IASOC - September 21-25, 2014

Aspects of cardiovascular pdrug discovery and

development :development :

Studies towards theStudies towards the discovery of Thrombin and

TAFIa inhibitors

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Case Study # 1y

SYNTHESIS OF NEW TETRAHYDRO-PYRROLO-PYRAZINONEPYRROLO PYRAZINONE CONTAINING ORALLY

BIOAVAILABLE THROMBINBIOAVAILABLE THROMBIN INHIBITORS

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COAGULATION CASCADE

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THROMBIN (fIIa)( )

Proteolytic Enzyme belonging to serine proteases familyy y g g p y

Cleaves soluble fibrinogen to fibrin and stabilizes clots by activationof factor XIII

Amplifies its own formation by activation of several coagulationfactors

(factors V et VIII)

Lowers its own formation by binding to thrombomodulin toti t t i C (i ti ti f f t V t VIII )activate protein C (inactivation of factors Va et VIIIa)

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MARKETED ANTITHROMBOTICS

INDIRECT INHIBITORS OF THROMBIN

HEPARIN (iv/sc) Polysulfated polysaccharide

Potentiate the activity of AT III (neutralisation of thrombin and factor Xa)

Thrombopenia

LMWH (iv/sc) Chemical or Enzymatic Depolymerisation Heparin

Same activity compared to Heparin

Coumarin derivatives (ex: warfarin)

Same activity compared to Heparin

A.V.K. (po) Inhibit vitamin K- dpendent factors

Orally active

High bleeding risk

DDI

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DDI

MARKETED ANTITHROMBOTICS

O

DIRECT INHIBITOR OF THROMBIN

N N

O

NO

N

N

NH

NH

NH

O

O

O

PRADAXA ( Dabigatran )B hi I lh iBoerhinger Ingelheim

Launched 2008

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TARGET PRODUCT PROFILE

P t t d l ti I hibit f Th bi Potent and selective Inhibitor of Thrombin

Orally Active (good bioavailability)

Active on venous / arterial thrombosis models

Without Food /Drug Interaction

Once a Day Administration

No prodrug No prodrug

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PEPTIDOMIMETIC CORES AS Phe-Pro ANALOG

N

O

NH2

O

N

O

O

NH2 OH

N

N

O

O

NH2 OH

OOH

O O

PFIZER ,NOVARTIS,

MERCK,ELI LILLY,

…. …

N

NNH2

O

N

OOH

SERVIER

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( Tetrahedron Lett. 2002, 43, 3499-3501)

PEPTIDOMIMETIC INHIBITORS

N

O

NH2

O N N

N

N

NH

N

NH2O

NH

H

NH2

O H

S 34211S 34211

Direct and Reversible Thrombin InhibitorSelectiveSelectiveOrally active ( F=80 %)t1/2 (1h)

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Pharmacological Profile of S 34840g

N

N

N

N

O

NH

ONH NH2O

BIOAVAILABILITY

IC50(nM)

SELECTIVITY

(nM)

COAGULATION H D R

BIOAVAILABILITY Dog Rat

t1/2 (h)

C(TT)2 (M)

3

100

34

> 33000

3 2

56

10

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CHEMICAL SYNTHESIS OF S 34840

CO2BzPhCH2OH

DMAP Boc O DIBAL(S)

(S)

NHO

CO2HNHO

CO2BzN

O

2

OO

DMAP Boc2O DIBALSOCl2(S)

(S)

80 %70 %98 %

N

CO2Bz

OH ON

CO2Bz

CH3O O NH

CO2BzMeOH APTS

TMSCN SnCl4

(S)(S)

(S)

(R/S) (R/S)(R/S)

98 %OHO

O CH3OO

HNC98 %85 %

NN

Cl

HCl gaz1)

NCl

O CO2Bz

ClCOCOCl (S)2)

55 %

N

Cl

N

Cl

+N

ClO CO2Bz

N NH2 NH

O CO2Bz

N(S)

