihyperglycemic activities were examined. The authors discovered that the benzylidene precursor of RG, compound 66, (Fig 2.1A), showed moderate antihyperglycemic activity. The chemical shift analysis of the position of the benzylidene protons of compound 66 confirmed the
Z-geometry about the double bond (60). Another study demonstrated that, compare to RG
(or BRL49653 ), compound 66 has a much weaker binding affinity to PPARγ. A
19 competitive binding assay using purified PPARγ protein and [3H]BRL49653 showed that .
[3H]BRL49653 was effectively displaced by an excess of unlabeled BRL49653,
indicating that binding was specific, while no competition was observed with compound
66 (61) (Fig.2.1B). It is suggested that the double bond between the TZD ring and the
phenyl group increases the rigidity of the whole molecule, thereby reducing the binding
affinity of the compound to PPARγ.
We subsequently synthesized the corresponding analogues of TG, CG, RG, and PG that contain a double bond between the phenyl group and TZD rings (Fig 2.2). To assess the activities of these derivatives to activate PPARγ, PC3 prostate cancer cells were treated with these compounds for 48 hours. The amounts of activated PPARγ in the nuclear extracts of the cells were than determined by ELISA (Fig 2.3A). Compared to their parent compounds and DMSO, these Δ2 series compounds are weak activators of
PPARγ (62). Similar results were obtained in MCF-7 cells. (Fig. 2.3B) (63).
2.2 Synthesis of TZD Compounds and Their Derivatives
The first method to synthesize TG was published in 1989 (scheme 2.1) (64). The
authors used racemic tetramethylchroman-2-carboxylic acid as starting material. After
reducing the carboxyl group and protecting the hydroxyl group on the chromanol ring,
p-chloronitrobenzene was used to construct the linked benzene group (compound 4). The
protecting group MOM (methoxymethyl) on the phenol was replaced by an acetate group
in this synthetic strategy and the nitro group was then able to be hydrogenated to an
20 amino group. Two additional steps were used to establish the TZD ring with this
synthetic strategy, eight steps are necessary to complete the final compound, TG, with
less than 10% total yield. Therefore, it was worthwile to modify the synthetic approach
by reducing the number of synthetic steps and increasing the total yield.
The same starting reagent was selected for this modified approach.
p-Fluorobenzaldehyde was applied to create the benzaldehye compound 11 (scheme 2.2) after reducing the carboxyl group and protecting the hydroxyl group on the chromanol ring by allyl bromide. The TZD ring was then constructed under a basic condition.
Subsequently, different conditions were utilized to deprotect and reduce the double bond together (for compound 9, TG) or to deprotect the allyl group (for compound 13, Δ2-TG).
This modified synthetic strategy decreases the number of synthetic steps from eight to five, improves the total yield, and permits the generation of either TG of Δ2-TG by using
two specific conditions. CG and its derivative, Δ2-CG, can also be synthesized by a
similar strategy (scheme 2.3). Since there is no hydroxyl groups on the cyclohexane ring
of CG, the protecting and deprotection steps which were used in the synthesis of TG can
be omitted.
21 A
O O
NH NH N N S N N S O O O O
Rosiglitazone (RG) Compound 66 or BRL 49653 or Δ2-RG
B
Fig. 2.1 A, Chemical structures of RG (or BRL49653) and its precursor compound 66
[( (Z )-5-[[4-[2-(methyl-2-pyridinylamino)ethoxy]phenyl]- methylenel-2,4-
thiazolidinedione)], which was renamed as Δ2-RG. B, Binding of [3H]BRL49653 to
PPARγ is specifically displaced by an excess of unlabeled BRL49653 but not compound
66. For each point, approximately 400 ng of purified bacterially expressed PPARγ was incubated with 500 nM of [3H]BRL49653 with carrier (dash, DMSO) or with the
unlabeled competitors: BRL49653 or compound 66, 50 μM. After incubation,
[3H]BRL49853 bound to PPARγ was separated from the free label and quantified by
liquid scintillation counting (61).
22 O O NH NH O S O S O O O O HO HO Δ2-TG
Troglitazone (TG) O O NH NH S S O O O O Δ2-CG Ciglitazone (CG)
O O
NH NH N N S N N S O O O O
Rosiglitazone (RG) Δ2-RG
O O
NH NH S S N O N O O O Pioglitazone (PG) Δ2-PG
Fig. 2.2 Chemical structures of troglitazone, ciglitazone, rosiglitazone, pioglitzone and their analogues with double bond between the phenyl group and the TZD rings, Δ2-TG,
Δ2-CG, Δ2-RG, and Δ2-PG.
23 A
B
Fig. 2.3 A, Δ-2-thiazolidenedione derivatives lack activity in PPARγ activation. PC-3 cells were exposed to individual test agents (10 μM) or DMSO vehicle in 10% fetal bovine serum–supplemented RPMI 1640 for 48 hours. Amounts of activated PPARγ in the resulting nuclear extracts were analyzed by PPARγ transcription factor ELISA kit.
Columns, mean; bars, SD (n = 3, *P < 0.01) (62). B, Similar result was shown in MCF-7 cells. Cells were exposed to individual test agents (10 μmol/L) or DMSO vehicle in 10% fetal bovine serum–supplemented DMEM/F12 for 48 hours.
24 O O O LiAlH O OH 4 OH NaH, MOMCl OH
HO HO MOMO
12 3
NO2 NO2 NaH H+ O O O O Ac2O Cl NO2 AcO MOMO 4 5 O
NH2 OEt O Cl NO2 H O O 2 O Pd/C AcO AcO O EtO 7 6 O O
HS NH NH O S O O S base O O O H2N O HO AcO
9, TG 8
Scheme 2.1 Synthesis of troglitazone (TG) (64).
25 O O O 1). Triflate anhydride, Py OTf O 1). LAH OH OH O O 2). NaH, O 2). K2CO3, O HO H Br O 1 10 F 11 O O O NH NH Pd/C O S S O NH O O O S O HO O O 13 (Δ2−TG) N H O H 12 2 Pd/C NH O S O O HO 9 (TG)
Scheme 2.2 Our modified synthetic strategy of TG and Δ2-TG.
26 O
NH O S O NaH LAH H O OH OH O O H N F H
O O
H2 NH NH S S O O Pd/C O O Δ2−CG CG
Scheme 2.3 Synthesis of CG and Δ2-CG.
27
CHAPTER 3
THIAZOLIDINEDIONES AND THEIR DERIVATIVES
DOWN-REGULATE CYCLIN D1
3.1 Effect of TZDs on Cyclin D1 Down-regulation Is Independent of PPARγ
Since it has been reported that some PPARγ ligands, such as TZDs, can induce
cyclin D1 degradation (56-57), and cyclin D1 overexpression is associated with breast
tumorgeinsis, these TZD compounds are potential therapeutic agents for breast cancer.
Using the ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cell lines,
we have obtained evidence demonstrating that TZD-mediated cyclin D1 down-regulation
is independent of PPARγ activation. First, endogenous PPARγ expression in
MDA-MB-231 cells was at least an-order-of-magnitude higher than that in MCF-7 cells
(Fig. 3.1A). This finding suggested that MDA-MB-231 should display a higher degree of
susceptibility to TG-mediated cyclin D1 down-regulation if this activity is
PPARγ-dependent. However, the result showed that MCF-7 cells were more sensitive to
TG treatment with respect to cyclin D1 ablation since the expression level of cyclin D1 is
totally abrogated in MCF-7 cells but is only moderately reduced in MDA-MB 231 cells at
40 μM TG treatment (Fig. 3.1 B). In addition, the effects of three different TZDs, TG, 28 CG, and RG (see Fig. 2.2), on cyclin D1 levels in MCF-7 cells were varied. TG and CG
at high doses were effective in reducing cyclin D1 levels (Fig. 3.2A). In contrast, RG
lacked appreciable effects at comparable concentrations (Fig. 3.2B) even though RG is
the most potent PPARγ activator of the three TZD compounds (40). The effect of
GW9662, a potent PPARγ antagonist (65-66), on TG-mediated cyclin D1 repression in
MCF-7 cells was also examined. Even at concentrations three orders-of-magnitude higher
than its IC50 for PPARγ binding, GW9662 had no appreciable effect on cyclin D1 expression and did not prevent TG-mediated cyclin D1 down-regulation (Fig. 3.3).
To further discern the role of PPARγ in TZD-induced cyclin D1 ablation, the
unsaturated derivatives of TG and CG, Δ2-TG and Δ2-CG, (Fig. 2.2) were compared to
their parent compounds for activity in MCF-7 cells. Since these two Δ2-series compounds are devoid of PPARγ activating effects (see chapter 2, Fig. 2.3B), they are
useful tools to separate the two activates, PPARγ activation and cyclin D1
down-regulation. Western blot analysis was performed to reveal the effects of TG, CG,
and their Δ2-counterparts on the expression of cyclin D1 and ERα in MCF-7 cells. The
results show that Δ2-TG and Δ2-CG, though devoid of PPARγ activity, were able to
reduce the expression levels of cyclin D1 and ERα in MCF-7 cells in a dose-dependent
manner with potency higher than that of TG and CG (Fig. 3.4). For example, the
minimum concentration required for the complete ablation of cyclin D1 was 30 μM for
both Δ2-TG and Δ2-CG, as compared to 40 and 50 μM for TG and CG, respectively. On
the other hand, the effect of these agents on ERα lagged behind that of cyclin D1,
29 requiring substantially higher concentrations to achieve the same extent of repression.
These four lines of evidence demonstrated that TZD-mediated cyclin D1 degradation in
MCF-7 cells is independent of PPARγ activation.
Fig. 3.5 displays the time course of cyclin D1 down-regulation by 40 μM TG and 30
μM Δ2-TG in MCF-7 cells. Both agents achieved complete ablation after 24 hours of treatment. However, semi-quantitative PCR shows that the mRNA level of cyclin D1 remained unaltered after the 24 h-exposure (Fig. 3.6), suggesting that TG- and
Δ2-TG-induced cyclin D1 ablation was mediated at the posttranscriptional level.
3.2 TG and Δ2-TG Facilitate Proteasome-Mediated Proteolysis of Cyclin D1
It has been reported that the effect of 15d-PGJ2 and CG on cyclin D1 repression was attributable to proteasome-mediated degradation (56, 67). Thus, the effects of three proteasome inhibitors (MG132, lactacystin, and epoxomicin) on TG and
Δ2-TG-facilitated cyclin D1 ablation in MCF-7 cells was examined. As shown in Fig. 3.7, all three proteasome inhibitors were effective in rescuing the TG and Δ2-TG -induced cyclin D1 repression.
As proteasome-facilitated proteolysis of cyclin D1 is preceded by ubiquitination (16),
we examined the formation of ubiquitinated cyclin D1in MCF-7 cells treated with 30 μM
TG or 20 μM Δ2-TG for 20 h. The cell lysates were exposed to cyclin D1 antibodies,
followed by protein-A beads. Equivalent amounts of the immunoprecipitated proteins
were subjected to Western blotting with either cyclin D1 or ubiquitin antibodies. As
shown in Fig. 3.8, while cyclin D1 expression was diminished in TG- and Δ2-TG-treated
30 MCF-7 cells (left panel; IP, anti-cyclin D1; IB, anti-cyclin D1), the extent of
ubiquitination of cyclin D1 increased as indicated by a complex ladder of ubiquitinated
cyclin D1 bands (right panel; IP, anti-cyclin D1; IB, antiubiquitin).
3.3 Investigation of the Mechanism of TG and Δ2-TG-mediated Cyclin D1
Degradation
The anti-proliferative activities of TG and other TZD compounds have been
demonstrated in various cancer cell lines (45-51). We thus also examined the effects of
TG and Δ2-TG on the growth of MCF-7 cells using the MTT assay (see Chapter 6,
Materials and Methods part). As shown in Fig. 3.9, Δ2-TG exhibits a more potent
anti-proliferation effect in MCF-7 cells than TG. While the IC50 for TG was 70 μM, the
IC50 for Δ2-TG was 57 μM.
Based on the observation of growth inhibiting activity of TG and Δ2-TG, we
assessed the effect of TG and Δ2-TG on cell cycle progression. The results are displayed in Fig. 3.10. The cell cycle distribution exhibited little change with any concentration of
TG or Δ2-TG compared to DMSO vehicle. Both reagents increased the proportion of cell in S phase from 20 to 40 μM at 24 hours. Since cyclin D1 serves as a key regulator in
cells in early to mid-G1 phase in the cell cycle, it was predicted that TG and Δ2-TG
should induce cell cycle arrest at G1 phase. A possible reason of this conflicting data
might be that these two compounds do not only mediate the expression of cyclin D1, but
also other proteins which regulate the cell cycle.
Consequently, we investigated whether TG or Δ2-TG also affects other cell cycle regulators in MCF-7 cells. MCF-7 cells were treated with different doses of TG and
31 Δ2-TG and the expression levels of cyclins D2, D3, A, B, and E, and cyclin-dependent kinases (CDKs) 2 and 4 were assessed (Fig. 3.11). While cyclin D2 and CDK4 showed a slight decrease in the expression levels, no appreciable effects were observed with the
other cyclins and CDKs. The results indicate that the ablative effect on cyclin D1 was
highly specific.
Evidence indicates that cyclin D1 ubiquitination could be facilitated by either a
GSK-3β-dependent or –independent pathway. In the GSK-3β-dependent pathway,
CDK-bound cyclin D1 undergoes GSK-3β-mediated phosphorylation at Thr286,
followed by translocation to the cytoplasm, where it undergoes proteasomal degradation
(26-27). Alternatively, free cyclin D1 can be ubiquitinated independently of GSK-3β,
although the exact mechanism remains elusive (28). In this study, we obtained two lines
of evidence to exclude the involvement of GSK-3β inTG- and Δ2-TG-facilitated cyclin
D1 degradation. First, the GSK-3β phosphorylation level remained unaltered in TG- and
Δ2-TG-treated MCF-7 cells (Fig. 3.12A). Second, cotreatment with the selective GSK-3β
inhibitor SB216763 could not rescue TG- or Δ2-TG-induced cyclin D1 ablation (Fig.
3.12B).
Ubiquitin-proteasomal proteolysis of cyclin D1 is also mediated by ubiquitin E3
ligase. It has been reported that the E3 ubiquitin ligase that regulates the G1/S transition
is the SCF complex which consists of four subunits, SKP1, CUL1, an F-box protein , and
ROC/Rbx/Hrt1 (68). ROC1 contains a RING finger domain that interacts with E2 and provides the ligase activity (69). SKP1 functions as an adapter to connect the CUL1
scaffold to the F-box protein (70). In many cases, F-box proteins are receptor subunits
32 that provide the substrate selectivity and interact with phosphorylated substrates. A
number of F-box proteins have been identified, and each interacts with specific substrates
(68). For example, the F-box protein required for cyclin E1 degradation is hCdc4/Fbw7
(71). Another F-box protein, SKP2, is attributed to the degradation p27 (72), p21, and cyclin A/CDK2 complex (73). Evidence has also shown that the p19SKP1/p45SKP2/CUL-1 complex is likely to function as a conserved ubiquitin E3 enzyme that regulates cyclin D1 in HaCat (human keratinocyte), RKO (human colorectal carcinoma), and HCT116
(human colon carcinoma) cells (25). To investigate whether TG and Δ2-TG-mediated cyclin D1 degradation involves influencing the expression of the p19SKP1/p45SKP2/CUL-1 complex, we used siRNA of SKP2 to ablate the expression of SKP2 in MCF-7 cells. As shown in Fig. 3.13, after transfecting SKP2 siRNA, the expression level of SKP2 was reduced. However, Western blot analysis shows that cyclin D1 level were not up-regulated in SKP2 siRNA-transfected MCF-7 cells, after 40 μM TG or 30 μM Δ2-TG treatment. In addition, drug treatment did not up-regulate the expression level of SKP2 in
MCF-7 cells (without siRNA transfection). The data suggest that SKP2 is not the target for TG and Δ2-TG in MCF-7 cells.
33
Fig. 3.1 A, Differential expression levels of PPAR γ in MDA-MB-231 and MCF-7 cells.
B, dose-dependent effect of TG on cyclin D1 repression in MDA-MB-231 and MCF-7
cells. Cells were treated with TG at the indicated concentrations in 5%
FBS-supplemented DMEM/Ham’s F-12 medium for 24h. These Western blots are representative of three independent experiments.
34
Fig. 3.2 A, Dose-dependent effect of TG and CG on cyclin D1 in MCF-7 cells. B, Lack of effect of RG on cyclin D1 in MCF-7 cells. MCF-7 cells were exposed to the individual agents at the indicated concentrations in 5% FBS-supplemented medium for 24 h, and the expression of cyclin D1 was evaluated by Western blot analysis.
35
Fig. 3.3 High doses of the PPARγ antagonist GW9662 have no effect on cyclin D1 expression (top) or troglitazone-mediated cyclin D1 ablation (bottom) in MCF-7 cells.
36
Fig. 3.4 Dose-dependent effect of TG, Δ2-TG, CG, and Δ2-CG on cyclin D1 and ERα
expression in MCF-7 cells. MCF-7 cells were exposed to the individual agents at the indicated concentrations in 5% FBS-supplemented medium for 24 h, and the expression
of cyclin D1 and ERα was analyzed by Western blot analysis (top and bottle). Signals
were quantitated by densitometry and normalized against β-actin measurements for
effects of TG and Δ2-TG (middle). Each data point represents mean ± S.D. (n = 3).
37
Fig. 3.5 Time-dependent effect of 40 μM TG and 30 μM Δ2-TG on cyclin D1 expression in MCF-7 cells.
38
Fig. 3.6 RT-PCR analysis of cyclin D1 mRNA in MCF-7 cells after exposure to 40 μM
TG or 30 μM Δ2-TG for 24 h. Signals were quantitated by densitometry and normalized against β-actin measurements (bottom). Each data point represents mean ± S.D.(n = 3).
39
Fig. 3.7 Dose-dependent effects of the proteasome inhibitors MG132, lactacystin, and epoxomicin on TG- and Δ2-TG-mediated cyclin D1 ablation. MCF-7 cells were exposed to 40 μM TG or 30 μM Δ2-TG in the presence of various concentrations of the proteasome inhibitor in 5% FBS-supplemented medium for 24 h, and the expression of cyclin D1 was analyzed by Western blot analysis.
40
Fig. 3.8 Cyclin D1 ubiquitination in TG- and Δ2-TG-treated MCF-7 cells. Cell were
treated with DMSO vehicle, 30 μM TG, or 20 μM Δ2-TG in 5% FBS-supplemented
DMEM/Ham’s F-12 medium for 20 h. Cell lysates were immunoprecipitated (IP) with anti-cyclin D1, and the immunoprecipitates were analyzed by Western blotting (WB) with anti-cyclin D1 or anti-ubiquitin.
41
Fig. 3.9 Dose-dependent effects of TG and Δ2-TG on viability of MCF-7 cells.
MCF-7cells were exposed to ΤG or Δ2-TG at the indicated concentrations in 5%
FBS-supplemented DMEM/Ham’s F-12 medium in 96-well plates for 24 h, and cell viability was assessed by MTT assay. Each data point represents the mean of six replicates.
42
Fig. 3.10 Cell cycle analysis following treatment with DMSO vehicle, TG or Δ2-TG in
MCF-7 cells. MCF-7cells were exposed to DMSO, TG or Δ2-TG at the indicated concentrations in 5% FBS-supplemented DMEM/Ham’s F-12 medium for 24 h.
43
Fig. 3.11 Dose-dependent effects of TG and Δ2-TG on the expression of cyclins and
CDKs. MCF-7 cells were exposed to the individual agents at the indicated concentrations in 5% FBS-supplemented DMEM/Ham’s F12 medium for 24 h, and the expression of various cell cycle-regulating proteins was analyzed by Western blot analysis.
