y oung i nnovators 2009 discovery, design, and synthesis of anti- metastatic lead phenylmethylene...

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.

Young Innovators 2009

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.


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.


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.


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.



• IDENTIFICATION OF STRUCTURES• Structure elucidation of isolated and synthetic

phenylmethylene hydantoins was based on different spectroscopic methods including 1D and 2D NMR techniques.



• 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.



• 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.



• 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.


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.



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


1 + CT1

Diluent

CT

PARACELLULAR PERMEABILITY ASSAY


1 + CT 1 CT V

SPHEROID DISAGGREGATION MODEL


Diluent 4

CoMFA ANALYSIS


Steric Contour Plots Electrostatic Contour Plots

ORTHOTOPIC TUMOR GROWTH


Diluent

PMH-1

PMH-4

SURVIVAL CURVES


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.


• 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.



• 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.



• 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.



• 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.



• 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.



• 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.



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.


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.


ACKNOWLEDGMENTS

Major Advisor

Dr. Khalid El Sayed

Advisory Committee Members

ULM College of Pharmacy

AAPS Organizing Committee


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.


CONTACT INFO

Mudit Mudit

College of Pharmacy

University of Louisiana at Monroe

1800 Bienville Drive, Room # 322

Monroe, LA – 71201

E-mail: [email protected]


y oung i nnovators 2009 discovery, design, and synthesis of anti- metastatic lead phenylmethylene...

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