y oung i nnovators 2009 discovery, design, and synthesis of anti- metastatic lead phenylmethylene...
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YOUNG INNOVATORS 2009
Discovery, Design, and Synthesis of Anti-Metastatic Lead Phenylmethylene Hydantoins
Mudit Mudit, Mohammad Khanfar, Anbalagan Muralidharan, Shibu Thomas, Girish Shah, Khalid El Sayed
Department of Basic Pharmaceutical SciencesCollege of Pharmacy
University of Louisiana at Monroe
ABSTRACT
• Prostate cancer is the most prevalent cancer form in men and constitutes the second leading cause of cancer deaths in men in United States.
• Later stage of prostate cancer progresses to life-threatening, androgen-independent and fatal metastatic forms.
• Currently few options are available for the treatment at this stage.
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ABSTRACT
• Marine natural products display extraordinary chemical and pharmacological scopes.
• The Red Sea sponge Hemimycale arabica afforded (Z)-5-(4-hydroxybenzylidene) hydantoin (1) and other known related hydantoins 2 and 3.
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ABSTRACT
• The natural phenylmethylene hydantoin (PMH-1) and the synthetic (Z)-5-(4- (ethylthio)benzylidene)hydantoin (4) showed potent in vitro anti-growth and anti-invasive properties against PC-3M prostate cancer cells.
• PMHs 1 and 4 also showed significant anti-metastatic activities in orthotopic xenograft and LPB-Tag transgenic mice models.
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ABSTRACT• To study the effect of electronic and lipophilic
parameters on the activity, a wide array of substituted PMHs (4-42) possessing electron-withdrawing (+σ), lipophilic (+π), electron-donating (-σ), and less lipophilic substituents (-π) were synthesized.
• The anti-invasive activities of all compounds were evaluated. Comparative Molecular Field Analysis (CoMFA) was then used to study the 3D QSAR.
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MATERIALS AND METHODS
• ISOLATION OF PHENYLMETHYLENE HYDANTOIN
• Secondary metabolites of the sponge H. arabica were extracted, and the eluents were purified by various normal and reversed phase flash, medium, and high pressure liquid chromatographic methods.
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MATERIALS AND METHODS
• IDENTIFICATION OF STRUCTURES• Structure elucidation of isolated and synthetic
phenylmethylene hydantoins was based on different spectroscopic methods including 1D and 2D NMR techniques.
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MATERIALS AND METHODS
• SYNTHESIS• Reflux condensation of substituted aromatic
aldehydes with hydantoin or 2-thiohydantoin, in presence of ethanolamine, afforded several analogs of phenylmethylene hydantoins, including H. arabica metabolites.
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MATERIALS AND METHODS
• IN VITRO ASSAYS• CELL-CELL ADHESION
PMH-1 and 4 were tested for their cell-cell adhesion ability. Trans Epithelial resistance assay was used to measure the integrity of the cellular monolayer. Paracellular permeability assay was used to measure the diffusion of TMR-dextran, 4kDa, across a cell layer.
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MATERIALS AND METHODS
• CELL GROWTH AND INVASION
Proliferation of PC cells was assessed by MTT assay. The effect of 1 and 4 on the migratory potential of PC-3M cells in spheroid disaggregation assay was examined. This model reliably predicts the relative metastatic potential of prostate cancer cell lines.
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MATERIALS AND METHODS• IN VIVO ASSAYS
• ORTHOTOPIC TUMOR GROWTH AND METASTASIS
To detect micrometastases of implanted tumor cells in mice, PC-3M cells stably transfected with DsRed-MCherry-Hyg-N1 were used. All cell lines expressed strong red fluorescence at a steady level over the duration of the observation period.
Young Innovators 2009
Young Innovators 2009
NH
NH
O
O
S
Semi-synthesis
NH
HN
O
O
NH
Br
NH
NH
O
O
HO 2
1
3
4
NH
NH
O
O
HO
TRANS EPITHELIAL RESISTANCE ASSAY
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1 + CT1
Diluent
CT
PARACELLULAR PERMEABILITY ASSAY
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1 + CT 1 CT V
SPHEROID DISAGGREGATION MODEL
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Diluent 4
CoMFA ANALYSIS
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Steric Contour Plots Electrostatic Contour Plots
ORTHOTOPIC TUMOR GROWTH
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Diluent
PMH-1
PMH-4
SURVIVAL CURVES
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0 10 20 30 40 50 60 700
25
50
75
100
Control
H
H2
Life Expectancy (Days)
14
SUMMARY OF KEY FINDINGS
• 1 and 4 acts by reversal of the actions of CT on TJs and AJs.