44
Fig. 3.12 Evidence that TG and Δ2-TG-induced cyclin D1 downregulation is independent
of GSK-3 β activation. A, the phosphorylation levels of GSK-3β remained unaltered in
MCF-7 cells treated with different doses of TG and Δ2-TG. B, the GSK-3 β inhibitor
SB216763 could not rescue TG- and Δ2-TG-induced cyclin D1 ablation. MCF-7 cells were exposed to the individual or combined agents at the indicated concentrations in 5%
FBS-supplemented DMEM/Ham’s F12 medium for 24 h.
45
Fig. 3.13 Western blot analysis of cyclin D1 and SKP2 in MCF-7 cells with or without
SKP2 siRNA transfection. MCF-7 cells were exposed to the individual agents at the indicated concentrations in 5% FBS-supplemented DMEM/Ham’s F12 medium for 24 h, and the expression of various cell cycle-regulating proteins was analyzed by Western blot analysis.
46
CHAPTER 4
DEVELOPMENT OF NOVEL Δ2-TG-DERIVED CYCLIN
D1-ABLATIVE AGENTS
4.1 Modification of Δ2-TG
We have demonstrated that the TG analogue Δ2-TG possesses the ability to down-regulate cyclin D1 without activating PPARγ. This finding prompted the notion that Δ2-TG could be used as a scaffold to develop novel cyclin D1 ablative agents. A series of Δ2-TG derivatives was synthesized based on modifications to three parts of the molecule. As shown in Fig. 4.1, the first modifications were at the hydroxyl group on the chromanol ring. Because the synthesis of TG requires the protection of the hydroxyl group, modification at this site was achieved by omitting the deprotection step and then determining if this protecting group affected activity. The second modifications were the substitutions at either or both of the hydrogen atoms on the benzene ring. These hydrogens were replaced by electronic withdrawing or donating groups of various sizes.
The final part of the molecule to be modified was the TZD ring. Alteration of the TZD ring helps to determine whether this structure is critical for the cyclin D1-ablative activity.
47 Scheme 4.1 is the general synthetic strategy for a series of Δ2-TG derivatives. After
reducing the starting material, tetramethylchroman-2-carboxylic acid, to the
corresponding alcohol compound, different protecting reagents, such as allyl bromide,
were selected to protect the hydroxyl group on the chromanol ring. Since the mild base,
K2CO3, was used, the deprotonation occurred only at the hydroxyl group on the phenyl
position. Another hydroxyl group close to the pyran ring was modified to a good leaving
group (OTf) to facilitate the connection with different aldehydes. The TZD ring or other
selected reagents, such as malononitril [CH2(CN)2], was finally constructed through the
condensation reaction with the aldehyde.
4.2 Structure-activity Relationship (SAR) Study of Δ2-TG Analogues
The first protecting group used for the modification of the phenol group was
dimethyl allyl. A series of compounds with this protecting group was synthesized with
different substitutions of the hydrogen atoms on the benzene ring (Table 4.1). The first
analogue of this series to be evaluated, TG-14, had the retained protecting group as its
only modification (entry 1). Compared to Δ2-TG, TG-14 exhibited much more potent
anti-proliferative (IC50: 14.5 μM for TG-14 vs. 57 μM for Δ2-TG) and cyclin D1-ablative
(IC50: 7 μM for TG-14 vs. 20 μM for Δ2-TG) activities in MCF-7 cells. This result showed that the protecting group on the phenol group of the Δ2-TG scaffold is critical for
the anti-proliferative and cyclin D1-ablative activities of the compounds. Therefore, all
subsequent derivatives retained the protecting groups. The substitutions at the X1 and X2 positions on the benzene ring also showed effects on cyclin D1-ablative activities. For example, when the X1 hydrogen was replaced with an electron-donating group, OMe,
48 cyclin D1-ablative activity was slightly enhanced (IC50: 5.6 μM for TG-15 vs. 7 μM for
TG-14) (entries 1 and 2). In contrast, if the X1 position was substituted with more bulky
groups, such as in TG-31, TG-32, and TG-33, the compounds lost their cyclin
D1-ablative activities (entries 5-7). Among this series of compounds, TG-16, TG-34, and
TG-35 (entries 2, 8, 9 respectively) exhibited enhanced cyclin D1-ablative activities.
However, there is no apparent strong correlation between the anti-proliferative (based on
IC50 values from MTT data) and cyclin D1-ablative activities of these compounds.
The second protecting group used to modify the phenol was an allyl group (Table
4.2). Based on the results of the dimethyl allyl series (Table 4.1), bulky groups were avoided in the modification of this series. Compared to TG-14 (entry 1), TG-6 is the better cyclin D1-ablative agent (entry 13). In addition, TG-28, which contains OMe at X1,
(entry 15, with allyl protection) is a more potent inducer of cyclin D1 degradation than its counterpart, TG-16 (entry 2, with dimethy allyl protection). TG-28 represents the best cyclin D1-ablative agent among these 19 Δ2-TG analogues (entries 1-19).
Another series of compounds that assisted in demonstrating the difference between the dimethyl allyl and allyl protecting groups is shown in Table 4.3. In this series, the benzene ring on the Δ2-TG scaffold was replaced with naphthalene. Comparing the counterparts TG-43 and TG-53 (entries 20 and 22), TG-53, which is protected by an allyl group, possesses the better activity in down-regulating cyclin D1 in MCF-7 cells.
Moreover, the modification of one oxygen on the TZD ring to sulfur (W = S, entry 21,
TG-46) did not improve the cyclin D1-ablative activity. Thus, the unaltered TZD ring was selected in most analogues.
49 Three other types of protecting groups were also investigated for their effects on
cyclin D1-ablative activity (Table 4.4, entries 23 or 24, 25, 26). Among these protecting
groups, only the phenyl (TG-12, entry 25) permitted retention of the ability to
down-regulate cyclin D1, while the other compounds containing cinnamyl (entries 23 and
24) and succinate (entry 26) protecting groups exhibited decreased activities.
The last modifications evaluated were to the TZD ring. These acid or ester groups
(Table 4.5, entries 28-32) can act as hydrogen bond donors or acceptors. Nevertheless,
most of the functional groups selected to modify the TZD ring did not improve the cyclin
D1-ablative activity. This finding suggests that the TZD ring may play a critical role in
the induction of cyclin D1 degradation by these compounds.
Among these 32 Δ2-TG derivatives, TG-28 represents the optimal cyclin D1 ablative agent. To examine whether the chiral center in the compound affects its activity
on cyclin D1 degradation, S- and R- forms of TG-28 were synthesized. As shown in
Table 4.6, the S- form of TG28 (STG-28) exhibited more potent activity to induce cyclin
D1-ablation (IC50: 1.8 μM for STG-28 vs. 7.5 μM for RTG-28). The three-dimensional
structures of the two compounds show that STG-28 is a more extended molecule which
may be the basis of the observed difference in the cyclin D1-targeted activities of these
two isomers.
4.3 Bioactivities of STG-28
Since STG-28 represents a structurally optimized agent with potency an
order-of-magnitude higher than that of Δ2-TG to induce cyclin D1-degradation, it is
essential to examine whether the regulation of cyclin D1 expression by this derivative
and its parent compounds (TG and Δ2-TG) occurs via the same mechanism. First, effects 50 of the compounds on other cell cycle regulators were examined (Fig. 4.2). Like the data for TG and Δ2-TG which is shown in Fig. 3.11, STG-28 did not exhibit obvious effects on the expression levels of cyclins D2, D3, A, B, and E, and CDKs 2 , 4, and 6.
We also determined whether STG-28-mediated cyclin D1 degradation is regulated
by ubiquitin-proteasomal proteolysis as well. As was the case for TG and Δ2-TG, the
proteasome inhibitor, MG132, rescued cyclin D1 from STG-28-induced repression (Fig.
4.3). Reverse transcriptase (RT)- PCR data showed that the mRNA level of cyclin D1
remained unaltered after 24 h-exposure (Fig. 4.4), suggesting that, like TG- and Δ2-TG,
STG-28-induced cyclin D1 ablation was mediated at the posttranscriptional level. Finally,
we examined the effect of STG-28 on GSK-3β. The results showed that
STG-28-facilitated cyclin D1 degradation is not GSK-3β dependent. Like TG and Δ2-TG,
STG-28 treatment did not alter GSK-3β phosphorylation level in MCF-7 cells (Fig. 4.5A),
and cotreatment with the selective GSK-3β inhibitor SB216763 could not rescue
STG-28-induced cyclin D1 ablation (Fig. 4.5B). Collectively, these results suggest that
STG-28-mediated cyclin D1 degradation involves a mechanism similar to that of its
parent compounds, TG and Δ2-TG.
51
Fig. 4.1 Three parts of Δ2-TG were modified. Part A, the phenol group was modified by different protecting groups. Part B, one or both hydrogen atoms on the benzene ring were replaced by different substituents. Part C, the TZD ring was substituted by other functional groups.
52 O
O O OH 1. LAH, THF OTf Y HO 2. K2CO3, protecting agents, Y O 3. Triflate anhydride, Py
O
X1 X O H 1
H O HO O
X2 Y O X2
K2CO3
X1 Z O 2,4 - thiazolidinedione O or other funciton group, Z Y piperidine O X2
Scheme 4.1 General synthetic procedure for Δ2-TG analogues.
53 O X1 NH O S O O X O 2
Compounds (μM) IC for IC for 50 50 ______Entry compound MTT WB X1 X2
(μM) (μM) 0 2.5 5 7.5 1 TG-14 H H 14.5 7 2 TG-16 OMe H 15 5.6 3 TG-17 Me H 14.5 7 4 TG-30 F H 12.5 7.2
CF 5 TG-31 3 H >50 >7.5
F 6 TG-32 H 19.5 >7.5
7 TG-33 H >50 >7.5 8 TG-34 Br Br 15.5 5.5 9 TG-35 NO2 H 38 5.8 10 TG-44 Br OMe 14.5 7 11 TG-45 OEt H 13 6.7 12 TG-88 Br H 14.5 7.5
Table 4.1 A series of compounds with dimethyl allyl protecting groups at the phenol position. IC50 for MTT represents 50% anti-proliferative activities in MCF-7 cells. The
MTT assay is described in Chapter 6. IC50 for WB represents the concentration of compound needed to induce 50% reduction in cyclin D1 levels as determined by Western
Blot analysis.
54 O X1 NH O S O O X O 2
IC50 for IC50 for Compounds (μM) Entry compound X1 X2 MTT WB ______(μM) (μM) 0 2.5 5 7.5 13 TG-6 H H 9 3 14 TG-27 Br H 28 >7.5 15 TG-28 OMe H 14.5 2.3 16 TG-29 Me H 23.5 3.6 17 TG-52 Me Me 10.5 7.5 18 TG-54 Br OMe 17.5 3.8 19 TG-55 OEt H 17 7.2
Table 4.2 A series of compounds with allyl protecting groups at the phenol position. See explanations for “IC50 for MTT” and “IC50 for WB” in the legend for Table 4.1.
55 O
NH O S O W Y O
IC50 for IC50 for Compounds (μM) Entry compound W Y MTT WB ______(μM) (μM) 0 2.5 5 7.5
20 TG-43 O 14.5 7.2
21 TG-46 S 37.3 7.5
22 TG-53 O 14.5 3.4
Table 4.3 Three compounds with allyl or dimethyl allyl protecting groups at the phenol position and replacement of the benzene group with naphthalene. See explanations for
“IC50 for MTT” and “IC50 for WB” in legend for Table 4.1.
56 O X1 NH O S O O Y O
IC50 for IC50 for Compounds (μM) Entry compound X1 Y MTT WB ______(μM) (μM) 0 2.5 5 7.5 23 TG-10 H 19 >7.5 24 TG-11 Br 28.5 >7.5 H 25 TG-12 16.7 4.5
26 TG-13 H >50 >7.5
Table 4.4 A series of compounds with different protecting groups at the phenol position.
See explanations for “IC50 for MTT” and “IC50 for WB” in legend for Table 4.1.
57 Br Z
O O
O
IC for 50 IC for Compounds (μM) Entry compound Z MTT 50 ______WB (μM) (μM) 0 2.5 5 7.5
27 TG-36 >50 >7.5
28 TG-37 34 >7.5
29 TG-38 >50 4.5
30 TG-39 46 >7.5
31 TG-41 >50 >7.5
32 TG-42 >50 >7.5
Table 4.5 Compounds with different functional groups at the TZD position. See explanations for “IC50 for MTT” and “IC50 for WB” in legend for Table 4.1.
58
IC50 for IC50 for Compounds (μM) Entry compound MTT WB ______(μM) (μM) 0 1 2.5 5 7.5 10 20 30 40
33 STG-28 15 1.8
34 RTG-28 17 7.5
O O MeO MeO NH NH O S O S O O O O O O
STG-28 RTG-28
Table 4.6 Anti-proliferative and cyclin D1-ablative activities of the S- or R-forms of
TG-28. The 3D-structures of both isomers are shown at the bottom. See explanations for
“IC50 for MTT” and “IC50 for WB” in legend for Table 4.1.
59
Fig. 4.2 Dose-dependent effects of STG-28 on the expression of cyclins and CDKs.
MCF-7 cells were exposed to the individual agents at the indicated concentrations in 5%
FBS-supplemented DMEM/Ham’s F12 medium for 24 h, and the expression of various cell cycle-regulating proteins was analyzed by Western blot analysis.
60
Fig. 4.3 Dose-dependent effects of the proteasome inhibitor MG132 on STG-28-mediated cyclin D1 ablation. MCF-7 cells were exposed to 10 μM STG-28 in the presence of various concentrations of the proteasome inhibitor in 5% FBS-supplemented
DMEM/Ham’s F12 medium for 24 h, and the expression of cyclin D1 was analyzed by
Western blot analysis.
61
Fig. 4.4 RT-PCR analysis of the mRNA levels of cyclin D1 in MCF-7 cells. MCF-7 cells were exposured to 5, 10, or 20 μM STG-28 in 5% FBS-supplemented DMEM/Ham’s F12 medium for 24 h.
62
Fig. 4.5 Evidence that STG-28-induced cyclin D1 downregulation is independent of
GSK-3 β activation. A, the phosphorylation levels of GSK-3β remained unaltered in
MCF-7 cells treated with different doses of STG-28. B, the GSK-3 β inhibitor SB216763 could not rescue STG-28-induced cyclin D1 ablation. MCF-7 cells were exposed to
STG-28 or combined with SB216763 in the presence of various concentrations in 5%
FBS-supplemented DMEM/Ham’s F12 medium for 24 h.
63
CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTION
5.1 Down-regulation of Cyclin D1 by Troglitazone and Its Analogues Is
PPARγ-Independent
Numerous studies have reported that the PPARγ agonists, TG and CG, are good
candidates for chemotherapy due to their anti-proliferative activities in cancer cells and, consequently, they have been used in clinical trails. However, whether their anti-tumor
activities are related to their effects on PPARγ is still under investigation. In our study,
we obtained several lines of evidence that the mechanism underlying TG- and
CG-mediated cyclin D1 down-regulation is PPARγ-independent. First, this cyclin
D1-ablative effect was not observed with the more potent PPARγ agonist, RG, at
comparable concentrations and could not be rescued by the PPARγ antagonist GW9662.
In addition, in spite of significantly higher PPARγ expression, MDA-MB-231 cells were
less susceptible to TG-mediated cyclin D1 ablation. Finally, the analogues of TG and CG,
Δ2-TG and Δ2-CG, although devoid of PPARγ activity, were able to induce cyclin D1
ablation with slightly higher potency than their parent compounds.
64 Furthermore, TG and Δ2-TG exhibit the same mechanism in down-regulating cyclin
D1 in MCF-7 cells. Our data indicate that both agents facilitated proteasomal proteolysis via a GSK-3β-independent mechanism. The RT-PCR data also showed that both TG and
Δ2-TG-induced cyclin D1 ablation was mediated at the posttranscriptional level.
Moreover, the effect of TG and Δ2-TG on cyclin D1 is highly specific since there are no considerable effects on many other cell cycle regulators. Two lines of evidence suggest that ERα might play a role in the TZD-promoted degradation of cyclin D1. First, the
ERα-negative MDA-MB-231 cells were more resistant than the ERα-positive MCF-7 cells to the cyclin D1-ablative effect of TG (Fig. 3.1). Second, cyclin D1 ablation was accompanied by a decrease in ERα expression in MCF-7 cells (Fig. 3.4). This
TZD-mediated down-regulation of cyclin D1 and ERα is reminiscent of action of the histone deacetylase inhibitor, trichostatin A (74). Trichostatin A has been shown to repress cyclin D1 and ERα expression, in part through the up-regulation of Skp2/p45, a regulatory component of the Skp1/Cullin/F-box complex implicated in the ubiquitination of cyclin D1. We have also examined whether TG and Δ2-TG have effects on Skp2 by using SKP2 siRNA-transfected MCF-7 cells. The findings revealed that TG and Δ2-TG did not exhibit an obvious effect on the expression level of SKP2 protein (Fig. 3.13).
5.2 Development of Cyclin D1 Ablative Agents
The separation of cyclin D1 ablation from the PPARγ activity of TG and its derivatives provides a rationale to use the structure of Δ2-TG as a platform to carry out lead optimization. A series of Δ2-TG analogues has been synthesized. Based on the structure-activity relationship study of this small library, it was concluded that the
65 protection group of the phenol position is critical for activity. TG-6, which contains an
allyl protecting group, exhibited potency that was an order-of-magnitude higher than that
of TG and Δ2-TG in facilitating cyclin D1 repression and inhibiting MCF-7 cell proliferation. In addition, it was determined that the size of the substitutes on the benzene ring (X1 and X2, Scheme 4.1) should be moderate to achieve optimal activity as bulky groups reduced the induction of cyclin D1 degradation. Among the compounds
synthesized, the best substitute on position X1 is methoxy group (OMe). Moreover, the
TZD ring might be essential for cyclin D1 ablation since the six other functional groups
tested decreased this activity of the compounds. Among the 32 derivatives in the library,
TG-28, which has an allyl protecting group and OMe substituted at X1, represents the most potent cyclin D1-ablative agent. In addition, the chiral center of TG-28 also affects the activity, as STG-28, the more extended isomer, exhibits better cyclin D1 down-regulating activity than RTG-28.
We further examined whether this new generation cyclin D1 ablative agent, STG-28,
facilitates cyclin D1 repression via a mechanism similar to that of its parent compounds,
TG and Δ2-TG. The results showed that like its parent compounds, STG-28 specifically
ablates cyclin D1 without effects on many other cell cycle regulators, facilitates
proteasomal proteolysis via a GSK-3β-independent pathway, and regulates cyclin D1 at
the posttranscriptional level.
5.3 Future Directions
Abundant evidence has solidly established cyclin D1 as an oncogene with an
essential pathogenetic role in breast cancer. However, the precise cellular mechanism
through which aberrant cyclin D1 expression drives human neoplasia is less well 66 understood. Antisense oligonucleotides, siRNA, or some small molecules which block
the expression of cyclin D1 specifically and directly have been applied in both in vitro
and in vivo studies (75). Our cyclin D1 ablative agents, therefore, also provide another
approach to elucidate the mystery.
The clinical relevance of the small-molecule cyclin D1 ablative agents in breast cancer therapy/prevention is multifold. First, cyclin D1 ablation provides specific protection against breast carcinogenesis. Because cyclin D1-deficient mice are resistant to breast cancers induced by the NEU and Ras oncogenes (33), it has been speculated that cyclin D1 ablative agents could be used to effectively treat human breast carcinomas that overexpress NEU or Ras. Second, it has been suggested that the overexpression of cyclin
D1 plays an essential role in, and is predictive of, the resistance to antiestrogens agents,
such as ICI 182780 and tamoxifen (17, 18). Thus, cyclin D1 ablation may be helpful in
overcoming antiestrogen resistance. Furthermore, the synergistic interaction between
flavopiridol and trastuzumab in inhibiting breast cancer cell proliferation was attributable,
in part, to the reduction of cyclin D1 expression (76). Thus, our novel cyclin D1 ablative
agents may be able to sensitize cells to the antiproliferative action of either CDK inhibition or Her-2/Akt inhibition. In addition, it has been demonstrated that retinoids
which repress cyclin D1 in different cancer cells could be effective chemopreventive agents (77-78). Since cyclin D1 overexpression is an apposite molecular marker that expressed early in the evolution of breast cancer, the cyclin D1 ablative agents should be good candidates as a chemopreventive agents.