+(S)84 %

N

N

Cl

N

N

NH

N

CO H(S)

H2 ; Pd/C

H2 ; Pd/CNaOH N

(S)85%N

HO CO2H

CH3

HO CO2H

(S)97 % 85%

N

NH

O

N

NH

NHBocN

NNH2

NHBoc

3

(S)HCl gaz

TBTU ; HOBT ; DIEA95 %O O H

N

82 %95 %

NH

O

N

ONH

NH2

N(S) ; 2 HCl

S 34840-1

SAR FROM S 34840

NN

NH

NN

OO

NH

NH2

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SUMMARY of SAR

N

NH

NN O CONHEt

HO CONH

S 35972S 35972

Potent and selective : IC 50 = 4nM , C(TT)2 = 0.24M Potent and selective : IC 50 4nM , C(TT)2 0.24M

Orally Active : F(Dog) = 85%

Without food interactionWithout food interaction

Orally active on venous and arterial models of thrombosis

Half-life = 4 h

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Half life = 4 h

Other scaffolds …..

IC50 (nM)

34

N

NH

O

N

N

N

NH

508

OO

NH

NH2

N

NH

O

N

ONH

N

NH2

5695

O H

N

N

NH

O

NH

N

375

OH

NH2

O

NO

NH

N

O

NH

810

ONH2

NN

NH

O

NH

N

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OH H

NH2

Metabolism Profile of S 35972

NO CONHEt

NH

ON

N

CONH

O CONHEt

S 35972

IC50 = 4 nMC(TT)2 = 0 24 MC(TT)2 = 0.24 M

t1/2 = 4 hF (Dog) = 85%

MF = 32%

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Metabolites of S 35972

N

NNN

O CONHEt(R/S) S 40240

IC 10 M

S 40243

IC 43 MO

H CONHOHIC50 = 10 nM

C(TT)2 = 0.22 Mt1/2 = 3h15

F(Dog) = 50%

IC50 = 43 nMC(TT)2 = 0.48 M

F(Dog) 50%MF = 65%

N

NNN

O CONHEtS 39733

IC50 = 1.8 nMO

NH

NCONH

O

50C(TT)2 = 0.11 M

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« Protected » S 35972

N

N

O

NH

NCONHF F

O CONHEt

OO

S 39858S 39858

IC50 = ndC(TT) = 0 19 MC(TT)2 = 0.19 M

F(Dog) = 18%MF = 67%

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Modification on RHS of S 35858od cat o o S o S 35858

N

NNH

NCONH

O CONHEt

O CONHF FO

S 39858IC50 = nd

C(TT) 0 19 MC(TT)2 = 0.19 MF = 18%

MF = 67% FN

NNN

S 40294

F F O

NH

NCONH

O

S 40294IC50 = 16 nM

C(TT)2 = 0.15 MF= 60%

MF 92%

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MF = 92%

Case Study # 2Case Study # 2

SYNTHESIS OF NEW TAFIaINHIBITORSINHIBITORS

( Thrombin Activable Fibrinolysis Inhibitor Activated )

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Role of TAFIa in Thrombosis

Lys Lys

IIa

Fibrinogen

TII

Fibrin

Plasmin

Lys

TmIIa

TAFIaFibrin degradation

productsPlasmin

Lys

TAFIa TAFIproducts

t-PA

plasminogenLys

Lys+

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Hit Compounds

Captopril inhibitor of TAFIaCaptopril inhibitor of TAFIa

V 811, V 812, M 326# 20 compounds

IC50 TAFIa (M)

# 20 compounds

N

OH

OHSH

OSHN

HOOC

O

SH

NH

SHSH

COOH

S 889725

S 991720

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Competitors

N

NH

N

N

SH

N

NH2(R)

NH2 COOH(S)

Pfizer (Phase I)

NCOOH(S)

Astra-Zeneca (Phase II)Pfizer (Phase I)UK-396082

Astra Zeneca (Phase II)AZD-9684

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Key Interactions in the Active Site