• CT significantly reduced TER. In contrast, 1 significantly increased TER of diluent and CT-treated PC-3M cultures, suggesting that 1 has stabilizing effects on TJs and it completely reverses the actions of CT on TER.
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• CT caused an almost 2-fold increase in paracellular permeability. In contrast, 1 decreased baseline paracellular permeability and abolished CT-induced increase.
• 1 caused a small but significant decline in the rate of baseline PC-3M cell proliferation. CT caused a modest but significant increase in PC-3M proliferation, and 1 remarkably reduced the CT-stimulated increase.
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SUMMARY OF KEY FINDINGS
• PC-3M cells displayed a high rate of invasion, and CT caused almost a 2-fold increase in invaded cells. 1 had a minimal effect on baseline invasion but almost attenuated the CT-stimulated increase.
• In the spheroid disaggregation assay both molecules inhibited the cell migration in a dose-dependent manner. 4 was approximately three times more potent than 1 in inhibiting PC-3M-CT+ spheroid dispersal.
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SUMMARY OF KEY FINDINGS
• Fluorograms as well as growth curves suggest that tumors of 1 and 4 -treated mice were remarkably smaller than diluent-treated mice and remained localized within the abdominal cavity. Both 1 and 4 significantly reduced incidents of metastases in several organs.
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SUMMARY OF KEY FINDINGS
• Diluent-treated mice showed large tumor cell colonies in lymph node, seminal vesicles, bone, lungs, and smaller tumor cell numbers in other organs including brain. Both 1 and 4 remarkably decreased metastasis of PC-3M-CT+ cells in most organs and reduced the size of tumor cell colonies in organs where metastases were present. Comparatively, 4 seemed more antimetastatic than 1.
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SUMMARY OF KEY FINDINGS
• Areas of high steric bulk tolerance were observed near the p-position of the benzylidene group in 4, therefore, bulky groups in p-position are favorable for the activity.
• Red contour was observed near m- and p-positions of the phenyl ring of 4, therefore, compounds possessing electronegative groups near these regions (high electron density) may show more activity.
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SUMMARY OF KEY FINDINGS
• In short, red contours near the p-position of the benzylidene group coincide with the sterically favorable green contours. Hence, substituents at these positions should be electron-donating but with high steric bulk for better activity.
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SUMMARY OF KEY FINDINGS
CONCLUSIONS• Hydantoins are rare bioactive natural products
class that can show future promise as potential drug candidates appropriate for development as possible treatment for metastatic prostate cancer.
• Among these hydantoins, 1 and 4 displayed potent and consistent anti-growth and anti-invasive properties when tested against androgen-independent highly invasive prostrate cancer PC-3M cells.
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CONCLUSIONS
• Hence, these findings strongly advocate that hydantoins inspired by marine sponge H. arabica secondary metabolites might have an excellent potential as anti-invasive small lead molecules appropriate for advance optimization as practicable treatment options for metastatic prostrate cancer.
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ACKNOWLEDGMENTS
Major Advisor
Dr. Khalid El Sayed
Advisory Committee Members
ULM College of Pharmacy
AAPS Organizing Committee
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Dr. Paul Sylvester Dr. Girish Shah Dr. Ronald Hill
REFERENCES
• Mudit M, Khanfar M, Muralidharan A, Thomas S, Shah GV, van Soest RW, El Sayed KA., Discovery, design, and synthesis of anti-metastatic lead phenylmethylene hydantoins inspired by marine natural products., Bioorg Med Chem. 2009 Feb 15;17(4):1731-8.
• Shah GV, Muralidharan A, Thomas S, Gokulgandhi M, Mudit M, Khanfar M, El Sayed K., Identification of a small molecule class to enhance cell-cell adhesion and attenuate prostate tumor growth and metastasis., Mol Cancer Ther. 2009 Mar;8(3):509-20.
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CONTACT INFO
Mudit Mudit
College of Pharmacy
University of Louisiana at Monroe
1800 Bienville Drive, Room # 322
Monroe, LA – 71201
E-mail: [email protected]
Young Innovators 2009