Although these TG-derived cyclin D1 ablative agents, have potential as
chemotherapeutic and/or chemopreventive agents, there are some concerns about these 67 compounds of which the primary issue is toxicity. TG was found to cause significant
hepatic toxicity and was withdrawn from the market in the year 2000. One compound in
our library, TG-88, has been examined for its ability to inhibit the growth of prostate
cancer xenografts and tolerance in nude mice in which it showed efficacy without
obvious toxicity (62). Despite the promising outcome, STG-28, the best cyclin D1
ablative agent in our series of TG derivatives, needs further in vivo study in breast cancer.
Another issue to be addressed is the elucidation of the precise mechanism of the
STG-28-mediated cyclin D1 degradation. Due to the complexity signal transduction
networks involving cyclin D1, the effects of STG-28 on specific proteins or genes which
regulate the ablation of cyclin D1 are still under investigation. The further study of the
mechanism is necessary since identification of a clear target is essential for drug development. Molecular modeling and combinatorial chemistry will be helpful tools for this drug discovery endeavor.
68
CHAPTER 6
EXPERIMETNATL METHODS AND MATERIAL FOR PART 1
6.1 Reagents
Troglitazone, ciglitazone, MG132 (proteasome inhibitor), lactacystin (proteasome inhibitor), GW9662 (Irreversible PPAR-γ antagonist), SB216763 (GSK-3β inhibitor),
and tryspin-EDTA solution (10×, T4174) were purchased from Sigma-Aldrich (St. Louis,
MO). Rosiglitazone and pioglitazone were prepared from the respective commercial
tablets by solvent extraction, followed by recrystallization or chromatographic
purification. Epoxomicin was a kind gift from Dr. Kyung Bo Kim (University of
Kentucky, Lexington, KY). Rabbit antibodies against p-glycogen synthase
kinase-3β (p-GSK3β) and mouse anti-cyclin D1 and anti-ubiquitin were purchased from
Cell Signaling Technology Inc. (Beverly, MA). Rabbit antibodies against ERα (sc-544),
CDK2, CDK4, cyclin A, cyclin B, cyclin D2, cyclin D3, cyclin E, p21, p27, SKP2 and
SKP2 siRNA, siRNA transfection regents (sc-29528), and siRNA transfection medium
(sc-36868) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse
69 monoclonal anti-actin was obtained from MP Biomedicals (Irvine, CA). MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] for cell viability
assay were purchased form TCI America, Inc. (Portland, OR) 6.2 Cell Culture
ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells were
obtained from the American Type Culture Collection (Manassas, VA) and were
maintained in DMEM/Ham’s F-12 medium supplemented with 10% fetal bovine serum
(FBS, Gibco) at 37 °C in a humidified incubator containing 5% CO2.
6.3 Cell Viability Analysis
The effect of individual test agents on cell viability was assessed by using the MTT
assay in six replicates. Cells were seeded and incubated in 96-well, flat-bottomed plates
in DMEM/Ham’s F-12 medium with 10% FBS for 24 h and were exposed to various
concentrations of test agents dissolved in DMSO (final DMSO concentration, 0.1%) in
5% FBS-supplemented DMEM/Ham’s F-12 medium. Controls received DMSO vehicle
at a concentration equal to that of drug-treated cells. The medium was removed and
replaced by 200 μL of 0.5 mM MTT in 10% FBS-containing RPMI 1640 medium, and
cells were incubated in the 5% CO2 incubator at 37 °C for 2 h. Supernatants were
removed from the wells, and the reduced MTT dye was solubilized in 200 μL /well
DMSO. Absorbance at 570 nm was determined on a plate reader.
6.4 Analysis of PPARγ Activation
The analysis was carried out by using a PPARγ transcription factor ELISA kit
(Active Motif, Carlsbad, CA), in which an oligonucleotide containing the peroxisome
70 proliferator response element was immobilized onto a 96-well plate. PPARs contained in
nuclear extracts bind specifically to this oligonucleotide and are detected through an
antibody directed against PPARγ. In brief, MCF-7 cells were cultured in DMEM/Ham’s
F-12 medium supplemented with 10% FBS and treated with DMSO vehicle or individual
test agents, 10 μM each, for 48 h. Cells were collected, and nuclear extracts were
prepared with a Nuclear Extract kit (Active Motif Inc., Carlsbad, CA). Nuclear extracts of the same protein concentration from individual treatments were subject to the PPARγ transcription factor ELISA according to the manufacturer’s instruction.
6.5 Western Blot Analysis
MCF-7 or MDA-MB-231 cells were seeded in 10% FBS-containing DMEM/Ham’s
F-12 medium for 24 h and treated with various agents as indicated. After individual
treatments for 24 h, both the incubation medium and adherent cells in T-25 or T-75 flasks
were scraped and collected by centrifugation at 2200 rpm for 10 min. The supernatants
were recovered, placed on ice, and triturated with 20 to 50 μL of a chilled lysis buffer
(M-PER Mammalian Protein Extraction Reagent; Pierce, Rockford, IL), to which was
added 1% protease inhibitor cocktail (set III; EMD Biosciences, Inc., San Diego, CA).
After a 30-min incubation on ice, the mixture was centrifuged at 16,100g for 3 min. Two
μL of the suspension was taken for protein analysis using the Bradford assay kit
(Bio-Rad, Hercules, CA); to the remaining solution was added the same volume of 2×
SDS-polyacrylamide gel electrophoresis sample loading buffer (100 mM Tris-HCl, pH
6.8, 4% SDS, 5% β-mercaptoethanol, 20% glycerol, and 0.1% bromphenol blue). The
mixture was boiled for 10 min. Equal amounts of proteins were loaded onto 10%
71 SDS-polyacrylamide gels. After electrophoresis, protein bands were transferred to
nitrocellulose membranes in a semidry transfer cell. The transblotted membrane was
blocked with Tris-buffered saline/0.1% Tween 20 (TBST) containing 5% nonfat milk for
90 min, and the membrane was incubated with the appropriate primary antibody in
TBST/5% nonfat milk at 4 °C overnight. After washing three times with TBST for a total
of 45 min, the transblotted membrane was incubated with goat anti-rabbit or anti-mouse
IgG-horseradish peroxidase conjugates (diluted 1:1000) for 1 h at room temperature and
washed four times with TBST for a total of 1 h. The immunoblots were visualized by enhanced chemiluminescence.
6.6 Coimmunoprecipitation/Western Blot
MCF-7 cells were cultured in 10% FBS-containing DMEM/Ham’s F-12 medium in
T-75 flask for 24 h. Cells were treated with DMSO vehicle, 30 μM troglitazone, or 20
μM M Δ2-TG in 5% FBS-containing DMEM/Ham’s F-12 medium for another 20 h.
Cells were rinsed with phosphate buffered saline at room temperature, scraped off the flask, transferred into centrifuge tubes, and centrifuged at 2200 rpm for 10 min to pellet the cells. The pellet was resuspended in ice-cold 0.5 mL of radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate,
150 mM NaCl, 1 mM EDTA, and 1% protease inhibitor cocktail) and gently mixed on an orbital shaker at 4 °C for 15 min, followed by centrifugation at 14,000g for 15 min to yield cell lysates. These cell lysates were treated with 100 μL of protein A-agarose bead slurry followed by brief centrifugation to remove nonspecific binding proteins. Equal amounts of proteins from these lysates, as determined by the Bradford assay, were mixed with anti-cyclin D1 in an orbital shaker at 23 °C for 2 h, followed by 100 μL of protein 72 A-agarose bead slurry at 4 °C for 12 h. The immunocomplex was collected by brief
centrifugation, washed four times with 800 μL of ice-cold radioimmunoprecipitation assay buffer, and suspended in 50 μL of 2× SDS sample loading buffer. The suspension was boiled for 10 min, cooled, and briefly centrifuged to remove the beads. Western blot
analysis was performed with anti-cyclin D1 or anti-ubiquitin as described above.
6.7 Reverse Transcriptase (RT)-PCR Analysis of mRNA Transcripts of Cyclin D1
Gene
MCF-7 cells were subject to total RNA isolation by using RNeasy mini kit (Qiagen,
Valencia, CA). RNA concentrations and quality were assessed spectrophotometrically by
measuring absorption at 260 nm. Aliquots of 20 μg of total RNA from each sample were reverse transcribed to cDNA using Omniscript RT Kit (Qiagen) according to
manufacturer’s instructions. The primers used were as follows: cyclin D1, forward,
5’-ATGGAACACCAGCTCCTGTGCTGC-3’, reverse, 5’-TCAGATGTCCACGTCCC
-GCACGT-3’; β-actin, forward, 5’-TCTACAATGAGCTGCGTGTG-3’, reverse,
5’-GGTCAGGATCTTCATGAGGT-3’. The reaction conditions were as follows: for
cyclin D1, (1) initial denaturation at 95 °C for 5 min; (2) 34 cycles of amplification (95
°C for 1 min, 65 °C for 1 min 45 s, and 72 °C for 1 min); and (3) a final extension step of
10 min at 72 °C; for β-actin, (1) initial denaturation at 95 °C for 3 min; (2) 40 cycles of amplification (95 °C for 30 s, 58 °C for 20 s, and 72 °C for 45 s); and (3) a final
extension step of 10 min at 72 °C. The PCR reaction products were separated electrophoretically in a 1.2% agarose gel and stained with ethidium bromide.
6.8 Cell Cycle Analysis
73 MCF-7 cells were seeded in T-75 flask with 10% FBS-containing DMEM/Ham’s
F-12 medium for 24 h and treated with various agents as indicated. 106 cells of each
sample were collected and resuspended in 500 μL of solution A (30 μM trypsin, 10 μM
EDTA in stock solution) and incubated in 37 °C for 30 mins, then followed by another 30 min incubation in 37 °C after adding 500 μL of solution B (500 μM trypsin inhibitor, 100
μM RNase A in stock solution). Then, 500 μL of solution C (50 μM propidium iodide,
1.16 μM spermine．4HCl in stock solution) was added and the cell samples were incubated on ice in the dark for 1h. The cell cycle analysis was performed using the BD
FACS Calibur system (Franklin Lakes, NJ). (Stock solution: 1 g trisodium citrate dehydrate, 1 mL NP-40, 522 mg spermine．4HCl, 60.5 mg Tris- hydroxymethyl-aminoethane in 1 L of H2O).
6.9 siRNA Transfection Procedure
MCF-7 cells were prepared one day before transfection. Cells were cultured in
DMEM/F12 medium containing 10% FBS, without antibiotic. After washing cells with sterile 1X PBS, trypsin was applied to detach the cells. Once cells begin to detach, dilute the suspension with a 3-fold volume of culture medium. Centrifuge at 1,500 rpm for 5 minutes. Remove medium and resuspend pellet in the appropriate medium without antibiotics. Transfer cells to 6 well tissue culture plates and overnight growth results in
40-50% confluency. Incubate cells at 37 °C in a 5% CO2 incubator.
The amount of the reagents were followed the procedure which provided by Santa
Cruz Biotechnology, Inc. (A) Mix appropriate amount of 10 μM SKP2 siRNA and siRNA Transfection Medium (sc-36868) gently and keep at room temperature for 5
74 minutes. (B) Mix appropriate amount of siRNA Transfection Reagent (sc-29528) and
siRNA Transfection Medium gently and keep at room temperature for 5 minutes. After 5
minutes, combine A and B to form siRNA-siRNA Transfection Reagent Complex. Mix
gently and then incubate at room temperature for 20 minutes. After incubation period,
add appropriate amount of siRNA Transfection Medium to the tube containing the
siRNA-siRNA Transfection Reagent Complex and mix gently. Just prior to transfection,
aspirate medium and wash cells once with 1 mL of fresh DMEM/F12 medium containing
10% FBS without antibiotics and remove. Overlay the diluted siRNA-siRNA
Transfection Reagent Complex onto the washed cells for each transfection and incubate
cells at 37 °C for 24 hours. Another 6 well plate with MCF-7 cells were cultured in the
same DMEM/F12 medium and incubate cells at 37 °C for 24 hours as blank. After
incubation, the media was removed and add 1 mL of normal growth medium containing
DMSO, 40 μM TG, or 30 μM Δ2-TG in 5% FBS culture medium to each well containing
transfected cells or blank. After another 24 hours incubation at 37 °C with 5% CO2, the cells was detached by tryspin and the further Western blot analysis was performed with anti-cyclin D1 or anti-SKP2 as described above.
6.10 Determination of IC50 Values
Data from cell viability and fluorescence polarization assays were analyzed by using
the CalcuSyn software (Biosoft, Ferguson, MO) to determine IC50 values, in which the calculation was based on the medium-effect equation [i.e., log(fa/fu) = mlog(D) - mlog(Dm), where fa and fu denote fraction affected and unaffected, respectively; m represents the Hill-type coefficient signifying the sigmoidicity of the dose-effect curve; and D and Dm are the dose used and IC50, respectively (62). 75 6.11 General Information on Chemical Methods
Reactions involving moisture-sensitive reagents were carried out under an inert
atmosphere of dry argon. Anhydrous solvents were dried by standard procedures:
Tetrahydrofuran (THF) and ether was distilled from sodium metal in the presence of
benzophenone under argon. Dichloromethane (CH2Cl2) was distilled from calcium hydride under argon. Silica gel column chromatography was performed using silica gel
60A (Merck, 230-400 Mesh). High-resolution electrospray ionization mass spectra were obtained on the Micromass QTOF Electrospray mass spectrometer at The Ohio State
Chemical Instrumentation Center. All the NMR spectra were recorded on a Bruker AC
250, Bruker DPX 250, or Bruker DRX 400 model spectrometer in CDCl3 solution. The
abbreviations s, d, t, q, m, dd, dt, and br are used for singlet, doublet, triplet, quartet,
doublet of doublets, doublet of triplets, and broad, respectively. Chemical shifts for 1H
NMR spectra are reported in ppm relative to residual solvent protons.
6.12 Synthetic Procedures of Δ2-TG, Δ2-TG Analogues, and Δ2-CG
(a) General synthetic procedures of Δ2-TG and its analogues (scheme 2.2 and
Schemem 4.1)
2-hydroxymethyl-2, 5, 7, 8-tetramethyl-chroman-6-ol. To a stirring solution of
o 0.78 g LiAlH4 (20 mmol) in 10 mL of THF at 0 C was added 5 g (20 mmol) of
6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (1) in 250 mL of THF
dropwise over a period of 1 hour. The solution was stirred at room temperature under
nitrogen for another 6 hours. After 6 hours, 1 mL of H2O, 1 mL of 1 N NaOH, and 2 mL
of H2O mixture was slowly added to the solution to quench the reaction. The solution was
76 stirred at room temperature for 1 hour, filtered out of solid, and concentrated. Purification
by flash silica gel chromatography (ethyl acetate/hexanes = 1/2) gave the product in 75%
(0.35 mg) yield.
(6-allyloxy-2, 5, 7, 8-tetramethyl-chroman-2-yl)methanol (10). The mixture of
2.0 mmol of 2-hydroxymethyl-2, 5, 7, 8-tetramethyl-chroman-6-ol, 4 mmol of allylic
bromide (or other appropriate protecting reagents in scheme 4.1 or table 4.4), and 3.0
mmol of K2CO3 in 10 mL of acetone was refluxed for 48 hrs. After cooling down in room
temperature, the solution was filtered and concentrated. Purification via flash silica gel
chromatography (ethyl acetate/hexanes = 1/5) gave 0.45 g of 10 in 81 % yield.
4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)benzoictrifluorometh
anesulfonic anhydride. 1.2 mmol triflate anhydride was added dropwise into the mixture
o of 1 mmol 10, 1.2 mmol pyridine, and 5 mL CH2Cl2 at 0 C. The solution was stirring at 0
oC for 30 min after all triflate anhydride was added. The solution was concentrated and purified via flash silica gel chromatography (ethyl acetate/hexanes = 1/10) gave product in 92 % yield.
4-(6-allyloxy-2, 5, 7, 8-tetramethyl-chroman-2-ylmethoxy)-benzaldehyde (11). A mixture of 4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)benzoictrifluoro- methanesulfonic anhydride (0.5 mmol) in 10 mL DMF was treated with 0.5 mmol NaH at
0 oC for 30 min, followed by 0.5 mL (4.7 mmol) 4-fluorobenzaldehyde (or appropriate
aldehyde in scheme 4.1) at room temperature overnight. The solution was poured into
water to quench the reaction, extracted with ethyl acetate and concentrated. Purification
via flash silica gel chromatography (ethyl acetate/hexanes = 1/10 ) gave 11 in 93 % yield.
77 (Z)-5-(4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)benzylidene)th
iazolidine-2,4-dione (12, TG-6). A mixture of aldehyde 11 (0.3 mmol),
4-thiazolidinedione, (0.6 mmol), catalytic amount of piperidine was refluxed in 5 mL
EtOH for 24 h and then concentrated. The oil product was dissolved in ethyl acetate and poured into water and acidified with AcOH. The solution was extracted with ethyl acetate and concentrated. Purification via flash silica gel chromatography (ethyl acetate/hexanes
= 1/5) gave 12 (TG-6) in 71 % yield.
(Z)-5-(4-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methoxy)benzylidene)thi
azolidine-2,4-dione (13, Δ2-TG). The mixture of 12 (100 mg, 0.2 mmol), Pd/C (10 mg), p-toluenesulfonic acid (10 mg), and methanol-water (4:1, 5 mL) was stirred under refluxed for 12 hrs, filtered, and concentrated. Purification via flash silica gel chromatography (ethyl acetate/hexanes = 1/5) gave 83 mg of 13 (Δ2-TG) in 91 % yield.
(b) Synthetic procedures of Δ2-CG (scheme 2.3)
(1-methyl-cyclohexyl)-methanol. To a stirring solution of 0.27 g LiAlH4 (20 mmol)
in 10 mL of THF at 0 oC was added 1 g (7.0 mmol) of 1-methyl-cyclohexanecarboxylic
acid in 50 mL of THF dropwise over a period of 1 hour. The solution was stirred at room
temperature under N2 for 6 hours. After 6 hours, 1 mL of H2O, 1 mL of 1 N NaOH and 2
mL of H2O was slowly added to the solution to quench the reaction. The solution was
stirred at room temperature for another hour and then filtered out of solid. The solution
was concentrated. Purification by flash silica gel chromatography (ethyl acetate/hexanes
= 1/2) gave the product in 71%.
4-(1-methyl-cyclohexylmethoxyl)-benzaldehyde. A solution of 0.6 g (4.6 mmol)
(1-methyl-cyclohexyl)-methanol in 10 mL DMF was treated with 0.12g (4.7 mmol) NaH 78 at 0 oC for 30 min, followed by 0.5 mL (4.7 mmol) 4-fluorobenzaldehyde at room
temperature overnight. The solution was poured into water to quench the reaction,
extracted with ethyl acetate and concentrated. Purification via flash silica gel
chromatography (ethyl acetate/hexanes = 1/10 ) gave 0.53 g of product in 52 % yield.
(Z)-5-[4-(1-methyl-cyclohexylmethoxyl)-benzylidene]-thiazolidine-2,4-dione
(Δ2-CG). A mixture of 4-(1-methyl-cyclohexylmethoxyl)-benzaldehyde (0.423 g 1.0 mmol), 4-thiazolidinedione, (0.20 g, 1.1 mmol), catalytic amount of piperidine was refluxed in 10 mL EtOH for 24 h and then concentrated. The oil product was dissolved in ethyl acetate and poured into water and acidified with AcOH. The solution was extracted with ethyl acetate and concentrated. Purification via flash silica gel chromatography
( ethyl acetate/hexanes = 1/5 ) gave 0.25 g of Δ2-CG in 79 % yield.
