Examination of the StructureExamination of the StructureExamination of the StructureExamination of the Structure----Activity Relationships of Inhibitors of Plasminogen Activator InhibitorActivity Relationships of Inhibitors of Plasminogen Activator InhibitorActivity Relationships of Inhibitors of Plasminogen Activator InhibitorActivity Relationships of Inhibitors of Plasminogen Activator Inhibitor----1111

Karen L. Sanders,1 Hasina Saraha,1 Mark Warnock,2 Jacqueline M. Cale,2 Daniel A. Lawrence,2 and Cory D. Emal1*; (1) Chemistry

Department, Eastern Michigan University, Ypsilanti, 48197; (2) Department of Internal Medicine, University of Michigan Medical

School, Ann Arbor, MI, 48109

Introduction/BackgroundIntroduction/BackgroundIntroduction/BackgroundIntroduction/Background Common Structural ThemesCommon Structural ThemesCommon Structural ThemesCommon Structural Themes

Many of the compounds displaying PAI-1 inhibition contain gallate or

digallate groups. The focus of our research has been centered on the

structure-activity relationship of different configurations of polyphenols.

This includes the manipulation of the carbamate handle and the number and

position of the hydroxyl groups on the inhibitory molecules.

Design and SynthesisDesign and SynthesisDesign and SynthesisDesign and Synthesis

The inhibition of plasminogen activator-inhibitor-1 (PAI-1) is

anticipated to increase our understanding of various human ailments

including diabetes, stroke, and atherosclerosis, for which high levels of

PAI-1 have been associated.(1) PAI-1 prevents certain serine proteases

from cleaving peptide bonds and thus is able to regulate various cellular

processes such as controlling the levels of other intracellular proteins,

such as tissue-type plasminogen activator (tPA) and urokinase-type

plasminogen activator (uPA).(2)

The focus of the reactions depicted in Scheme 1 is the production of

compounds that investigate the effect of manipulating the carbamate handle

on either a gallate or protocatechuate coupled species. In concert, the effect

on inhibition which the change in either the number or position of the

hydroxyl groups that compose the gallate core was examined. A series of

five-six reactions are required to synthesize the desired compounds. These

reactions were repeated as necessary using different attachments. The last

step which entailed the removal of the benzyl protecting groups utilized

Pd/C 10%. Attempts were made to remove these protecting groups with an

Goal: to determine the effect of manipulating the carbamate handle and

the number and position of the hydroxyl groups on the gallate core.

Effect of modifying the gallate to a protocatechuate on PAI-1 inhibition:

IC50 = 4.69 µm

Fibrinolysis: Green arrows indicate a

stimulatory effect, and red arrows

indicate an inhibitory effect.

Therefore, the goal of this research is to inhibit the inhibitor of fibrinolysis,

the process which leads to the break down of blood clots.

Inhibition ResultsInhibition ResultsInhibition ResultsInhibition Results

Effect of manipulating the carbamate handle on PAI-1 inhibition:

IC50 = 0.048 µm

IC50 = 4.75 µm

IC50 = 0.02 µm

IC50 = 0.159 µm

IC50 = 9.6 µm

IC = 0.022 µm

IC50 = 0.105 µm

IC = 0.0108 µm

IC50 = 0.104 µm

IC50 = 0.062 µm

IC50 = 0.029 µm

A high-throughput screen was conducted by Daniel Lawrence and

coworkers at the University of Michigan against a large library of a variety

of organic compounds. Some of these compounds proved to be effective

PAI-1 inhibitors. Many of these compounds contain gallate or digallate

groups. Similar compounds containing related phenolic structures did not

show inhibitory activity versus PAI-1. Molecules were synthesized

containing varying numbers of gallates which proved to be effective PAI-1

inhibitors.

Future DirectionsFuture DirectionsFuture DirectionsFuture Directions- Test the inhibitory effect of electronically larger species attachments.

- Synthesize inhibitors with asymmetric gallate attachments.

- Incorporate R-NH2 attachments.

ReferencesReferencesReferencesReferences

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements- The remaining members of the Emal and Lawrence research groups

Funding from:

- Eastern Michigan University

- Camille and Henry Dreyfus Foundation

- National Institutes of Health

1.) Ren, Y., Himmeldirk, K., Chen, X. J. Med. Chem. 2006, 49, 2829-2837.2.) Wang et al. Biochemistry. 1996, 35 (51), 16443-16448.3.) Miyazaki, H.; Ogiku, T.; Hiroshi, S.; Moritani, Y.; Ohtanl, A. Chem. Pharm. Bull. 2009, 57 (9) 979-985.4.) Cale, J. M.; Li, S.; Warnock, M.; Su, E. J.; North, P. R.; Sanders, K. L.; Puscau, M. M.; Emal, C. D.; Lawrence,

D. A. Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor-1. J.Bio. Chem. 2010 In Press

Scheme 1: Synthesis of differing PAI-1 inhibitory molecules

Pd/C 10%. Attempts were made to remove these protecting groups with an

encapsulated version, Pd Encat 30 Palladium Acetate Microencapsulated in

Polyurea Matrix 0.4 mmol Pd/g. It was done with the aim of eliminating

the highly toxic palladium from in vivo studies.(4)

Activity towards PAI-1 is very sensitive to the identity of the

carbamate handle. Carbamate handles which are composed of electron-rich

Species result in a increased inhibition of PAI-1. The modification of the gallate

core to a protocatechuate results in a positive increase in the inhibition of the

serpin.

O O

O O

HO

HO

OH

OH

OH

OH

O O

HO

OH

OH

the process which leads to the break down of blood clots.

The complexity of the PAI-1 structure has the potential to possess several

binding sites to a wide variety of inhibitory molecules. Development of

therapeutic agents that act as selective inhibitors of PAI-1 may provide an

approach to treat these ailments. Recent reports have noticed inhibitive

properties in furan-2-one and pyrrolin-2-one derivatives.(3) However,

these known accounts have reported the synthesis of inhibitors that bind to

PAI-1 with a low affinity and fail to inhibit PAI-1 when vitronectin, a

cofactor of PAI-1 is present.

A furan-2-one/pyrrolin-2-one derivative.

ConclusionConclusionConclusionConclusion

IC50 = 0.025 µm

IC50 = 0.02 µm

IC50 = ? µmIC50 = ? µm

IC50 = 0.022 µm

IC50 = 0.027 µm

IC50 = 0.0108 µm

IC50 = 9.6 µm

IC50 = 0.37 µm