Peptidomimetic InhibitorsPeptidomimetic Inhibitors : :An Integrated Synthetic and Theoretical An Integrated Synthetic and Theoretical

Approach to their DesignApproach to their Design

Everardo Macias, Patrick Tomboc

Eamonn F. Healy,

Chemistry Department,

St. Edward’s University,

Austin TX 78704

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AbstractAbstractPeptidomimetics represent a powerful approach to pharmaceutical treatments based on enzymatically controlled reactions. Peptidomimetics are simply small organic molecules that serve to mimic the transition state of the natural substrate and thus serve to competitively inhibit the enzyme process. We are focusing on the design and synthesis of inhibitors for the serine protease thrombin. Thrombin plays a critical role in the formation of insoluble fibrin that can lead to life threatening medical conditions. Our synthetic scheme utilizes hydroxy-aldehydes in the synthesis of polypeptide isoteres for active site inhibition. QSAR studies aid in the understanding of the steric and hydrophobic requirements of the enzymaticbinding sites.

EnzymeEnzyme Thrombin, a sub-class of the

hydrolyases is a serine protease that promotes blood clotting

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

Enzyme BindingSiteEnzyme BindingSite

In addition thrombin has three binding sites, labelled as S1, S2 and S3, that determine the strength and specificity of binding

The lipophilicity of S3 has been well determined

PeptidomimeticsPeptidomimetics Small peptide-like

molecules that mimic transition state of substrate and work by competitive inhibiting binding of the natural substrate

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

NNH

O NH2

O

O

NH

Ph

N

NH-BuO

OH

R

HYDROXYETHYLAMINE ISOTERE NATURAL PEPTIDE

MIMICS

O

NH

Saquinavir

Project DesignProject Design Design a polypeptide

isotere based on a natural thrombin substrate (Phe-Pro-Arg tripeptide), shown on the right

Optimize a generalized scheme for isotere synthesis

Model the S2 and S3 steric and hydrophobic requirements

H2N

O

N

HN

NH

NH2

NH

O

O

Gly216

Ser195

Asp189

S3

S2

S1

His57

Asp102

ProjectProject

Use Quantitative Structure-Activity Relationships (QSAR) to identify optimum R2 and R3 binding fragments

Synthesize the isotere, shown in red on the right, designed to mimic the serine-195 mediated transition state

Gly216

Ser195

Asp189

S3

S2

S1

His57

Asp102

H2N

O

R3

NH

R2

R1

NH

O

OH

N

Retrosynthesis &Retrosynthesis &R NR2

NH2

OH

generalized isotere

R

NH2

OH

O

H

R

NH2

O

HR

NH2

O

OH

aminoacid

R

NH2

O

H

R

NH2

O

OH [H] +

S

N

Me3Si

1. BuLi

2. Me3SiCl

2-TST

R

NH2

OH

S

N1. CF3SO3Me

2. NaBH4

R

NH2

OH

N

S

Me

CuCl2

R

NH2

OH

O

H

+ HNR2

NaBH3CN

R

NH2

OH

NR2

S

N

Br

SynthesisSynthesis

ResultsResults

O

HR

S

N

Me3Si

1. BuLi

2. Me3SiCl

2-TST

R

OH

S

N

1. CF3SO3Me

2. NaBH4

3. CuCl2

S

N

Br

YIELD

IR

NMR

1.

2.

R = CH3

+ 2-TSTBu4NF

CH2Cl2

YIELD

IR

NMR

R

OH

S

N

3.R = CH3

R = CH3

R

OH

H

O

R = CH3

YIELD

IR

NMR

>70%

3100 cm-1, sharp; 2950 cm-1 ; 1610 cm -1, weak

7.7-7.8, multiplet, 2H ; 0.4, singlet, 9H

7.8-7.9, multiplet, 2H ; 3.6,multiplet,1H

1.2-1.5, multiplet, 4H

Structure-Activity Results from Ref 2Structure-Activity Results from Ref 2

R3 R2 Ki (nm)

CH3 benzyl (S) 17

CH3 phenethyl (R) 550

CH3 phenethyl (S) 235

CH3 phenylpropyl (R) 100

CH3 phenylpropyl (S) 4

H benzyl (R) 1112

H benzyl (S) 8

S3

S2

S1

HN

NH

NH2

NH

O

OX

S

N

O

R2

R3

QSARQSARQuantitative structure-activity relationships (QSAR) represent an attempt to correlate structural or property descriptors of compounds with their activities. These physicochemical descriptors, including parameters describing hydrophobicity, topology, electronic effects and steric effects, can be determined empirically or by computational methods. Once a correlation between structure and activity/property is found, new compounds can be screened to select those with the desired properties. Activities in which QSAR has found wide application include biological assays, chemical measurements, environmental risk assessment and de novo drug design.

QSAR MethodologyQSAR MethodologyLinear regression analysis of the predicted versus observed activity/property is most commonly used to develop the QSAR relationship. The higher the r2 value the better the fit. The technique of leave-one-out cross-validation, quantified as rCV, is used to assess the predictive power of the QSAR model.

Two parameters, molecular connectivity and valence connectivity, developed by Kier and Hall were used extensively in this study. While these are fundamentally topological parameters they have been shown to contain electronic as well as structural information.