1 6.13 Nomenclatures, H NMR (Proton Nuclear Magnetic Resonance), and HRMS
(high Resolution Mass Spectrometry) Characterizations of Δ2-TG, Δ2-CG, and
Δ2-TG Analogues.
(Z)-5-(4-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)methoxy)benzylidene)thi
1 azolidine-2,4-dione (13, Δ2-TG). H NMR (250 MHz, CDCl3) 1.39 (s, 3H), 1.75-1.93
(m, 1H), 2.06-2.14 (m, 10H), 2.61 (t, 2H, J = 5.80 Hz), 3.94 (d, 1H, J = 9. 47Hz), 4.02 (d,
1H, J = 9.47 Hz), 6.02-6.17 (m, 1H), 6.99 (d, 2H, J = 8.57 Hz), 7.42 (d, 2H, J = 8.57
Hz), 7.79 (s, 1H), 8.46 (s, 1H); HRMS (ESI, m/z) calcd for C24H25NO5SNa (M+Na):
462.1345, found: 462.1317.
79 (Z)-5-[4-(1-methyl-cyclohexylmethoxyl)-benzylidene]-thiazolidine-2,4-dione
1 (Δ2-CG). H NMR (250 MHz, CDCl3) 1.02 (s, 3H), 1.23-1.47 (brs, 10H), 3.76 (s, 2H,),
6.97 (d, 2H, J = 8.37 Hz), 7.42 (d, 2H, J = 8.37 Hz), 7.80 (s, 1H), 8.28 (s, 1H), HRMS
+ (ESI, m/z) calcd for C18H21NO3SNa (M Na): 354.1134, found: 354.1147.
(Z)-5-(4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)benzylidene)th
1 iazolidine-2,4-dione (12, TG-6). H NMR (250 MHz, CDCl3) 1.30 (s, 3H), 1.83-1.93 (m,
1H), 2.04-2.16 (m, 10H), 2.58 (t, 2H, J = 5.80 Hz), 3.95 (d, 1H, J = 8.75 Hz), 4.03 (d, 1H,
J = 8.75 Hz), 4.15 (d, 2H, J = 11.54 Hz), 5.23 (d, 1H, J = 10.13 Hz), 5.41 (d, 1H, J =
18.21 Hz), 6.02-6.17 (m, 1H), 7.10 (d, 2H, J = 9.78 Hz), 7.48 (d, 2H, J = 9.78 Hz), 7.79
(s, 1H), 8.56 (s, 1H) ;HRMS (ESI, m/z) calcd for C27H29NO5SNa (M+Na): 502.1658,
found: 502.1685.
(Z)-5-(4-((6-(cinnamyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)benzylide
1 ne)thiazolidine-2,4-dione (TG-10). H NMR (250 MHz, CDCl3) 1.41 (s, 3H), 1.83-1.94
(m, 1H), 2.02-2.20 (m, 10H), 2.62 (t, 2H, J = 5.95Hz), 3.95 (d, 1H, J = 8.92 Hz), 4.04 (d,
1H, J = 8.92 Hz), 4.34 (d, 2H, J = 5.76), 6.40-6.51 (m, 1H), 6.73 (d, 1H, J = 16.00 Hz),
7.01 (d, 2H, J = 8.73Hz), 7.25-7.50 (d, 7H), 7.79 (s, 1H), 8.54 (brs, 1H). HRMS (ESI,
+ m/z) calcd for C33H33NO5SNa (M Na): 578.1971, found: 578.1966.
(Z)-5-(3-bromo-4-((6-(cinnamyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)
1 benzylidene)thiazolidine-2,4-dione (TG-11). H NMR (250 MHz, CDCl3) 1.45 (s, 3H),
1.89-1.98 (m, 1H), 2.02-2.19 (m, 10H), 2.58-2.64 (m, 2H), 4.01 (d, 1H, J = 9.10 Hz),
4.08 (d, 2H, J = 9.10 Hz), 4.34 (d, 2H, J = 5.78), 6.40-6.51 (m, 1H), 6.73 (d, 1H, J =
16.00 Hz), 6.96 (d, 1H, J = 8.67Hz), 7.25-7.43 (d, 6H), 7.68 (s, 1H), 7.70 (s, 1H), 8.07 80 + (brs, 1H). HRMS (ESI, m/z) calcd for C33H32BrNO5SNa (M Na): 656.1076, found:
656.1057.
(Z)-5-(4-((6-(benzyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)-3-bromobe
1 nzylidene)thiazolidine-2,4-dione (TG-12). H NMR (250 MHz, CDCl3) 1.42 (s, 3H),
1.84-1.95 (m, 1H), 2.04-2.20 (m, 10H), 2.62 (t, 2H), 3.95 (d, 1H, J = 9.28), 4.01 (d, 1H, J
= 9.28 Hz), 4.67 (s, 2H), 7.01 (d, 2H, J = 8.85Hz), 7.35-7.50 (m, 7H), 7.79 (s, 1H), 8.69
+ (brs, 1H). HRMS (ESI, m/z) calcd for C31H31NO5SNa (M Na): 552.1815, found:
552.1839.
(Z)-4-(2-((2-bromo-4-((2,4-dioxothiazolidin-5-ylidene)methyl)phenoxy)methyl)-
2,5,7,8-tetramethylchroman-6-yloxy)-4-oxobutanoic acid (TG-13). 1H NMR (250
MHz, CDCl3) 1.36 (s, 3H), 1.81-2.10 (m, 11H), 2.62 (m, 2H), 2.77-2.82 (m, 2H),
2.88-2.92 (m, 2H), 3.95 (d, 1H, J = 9.15), 4.02 (d, 1H, J = 9.15 Hz), 6.96 (d, 2H, J = 6.55
Hz), 7.40 (d, 2H, J = 6.55 Hz), 7.77 (s, 1H), 8.43 (brs, 1H). HRMS (ESI, m/z) calcd for
+ C28H29NO8SNa (M Na): 562.1506, found: 562.1480.
(Z)-5-(4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl)methoxy
1 )benzylidene)thiazolidine-2,4-dione (TG-14). H NMR (250 MHz, CDCl3) 1.40 (s, 3H),
1.63 (s, 3H), 1.69 (s, 3H), 1.88-1.93 (m, 1H), 2.04-2.18 (m, 10H), 2.61 (t, 2H, J = 6.75),
3.95 (d, 1H, J = 9.31 Hz), 4.03 (d, 1H, J = 9.31 Hz), 4.15 (d, 2H, J = 7.04), 5.58 (t, 1H, J
= 7.20), 7.01 (d, 2H, J = 8.80 Hz), 7.42 (d, 2H, J = 8.80Hz), 7.90 (s,1H), 8.84 (s,
+ 1H).HRMS (ESI, m/z) calcd for C29H33NO5SNa (M Na): 530.1971, found: 530.1975
(Z)-5-(3-methoxy-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-
1 yl)methoxy)benzylidene)thiazolidine-2,4-dione (TG-16). H NMR (250 MHz, CDCl3)
1.42 (s, 3H), 1.69 (s, 3H), 1.78 (s, 3H), 1.86-1.93 (m, 1H), 2.01-2.17 (m, 10H), 2.61 (t, 81 2H, J = 6.84 Hz), 3.87 (s, 3H), 4.00 (d, 1H, J = 9.52 Hz), 4.09 (d, 1H, J = 9.52 Hz), 4.14
(d, 2H, J = 6.84 Hz), 5.58 (t, 1H, J = 7.14 Hz), 6.96-7.06 (m, 3H), 7.76 (s, 1H). HRMS
+ (ESI, m/z) calcd for C30H35NO6SNa (M Na): 560.2077, found: 560.2086.
(Z)-5-(3-methyl-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl
1 )methoxy)benzylidene)thiazolidine-2,4-dione (TG-17). H NMR (250 MHz, CDCl3)
1.44 (s, 3H), 1.69 (s, 3H), 1.78 (s, 3H), 1.86-1.94 (m, 1H), 2.02-2.20 (m, 10H), 2.26 (s,
3H), 2.61 (t, 2H, J = 3.19 Hz), 3.95 (d, 1H, J = 9.34 Hz), 4.03 (d, 1H, J = 9.34 Hz), 4.17
(d, 2H, J = 6.88 Hz), 5.58 (t, 1H, J = 5.45 Hz), 6.88 (d, 1H, J = 9.22), 7.27 (s, 1H), 7.29 (d,
1H, J = 9.22 Hz), 7.76 (s, 1H), 8.24 (brs,1H). HRMS (ESI, m/z) calcd for C30H35NO5SNa
(M+Na): 544.2128, found: 544.2134.
(Z)-5-(4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)-3-bromobenz
1 ylidene)thiazolidine-2,4-dione (TG-27). H NMR (250 MHz, CDCl3) 1.47 (s, 3H),
1.94-2.19 (m, 11H), 2.60-2.64 (m, 2H), 4.00 (d, 1H, J = 9.20 Hz), 4.07 (d, 1H, J = 9.20
Hz), 4.16 (d, 2H, J = 5.45 Hz), 5.23 (d, 1H, J = 10.42 Hz), 5.41 (d, 1H, J = 17.19 Hz),
5.98-6.11 (m, 1H), 6.96 (d, 1H, J = 8.66 Hz), 7.36 (dd, 1H, J = 8.66, 2.13 Hz), 7.67 (d,
1H, J = 2.13 Hx), 7.70 (s, 1H), 8.30 (brs, 1H). HRMS (ESI, m/z) calcd for
+ C27H28BrNO5SNa (M Na): 580.0763, found: 580.0763.
(Z)-5-(4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)-3-methoxybe
1 nzylidene)thiazolidine-2,4-dione (TG-28). H NMR (250 MHz, CDCl3) 1.42 (s, 3H),
1.88-1.92 (m, 1H), 2.04-2.18 (m, 10H), 2.61 (t, 2H, J =7.16 Hz), 3.88 (s, 3H), 3.98 (d, 1H,
J = 9.91 Hz), 4.09 (d, 1H, J = 9.91 Hz), 4.16 (d, 2H, J = 5.47 Hz), 5.23 (d, 1H, J = 10.42
Hz), 5.40 (d, 1H, J = 17.18 Hz), 6.03-6.15 (m, 1H), 6.96-7.06 (m, 3H), 7.76 (s, 1H), 8.34
+ (s, 1H). HRMS (ESI, m/z) calcd for C28H31NO6SNa (M Na): 532.1764, found: 532.1762. 82 (Z)-5-(4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)-3-methylbenz
1 ylidene)thiazolidine-2,4-dione (TG-29). H NMR (250 MHz, CDCl3) 1.43 (s, 3H),
1.89-1.94 (m, 1H), 2.05-2.20 (m, 10H), 2.26 (s, 3H), 2.61 (t, 2H, J =6.46 Hz), 3.95 (d, 1H,
J = 9.27 Hz), 4.02 (d, 1H, J = 9.27 Hz), 4.16 (d, 2H, J = 5.49 Hz), 5.23 (d, 1H, J = 10.38
Hz), 5.41 (d, 1H, J = 17.23 Hz), 6.04-6.15 (m, 1H), 6.88 (d, 1H, J = 8.80 Hz), 7.27 (s,
1H), 7.29 (d, 1H, J = 8.0 Hz), 7.76 (s, 1H). HRMS (ESI, m/z) calcd for C28H31NO5SNa
(M+Na): 516.1815, found: 516.1837.
(Z)-5-(3-fluoro-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl)
1 methoxy)benzylidene)thiazolidine-2,4-dione (TG-30). H NMR (250 MHz, CDCl3)
1.42 (s, 3H), 1.69 (s, 3H), 1.78 (s, 3H), 1.87-1.93 (m, 1H), 1.96 (s, 3H), 2.01-2.17 (m,
7H), 2.61 (t, 2H, J = 6.50 Hz), 4.02 (d, 1H, J = 9.49 Hz), 4.10 (d, 1H, J = 9.49 Hz), 4.15
(d, 2H, J = 7.22 Hz), 5.57 (t, 1H, J = 6.87 Hz), 7.05 (t, 1H, J = 8.25 Hz), 7.17-7.23 (m,
+ 2H), 7.72 (s,1H), 8.46 (brs, 1H). HRMS (ESI, m/z) calcd for C29H32FNO5SNa (M Na):
548.1877, found: 548.1889.
(Z)-5-((6-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl)methoxy
)-4'-(trifluoromethyl)biphenyl-3-yl)methylene)thiazolidine-2,4-dione (TG-31). 1H
NMR (250 MHz, CDCl3) 1.40 (s, 3H), 1.69-1.93 (m, 8H), 2.00 (s, 3H), 2.12 (s, 3H), 2.22
(s, 3H), 2.47-2.58 (m, 2H), 4.02 (s, 2H, J = 9.49 Hz), 4.15 (d, 2H, J = 7.01 Hz), 5.57 (t,
1H, J = 7.12 Hz), 7.08 (d, 1H, J = 8.75 Hz), 7.46 (s, 1H), 7.48 (d, 1H, J = 8.75Hz), 7.65 (s,
+ 4H), 7.82 (s,1H), 8.61 (brs, 1H). HRMS (ESI, m/z) calcd for C36H36F3NO5SNa (M Na):
674.2154, found: 674.2165.
(Z)-5-((4'-fluoro-6-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-y
l)methoxy)biphenyl-3-yl)methylene)thiazolidine-2,4-dione (TG-32). 1H NMR (250 83 MHz, CDCl3) 1.27 (s, 3H), 1.69-1.94 (m, 8H), 2.00 (s, 3H), 2.10 (s, 3H), 2.17 (s, 3H),
2.46-2.61 (m, 2H), 3.99 (s, 2H), 4.14 (d, 2H, J = 6.96 Hz), 5.58 (t, 1H, J = 7.14 Hz),
7.02-7.10 (m, 3H), 7.41-7.52 (m, 4H), 7.81 (s, 1H), 8.30 (brs, 1H). HRMS (ESI, m/z)
+ calcd for C35H36FNO5Na (M Na): 624.2190, found: 624.2206.
(Z)-5-((4'-ethyl-6-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl)
methoxy)biphenyl-3-yl)methylene)thiazolidine-2,4-dione (TG-33). 1H NMR (250
MHz, CDCl3) 1.21-1.30 (m, 6H), 1.69-1.95 (m, 8H), 2.00 (s, 3H), 2.07 (s, 3H), 2.17 (s,
3H), 2.53-2.57 (m, 2H), 2.69 (q, 2H, J = 7.45 Hz), 3.99 (s, 2H), 4.14 (d, 2H, J = 6.80 Hz),
5.57 (t, 1H, J = 7.03 Hz), 7.03 (d, 1H, J = 8.65 Hz), 7.05-7.24 (m, 2H), 7.38-7.47 (m, 4H),
+ 7.82 (s, 1H), 8.59 (brs, 1H). HRMS (ESI, m/z) calcd for C37H41NO5SNa (M Na):
634.2597, found: 634.2634.
(Z)-5-(3,5-dibromo-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-
1 2-yl)methoxy)benzylidene)thiazolidine-2,4-dione (TG-34). H NMR (250 MHz, CDCl3)
1.24 (s, 3H), 1.68 (s, 3H), 1.77 (s, 3H), 1.81-1.88 (m, 2H), 2.03 (s, 3H), 2.12 (s, 3H), 2.16
(s, 3H), 2.57-2.72 (m, 2H), 3.90 (d, 2H, J = 14.00 Hz), 4.11 (d, 2H, J = 11.98 Hz), 5.57
(m, 1H), 6.18 (s, 1H), 7.59 (s, 1H), 7.67 (s, 1H), 8.14 (s, 1H).HRMS (ESI, m/z) calcd for
+ C29H31Br2NO5SNa (M Na): 688.0161, found: 688.0161.
(Z)-5-(3-nitro-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl)
1 methoxy)benzylidene)thiazolidine-2,4-dione (TG-35). H NMR (250 MHz, CDCl3)
1.23 (s, 3H), 1.68 (s, 3H), 1.77 (s, 3H), 1.80-1.90 (m, 1H), 2.02-2.21 (m, 10H), 2.62 (m,
2H), 3.91-4.26 (m, 6H), 5.56 (t, 1H, J = 4.84 Hz), 7.30 (d, 1H, J = 8.55 Hz),7.70 (d, 1H, J
= 8.55 Hz), 7.84 (s, 1H), 8.26 (s, 1H), 10.08 (s, 1H). HRMS (ESI, m/z) calcd for
+ C29H32N2O7S2Na (M Na): 575.1822, found: 575.1824. 84 (E)-3-(3-bromo-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl
1 )methoxy)phenyl)acrylic acid (TG-36). H NMR (300 MHz, CDCl3) 1.41 (s, 3H), 1.71
(s, 3H), 1.79 (s, 3H), 1.90-1.98 (m, 1H), 2.02 (s, 3H), 2.12 (s, 3H), 2.16-2.2 (m, 4H), 2.61
(q, 2H, J = 5.05 Hz), 4.04 (d, 1H, J= 6.43 Hz), 4.15 (m, 3H), 5.57 (t, 1H, J = 8.2 Hz), 6.98,
(d, 1H, J = 12 Hz), 7.58 (s, 1H), 7.88 (d, 1H, J = 9.0 Hz), 7.66 (s, 1H), 8.06 (s,
+ 1H).HRMS (ESI, m/z) calcd for C29H31BrN2O3Na (M Na): 557.1410, found: 557.1402.
(E)-3-(3-bromo-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl
1 )methoxy)phenyl)acrylic acid (TG-37). H NMR (300 MHz, CDCl3) 1.48 (s, 3H), 1.58
(s, 3H), 1.68 (s, 3H), 1.89-1.98 (m, 1H), 2.05 (s, 3H), 2.12 (s, 3H), 2.16-2.2 (m, 4H), 2.62
(q, 2H, J = 4.88 Hz), 3.97 (d, 1H, J= 6.43 Hz), 4.05 (d, 1H, J = 6.90 Hz), 4.15 (d, 2H, J =
6.02 Hz), 5.58 (t, 1H, J = 8.1 Hz), 6.29, (d, 1H, J = 17.7 Hz), 6.88 (d, 1H, J = 8.7 Hz),
7.39 (d, 1H, J = 8.4 Hz), 7.61 (s, 1H), 7.66 (s, 1H), 7.75 (s, 1H). HRMS (ESI, m/z) calcd
+ for C28H33BrO5Na (M Na): 551.1404, found:551.1040.
Ethyl-3-(3-bromo-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-
yl)methoxy)phenyl)-3-hydroxy-2,2-dimethylpropanoate (TG-38). 1H NMR (300 MHz,
CDCl3) 1.09 (s, 3H), 1.11 (s, 3H), 1.27 (t, 3H, J = 9.0 Hz), 1.47 (s, 3H), 1.69 (s, 3H), 1.78
(s, 3H), 1.89-1.98 (m, 1H), 2.05 (s, 3H), 2.12 (s, 3H), 2.16-2.2 (m, 4H), 2.62 (brs, 2H),
3.29 (brs, 1H), 3.91 (d, 1H, J= 6.43 Hz), 3.98 (d, 1H, J = 6.90 Hz), 4.15 (m, 4H), 4.78 (s,
1H), 5.57 (t, 1H, J = 7.14 Hz), 6.82 (d, 1H, J = 8.49 Hz), 7.16 (d, 1H, J = 8.49 Hz), 7.51
+ (s, 1H). HRMS (ESI, m/z) calcd for C32H43BrO6Na (M Na): 625.2135, found:625.2172.
