Synthesis and Retrosynthesis of Peptidomimetic Inhibitors - Thrombin Presented by: Kevin Condel

Synthesis and Retrosynthesis of Peptidomimetic Inhibitors -

Thrombin

Presented by:

Kevin Condel

Overview / Terminology• Goal: To design and synthesize a peptidomimetic that competes to inhibit the enzyme thrombin.

• Thrombin is part of a cascade leading to the formation of insoluble fibrin, a material found in blood clots. Unregulated clotting may lead to cardiac arrest or stroke.

• A Peptidomimetic is any small organic molecule that mimics the transition state of a natural substrate.

• Peptidomimetics competitively inhibit the enzyme process, preventing the natural reaction from occurring.

– i.e. - The peptidomimetic binds more readily to thrombin than the substrate.

• Hydroxy-aldehydes are important components of peptidomimetic inhibitors of the thrombin system. This work involves the development of a simple, yet effective protocol for the generation of hydroxy-aldehydes.

Thrombin• Blood clotting must be regulated.

• Errors in blood clotting lead to: – cardiac arrest (in the heart)– stroke (in the brain)

• Thrombin begins inactive and is shown on the bottom-left.

• Inactive thrombin has extra domains, colored blue, which are clipped off during activation.

• The purple atoms are Ca2+ ions, bound to modified glutamate amino acids.– The strong (+) charge on these ions

tether the protein to the surfaces of blood vessels, holding thrombin and localizing it to one spot.

Thrombin• Since inactive thrombin is held, blood clots

will generally not spread to other areas.

– Only the thrombin adjacent to the damage will be activated.

• Activated thrombin (the upper structure shown here) lasts only seconds, serving also to limit the clot to the area of damage.

• Thrombin is simply part of a cascade which serves to synthesize the cross-linked fibrin polymers found in blood clots.

Click to enlarge

Thrombin as an Enzyme

• Thrombin has an active site consisting of the catalytic triad: Ser 195, His 57, and Asp 102

Enzyme Binding Site• In addition to the active site, thrombin

has three binding sites, labeled as S1, S2 and S3, that determine the strength and specificity of binding

• The lipophilicity of S3 has been well determined

• Lipophilicity represents the affinity of a molecule or moiety for a lipophilic environment (i.e. hydrophobicity)

Inhibition of the Active Site

• Leeches synthesize proteins that block thrombin (and other enzymes), stopping the formation of the clot.

• One example, a protein called hirudin, is shown here on the left in blue. Notice how it fits the active

site of thrombin perfectly.

Peptidomimetics

• Small peptide-like molecules that mimic transition state of substrate and work by competitively inhibiting the binding of the natural substrate (ex. Hirudin from the leech)

• Peptide analog must be stable.

• Drug must be a reversible inhibitor of the enzyme but can be irreversible if the

enzyme is unique to the disease.

Saquinavir

Project Design• Goals:

– Design a polypeptide isotere based on a natural thrombin substrate (natural Phe-Pro-Arg tripeptide shown below)

– Optimize a generalized scheme for isotere synthesis

– Model the S2 and S3 steric and hydrophobic requirements

• All reactions required an anhydrous environment.

• Nitrogen steadily flushed through the system to exclude water vapor.

• Various temperatures were achieved as follows:– -78C (Dry Ice + Acetone)

– 0C (Ice Bath)

Project Setup

S

N

Br 1. BuLi2. TMSCl

S

N

SiMe 3

S

N

R

OR

OH

R CHO

OR2-TST

CHOunmasking

RCHO

OR

OH

OverallReaction

2-TST

S

N

Br Bu- + Li+

S

N

Li

Me3Si Cl

Bu Br

S

N

SiMe3

+ Li Cl

S

N

SiMe3

1. 4.22ml n-Butyl-Lithium was injected into a round bottom flask containing aswirling solution of 50 ml ether through the septum using the syringe

2. 2.33ml 2-Bromothiazole in 50 ml of ether slowly added over 30 min via aseparatory funnel into a controlled -78ºC nitrogenous environment.

3. Mixture allowed to stir for 30 min

4. 4.33ml (CH3)SiCl in 50 ml ether added drop wise for 30 min via the sep funnel

2-TST NMR Identification

S

N

SiMe3

C

O

OHLiAlH4

0ºC RT

1. 7.33g Benzoic Acid was added under inert nitrogenous conditions to a flask containing 15 mL Tetrahydrofuran (THF).

2. Under 0ºC conditions, 45 mL of pre-cooled 1.0 M Lithium Aluminum Hydride(LiAlH4) in THF was added dropwise with vigorous stirring.

Benzaldehyde

CH2 OH

CH2 OH

(No isolation)

PCC

RT, 6-12 h

C O

H

3. After the hydrogen had evolved, the solution was cooled to room temp. and stirred for 30 min.

4. In a separate flask under the same nitrogenous conditions, 14.3 g Pyridinium Chlorochromate (PCC) was added to 100mL Methylene Chloride and stirred into solution.

Benzaldehyde

CH2 OH

CH2 OH

(No isolation)

C

O

OHLiAlH4

0ºC RT

PCC

RT, 6-12 h

C O

H

CH2 OH

CH2 OH

(No isolation)

C

O

OHLiAlH4

0ºC RT

PCC

RT, 6-12 h

C O

H

5. The alkoxyaluminum salt in THF created by mixing LiAlH4 with benzoic acid was next added dropwise at room temperature via a separatory funnel

6. The reaction mixture was stirred for 12 hours at room temperature, diluted with diethyl ether, and filtered and washed to remove the supernatant liquid. The ether was then distilled from the filtrate to obtain benzaldehyde.

