Towards the Development of Non-Toxic Therapeutics in the Fight Against Cancer Open Access

Baillie, Mark (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/ks65hd03c?locale=en%255D
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Abstract

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Towards the Development of Non-Toxic Therapeutics in the Fight Against Cancer

Chapter 1: Synthesis and Application of Novel Tail-Modified Enigmol Analogs. Sphingolipids are a set of signaling molecules that are intricately involved in cancer progression. Enigmol (2 S-amino-octadecane- 3 S,5 S-diol) is a sphingoid base derivative that reregulates apoptosis in cancer cells by interacting with the sphingolipid signaling pathways. Herein we report the synthesis of a set of Enigmol analogs featuring fluorination and rigidification of the tail. Geometric alterations and rigidification between C-11 and C-12
decreased efficacy of these compounds to induce cell death in prostate cancer cell lines (PC-3 and LNCaP) when compared to Enigmol, while compounds with fluorination at multiple points in the tail retained anticancer activity in vitro. Pharmacokinetic properties and a biological distribution profile of the C-18 trifluoromethyl analog 2 (R = CF3) dosed orally in rats at 10 mg/kg showed nearly three fold increase in plasma levels and tissue concentrations as compared with Enigmol. Most notable was the substantial increase in bioavailability, which was found to be 76% for 2 as compared to 11% for Enigmol. Trifluoromethyl-containing compound 2 is a promising lead molecule that we plan to further pursue as an anti-cancer compound for the clinical treatment of prostate cancer. Chapter 2: Following in the Steps of Otto Warburg. Design and Synthesis of Potential Pyruvate Kinase Inhibitors. Chapter 3: Design and Synthesis of Novel CXCR4 Modulators for Treatment of Macular Degeneration.

Table of Contents




1 Table of Contents

Chapter 1: Synthesis and Application of Novel Tail-Modified Enigmol Analogs ......................... 1
1.1
Introduction ...................................................................................................................... 1
1.1.1
Background and Significance ................................................................................... 1
1.1.2
Developing a Cancer Therapeutic ............................................................................. 5
1.1.3
19F MRI Application ............................................................................................... 11
1.2
Synthesis of Target Compounds .................................................................................... 12
1.2.1
Liebeskind-Srogl Route and Side Reaction Investigation ...................................... 12
1.2.2
Synthesis Via Aldol Reaction ................................................................................. 20
1.3
Biological Evaluation ..................................................................................................... 28
1.3.1
In vitro Evaluation of Enigmol Analogs Against Prostate Cancer ......................... 28
1.3.2
Pharmacokinetic Evaluation ................................................................................... 29
1.3.3
19F MRI Study Results ............................................................................................ 32
1.4
Discussion ...................................................................................................................... 35
1.5
Conclusions .................................................................................................................... 37
1.6
Methods and Experimental Protocol .............................................................................. 38
1.6.1
Experimental Procedure for the Synthesis of Tail-modified Enigmols: ................. 38
1.6.2
Biological Protocols ................................................................................................ 75
Chapter 2: Following in the steps of Otto Warburg. Design and Synthesis of Potential Pyruvate
Kinase Inhibitors ............................................................................................................................. 1
2.1
Introduction .................................................................................................................... 82
2.1.1
Background and Significance ................................................................................. 82
2.1.2
Design of Novel PK Inhibitors ............................................................................... 85
2.2
Results ............................................................................................................................ 87
2.2.1
Synthesis of Novel PKI's ........................................................................................ 87
2.2.2
Chemical Routes Attempted to Obtain the Difluorophosphonate PKI's ................ 90
2.3
Biological Evaluation ..................................................................................................... 92
2.3.1
In vitro Cell-based Assays ...................................................................................... 92
2.3.2
In vitro Cell-free Assay ........................................................................................... 94
2.4
Discussion ...................................................................................................................... 95
2.5
Conclusion ...................................................................................................................... 96
2.6
Methods and Experimental Protocol .............................................................................. 96
2.6.1
Experimental Protocol for Synthesis ...................................................................... 96



2.6.2
Biological Evaluation of anti-cancer agents ......................................................... 107
Chapter 3: Design and Synthesis of Novel CXCR4 Modulators for Treatment of Macular
Degeneration ................................................................................................................................. 82
3.1
Introduction .................................................................................................................. 109
3.1.1
Background and Significance ............................................................................... 109
3.1.2
Developing a CXCR4 Modulator ......................................................................... 114
3.2
Results .......................................................................................................................... 117
3.2.1
Synthesis of Novel WZ-41 Analogs ..................................................................... 117
3.3
Biological Evaluation ................................................................................................... 119
3.3.1
In vitro Cell-based TN-14003 Binding Assay Results ......................................... 119
3.3.2
In vitro Vasculature Formation Assay Results ..................................................... 122
3.4
Conclusions .................................................................................................................. 123
3.5
Methods and Experimental Protocol ............................................................................ 124
3.5.1
Experimental Procedure for the Synthesis of Novel WZ-41 Analogs .................. 124
3.5.2
Experimental Protocol for TN-14003 Binding Assay .......................................... 138