3-(3-bromo-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl)met
hoxy)phenyl)-3-hydroxy-2,2-dimethylpropanoic acid (TG-39). 1H NMR (300 MHz,
CDCl3) 1.12 (s, 3H), 1.13 (s, 3H), 1.47(s, 3H), 1.69 (s, 3H), 1.78(s, 3H), 1.89-1.98 (m, 85 1H), 2.05 (s, 3H), 2.13-2.21 (m, 7H), 2.62 (brs, 2H), 3.92 (d, 1H, J = 9.75 Hz), 4.00 (d,
1H, J= 9.75 Hz), 4.14 (d, 2H, J = 6.90 Hz), 5.57 (t, 1H, J = 7.14 Hz), 6.82 (d, 1H, J = 8.49
Hz), 7.16 (d, 1H, J = 8.49 Hz), 7.51 (s, 1H). HRMS (ESI, m/z) calcd for C30H39BrO6Na
(M+Na): 597.1822, found: 597.1836.
Diethyl-2-(3-bromo-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-
1 2-yl)methoxy)benzylidene)malonate (TG-41). H NMR (300 MHz, CDCl3) 1.28-1.34
(m, 6H), 1.46 (s, 3H), 1.69 (s, 3H), 1.78 (s, 3H), 1.91-1.98 (m, 1H), 2.04 (s, 3H),
2.13-2.20 (m, 10H), 2.58-2.64 (m, 2H), 3.95 (d, 1H, J = 9.10 Hz), 4.30 (d, 1H, J = 9.10
Hz), 4.14 (d, 2H, J = 7.07 Hz), 4.26-4.35 (m, 4H), 5.58 (t, 1H, J = 7.05 Hz), 6.85 (d, 1H, J
= 8.66 Hz), 7.33 (dd, 2H, J = 8.66, 2.21 Hz), 7.56 (s, 1H), 7.65 (d, 1H, J = 2.21 Hz).
+ HRMS (ESI, m/z) calcd for C33H41BrO7Na (M Na): 651.1927, found: 651.1923.
2-(3-bromo-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl)met
hoxy)benzylidene)malonic acid (TG-42). (250 MHz, CDCl3) 1.41 (s, 3H), 1.69 (s, 3H),
1.77 (s, 3H), 1.93-2.17 (m, 11H), 2.10-2.19 (m, 7H), 2.58-2.70 (m, 2H), 4.04-4.16 (m,
4H), 5.48 (t, 1H, J = 5.91 Hz), 6.82 (d, 1H, J = 6.45 Hz), 7.46 (s, 1H), 7.70 (d, 1H, J =
+ 15.91 Hz), 7.83 (s, 1H).HRMS (ESI, m/z) calcd for C29H31BrO7Na (M Na): 595.1301, found: 595.1282.
(Z)-5-((4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl)methoxy
)naphthalen-1-yl)methylene)thiazolidine-2,4-dione (TG-43). 1H NMR (250 MHz,
CDCl3) 1.53 (s, 3H), 1.70 (s, 3H), 1.79 (s, 3H), 1.95-2.03 (m, 1H), 2.08(s, 3H), 2.17-2.27
(m, 7H), 2.67 (t, 2H, J = 6.72 Hz), 4.12-4.23 (m, 4H), 5.59 (t, 1H, J = 6.94 Hz), 7.00 (d,
86 1H, J = 8.24 Hz), 7.53-7.66 (m, 3H), 8.10 (d, 1H, J = 7.98 Hz), 8.33 (d, 1H, J = 8.17 Hz),
+ 8.55 (s, 1H), 8.73 (s, 1H). HRMS (ESI, m/z) calcd for C33H35NO5SNa (M Na): 580.2128,
found: 580.2133.
(Z)-5-(3-bromo-5-methoxy-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)ch
roman-2-yl)methoxy)benzylidene)thiazolidine-2,4-dione (TG-44). 1H NMR (250 MHz,
CDCl3) 1.50 (s, 3H), 1.69(s, 3H), 1.78(s, 3H), 1.85-1.96(m, 1H), 2.02 (s, 3H), 2.14-2.27
(m, 7H), 2.63-2.72 (m, 2H), 3.81 (s, 3H), 4.01 (d, 1H, J = 9.01 Hz), 4.08 (d, 1H, J = 9.01
Hz), 4.15 (d, 2H, J = 6.98 Hz), 5.58 (t, 1H, J = 7.19 Hz), 6.90 (s, 1H), 7.27 (s, 1H), 7.68
+ (s, 1H), 8.60 (s, 1H). HRMS (ESI, m/z) calcd for C30H32BrNO6SNa (M Na): 638.1182,
found: 638.1190.
(Z)-5-(3-ethoxy-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl
1 )methoxy)benzylidene)thiazolidine-2,4-dione (TG-45). H NMR (250 MHz, CDCl3)
1.40-1.46 (m, 6H), 1.70 (s, 3H), 1.79 (s, 3H), 1.87-1.93 (m, 1H), 2.01 (s, 3H), 2.09-2.17
(m, 7H), 2.62 (t, 2H, J = 6.97 Hz), 3.97-4.16 (m, 6H), 5.58 (t, 1H, J = 7.12 Hz), 6.97-7.08
+ (m, 3H), 7.76(s, 1H), 8.59 (s, 1H). HRMS (ESI, m/z) calcd for C31H37BrNO6SNa (M Na):
574.2342.
(Z)-5-((4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl)methox y)naphthalen-1-yl)methylene)-2-thioxothiazolidin-4-one (TG-46). 1H NMR (250 MHz,
CDCl3) 1.54 (s, 3H), 1.70 (s, 3H), 1.79 (s, 3H), 1.96-2.04 (m, 1H), 2.08 (s, 3H),
2.15-2.29(m, 7H), 2.67(t, 2H, J = 6.29 Hz), 4.09-4.24 (m, 4H), 5.59 (t, 1H, J = 5.90 Hz),
87 6.90 (d, 1H, J = 8.30 Hz), 7.53-7.68 (m, 3H), 8.13 (d, 1H, J = 8.30 Hz), 8.33 (d, 1H, J =
+ 8.20 Hz), 8.38 (s, 1H), 9.74 (s, 1H). HRMS (ESI, m/z) calcd for C33H35NO4S2Na (M Na):
625.2135, found: 625.2172.
(Z)-5-(4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)-3,5-dimethylb
1 enzylidene)thiazolidine-2,4-dione (TG-52). H NMR (250 MHz, CDCl3) 1.43 (s, 3H),
1.92-1.98 (m, 1H), 2.08-2.16 (m, 10H), 2.31 (s, 6H), 2.69 (m, 2H), 3.79 (s, 2H), 4.17 (d,
1H, J = 1.31 Hz), 5.23 (d, 1H, J = 12.16 Hz), 5.41 (d, 1H, J = 15.51 Hz), 6.05-6.14 (m,
1H), 7.14 (s, 2H), 7.73 (s, 1H), 8.21 (s, 1H). HRMS (ESI, m/z) calcd for C29H33NO5SNa
(M+Na): 530.1972, found: 530.1976.
(Z)-5-((4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)naphthalen-1-
1 yl)methylene)thiazolidine-2,4-dione (TG-53). H NMR (250 MHz, CDCl3) 1.54 (s, 3H),
1.98-2.01 (m, 1H), 2.03 (s, 3H), 2.13 (s, 3H), 2.16 (s, 3H), 2.17-2.23 (m, 1H), 4.13-4.23
(m, 4H), 5.23 (d, 1H, J = 10.96 Hz), 5.41 (d, 1H, J = 17.37 Hz), 2.02-2.16 (m, 1H), 6.90
(d, 1H, J = 7.96 Hz), 7.53-7.66 (m, 3H), 8.08 (d, 1H, J = 4.08 Hz), 8.15 (s, 1H), 8.31 (d,
+ 1H, J = 4.08 Hz), 8.54 (s, 1H). HRMS (ESI, m/z) calcd for C31H31NO5SNa (M Na):
552.1815, found: 552.1812.
(Z)-5-(4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)-3-bromo-5-m
1 ethoxybenzylidene)thiazolidine-2,4-dione (TG-54). H NMR (250 MHz, CDCl3) 1.55
(s, 3H), 1.80-1.98 (m, 1H), 2.02 (s, 3H), 2.13-2.27 (m, 7H), 3.75 (s, 3H), 4.05 (q, 2H, J =
88 9.6 Hz), 4.15 (d, 2H, J = 4.45 Hz), 5.22 (d, 1H, J = 10.42 Hz), 5.41 (d, 1H, J = 17.4 Hz),
6.04-6.15 (m, 1H), 6.89 (s, 1H), 7.26 (s, 1H), 7.65 (s, 1H), 8.18 (s, 1H). HRMS (ESI, m/z)
+ calcd for C28H30BrNO6SNa (M Na): 610.0869, found: 610.0903.
(Z)-5-(4-((6-(allyloxy)-2,5,7,8-tetramethylchroman-2-yl)methoxy)-3-ethoxybenz
1 ylidene)thiazolidine-2,4-dione (TG-55). H NMR (250 MHz, CDCl3) 1.45 (s, 3H), 1.58
(s, 3H), 1.85-1.95 (m, 1H), 2.02 (s, 3H), 2.12-2.25 (m, 7H), 2.60 (m, 2H), 3.97-4.31 (m,
6H), 5.23 (d, 1H, J = 8.55 Hz), 5.40 (d, 1H, J = 13.25Hz), 6.05-6.16 (m, 1H), 6.94-7.10
+ (m, 3H), 7.76 (s, 1H), 8.31 (s, 1H). HRMS (ESI, m/z) calcd for C29H33NO6SNa (M Na):
546.1921, found: 546.1907.
(Z)-5-(3-bromo-4-((2,5,7,8-tetramethyl-6-(3-methylbut-2-enyloxy)chroman-2-yl)
1 methoxy)benzylidene)thiazolidine-2,4-dione (TG-88). H NMR (250 MHz, CDCl3)
1.40 (s, 3H), 1.69 (s, 3H), 1.78 (s, 3H), 1.91-1.97 (m, 1H), 2.02-2.21 (m, 10H), 2.61 (m,
2H), 3.94 (d, 1H, J = 9.48 Hz), 4.03 (d, 1H, J = 9.48 Hz), 4.14 (d, 2H, J = 6.84 Hz), 5.58
(t, 1H, J = 7.06 Hz), 6.96 (d, 1H, J = 8.63 Hz), 7.38 (dd, 1H, J = 8.63, 2.04 Hz), 7.67 (d,
1H, J = 2.04Hz), 7.79 (s,1H), 8.56 (brs, 1H). HRMS (ESI, m/z) calcd for
+ C29H32BrNO5SNa (M Na): 608.1076, found: 608.1094.
89
PART2
VITAMIN E AND ITS ANALOGUES INDUCE APOPTOSIS IN
PROSTATE CANCER CELLS IN PART THROUGH INHIBITION
OF BCL-2/BCL-XL FUNCTIONS
90
CHAPTER 7
INTRODUCTION
7.1 Vitamin E and Cancer
In spite of improvements in hormonal and chemotherapeutic approaches, prostate
cancer is still the second leading cause of cancer mortality in men in the United States.
Initially, prostate tumor growth is characterized as androgen-dependent. Patients at this
stage are generally treated by androgen ablation if they were not first cured by prostatectomy or radiation treatment of the primary tumor while locally restricted to the prostate capsule. Withdrawal of androgens by chemical or physical castration usually
leads to a temporary regression of the disease. However, decreased levels of androgens
favor the selection and growth of cancer cells that are androgen-independent in most
cases. So far, there has been no effective therapy for the treatment of
androgen-independent prostate cancer (79). Therefore, it is essential to develop new
nontoxic but effective therapeutic agents to increase the survival and reduce side effects
in prostate cancer patients. In this regard, vitamin E gained considerable attention in the
past two decades as a potential antineoplastic agent with high selectivity for malignant
cells and low toxicity.
91 Evidence has shown that vitamin E, a fat-soluble antioxidant, might offer cellular
protection against diverse free radicals which can damage DNA, and certain analogues of
vitamin E can cause apoptotic death of a variety of cancer cell lines. Thus, vitamin E may
represent a novel class of chemopreventive or chemotherapeutic agent. Numerous studies
have been conducted to determine whether vitamin E or its analogues can be used either
directly as an antineoplastic agent or indirectly through the augmentation of chemotherapy effects (80).
Vitamin E is a generic term referring to a group of naturally occurring and
synthetic tocopherol and tocotrienol derivatives. At least eight naturally occurring
compounds, including α−, β−, δ−, and γ−tocopherols and α−, β−, δ−, and γ−tocotrienols have been identified in vitamin E (Fig 7.1). Structurally, tocopherols and tocotrienols
share some similarities consisting of a common chromanol ring and a phytyl chain
attached to the chromanol ring. The fundamental difference between these two groups of
compounds is that tocopherols have a completely saturated phytyl tail, whereas the
tocotrienols are characterized by a partly unsaturated isoprenoid side chain. γ−Tocopherol
is the principal form of vitamin E found in human diets, while α−tocopherol acetate and
synthetic isomers of α−tocopherols are the major forms in vitamin E supplements. Some acetate and succinate derivatives of tocopherols and tocotrienols have been used in vitamin E supplements because of their increased stability in air, but these compounds are not active antioxidants until they are hydrolyzed in metabolic systems (81-83).
Although all vitamin E family compounds, except these acetate and succinate
analogues, exhibited antioxidant activity (84), their abilities to inhibit the proliferation of
malignant cells are tremendously different. Tocotrienols and tocopherol succinate, in 92 comparison to tocopherols, are much more potent inhibitors of cell growth and/or
inducers of apoptosis in certain cancer cell lines (85-96). For instance, Sylvester and coworkers demonstrated that up to 120 µM α− and γ−tocopherols failed to inhibit cell
proliferation of highly malignant mouse mammary epithelial cells over a 5-day culture
period. In contrast, IC50 values for growth inhibition for δ−tocopherol, α−tocotrienol,
γ−tocotrienol, and δ−tocotrienol are 23, 5, 4, and 3 µM respectively (85). α−Tocopheryl
(or vitamin E) succinate (α−TOS) is a relatively new vitamin E analogue with promising
anticancer activity. Initially applied in cancer research in 1995, α−TOS exhibited higher
potency than tocotrienols to induce apoptosis in many human tumor cells in vitro (81). It
is noteworthy that treatment with α−TOS had little or no effect on normal mammary
epithelial cell viability (85, 97-98).
The precise mechanism for the antiproliferative property of tocotrienols and
α−TOS has not been fully elucidated. However, the most promising anti-carcinogenic
property of α−TOS, its pro-apoptotic function, has been extensively studied. Some
proposed proapoptotic mechanisms of vitamin E analogues in vitro are summarized in
Table 7.1.
7.2 Bcl-2 Family and Its Signaling Pathways
Apoptosis, or programmed cell death, is essential for development, tissue
homeostasis, and protection against pathogens. Two major intracellular apoptosis
signaling pathways have been identified, the death receptor and the mitochondrial pathways. The death receptor pathway is triggered by binding of a ligand, such as Fas, 93 TNFα, and TRAIL, to the receptor in the plasma membrane. The mitochondrial pathway is probably activated by diverse stimuli, such as reactive oxygen species, UV or gamma irradiation, growth factor deprivation, kinase inhibitors, and cytotoxic compounds.
Bcl-2 (B-cell lymphoma gene 2) family proteins play key roles in regulating the mitochondrial apoptosis pathway (121). This family includes more than 20 members, all of which share at least one conserved Bcl homology (BH) domain (Fig. 7.2) and possess either pro- or anti-apoptotic activities (122). The anti-apoptotic proteins such as Bcl-2,
Bcl-XL, Bcl-W, Mcl-1, and A-1 which potently suppress cell death contain all four conserved domains (BH1-4) as well as the hydrophobic C-terminal domain. The pro-apoptotic members can be divided into two groups, the multidomain proteins, including Bax, Bak, and Bok, and “BH3-domain only” group, such as Bid, and Bad. Most multidomain members contain BH1-3 and the C-terminal hydrophobic domains, while
“BH-3 domain only” proteins only possess the BH3 domain.
The precise mechanism of how the Bcl-2 family controls apoptosis is still under study. One model indicates that anti-apoptotic Bcl-2 members might antagonize Apaf1
(apoptotic protease-activating factor 1) which functions to interact with cytochrome c and further activates caspase-9 to induce apoptosis. The main function of Bcl-2 and its anti-apoptotic homologues is to guard the integrity of mitochondrial membrane. They inhibit apoptosis through preventing oligomerization of Bax/Bak, which are able to insert into the outer mitochondrial membrane. Once apoptosis is induced, “BH3-domain only” proteins will damage the integrity of the mitochondrial membrane. The multidomain proteins further the process by forming a channel to trigger the release of several proteins
94 from the intermembrane space, including cytochrome c, Diablo/Smac, and Omi/HtrA2.
These proteins will neutralize the IAPs (inhibitor of apoptosis proteins) which inhibit
processed caspases (Fig. 7.3) (122). In addition, Bax and Bak are presumed to not only
damage the mitochondrial but also to induce an unknown protein (“activator”) to the
activation of caspase-12. Caspase-12 can process other caspases in the absence of Apf1
or cytochrome c. Once the effector caspase-3, -7, and -6 have been processed, they most
likely feedback to create more damage to the organelles (Fig.7.4) (123).
7.3 Vitamin E Analogues Target the Bcl-2 Proteins for Chemotherapy
Since defective apoptosis in human cancers often results from overexpression of
anti-apoptotic proteins in the Bcl-2 protein family, particularly Bcl-2 and Bcl- XL, a great deal of effort is being invested in the development of novel agents to inhibit the expression or function of these proteins. At least three therapeutic strategies which focus on ablation of expression or function of Bcl-2 pro-apoptotic proteins have been reported.
One approach, which applies antisense oligonucleotides directed against Bcl-2 mRNA, has reached Phase III clinical trials for melanoma, myeloma, acute myeloid leukemia, and chronic lymphocytic leukemia (124). Although Bcl-2 antisense agents exhibited some nonspecific toxicity due to the phosphorothiorate backbone structure of the antisense molecule, gene therapy is still a promising approach to kill certain types of cancer cells or to sensitize tumor cells to other chemotherapy.
Inhibition of the function of Bcl-2/Bcl-XL with BH3 domain peptides is the
second therapeutic strategy. Because the BH3 domain of pro-apoptotic Bax or Bak is
required for heterodimerization with Bcl-2 or Bcl-XL to induce apoptosis, peptides
representing the Bax or Bak BH3 domain have been designed to inhibit the pro-survival 95 function of Bcl-2 / Bcl-XL. Some experiments using such peptides to antagonize the
function of these anti-apoptotic proteins have yielded promising results. For example, the
Bak BH3 peptide complexed with ANT (the 16-amino-acid antennapedia peptide which
is frequently used to facilitate the delivery peptides into the cells) rapidly achieved entry
into HeLa cells and produced an apoptotic response within 2-3 hours (125). The early
challenges of targeting Bcl-2 and Bcl-XL with peptides, such as poor solubility, limited cellular permeability, and proteolytic degradation owing to their high molecular weight have been mitigated by employing transduction domains such as ANT to facilitate entry into cells. The enhancement of BH3 peptide stability through chemical modifications and the development of peptidomimetics represent other potential approaches for improving the drug-like properties of these peptides (126).
Compared to peptide-based anticancer agents, small molecule antagonists to
Bcl-2/Bcl-XL offer several advantages including the more rapid selection and refinement
of small molecules for optimal solubility, cell permeability, stability, binding affinity, and activity. In addition, recent advances have made it possible to rapidly screen larger numbers of compounds in exciting chemical libraries. Combinational chemistry is able to accomplish the modification and optimization of promising lead compounds, and computational methods and knowledge of the three-dimensional structures of
Bcl-2/Bcl-XL have been used to assist the modification of small molecule inhibitors.
More than a dozen compounds have been reported to inhibit the function of Bcl-2 and
Bcl-XL. For instance, HA14-1 (ethyl 2-amino-6bromo-4-(1-cyano-2-ethoxy-2ozoethyl)-
4H-chromene-3-carboxylate), the first small organic molecule which is capable of
binding Bcl-2, was identified by computer-aided screening of a chemical library (193,833 96 compounds were screened) (127). The chemical structure and molecular model for the
complex of HA14-1 with the Bcl-2 surface pocket are shown in Fig. 7.5 (128).