Connectivity Indices

ResultsResults

Preliminary results, summarized by the plot of the predicted (y-axis) versus experimental (x-axis) binding constants, clearly show that a functional predictive model of the steric and electronic requirements of the S2 and S3 binding sites can be constructed. Initial results seem to indicate the energetic descriptors are more useful as predictors than simple topological properties. This work continues.

ResultsResultschemical sample connecti

vity

index 0

log P energy

steric

(kcal/mo

molar

refracti

vity

Binding

Constant

Ki (nM)

(S)CH3-benzyl-BenzoThia

zole(S)

23.001 2.901 10.201 131.883 18.000

(R)CH3-phenylethl-BenzoT

hiazole

23.709 3.001 22.865 136.599 550.000

(R)CH3-phenylpropyl-Thia 21.847 2.610 40.858 125.072 100.000

(S)CH3-phenylpropyl-Thia 21.847 2.610 35.824 125.072 6.000

(R)CH3-3Fquin-Thiazole 26.372 3.188 29.088 135.393 150.000

(S)CH3-3Fquin-BenzoThia

zole(S)

28.941 4.272 21.630 151.406 18.000

(S)CH3-3Fquin-Thiazole 26.372 3.188 30.545 135.393 10.000

(S)CH3-benzyl-Thiazole2(

S)

20.433 1.817 23.388 115.870 16.000

F = -47.976*B - 1605.799*C + 13.368*D

+ 123.102*E - 10621.018 r^2 = 0.925

rCV^2 = 0.119

-10.954

544.609

83.202

15.910

50.041

58.192

69.520

57.480

Graph 3. Ki-QSAR-W/O-Outlier3

Binding Constant Ki (nM)

F = -47.976*B - 1605.799*C + 13.368*D + 123.102*E - 10621.018 r^2 = 0.925 rCV^2 = 0.119

-100 0 100 200 300 400 500 600

-100

0

100

200

300

400

500

600

1

2

3

4

567

8

F = -47.976*B - 1605.799*C + 13.368*D + 123.102*E - 10621.018 r^2 = 0.925 rCV^2 = 0.119 =0.925 *Binding Constant Ki (nM)+ 8.128; r ^ 2 = 0.925

F = -47.976*B - 1605.799*C + 13.368*D + 123.102*E - 10621.018 r^2 = 0.925 rCV^2 = 0.119

1 (S)CH3-benzyl-BenzoThiazole(S)

2 (R)CH3-phenylethl-BenzoThiazole

3 (R)CH3-phenylpropyl-Thia

4 (S)CH3-phenylpropyl-Thia

5 (R)CH3-3Fquin-Thiazole

6 (S)CH3-3Fquin-BenzoThiazole(S)

7 (S)CH3-3Fquin-Thiazole

8 (S)CH3-benzyl-Thiazole2(S)

ResultsResultschemical sample connecti

vity

index 0

log P energy

steric

(kcal/mo

molar

refracti

vity

Binding

Constant

Ki (nM)

(S)CH3-benzyl-BenzoThia

zole(S)

23.001 2.901 10.201 131.883 18.000

(R)CH3-phenylethl-BenzoT

hiazole

23.709 3.001 22.865 136.599 550.000

(S)CH3-phenylethl-BenzoT

hiaz.58

23.709 3.001 23.749 136.599 235.000

(R)CH3-phenylpropyl-Thia 21.847 2.610 40.858 125.072 100.000

(S)CH3-phenylpropyl-Thia 21.847 2.610 35.824 125.072 6.000

(R)CH3-3Fquin-Thiazole 26.372 3.188 29.088 135.393 150.000

(S)CH3-3Fquin-BenzoThia

zole(S)

28.941 4.272 21.630 151.406 18.000

(S)CH3-3Fquin-Thiazole 26.372 3.188 30.545 135.393 10.000

(S)CH3-benzyl-Thiazole2(

S)

20.433 1.817 23.388 115.870 16.000

F = -29.160*B - 1116.702*C + 9.582*D

+ 84.612*E - 7350.105 r^2 = 0.731

rCV^2 = -0.247

-3.367

384.872

393.343

72.782

24.551

55.110

53.507

69.072

53.128

Graph 2. Ki-QSAR-all

Binding Constant Ki (nM)

F = -29.160*B - 1116.702*C + 9.582*D + 84.612*E - 7350.105 r^2 = 0.731 rCV^2 = -0.247

-100 0 100 200 300 400 500 600

-100

0

100

200

300

400

500

1

23

4

5

67

8

9

F = -29.160*B - 1116.702*C + 9.582*D + 84.612*E - 7350.105 r^2 = 0.731 rCV^2 = -0.247 =0.731 *Binding Constant Ki (nM)+ 33.028; r ^ 2 = 0.731

F = -29.160*B - 1116.702*C + 9.582*D + 84.612*E - 7350.105 r^2 = 0.731 rCV^2 = -0.247

1 (S)CH3-benzyl-BenzoThiazole(S)

2 (R)CH3-phenylethl-BenzoThiazole

3 (S)CH3-phenylethl-BenzoThiaz.58

4 (R)CH3-phenylpropyl-Thia

5 (S)CH3-phenylpropyl-Thia

6 (R)CH3-3Fquin-Thiazole

7 (S)CH3-3Fquin-BenzoThiazole(S)

8 (S)CH3-3Fquin-Thiazole

9 (S)CH3-benzyl-Thiazole2(S)

ReferencesReferences 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

Acknowledgements We gratefully acknowledge the support of the Welch

Foundation in the form of a Departmental Research Grant