Benzaldehyde

C O

H

Benzaldehyde NMR Identification

Amino Acid Reduction Methodology (Theoretical)

• Use benzaldehyde synthesis schematic to reduce the amino acid argenine

• When reduced argenine is combined with 2-TST it creates the Arg side chain of the Phe-Pro-Arg tripeptide peptidomimetic

• Unmasking protocol removes thiazole ring and replaces it with CHO group, creating the active site inhibitor.

• Phe-Pro addition will be conducted in future experimentation.

Click to enlarge

Reduction of Argenine (Novel Approach)

1. 12.64g Argenine was added under inert nitrogenous conditions to a flask containing 20 mL Methyl Sulfoxide as a solvent.

2. Under 0ºC conditions, 45 mL of pre-cooled 1.0 M Lithium Aluminum Hydride(LiAlH4) in THF was added dropwise with vigorous stirring.

(No isolation of alkoxy-salt)

+

C

CH3N

NH

NH2

H2N+

O

OH

LiAlH4

0ºC RT

CH2 OH

+

C

H3N

NH

NH2

H2N+

C O

H

+

C

H3N

NH

NH2

H2N+

PCC

0ºC 2 h RT 6-12 h


3. After the hydrogen had evolved (2 h), the solution was cooled to room temp. and stirred for 30 min. The intermediate was not isolated.

4. In a separate flask under the same nitrogenous conditions, 14.3 g Pyridinium Chlorochromate (PCC) was added to 100mL Methylene Chloride and stirred into solution.


+

C

CH3N

NH

NH2

H2N+

O

OH

LiAlH4

0ºC RT

CH2 OH

+

C

H3N

NH

NH2

H2N+

C O

H

+

C

H3N

NH

NH2

H2N+

PCC

0ºC 2 h RT 6-12 h



+

C

CH3N

NH

NH2

H2N+

O

OH

LiAlH4

0ºC RT

CH2 OH

+

C

H3N

NH

NH2

H2N+

C O

H

+

C

H3N

NH

NH2

H2N+

PCC

0ºC 2 h RT 6-12 h

5. The intermediate in Methyl Sulfoxide created by mixing LiAlH4 with argenine was next added dropwise at room temperature via a separatory funnel.

6. Mixture stirred for 12 hours at room temperature, diluted with diethyl ether, filtered and washed. The ether was then distilled from the filtrate to obtain the aldehyde of argenine.

Methodological Problems with Argenine Reduction

• Need a nonpolar, nonreactive solvent to dissolve argenine without interfering with the reaction.

• Methyl Sulfoxide NOT efficient as a solvent for this reaction due to its exothermicity.

– i.e (Broken Manifold and intense sulfur scent)

• Length of complete reaction and temperature requirements are dependent on the solvent used to dissolve argenine.

– With Methyl Sulfoxide, it is proposed that 0ºC conditions must exist for at least two hours prior to addition of PCC.

Retrosynthesis and Unmasking Protocol Mechanism (Theory)

C

O

H

+

C

H3N

NH

NH2

H2N+

Aldehyde of Argenine

Me3Si S

N

2-TST

RT, 4 h

RT, 4 h

C

OH

+

C

H3N

NH

NH2

H2N+

S

N

Intermediate

NaBH4

CF3SO3Me4

C

OH

+

C

H3N

NH

NH2

H2N+

S

N

Me


C

OH

+

C

H3N

NH

NH2

H2N+

S

N

Previous research has determined that CF3SO3Me4 is added toN-methylate the thiazole ring. NaBH4 is added to reduce the mixtureand break the pi bonds.

Cu++ / Water

C

OH

+

C

H3N

NH

NH2

H2N+

S

N

Me

C

OH

+

C

H3N

NH

NH2

H2N+

O

HC


Cu++ and water are added to hydrolyze the system, removing the thiazole ring and adding yet another aldehyde.

C

OH

+

C

H3N

NH

NH2

H2N+

O

HC


HNR2

NaBH3CN

HNR2 is added as part of a dehydration reaction to remove waterand add NR2 to the molecule. This NR2 represents the part of the molecule that will be interacting with the active site of thrombin.

C

OH

+

C

H3N

NH

NH2

H2N+

CH2 NR2

References• Alessandro Dondoni, et al.; Synthesis of TSTs and Reactions with Carbonyl

Compounds; J. Org. Chem. 1988, 53, 1748-1761• Benoit Bachand , et al.; Synthesis and Structure-Reactivity of Potent Bicyclic

Lactam Thrombin Inhibitors; Bioinorg. & Med. Chem. 1999, 9, 913-918• Jin Soon Cha, et al.; Preparation of Aldehydes from Carboxylic Acids by

Reductive Oxidation with Lithium Aluminum Hydride and Pyridinium Chlorochromate or Pyridinium Dichromate; Bull. Korean Chem Soc. 1999, Vol. 20, No. 4

Acknowledgements

• We gratefully acknowledge the support of the Welch Foundation in the form of a Departmental Research Grant

Questions?

2-TST Reaction

NMR Identification

Benzaldehyde Reaction

NMR Identification

Thrombin

Structure

Inhibition

Catalytic Triad

Binding Sites

Peptidomimetics

Project Goals

Apparatus

Overall Reaction

Theory

Method

Argenine Reduction

Problems

Retro-synthesis

Click to view natural substrate

Proposed Peptidomimetic

Click to view proposed peptidomimetic

Natural Substrate

PCC Pyridinium Chlorochromate

NH+CrO3Cl-

Documents

Synthesis and Retrosynthesis of Peptidomimetic Inhibitors - Thrombin Presented by: Kevin Condel