Subsequent fluorescence polarization (FP) assays verified that HA14-1 competed with
Bak BH3 peptide for binding to Bcl-2. The challenge of using small molecules in this
context is that it is unclear whether the compounds specifically bind only to
anti-apoptotic Bcl-2 members or also antagonize other pro-apoptotic proteins. Therefore,
more studies are necessary to further characterize the mechanism of apoptosis and to
assess any potential side effects of these agents. Through the application of combinatorial and medicinal chemistry, more potent and more specific small molecules that specifically inhibit Bcl-2/Bcl-XL are likely to be discovered. These compounds may also provide new insight into the mechanism of Bcl-2 family members.
Since redox-silent α-TOS, but not α−tocopherols, showed potent activity to
induce apoptosis in cancer cells, it has been suggested that the principal anticancer
mechanism of vitamin E analogues does not depend on their antioxidative capabilities
(129). The mechanism by which these analogues induce apoptosis can vary among
different cancer cell lines and different analogues. Among the mechanisms implicated in
the anticancer effects of vitamin E analogues, such as modulation of PKC, TGF-β, Fas, or
JNK signaling pathways, initiation of cell cycle arrest, or induction of cell differentiation,
none of these mechanisms alone may be sufficient to trigger apoptosis. Thus, it is
suggested that the vitamin E analogues induce apoptosis by affecting multiple signaling
pathways, or by primarily modulating mitochondria-related proteins such as Bcl-2 family
members directly. Bcl-2 has a high frequency of overexpression in advanced forms of
prostate carcinoma and there is general agreement that Bcl-2 is involved in prostate 97 cancer progression (79). Some studies have indicated that the pro-apoptotic activities of
vitamin E analogues in cancer cells can be mediated by effects on the pro- and anti-apoptotic proteins of the Bcl-2 family (116, 130). Therefore, small molecules, such as vitamin E analogues, targeting Bcl-2 family members and inducing mitochondria-dependent apoptosis may represent a promising therapeutic strategy.
98
Fig. 7.1 Chemical structures of tocopherols and tocotrienols
99 Proposed Comments Type of cell lines (ref)
mechanism
Antioxidant Vitamin E reduces lipid peroxidation and None (83)
inhibits the formation of powerful
mutagenic nitric oxide species in turn to
prevent oxidant damage to DNA.
PKC inhibition PKC inhibits apoptosis by modulating Fas Vascular smooth muscle
ligand expression and upregulating Bcl-2 (99-101)
(102) Jurkat T lymphoma (102)
Leukemia (HL-60) (103)
TGF-β *TGF-β inhibits the activity of G1 B lymphoma (RL) (104)
pathway cyclin-CDK complexes, thereby inducing a
G1 cell cycle block; involves transcriptional
induction of p21Waf1/Cip1 (104)
*α-TOS activates TGF-β pathways in Breast (MDA-MB-435)
TGF-β nonresponsive cancer cells via (105-106)
activation of JNK signaling pathway (81, Gastric (SGC-7901) (107)
105-106) Prostate (LNCaP, PC-3, and DU-145) (108)
Continued
Table 7.1 Summary of some proposed proapoptotic mechanisms of vitamin E analogues
in vitro.
100 Table 7.1 continued
Fas pathway *Fas initiates a signal-transduction cascade Prostate (LNCaP; PC-3) (90)
leading to apoptosis (96) Gastric cancer (96)
*α-TOS can restore Fas signaling in Fas Breast (MDA-MB-231) (109)
nonresponsive cancer cells (81, 90, 96, Breast (MCF-7,
109-110) MDA-MB-231,
MDA-MB-435 ) (110)
MAPK/JNK *MAPK/JNK pathways are involved in the
pathway control of various cellular processes
including growth, differentiation, survival
and death (81)
*JNK phosphorylates c-Jun which triggers Gastric (SGC-7901) (95)
apoptosis (95) Breast (MCF-7,
*α-TOS upregulates expression of JNK (95, MDA-MB-435) (111-112)
111-112) Gastric (SGC-7901) (95)
AR function α-TOS can suppress AR expression by Prostate (LNCaP) (113)
inhibition means of transcriptional and
posttranscriptional modulation. (113)
Continued
101
Table 7.1 continued
Mediate *Bcl-2 family includes pro-apoptotic
mitochondria-related members, such as Bax and Bid, and
apoptosis pathway anti-apoptotic members, such as Leukemia (HL-60) (89) Bcl-2.
*α-TOS activates caspases-3, 6, 8, and 9
and triggeres the reaction leading to the Breast (MCF-7, cleavage of Bid-induce apoptosis (89) MDA-MB-435 ) (114) *α-TOS induces Bax conformational Murine mammary (+SA) change and thereby triggers apoptosis. (115) (114)
*tocotrienol-induced apoptosis in +SA
cells is mediated through activation of the Jurkat (Bcl-2 caspase-8 pathway and independent of over-expressing, DN, caspase-9 (115) CrmA), THP-1, murine *α-TOS-induced apoptosis involves leukemia (NSF/N2.H7,
caspase-3 activation, and both lysosomal S70A, and S70E), M1-1a,
and mitochondrial destabilization (117) and breast (MCF-7,
MCF7-C3) (116
Jurkat T (117)
102 Continued
Table 7.1 continued
Cell cycle *α-TOS blocks cell cycle of HL-60 cells at Leukemia (HL-60) (89)
arrest G2/M phase (89)
*α-TOS cause LNCaP cells arrest at G1 Prostate (LNCaP) (91)
(91)
*α-TOS induces inhibition of DNA Breast (MDA-MB-435) (105)
synthesis and a G0/G1 cell cycle arrest (105)
*γ-tocotrienol causes B16 murine Murine melanoma (B16) melanoma cell cycle arrest at G1 phase (118) (118)
Induction of α-TOS induces differentiation of human Breast (MCF-7,
differentiation MCF-7 and MDA-MB-435 breast cancer MDA-MB-435, and
cells but not normal human mammary MCF-10A) (119-120)
epithelial cells, MCF-10A cells (119)
103
Fig. 7.2 Three subfamilies of Bcl-2-related proteins. “TM” refers to a hydrophobic region in the carboxyl terminus (122).
104
Fig. 7.3 The model for Bcl-2 survival activity in mammals. Bcl-2 and its anti-apoptotic homologues guard mitochondrial membrane integrity until neutralized by a
“BH3-domain only” protein. Bax and Bak then form homo-oligomers within the mitochondrial membrane, resulting in the release of cytochrome c, which activates Apaf1, allowing it to bind to and activate caspases-9. Other pro-apoptotic molecules that exit the mitochondrial include Omi and Diablo, which antagonize inhibitor of apoptosis proteins
(IAPs). Apaf1, apoptotic protease-activating factor 1; cyt c, cytochrome c (122).
105
Fig. 7.4 Caspases inhibitor model for Bcl-2 function. “BH3-domain only” proteins can inhibit Bcl-2 anti-apoptotic members and further activate Bax and Bak. Oligomerization of Bax and Bak then produces damage to the organelles that amplifies the proteolytic cascade. Apaf1, apoptotic protease-activating factor 1; cyt c, cytochrome c (122).
106
Fig 7.5 A Structure of HA14-1, a small molecule that binds Bcl-2 protein. Note that
HA14-1 has two chiral centers (marked by asterisks) located at the C4 position and the carbon atom attached with the cyano group, respectively. B Structural model for the complex of HA14-1 with the Bcl-2 surface pocket as predicted by computer docking calculation (128).
107
CHAPTER 8
PROJECT DESIGN
8.1 Aims and Project Design
Evidence has shown that vitamin E or α-TOS (α- tocopheryl succinate)-induced
apoptosis in certain cancer cell lines is regulated by the mitochondrial pathway.
Specifically, data indicates that α-TOS-induced apoptosis involves Bax translocation
from the cytosol to the mitochondria, cytochrome c release, and processing of caspase-9
and -3 but not caspase-8 to active forms in MDA-MB-435 breast cancer cells (114). In
addition, Jurkat cells became more susceptible to α-TOS treatment after Bcl-2 was
downregulated by antisense ODN (oligodeoxynucleotide) treatment indicating that Bcl-2
family proteins are important for α-TOS-induced apoptosis (116). In γ-tocotrienol-treated
MDA-MB-231 breast cancer cells, the mitochondria were disrupted but the expression of
Bax and Bcl-2 (mRNA and protein) did not change (129). Based on these results, it is proposed that vitamin E and α-TOS trigger apoptosis through binding with Bcl-2/Bcl- XL protein.
Structurally, the molecules of tocopherols, tocotrienols, and α-TOS can be divided into three functionally distinct moieties, based on their possible roles in apoptosis 108 induction (Fig. 8.1) (131). The first domain is the hydrophobic domain, which comprises
the phytyl chain and is essential for docking of the agent into cell membranes and lipoproteins. This domain was also shown to be important for the pro-apoptotic function
of vitamin E analogues, since α-trolox succinate (Fig. 8.2), a water-soluble analogue
of α-TOS lacking the phytyl chain, failed to induce apoptosis. Evidence has also
indicated that the length of the phytyl side chain may also affect their bioactivities (132).
Domain II, or the signaling domain, is responsible for the PKC inhibitory activity of
vitamin E analogues. It has been reported that the varying number and position of methyl
substitutions on the chromanol ring (as occurs in α−, β−, δ−, and γ− tocopherols) affect
the PKC inhibitory activities (99) and tumor cell antiproliferative activities of these
analogues (82). The third domain, or functional domain, is responsible for the antioxidant
and apoptotic activities of vitamin E analogues. The phenol group on the chromanol ring
is required for its radical scavenging function (81). Thus, any derivatives without a
phenol group, such as α-TOS, fail to act as an antioxidant. In contrast, a charged moiety,
such as succinate, is critical for their pro-apoptotic properties in vitro (82). It is noteworthy that this anti-proliferative activity may be enhanced by replacing the ester linkage with an ether linkage between the phenol group with any charged side chain (82), such as [2,5,7,8-tetramethyl-2R-(4’R, 8’R, 12-trimethyltridecyl) chroman-6-yloxyacetic
acid, α−ΤΕΑ, or by reducing the length of the side chain from a succinate to a malonate
or oxalate group (133) (Fig. 8.2). Moreover, the ether-linked analogues of vitamin E
provide another option for anticancer agent development since it will mitigate the administration problem of a hydrolysable succinate group (82).
109 Various vitamin E analogues have been designed and synthesized for different
goals such as improvement of anti-proliferative activity (132) or water-solubility (134).
The relationship between the different functional groups on the three domains of these
vitamin E derivatives and their pro-apoptotic activities has also been reported (132). The
data showed that α-TOS is the most potent apoptotic inducer among four tocopheryl
succinate forms (α−, β−, δ−, and γ−) in selected cancer cell lines. A free carboxylic
group on domain III is pivotal since α-TOS lost its pro-apoptotic activity after
esterification. Also, a reduction in the length of the aliphatic side chain from three to two
isoprenyl units (γ-T3H and γ-T2H, Fig. 8.3A) enhanced apoptogenic activity. Moreover,
the existence of the chromanol ring in the structure is required for pro-apoptotic activity
since phytyl succinate (Fig. 8.3B) failed to induce apoptosis (132). Our new strategy for
development of novel analogues of vitamin E in this project is based on these studies.
8.2 Synthesis of Vitamin E Analogues
Among the known synthetic vitamin E analogues, just one of them, γ-T2H, has been reported to be modified at the hydrophobic domain. Therefore, we first propose to investigate whether the length of the phytyl side chain influences the apoptogenic activity
of vitamin E derivatives. Ten vitamin E analogues were prepared and used in this study.
α−Tocopherol, α−TOS, and 2,2,5,7,8-pentamethyl-6-chromanol (VEA-1) are
commercially available (Table. 8.1). The synthetic procedures for the other seven analogues are described in Scheme 8.1. The compound containing only a chromanol head but without a phytyl side chain, VEA-2, can be obtained by treating VEA-1 with succinic anhydride under basic conditions. The synthetic methods for the other six analogues are
110 similar. After esterification of the acid group of the starting material,
tetramethylchroman-2-carboxylic acid, the phenol position can be protected by benzyl
bromide. Following reduction of the ester to aldehyde by DIBAL, the Wittig reaction was
employed to increase the length of the phytyl side chain. Products with two isopranyl
units (VEA-3) and one isopranyl unit (VEA-4) can be constructed after reduction of the
C-C double bond which was generated by the Wittig reaction. Succinic anhydride was
used to add succinate to the functional domain on VEA-3 and VEA-4 to generate VEA-5
and VEA-6. VEA-7 and VEA-8, which possess an ether linkage instead of an ester
linkage, were obtained by substitution reactions with VEA-3 and VEA-4 under basic
conditions.
These compounds with different phytyl chain lengths and with or without a
succinate group are useful for determining whether the hydrophobic and/or functional
domains play a key role in the pro-apoptotic activities of these agents. Moreover, the
ether-linked compounds not only improve the stability of these compounds in the
digestive tract, but also may increase their abilities to induce apoptosis in prostate caner cell lines.
111 O O HO O O α-TOS
Functional Signalling Domain III Domain II Hydrophobic Domain I
Fig. 8.1 Domains in vitamin E analogues
112 O O HO O O COO-
α-Trolox Succinate
O O HO
O
α-TEA
HO O
O O O
α-Tocopheryl malonate
O O HO O O
α-Tocopheryl oxalate
Fig. 8.2 Some vitamin E analogues
113 (A) HO
O
γ-geranylchromanol (γ-T2H) apoptotic activity: 55.8 %
HO
O
γ-tocotrienol (γ-T3H) apoptotic activity: 38.2 %
(B)
O O
O O
phytyl succinate (PYS)
Fig. 8.3 A Chemical structures of γ-T2H and γ-T3H. The apoptotic activity was assessed in Jurkat cells (5x105 per mL) exposed to individual VE analogues at 50 μM for 12 h.
The annexin V-FITC binding method was used, and the numbers indicate the percentage
of the cells positive for annexin V (135). B Structure of phytyl succinate
114
Table 8.1 Structures of vitamin E analogues which are used in this study.
115 O O O O O O HO O HO Py O VEA-1 VEA-2
O O CHO O 1. MeOH, p-TsOH CO2Me DIBAL, Ether CO2H
2. K2CO3, BnBr BnO HO BnO 1 O R O O O O R HO O O 1. Ph3PCH2RBr O 2. Pd/C, H2 HO Py VEA-5 R=
1. BrCH2CH2CO2H VEA-6 R= VEA-3 R= KOH, DMF
VEA-4 R= 2.HCl O R O
HO O
VEA-7 R=
VEA-8 R=
Scheme 8.1 Synthetic procedures of VEA-1 to VEA-8. DIBAL = diisobutylaluminium hydride. Py = pyridine.
116
CHAPTER 9
VITAMIN E ANALOGUES INDUCE APOPTOSIS THROUGH
INHIBITING BCL-2 /BCL-XL FUNCTION
9.1 Vitamin E Analogues Induce Apoptosis in Prostate Cancer Cells
We first examined the dose-dependent growth inhibition effects of the ten vitamin E analogues by MTT assay in androgen-independent PC-3 (p53-/-) and androgen-dependent
LNCaP (p53+/+) prostate cancer cell lines, and the results are shown in Table. 9.1. Both
α-tocopherol and VEA-3, which contains a phytyl side chain that is one isopranyl unit shorter than that of α-tocopherol and lacks a succinate group at the phenol position, failed to induce apoptosis in either cancer cell line even at a high concentration (100 μM). The two compounds without a phytyl side chain, VEA-1 and VEA-2, also did not induce apoptosis in these cell lines. In contrast, α-TOS inhibited cell proliferation by 50% at
40 μM for PC-3 cells and 15.2 μM for LNCaP cells under serum-free conditions after 24 hours treatment. Compared to α-TOS, the other five novel synthetic vitamin E analogues exhibited better pro-apoptotic activities. The two compounds with phytyl side chains containing two isopranyl units, VEA-5 and VEA-7, were more potent than their counterparts with shorter phytyl side chains (VEA-6 and VEA-8). Similar to the
117 conclusions of a previous study of vitamin E analogues (132), our results also indicate
that a carboxylic group, such as succinate or propanoic acid groups (on VEA-7 and
VEA-8), are critical for anti-proliferative activity, since α-tocopherol and VEA-3 do not
exhibit any pro-apoptotic activity. The phytyl side chain also plays a role in the induction
apoptosis in prostate cancer cells, because VEA-2 lost antiproliferative activity in both
cell lines even though it contains a succinate group. In addition, VEA-5 showed better
apoptosis-inducing activity than α-TOS. Our results are the first to show that compounds
with two isopranyl units in the phytyl side chain possess better anti-proliferative activity,
and that the replacement of the ester linkage at the phenol position to an ether linkage
improves the apoptogenic activity as well. Furthermore, LNCaP cells were shown to be
more susceptible than PC-3 cells to these vitamin E analogues. VEA-4, however, is a special case among these ten vitamin E analogues, as it displays good apoptogenic activity despite the absence of a succinate group and the presence of just one isopranyl unit in the phytyl side chain.
The occurrence of apoptotic death in these vitamin E analogues-treated PC-3 cells
was determined by evaluating DNA fragmentation as shown in Fig. 9.1, cytochrome c
release as shown in Fig 9.2, and poly(ADP-ribose) polymerase (PARP) cleavage as
shown in Fig. 9.3. DNA fragmentation is very typical of the apoptotic process with
generation within the nucleus of a series of multiples of a 180 bp DNA subunit, through
the action of a Ca2+/Mg2+-dependent endonuclease with cleaves DNA in the linker region
between nucleosome cores. The formation of nucleosomal DNA was measured in PC-3
cells that were treated with α-TOS or VEA-7 at indicated concentrations for 24 hours.
The fragmentation of DNA in PC-3 cells exhibited an apparent increase after 50 μM 118 α-TOS or 15 μM VEA-7 treatments indicating that VEA-7 is a better apoptotic inducer
than α-TOS (Fig. 9.1). As mentioned in Chapter 7, cytochrome c will be released from mitochondria into the cytoplasm after the loss of mitochondrial membrane integrity resulting in further apoptotic signaling. As shown in Fig. 9.2, cytochrome c was released
in PC-3 cells after α-TOS and VEA-7 treatment in dose- and time-dependent manners. In
addition, both of these agents induced PARP cleavage as shown by Western blotting
analysis (Fig. 9.3). The PARP antibody detects endogenous levels of full length PARP
(116 kDa), as well as the large fragment (89 kDa) of PARP generated from
caspase-mediated cleavage.
9.2 Vitamin E Analogues Induce Apoptosis through Inhibiting Bcl- XL Function
To examine the effects of α-TOS and VEA-7 on Bcl-2 family proteins, we assessed
the dose-dependent effect of both agents on the expression levels of different Bcl-2
family members in PC-3 cells, including Bcl-XL, Bcl-2, Bax, Bak, Bad, and Bid. Figure
9.4 shows that, with the exception of a decrease in Bad expression, exposure of PC-3
cells to α-TOS or VEA-7 at the indicated concentrations did not cause an appreciable
change in the expression level of any of these Bcl-2 members.
In light of the recent discovery of small-molecule Bcl-2 or Bcl- XL inhibitors that disrupt BH3 domain–mediated interactions with pro-apoptotic Bcl-2 members (136), we investigated the in vitro effects of α-TOS and VEA-7 on the anti-apoptotic function of
Bcl-XL. It is well understood that when Bcl-XL and Bcl-2 form heterodimers via the BH3 domain with pro-apoptotic Bcl-2 members such as Bak, their anti-apoptotic functions will
be inhibited. Therefore, a well-established competitive FP analysis (FP) was applied to
119 demonstrate the effects of vitamin E analogues on the binding of a Bak BH3 domain
peptide to Bcl-XL. The results from the competitive FP analysis suggest that some of
these vitamin E analogues inhibited the anti-apoptotic functions of Bcl-XL by disrupting
the BH3 domain–mediated interactions with pro-apoptotic Bcl-2 members (Fig 9.5).