Figure 1: Common structural examples of the sphingolipid family. Sphingoid bases sphinganine
and sphingosine showed above the sphingolipids ceramide and further functionalized derivative
sphingomyelin. ................................................................................................................................ 2
Figure 2: Highly simplified de novo sphingolipid synthetic pathway. In the ceramide/S1P
rheostat, ceramide and sphingosine inhibit cell growth while S1P increases cell growth. ............. 3
Figure 3: Pictorial representation of the ceramide/S1P rheostat9 for a healthy cell (A) and a
cancerous cell (B). .......................................................................................................................... 4
Figure 4: Conception of an anti-cancer agent, Enigmol, which drew from sphingosine and
fumonisin B1. .................................................................................................................................. 6
Figure 5: Mouse xenograft study comparing the efficacy of Enigmol to hormone ablation in
androgen-dependant tumors (A) or docetaxel in androgen-independent tumors (B).23 .................. 8
Figure 6: Proposed sites for modification on the tail of Enigmol. .................................................. 9
Figure 7: Proposed synthetic targets probing the affect of fluorination and geometric restrictions
in the tail of Enigmol. ................................................................................................................... 11
Figure 8: Rationalization for the loss of stereochemistry based on the formation of the aromatic
intermediate 65. To capitalize on the equilibrium, we proposed the chiral directing group
attached to the nitrogen (67). ........................................................................................................ 18
Figure 9: Pharmacokinetic data comparing mean plasma concentrations of Enigmol to CF3-
enigmol (2) over 24 hours. ............................................................................................................ 30
Figure 10: Mean rat tissue, plasma, and RBC concentrations at 24 hours post administration of
Enigmol verses trifluoromethyl-enigmol 2 (10 mg/kg P.O. dose). Analysis performed by the
DMPK lab, EIDD. ......................................................................................................................... 32
Figure 11: 1H (left) and 19F (right) MRI images of rat brain, dosed with 2. ................................. 34
Figure 12: Structures of molecules that take advantage of the dependence of a cancer cell on
glycolysis. ..................................................................................................................................... 84



Scheme 13: Conversion of phosphoenolpyruvate (PEP) to pyruvate, catalyzed by Pyruvate
Kinase. This reaction results in the net gain of one unit of ATP by the consumption of ADP. ... 84
Figure 14: Comparison of phosphoenolpyruvate (PEP) with our proposed phosphonate PKI's.
The red bond highlights the removal of the cleavable P-O bond, replaced by a P-C bond. Blue
atoms show site of halogenations, which hold potential to tune PK isoform specificity. ............ 85
Figure 15: Specific examples of proposed pro-drug PKI's. ......................................................... 86
Figure 16: PEP analog pro-drugs 4-8 previously reported in literature,65 synthesized for
comparison in this study. 3-BrPA and its ester derivative are commercially available. .............. 86
Figure 17: Pyruvate Kinase inhibition assay performed with the 2 tri-acid compounds 33 and 34.
....................................................................................................................................................... 95
Figure 18: Cross section of the human eye. ................................................................................ 109
Figure 19: A schematic representation of the cornea, showing the possible pathways for
molecules to diffuse through to the anterior chamber.81 ............................................................. 111
Figure 20: Predictive relationship between solute distribution coefficient Φ and permeability of
the individual layers (A) and the whole cornea (B). Molecular radius is represented by line
type.81 These trends for over 150 compounds were calculated based on models determined and
tested in a composite porous medium.81 ..................................................................................... 112
Figure 21: Structure of the bicyclam compound AMD3100, an inhibitor of CXCR4. .............. 114
Figure 22: Lead CXCR4 antagonists developed in our laboratory by Weiqiang Zhan. ............. 115
Figure 23: A) CXCL12 displacing peptide TN14003 used in the competitive binding assay. B)
Bioassay used to measure interaction of novel compounds with CXCR4 on MB-231 breast
cancer cells. Novel compound (green) competes with high affinity biotinylated-peptide
TNF14003 (blue/purple). After washing away the unbound substrate, streptavadin-rhodamine
conjugate complexes with biotinylated peptide. Inhibition of peptide binding by drug dampens
fluorescent signal output. ............................................................................................................ 121

Table 1: Optimization of reaction conditions for cyclization utilizing Liebeskind-Srogl
conditions. Followed by LCMS. Yield reported was isolated yield. (-) means isolation not
attempted. ...................................................................................................................................... 17
Table 2: Survey of oxidative conditions for removal of N-benzyl protecting group from 35a. All
reaction progress was followed by LCMS. No inert atmosphere or exclusion of water was
utilized in these small scale reactions (20 - 80 mg). .................................................................... 25
Table 3: Optimized conditions for oxidative removal of N-benzyl protecting group with Dess
Martin periodinane. ....................................................................................................................... 25
Table 4: Efficacy of Enigmol analogs against prostate cancer cells for 24 h, and cytotoxicity was
assessed by WST-1 assay. cLogP calculated with QuikProp on Maestro. ................................... 28
Table 5: Mean pharmacokinetic parameters for Enigmol and CF3-enimgol (2). Performed by the
DMPK lab, EIDD. ......................................................................................................................... 30
Table 6: Mean rat tissue concentrations of Enigmol and trifluoromethyl-enigmol 2 shown in
Figure 10. ...................................................................................................................................... 32
Table 7: Summary of cancer cell growth inhibition assays results. IC50's were calculated for
various synthetic PKI's in breast (MDA-MB231) and prostate (PC-3) cancer cells in culture. .. 93
Table 8: Computational and biological testing values for WZ-41 and analogues. cLogP values
were calculated with QuikPro in Maestro. .................................................................................. 121
Table 9: Tubule formation assay results from S.R.I. .................................................................. 123

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