Except for VEA-4 and α-TOS, the results of FP analysis of the other vitamin E analogues
paralleled their cell viability data. For example, VEA-7 (IC50 = 7 μM) exhibited higher
binding affinity to Bcl-XL than VEA-8 (IC50 = 25 μM), as well as better pro-apoptotic
activity than VEA-8 (IC50 = 8.2 μM for VEA-7 and 16.7 μM for VEA-8) in PC-3 cells. In
addition, inefficient apoptotic inducers, such as α-tocopherol, VEA-1, VEA-2, and
VEA-3, also showed poor interactions with Bcl-XL. However, VEA-4, which displayed
anti-proliferative activity (IC50 = 13.5 μM) did not show binding affinity to Bcl-XL even at a high concentration (50 μM), indicating that VEA-4 inhibits cell proliferation by a different mechanism. In contrast, α-TOS exhibited binding affinity to Bcl-XL comparable
to that of VEA-5, VEA-6, and VEA-8, but its pro-apoptotic activity was weaker than
those of these three agents. Thus, this finding suggests that the effects of these
Bcl-XL-active analogues on cell proliferation may also involve regulation of other
signaling pathways.
LNCaP cells were shown to exhibit lower Bcl-XL expression levels than PC-3 cells
(139), which may be the basis for the differences between these two cell lines in their
susceptibilities to the apoptotic effects of α-TOS and other vitamin E analogues. To
further confirm that the inhibition of Bcl-XL functions plays a key role in apoptosis induction by these agents, we examined the impact of Bcl-XL overexpression on
120 susceptibility to the apoptotic effects of α-TOS and other vitamin E analogues in LNCaP
cells. The Bcl-XL-overexpressing LNCaP cell line, B3-LNCaP, was used for this study.
The relative expression levels of Bcl-XL in LNCaP and B3-LNCaP cells were demonstrated in our previous work (62). Table 9.2 lists the IC50 values for
anti-proliferative activities of our series of vitamin E analogues in both LNCaP cell lines.
Although the overexpression of Bcl-XL in B3-LNCaP cells provided partial protection
against vitamin E analogue-mediated apoptosis, this effect was not remarkable. For
example, the IC50 for VEA-7 after 24 hours of treatment was 2.8 μM in LNCaP cells,
while that for B3-LNCaP cells was 6.2 μM. Therefore, another mechanism(s) might be
also involved in the induction of apoptosis in LNCaP cells.
9.3 Effect of α-TOS and VEA-7 on Intracellular Bcl-2 and Bcl-XL Binding to Bak
It has been reported that Bcl-2 and Bcl-XL sequester Bax, Bak, and other
pro-apoptotic Bcl-2 members through BH3 domain-mediated heterodimerization, thereby
abrogating their pro-apoptotic effects (137-138). Therefore, the Bcl-2/Bax heterodimer
has been used as a target for drug development since it may provide a therapy particularly
suited to tumor cells for which resistance to conventional therapy is associated with
elevated expression of Bcl-2. For instance, synthetic peptide sequences derived from the
BH3 domain of pro-apoptotic Bcl-2 family members was induced in prostate tumor cells
to block the heterodimerization of Bcl-2/Bax resulting in apoptosis in up to 40% of cells
at 48 hours after introduction (138). To validate the mode of action of α-TOS and VEA-7, we assessed the effects of the agents on the dynamics of Bcl-2/Bak and Bcl-xL/Bak interactions in PC-3 cells. Lysates from PC-3 cells treated with α-TOS or VEA-7
121 vis-a`-vis DMSO for 24 hours were immunoprecipitated with antibodies against Bcl-2 or
Bcl-XL. Probing of the immunoprecipitates with anti-Bak antibodies by Western blotting indicated that the level of Bak associated with Bcl-2 and Bcl-XL was significantly
reduced in drug-treated cells compared with the DMSO control (Fig. 9.6). This
immunoprecipitation study revealed that both α-TOS and VEA-7 inhibited the
interactions of Bcl-XL and Bcl-2 with the Bak BH3-domain peptide. We further showed
that treatment of PC-3 cells with α-TOS or VEA-7 led to caspase-9 activation in a
dose-dependent manner (Fig. 9.7). Caspase-9 is a critical component of the intrinsic
apoptotic pathway, which is activated by Apaf-1 in the apoptosome, a large complex
assembled in response to release of cytochrome c from the mitochondria. Caspase-9
cleaves and activates effector caspases, predominantly caspase-3, resulting in the demise
of the cell. These findings indicate that α-TOS and VEA-7 inhibit the interactions of
Bcl-XL and Bcl-2 with Bak, and further induce cytochrome c release, caspase-9 activation, and PARP cleavage.
9.4 Vitamin E Analogues Induce Apoptosis Selectively in Prostate Cancer Cells but
Not in Normal Prostate Epithelial Cells (PrEC)
Vitamin E and its analogue, α-TOS, gained considerable attention as potential
anticancer agents with high selectivity for malignant cells and low toxicity. Evidence
showed that 50 μM α-TOS for 12 hours triggered apoptosis in 28-65% of certain cancer
cell lines, while some normal cell types, such as normal skin fibroblasts, were resistant to
the treatment (140). To investigate whether our series of vitamin E analogues also exhibit
high selectivity to induce apoptosis in prostate cancer cells, primary normal prostate
122 epithelial cells, PrEC, were used to determine their sensitivity to vitamin E analogues.
As shown in Fig 9.8, compared to PC-3 or LNCaP cells, PrEC cells showed no susceptibility to apoptosis induced by α-TOS or other vitamin E analogues. The finding suggests that α-TOS and these vitamin E analogues exhibit a selective toxicity for malignant prostate cells. Thus, our vitamin E analogues, especially VEA-7, which is a more potent pro-apoptotic agent than α-TOS, are promising anticancer agents.
123 IC (μM) IC (μM) compounds structures 50 50 in PC3 cells in LNCaP cells α-tocopherol >100 >100 (VE) α-TOS 40.0 15.2
VEA-1 >100 >100
VEA-2 >100 >100
VEA-3 >100 >100
VEA-4 13.5 7.2
VEA-5 9.1 3.8
VEA-6 18.8 8.1
VEA-7 8.2 2.8
VEA-8 16.7 6.9
Table. 9.1 Structures and anti-proliferative activities in PC-3 and LNCaP cells of the ten vitamin E analogues used in this study. IC50 represents the concentration of a drug that is required for 50% inhibition of cell viability in vitro. PC-3 or LNCaP cells were exposed to each vitamin E analogue in serum-free RPMI 1640 medium in 96-well plates for 24 hours and cell viability was assessed by MTT assay which is described in Chapter 11.
124
Fig. 9.1 Formation of nucleosomal DNA in PC-3 cells that were treated with α-TOS or
VEA-7 at the indicated concentrations for 24 hours. DNA fragmentation was quantitatively measured by a cell death detection ELISA kit.
125
Fig 9.2 Dose- and time-dependent effects of α-TOS and VEA-7 on cytochrome c release
in PC-3 cells. The cells were treated with either agent at the indicated concentrations for
the indicated time periods in serum-free RMPI 1640 medium and mitochondria-free lysates were prepared. Equivalent amounts of protein from individual lysates were electrophoresed and probed by Western blotting with anti–cytochrome c and β-actin antibodies.
126
Fig. 9.3 PARP cleavage by α-TOS and VEA-7 in PC-3 cells. PARP proteolysis to the apoptosis-specific 85-kDa fragment was monitored by Western blotting. Cells were treated with α-TOS or VEA-7 with at the indicated concentrations in serum-free RPMI
1640 medium for 24 hours.
127
Fig 9.4 Effect of α-TOS and VEA-7 on the expression levels of Bcl-2 family members in
PC-3 cells. Cells were exposed to each reagent at the indicated concentrations in serum-free RPMI 1640 medium for 24 hours.
128
Fig 9.5 Differential inhibition of BH3 domain-mediated protein interactions of Bak BH3 peptide with Bcl-XL by vitamin E analogues. Displacement of Flu-Bak BH3 peptide from
Bcl-XL by α-TOS and VEA-7 (upper). IC50 values of ten vitamin E analogues for
inhibiting the BH3-mediated protein interactions (bottom).
129
IC50 (μM) IC50 (μM) compounds in LNCaP cells in B3-LNCaP cells α-tocopherol >100 >100 (VE)
α-TOS 15.2 23.1
VEA-1 >100 >100 VEA-2 >100 >100
VEA-3 >100 >100 VEA-4 7.2 13.1
VEA-5 3.8 7.8 VEA-6 8.1 12.2
VEA-7 2.8 6.2 VEA-8 6.9 10.5
Table 9.2 Ectopic Bcl-XL protects LNCaP cells from vitamin E analogue-induced suppression of proliferation. B3-LNCaP is a Bcl-XL overexpressing prostate cancer cell line. IC50 represents the concentration of a drug that is required for 50% inhibition of cell viability in vitro. LNCaP or B3-LNCaP cells were exposed to each vitamin E analogue in serum-free RPMI 1640 medium in 96-well plates for 24 hours and cell viability was assessed by MTT assay which is described in Chapter 11.
130
Fig. 9.6 α-TOS and VEA-7 trigger apoptotic death in PC3 cells by inhibiting heterodimer formation of Bcl-2 and Bcl-XL with Bak. Effect of α-TOS and VEA-7 on the dynamics of Bcl-2/Bak (upper) and Bcl-xL/Bak (bottom ) interactions in PC-3 cells. PC-3 cells were treated with 40 μM α-TOS or 10 μM VEA-7 for 24 hours, and cell lysates were immunoprecipitated with anti-Bcl-2 or anti-Bcl-xL antibodies. The immunoprecipitates were probed with anti-Bak antibodies by Western blot analysis (WB) which is described in Chapter11.
131
Fig. 9.7 Dose-dependent effect of α-TOS and VEA-7 on caspase-9 activation in PC-3 cells. PC-3 cells were treated with α-TOS or VEA-7 in serum-free RPMI1640 medium at the indicated concentrations for 24 hours. Caspase-9 antibodies recognize the large
subunits (35 and 37 kDa).
132
IC50 (μM) IC50 (μM) IC50 (μM) compounds in PrEC cells in PC3 cells in LNCaP cells
α-TOS >100 40.0 15.2
VEA-4 >50 13.5 7.2
VEA-5 >40 9.1 3.8
VEA-6 >50 18.8 8.1 VEA-7 >40 8.2 2.8 VEA-8 >50 16.7 6.9
Fig. 9.8 Normal prostate epithelial cells (PrEC) are resistant to vitamin E analogue-induced suppression of proliferation. IC50 represents the concentration of a drug
that is required for 50% inhibition of cell viability in vitro. PrEC cells were cultured in
96-well plates at the recommended seeding density in optimized medium for three days before drug treatment. Cells were then exposed to each vitamin E analogue in optimized medium in 96-well plates for 18 hours after which cell viability was assessed by MTT assay. Cell culture conditions and the MTT assay are described in Chapter 11.
133
CHAPTER 10
CONCLUSIONS AND FUTURE DIRECTIONS
10.1 Vitamin E Analogues Induce Apoptosis in Part by Inhibiting Bcl-2/Bcl-XL
Function in Prostate Cancer Cells
Mounting evidence shows that vitamin E and its derivatives, such as α-TOS, are able to induce malignant cells to undergo apoptosis in vitro and in vivo (140). The mechanism of this growth inhibitory effect in cancer cells, however, is still under investigation. Our study demonstrated that α-TOS and some analogues of α-tocopherol induce apoptosis in PC-3 and LNCaP prostate cancer cells, at least in part, through inhibition of Bcl-2/Bcl-XL functions. The growth inhibition data in PC-3 and LNCaP
cells reveal that the carboxylic acid group, such as succinate, at the phenol position and the length of the isoprenoid side chain on these vitamin E analogues are essential for their anti-proliferative activities. Analogues without a carboxylic group at the phenol position, such as α-tocopherol and VEA-3, did not show any apoptosis-inducing activities up to
100 μM in both PC-3 and LNCaP cells. Analogues without an isoprenoid side chain, such as VEA-1 and VEA-2, also failed to induce apoptosis. Moreover, the derivatives with two-unit isopranyled side chains (VEA-5 and VEA-7) were more potent than those with
134 three (α-TOS) or one (VEA-6 and VEA-8) isopranyl units. The ether linkage at the
phenol position also plays a role in improving anti-proliferative activities. VEA-7 and
VEA-8, which possess an ether linkage instead of an ester linkage, are more potent than
their counterparts VEA-5 and VEA-6. The structures of these analogues are shown on
Table 9.1. Furthermore, assessment of DNA fragmentation, cytochrome c release, and
PARP cleavage demonstrated that inhibition of PC-3 cell proliferation by these vitamin E analogues is mediated through triggering apoptosis.
The apoptogenic mechanism of these vitamin E analogues was further investigated
using FP analysis, which revealed that induction of apoptosis in PC-3 cells by α-TOS and
VEA5-8 correlates with their abilities to inhibit BH3 peptide binding to Bcl-XL.
Moreover, immunoprecipitation experiments and evaluation of caspase 9 cleavage showed that α-TOS and VEA-7 compete with pro-apoptotic Bcl-2 member, Bak, for binding with Bcl-2 and Bcl-XL resulting in apoptosis. These findings are consistent with
our observation that the expression levels of most Bcl-2 members, including Bcl-2,
Bcl-XL, Bax, Bak, and Bid, remain unaffected by treatment with these agents.
It should be noted that Bcl-XL overexpression offers only slight protection against
apoptosis in LNCaP cells treated with vitamin E analogues. Thus, the FP data alone
cannot completely elucidate the relationship between binding affinity to Bcl-XL and
anti-proliferative activities of α-TOS and VEA-4. Although several lines of evidences
have attributed induction of apoptosis by α-TOS and some vitamin E analogues to their binding with Bcl-2/Bcl-XL, the precise mechanism remains to be fully clarified.
135 10.2 Development Bcl-2/Bcl-XL Inhibitors Based on Vitamin E Scaffold
To more fully explore the mechanism of vitamin E anlologue-induced apoptosis in cancer cells and to facilitate the identification of potent derivatives with therapeutic potential, a larger library of vitamin E analogues should be established. A parallel synthesis approach would facilitate the creation of a larger library (around seventy compounds) of vitamin E analogues. The main structure of tocopherols (compound 14,
Scheme 10.1), the chromanol ring, can be constructed by applying the synthetic route published by Muller (141). The protected and reduced synthetic blocks 15α and 15γ can be obtained after three more steps. Based on the previous study (132), only α−and γ− analogues are considered.
Three different lengths of phytyl chains can be created in vitamin E analogues after 15α and 15γ react with the synthetic blocks 18a-c (Scheme 10.2). Compounds 17b and 17c are not commercially available, but can be generated by bromination of respective alcohols 16b and 16c. The phosphoium salts 18a-c are ready for the use of
Witting olefination with building blocks 15α and 15γ. Six compounds 19a-c (α and
γ forms) have been obtained so far and the future hydrogenation can double the size of library. The major part of vitamin E has been finished, and all these protected (19a-c and
20a-c) or further deprotected (21a-f, Scheme 10.3) analogues can be tested by various bioassays.
Finally, the creation of different functional groups at domain III of the α-TOS analogues can be completed by the reactions of 21a-f with the selected five building blocks 22-26. The selection of the five building blocks 22-26 is based on the previous
136 literature (132-133). Succinyl is the most studied charged functional group in vitamin E
derivatives, such as α-TOS, and it can be synthesized through the reaction of 21a-f and succinate anhydride (compound 22). Maleyl (compound 28), oxalyl (compound 29), and
malonyl (compound 30) tocopherols have been reported that possess higher pro-apoptotic
activities than α-TOS. The ether-linkage compounds (31a-f and 32a-f) are also chosen
because the novel α-tocopherol analogue, α-TEA (Fig. 8.2) and VEA-7, exhibited more
potent antiproliferative property than α-TOS in MDA-MB-435 breast cancer cells (82)
and PC-3 prostate cancer cells respectively (Table 9.1) (Scheme 10.3).
This library of vitamin E analogues can be further extended by increasing
building blocks 18 and/or changing functional groups at the succinate site. (Changing the
chromanol ring was not considered at the beginning since this is the most characteristic
part of vitamin E). The additional structural modification of these vitamin E analogues
will be based on the results of bioactivity investigations. For example, if 32e, another the ether-linkage compound, possesses more potent pro-apoptotic activity than VEA-7, other ether-linkage compounds with different carbon numbers can be designed.
Parallel synthesis is a more efficient approach to building a larger library of vitamin E analogues. Such a library would not only provides more information about the
structure-activity relationships of these compounds, but also help to clarify the precise
mechanism by which these vitamin E analogues-induced apoptosis. In addition, molecular modeling should also be utilized to identify the possible binding sites of these
compounds with Bcl-2 and Bcl-XL proteins. The assistance of molecular modeling and a
moderate-sized library of vitamin E analogues will enhance the potential to discover
more potent Bcl-2/Bcl-XL inhibitors. 137 10.3 Future Directions
α-TOS has been shown to induce apoptosis in a multitude of malignant cell types without apparent toxicity in normal cells (131). Indeed, our own results show that normal prostate epithelial cells (PrEC) are insensitive to the inhibitory effects of α-TOS and our synthetic vitamin E analogues (Fig. 9.8). In addition, intraperitoneal administration of
α-TOS reduced the growth of certain tumors in xenograft-bearing athymic nude mice, whereas oral or subcutaneous administration in rodents was ineffective suggesting that most of α-TOS may be hydrolyzed before reaching the target tissue (142). Thus, our novel synthetic vitamin E analogue, VEA-7, holds some promise as a potential chemotherapeutic drug since it is a more potent apoptosis inducer than α-TOS and is not subject to hydrolysis after oral administration. In vivo studies are necessary to confirm this postulate.
It has been reported that α-TOS enhanced the growth-inhibitory effects of several chemotherapeutic agents on cancer cells. For example, α-TOS improved the inhibitory effects of adriamycin on human prostate carcinoma cells, and cisplatin and tamoxifen on human melanoma cells (142). Therefore, inclusion of VEA-7 in combination regimens with different chemotherapeutic agents should be explored as a potential approach in the treatment of cancer.
Another application of vitamin E analogues is in the context of chemoprevention.
Preclinical, epidemiological, and phase III data from randomized, placebo-controlled
138 clinical trials suggest that vitamin E has potential efficacy in prostate cancer prevention
(143). Thus, it is worthwhile to investigate whether the analogues of vitamin E can
provide better preventive effects on prostate cancer.
Finally, vitamin E is under investigation, not only as a cancer therapeutic agent, but also as a potential anti-atherogenic agent. Thus, the vitamin E analogues might also be employed to treat atherosclerosis. It is suggested that the redox activity of α-tocopherol plays a pivotal role in preventing the development of atherosclerosis. Therefore, vitamin
E analogues in the library which possess a free phenol group on the chromanol ring, such as VEA-3, might be candidates worthy of investigation as anti-atherogenic agents.
139 R1 HO O R1 OH HO O MgBr O O O OH O O THF BF3.Et2O/dioxane 14
R1 R1 MeOH, TsOH HO O TBSO O TBS-Cl, Imidazole OMe OMe Reflux O DMF O
DIBAL R1 TBSO O o CH Cl , -60 C 15α, R1=CH3 2 2 H O 15γ, R1=H
15
Scheme 10.1 Designed synthetic procedures to construct the main structure of vitamin E, the chromanol ring. TBS-Cl represents tert-butyldimethylsilyl chloride. DIBAL represents diisobutylaluminium hydride.
140 Br PPh3 Br Ph3P Toulene, reflux 17a 18a
PPh PPh3, CBr4 3 Br HO Br Ph3P n CH Cl n Toulene, reflux n 16 2 2 17 18 16b, n=1 17b, n=1 18b, n=1 16c, n=2 17c, n=2 18c, n=2
R R R1 1 1 TBSO O TBSO TBSO 18a-c H2, 10% Pd/C H - + O O O t-BuO Na , THF n Hexane n 15 19 20 19a, n=1 20a, n=1 19b, n=2 20b, n=2 19c, n=3 20c, n=3
Scheme 10.2 Designed synthetic procedures to construct saturated or unsaturated phytyl side chains with different lengths. Py represents pyridine.
141 O R1 HO O O O R2 27a-f, succinate O R1 HO O O O O O O R O O 2 Py 28a-f, maleate 22 23 O
Py
R O 1 R Cl TBSO 1 Cl HO O R R2 TBAF O 1 O R2 24 O O HO THF, 0 oC O O R2 19 or 20 21a-f Cl O 29a-f, oxalate O
21a-c R2 = O 25 Cl n NaH Br 21a, n = 1 O R1 21b, n = 2 26 HO O 21c, n = 3 O O O R2 O R1 21d-f R2 = O n O 30a-f, malonate 21d, n = 1 O R2 21e, n = 2 NaOH(aq) 21f, n = 3 31a-f O R1 HO O
O R2
32a-f
Scheme 10.3 Designed synthetic procedures to create different carboxylic groups at the phenol position of tocopherols or tocotrienols.
142
CHAPTER 11
EXPERIMETNATL METHODS AND MATERIAL FOR PART 2
11.1 Reagents
α-Tocopherol, α-tocopheryl succinate, 2,2,5,7,8-pentamethyl-6chromanol (VEA-1)
and all required chemical reagents for the synthesis of vitamin E analogues were purchased from Sigma (St. Louis, MO). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
-2H-tetrazolium bromide)] for cell viability assay were purchased form TCI America, Inc.
(Portland, OR). The Cell Death Detection ELISA kit was purchased from Roche
Diagnostics (Mannheim, Germany). Rabbit antibodies against Bcl-xL, Bax, Bak, Bid,
PARP, cleaved caspases-9, and caspase-9 were purchased from Cell Signaling
Technology, Inc. (Beverly, MA). Rabbit antibodies against Bad, cytochrome c, and
mouse anti-Bcl-2 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse
monoclonal anti-actin was from ICN Biomedicals, Inc. (Costa Mesa, CA).
11.2 Cell Culture
LNCaP androgen-dependent (p53+/+) and PC-3 androgennonresponsive (P53-/-) prostate cancer cells were obtained from the American Type Culture Collection (Manassas, VA).
PC-3, LNCaP, and stable clone B3-Bcl-xL-overexpressing LNCaP cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified 143 incubator containing 5% carbon dioxide. Normal human prostate epithelial (PrEC) cells were purchased from Cambrex Bio Science Walkersvill, Inc. (East Tutherford, NJ) within
CloneticsTM Prostate Epithelial Cell Systems. Cells were maintained in Prostate Epithelial
Cell Basal Medium (CC-3166) which contains growth supplements: BPE, hydrocortisone, hEGF, epinephrine, transferrin, insulin, retinoic Acid, triiodothyronine, and GA-1000 at
37 °C in a humidified incubator containing 5% carbon dioxide. The recommended seeding density for subculture is 2,500 cells/cm2. It takes 6-9 days from subculture to confluent monlayer.
11.3 Cell Viability Analysis
The effect of individual test agents on cell viability was assessed by using the MTT assay in 6 to 12 replicates. PC3, LNCaP, and B3-LNCaP cells were seeded and incubated in 96-well, flat-bottomed plates in RPMI 1640 supplemented with 10% FBS medium for
24 hours. PrEC cells were seeded at recommend density in 96-well, flat-bottomed plates in Prostate Epithelial Cell Basal Medium with growth supplements for 3 days. All cells and were exposed to various concentrations of test agents dissolved in DMSO or ethanol
(α-tocopherol, α-TOS, VEA-1, and VEA-2) with final concentration, 0.1% in serum-free
RPMI 1640 for PC3, LNCaP, and B3-LNCaP cells or in Prostate Epithelial Cell Basal
Medium with growth supplements for PrEC cells respectively. Controls received DMSO or ethanol vehicle at a concentration equal to that in drug-treated cells. The medium was removed, replaced by 200 μL of 0.5 mM MTT in 10% FBS–containing RPMI 1640, and cells were incubated in the carbon dioxide incubator at 37 °C for 2 hours. Supernatants were removed from the wells and the reduced MTT dye was solubilized in 200 μL/well
DMSO. Absorbance at 570 nm was determined on a plate reader. 144 11.4 Apoptosis Detection by ELISA
Induction of apoptosis was assessed with a Cell Death Detection ELISA kit (Roche
Diagnostics) following the manufacturer’s instruction. This test is based on the
quantitative determination of cytoplasmic histone–associated DNA fragments in the form
of mononucleosomes and oligonucleosomes after induced apoptotic death. In brief, 1 ×
106 cells were cultured in a T-25 flask in 10% FBS–containing medium for 24 hours, and
were treated with the test agents at various concentrations in serum-free medium for 24
hours. Both floating and adherent cells were collected; cell lysates equivalent to 5 × 105 cells were used in the ELISA.
11.5 Western Blot Analysis of Cytochrome c Release into the Cytoplasm
Cytosolic-specific, mitochondria-free lysates were prepared according to an established procedure (139). In brief, after individual treatments for 24 hours, both the incubation medium and adherent cells in T-75 flasks were collected and centrifuged at
600 × g for 5 minutes. The pellet fraction was recovered, placed on ice, and triturated with 100 μL of a chilled hypotonic lysis solution [50 mM PIPES-KOH (pH 7.4) containing 220 mM mannitol, 68 mM sucrose, 50 mM KCl, 5 mM EDTA, 2 mM MgCl2,
1 mM dithiothreitol (DTT), and a mixture of protease inhibitors including 100 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 80 nM aprotinin, 5μM bestatin, 1.5 μM E-64 protease inhibitor, 2 μM leupeptin, and 1 μM pepstatin A]. After a 45-minute incubation on ice, the mixture was centrifuged at 600 × g for 10 minutes. The supernatant was collected in a microcentrifuge tube, and centrifuged at 14,000 rpm for 30 minutes. An 145 equivalent amount of protein (50 μg) from each supernatant was resolved in 15 %
SDS-polyacrylamide gel. Bands were transferred to nitrocellulose membranes and analyzed by immunoblotting with anti–cytochrome c antibodies as described below.
11.6 Immunoblotting
Cells were seeded in 10% FBS-containing RPMI-1640 medium for 24 h and treated
with various agents as indicated. After individual treatments for 24 h, both the incubation
medium and adherent cells in T-25 or T-75 flasks were scraped and collected by centrifugation at 2200 rpm for 10 min. The supernatants were recovered, placed on ice, and triturated with 20 to 50 μL of a chilled lysis buffer (M-PER Mammalian Protein
Extraction Reagent; Pierce, Rockford, IL), to which was added 1% protease inhibitor cocktail (set III; EMD Biosciences, Inc., San Diego, CA). After a 30-min incubation on ice, the mixture was centrifuged at 16,100 × g for 3 min. Two μL of the suspension was taken for protein analysis using the Bradford assay kit (Bio-Rad, Hercules, CA); to the remaining solution was added the same volume of 2× SDS-polyacrylamide gel electrophoresis sample loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 5%
β-mercaptoethanol, 20% glycerol, and 0.1% bromphenol blue). The mixture was boiled for 10 min. Equal amounts of proteins were loaded onto 8-12% SDS-polyacrylamide gels.
After electrophoresis, protein bands were transferred to nitrocellulose membranes in a semidry transfer cell. The transblotted membrane was blocked with Tris-buffered saline/0.1% Tween 20 (TBST) containing 5% nonfat milk for 90 min, and the membrane was incubated with the appropriate primary antibody in TBST/5% nonfat milk at 4 °C overnight. After washing three times with TBST for a total of 45 min, the transblotted
146 membrane was incubated with goat anti-rabbit or anti-mouse IgG-horseradish peroxidase
conjugates (diluted 1:1000) for 1 h at room temperature and washed four times with
TBST for a total of 1 h. The immunoblots were visualized by enhanced
chemiluminescence.
11.7 Competitive Fluorescence Polarization Assay
The binding affinity of the test agent to Bcl-XL was analyzed by a competitive fluorescence polarization assay in which the ability of the agent to displace the binding of a Bak BH3–domain peptide to either Bcl-2 or Bcl-XL was determined. Flu-BakBH3, a
Bak-BH3 peptide labeled at the NH2 terminus with fluorescein, was purchased from
Genemed Synthesis (San Francisco, CA). COOH-terminal-truncated, His-tagged Bcl- XL was purchased from EMD Biosciences (San Diego, CA) and soluble glutathione
S-transferasefused Bcl-2 was obtained from Santa Cruz Biotechnology. The KD determination was carried out in a dual–path length quartz cell with readings taken at
Eem 480 nm and Eex 530 nm at room temperature using a luminescence spectrometer according to an established procedure (144).
11.8 Determination of IC50 Values
Data from cell viability and fluorescence polarization assays were analyzed by using
the CalcuSyn software (Biosoft, Ferguson, MO) to determine IC50 values, in which the
calculation was based on the medium-effect equation [i.e., log(fa/fu) = mlog(D) -
mlog(Dm), where fa and fu denote fraction affected and unaffected, respectively; m represents the Hill-type coefficient signifying the sigmoidicity of the dose-effect curve; and D and Dm are the dose used and IC50, respectively (62).
11.9 Co-immunoprecipitation 147 PC3 cells treated with 40 μM α-TOS or 10 μM VEA-7 for 24 hours were scraped off the
flask, transferred into centrifuge tubes, and centrifuged at 2200 rpm for 10 min to pellet the cells. The pellet was resuspended in ice-cold 0.5 mL of radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate,
150 mM NaCl, 1 mM EDTA, and 1% protease inhibitor cocktail) and gently mixed on an orbital shaker at 4 °C for 15 min, followed by centrifugation at 14,000× g for 15 min to yield cell lysates. These cell lysates were treated with 100 μL of protein A-agarose bead slurry followed by brief centrifugation to remove nonspecific binding proteins. Equal amounts of proteins from these lysates, as determined by the Bradford assay, were mixed with anti-Bcl-2 or anti Bcl-XL antibodies in an orbital shaker at 23 °C for 2 h, followed
by 100 μL of protein A-agarose bead slurry at 4 °C for 12 h. The immunocomplex was
collected by brief centrifugation, washed four times with 800 μL of ice-cold
radioimmunoprecipitation assay buffer, and suspended in 50 μL of 2× SDS sample
loading buffer. The suspension was boiled for 10 min, cooled, and briefly centrifuged to
remove the beads. Western blot analysis with antibodies against Bak as described above.
11.10 General Information on Chemical Methods
Reactions involving moisture-sensitive reagents were carried out under an inert
atmosphere of dry argon. Anhydrous solvents were dried by standard procedures:
Tetrahydrofuran (THF) and ether was distilled from sodium metal in the presence of
benzophenone under argon. Dichloromethane (CH2Cl2) was distilled from calcium hydride under argon. Silica gel column chromatography was performed using silica gel
60A (Merck, 230-400 Mesh). High-resolution electrospray ionization mass spectra were
148 obtained on the Micromass QTOF Electrospray mass spectrometer at The Ohio State
Chemical Instrumentation Center. All the NMR spectra were recorded on a Bruker AC
250, Bruker DPX 250, or Bruker DRX 400 model spectrometer in CDCl3 solution. The
abbreviations s, d, t, q, m, dd, dt, and br are used for singlet, doublet, triplet, quartet,
doublet of doublets, doublet of triplets, and broad, respectively. Chemical shifts for 1H
NMR spectra are reported in ppm relative to residual solvent protons.
11.11 Synthetic Procedures of Vitamin E Analogues (scheme 8.1)
The detail synthetic procedures for 6-(benzyloxy)-2,5,7,8-tetramethyl
-chroman-2-carbaldehyde from starting material 6-hydroxy-2, 5, 7, 8-tetramethylchroman
-2-carboxylic acid (compound 1) are published (145).
4-oxo-4-(2,2,5,7,8-pentamethylchroman-6-yloxy)butanoic acid (VEA-2).
2,2,5,7,8-Pentamethyl-6-chromanol (VEA-1) were dissolved in dry pyridine, and
succinate anhydride were added in a 50% molar excess. The solution was purged with
argon and refluxed for 12 hours. The reaction mixture was poured into 1 M HCl and
dried with Na2SO4. After solvent evaporation, the crude product was purified by flash
chromatography and gave the product in 75% yield.
2-(4,8-dimethylnonyl)-2,5,7,8-tetramethylchroman-6-ol (VEA-3) and 2,5,7,8- tetramethyl-2-(4-methylpentyl)chroman-6-ol (VEA-4). A mixture of triphenylphosphane (PPh3) and commercial available 1-Bromo-3,7-dimethyloctane (for
VEA-3) or 1-Bromo-3-methylbutane (for VEA-4) (1:1) was dissolved in toluene and
reflux overnight. The phosphonium salts were recrystallized and react with
6-(benzyloxy)-2,5,7,8 -tetramethylchroman-2-carbaldehyde in tetrahydrofuran (THF) at 149 reflux condition for 3 hours. After hydrogenation by stirring with equal amount 10%
Pd/C in dry ethanol in room temperature overnight, the products, VEA-3 or VEA-4 were
purified via flash silica gel chromatography in 55 and 60% yield respectively.
4-(2-(4,8-dimethylnonyl)-2,5,7,8-tetramethylchroman-6-yloxy)-4-oxobutanoic
acid (VEA-5) and 4-oxo-4-(2,5,7,8-tetramethyl-2-(4-methylpentyl)chroman
-6-yloxy)butanoic acid (VEA-6). VEA-3 (for VEA-5) or VEA-4 (fro VEA-6) was
dissolved in dry pyridine, and succinate anhydride were added in a one equivalent excess.
The solution was purged with argon and refluxed for 12 hours. The reaction mixture was
quenched by 1 M HCl and dried with Na2SO4. After solvent evaporation, the crude product was purified by flash chromatography in 85 and 90% yield respectively.
3-(2-(4,8-dimethylnonyl)-2,5,7,8-tetramethylchroman-6-yloxy)propanoic acid
(VEA-7) and 3-(2,5,7,8-tetramethyl-2-(4-methylpentyl)chroman-6-yloxy)propanoic
acid (VEA-8). The mixture of 2 equivalents 3-Bromo-propionic acid, 1 equivalent
VEA-3 (for VEA-7) or VEA-4 (for VEA-8) and 4 equivalents KOH was dissolved in
dimethylformamide (DMF). The reaction was performed at 50-60 oC overnight. After
quenching by 1 M HCl, the products were purified by flash chromatography in 30 and
33% yield respectively.
1 11.12 Nomenclatures, H NMR (Proton Nuclear Magnetic Resonance), and HRMS
(High Resolution Mass Spectrometry) Characterizations of Vitamin E Analogues
4-oxo-4-(2,2,5,7,8-pentamethylchroman-6-yloxy)butanoic acid (VEA-2). 1H
NMR (300 MHz, CDCl3) 1.26 (s, 6H), 1.76 (t, 2H, J = 8.15 Hz), 1.95 (s, 3H), 1.99 (s,
3H), 2.06 (s, 3H), 2.56 (t, 2H, J = 8.03 Hz), 2.81 (t, 2H, J = 5.11 Hz), 2.89 (t, 2H, J = 7.48
Hz); HRMS (ESI, m/z) calcd for C18H24O5Na (M+Na): 343.1516, found: 343.1513. 150 2-(4,8-dimethylnonyl)-2,5,7,8-tetramethylchroman-6-ol (VEA-3). 1H NMR (300
MHz, CDCl3) 0.83 (d, 3H, J = 6.49 Hz ), 0.84 (d, 6H, J = 6.60 Hz), 0.99-1.59 (m, 14H),
1.28 (s, 3H), 1.71-1.81 (m, 2H,), 2.09 (s, 6H), 2.14 (s, 3H), 2.58 (t, 2H, J = 6.91 Hz), 4.14
(s, 1H); HRMS (ESI, m/z) calcd for C24H38O2Na (M+Na): 381.2764, found: 381.2793.
2,5,7,8- tetramethyl-2-(4-methylpentyl)chroman-6-ol (VEA-4). 1H NMR (300
MHz, CDCl3) 0.84 (d, 6H, J = 6.60 Hz ), 1.11-1.18 (m, 2H), 1.21 (s, 3H), 1.33-1.58 (m,
5H), 1.71-1.82 (m, 2H), 2.09 (s, 6H), 2.14 (s, 3H), 2.58 (t, 2H, J = 6.84 Hz), 4.14 (s, 1H);
HRMS (ESI, m/z) calcd for C19H28O2Na (M+Na): 311.1981, found: 311.1993.
4-(2-(4,8-dimethylnonyl)-2,5,7,8-tetramethylchroman-6-yloxy)-4-oxobutanoic
1 acid (VEA-5). H NMR (300 MHz, CDCl3) 0.83 (d, 3H, J = 6.42 Hz ), 0.84 (d, 6H, J =
6.59 Hz), 0.99-1.14 (m, 2H), 1.21 (s, 3H), 1.22-1.54 (m, 12H), 1.71-1.79 (m, 2H,), 1.95 (s,
3H), 1.99 (s, 3H), 2.06 (s, 3H), 2.56 (t, 2H, J = 6.91 Hz), 2.80 (t, 2H, J = 6.50 Hz), 2.91 (t,
2H, J = 7.51 Hz); HRMS (ESI, m/z) calcd for C28H44O5Na (M+Na): 483.3081, found:
483.3093.
4-oxo-4-(2,5,7,8-tetramethyl-2-(4-methylpentyl)chroman-6-yloxy)butanoic acid
1 (VEA-6). H NMR (300 MHz, CDCl3) 0.85 (d, 6H, J = 6.61 Hz ), 1.11-1.18 (m, 2H),
1.21 (s, 3H), 1.36-1.58 (m, 5H), 1.71-1.82 (m, 2H), 1.95 (s, 3H), 1.99 (s, 3H), 2.06 (s,
3H), 2.56 (t, 2H, J = 6.78 Hz), 2.81 (t, 2H, J = 6.43 Hz), 2.92 (t, 2H, J = 7.22 Hz); HRMS
(ESI, m/z) calcd for C23H34O5Na (M+Na): 413.2298, found: 413.2293.
3-(2-(4,8-dimethylnonyl)-2,5,7,8-tetramethylchroman-6-yloxy)propanoic acid
1 (VEA-7). H NMR (300 MHz, CDCl3) 0.83 (d, 3H, J = 6.43 Hz ), 0.84 (d, 6H, J = 6.56 151 Hz), 0.99-1.13 (m, 2H), 1.20 (s, 3H), 1.23-1.57 (m, 12H), 1.70-1.82 (m, 2H,), 2.01 (s,
3H), 2.10 (s, 3H), 2.15 (s, 3H), 2.53 (t, 2H, J = 6.97 Hz), 2.81 (t, 2H, J = 6.22 Hz), 3.92 (t,
2H, J = 6.29 Hz); HRMS (ESI, m/z) calcd for C27H44O4Na (M+Na): 455.3132, found:
455.3137.
3-(2,5,7,8-tetramethyl-2-(4-methylpentyl)chroman-6-yloxy)propanoic acid
1 (VEA-8). H NMR (300 MHz, CDCl3) 0.85 (d, 6H, J = 6.63 Hz ), 1.11-1.18 (m, 2H),
1.20 (s, 3H), 1.23-1.56 (m, 5H), 1.72-1.79 (m, 2H), 2.02 (s, 3H), 2.10 (s, 3H), 2.15 (s,
3H), 2.54 (t, 2H, J = 6.66 Hz), 2.81 (t, 2H, J = 6.21 Hz), 3.92 (t, 2H, J = 6.27 Hz); HRMS
(ESI, m/z) calcd for C22H34O4Na (M+Na): 385.2349, found: 385.2365.
152
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