His 159

Zn2+

Glu 162

Zinc BindingGroup

His 288

Acid FunctionAmino Function

Arg 235 Arg 217

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Asp 348

Hit to Lead Exploration

COOHSH

O

NH

IC50 TAFIa (M)

O S 43970 30

NH

COOHNH2

NH

SHSH

COOH> 30

NH

COOHSH

OS 991720

N

S 46321> 30

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30

S 43621-1 Binding Mode

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Hit to Lead Optimisation

IC50 TAFIa (M)

n

NH

COOHSH

O

NH2

SHNH2 n

COOH

S 43970

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Lead Optimisation

IC50 TAFIa (M)

SHNH2 COOH

SH

NH2 COOH

S 457080.014

S 449000.17

SH

COOHNH2

S 453771 65

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1.65

S 44900-1 Binding Mode g

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S 44900-1 / S 45708-1Enantiomers SeparationEnantiomers Separation

SH

S 456462.03

IC50 TAFIa (M)

NH2 COOH

S 456470.081

S 449000 17

Syn-rac

0.17

SHNH

S 469115

NH2 COOH

S 469130 005

S 457080 014

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0.0050.014

Lead Optimisation : SAR Results

SHNH2 COOH2 COOH

Thiol function replacementsS 46913

Basic side chain modifications

• N methyl amine

Thiol function replacements

• Carboxylic Acid

• Hydroxamic Acid• N-methyl amine

• N,N-dimethyl amine

• Cyclic amine

Hydroxamic Acid

• Retro-hydroxamic Acid

• Triazoley

• Pyridine

IC > M

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IC50 > M

Stereoselective synthesis of S 46913

OH

tBuOH Pyridine tBuOH - NaH Cat*CO2tBu

O

COCl CO2tButBuO2CClOC

O

90%93%

tBuOH - Pyridine Toluène Toluène

90 %

OCO2tBu

H

OCO2tBu

OH

OCO2tBu

Br

OHCO2tBu

Br

H

(+)DIPCl NaBH(OAc)3 THF

82%ee > 99%

Ph3P - NBS CH2Cl2

87%de > 99%

43%

O

Boc2NH - Cs2CO3 - DMFOH

CO2tBuHO

SOO

CO2tBuHS

CO2tBuO

H MsCl-THF-TEA AcSK-Isopropanol

ee 99%

2 2 3

NBoc2NBoc2

2

NBoc255 % 87% 28%

SCO2H

OH

SHCO2HHHCl/Dioxane 4M

76%

HCl aq 4M

95%

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NH2NH2

76%HCl

95%HCl

ACKNOWLEDGEMENTS

CHEMISTRY PHARMACOLOGY

Guillaume DE NANTEUIL

Bernard PORTEVIN

Tony VERBEUREN

Alain RUPIN

Alain BENOIST

Danièle HENO

Sophie CORNET

Marie-Odile VALLEZ

Philippe MENNECIER

Isabelle RICHARD

Camille POINDRON

Aurélie MOTHE

Christian MERIAUX

A F i GUILLOUZIC

Christine MAUCLAIR

Anne Françoise GUILLOUZIC

Nicolas BOYER

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Milestones of our companyp y

Fi i i h

Servier crosses the threshold of  75% of its 

turnover made worldwide

First affiliates abroad : SpainBrazil

Portugal

First operations with China and Russia

worldwide

Portugal

Fi t ffili t i A i

Construction of Arklow production plant 

3 new chemical entities : PROTELOS

PROCORALAN VALDOXAN

Doctor Servier founded“ LES LABORATOIRES SERVIER”

in Orléans (FRANCE)

First affiliates in Asia :Hong Kong,Malaysia,Singapore

(Ireland) 21.000 employees

in Orléans (FRANCE)9 employees

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Servier : Research Orientations

Epigeneticsp gAngiogenesisProliferation / Differention PathwaysCell metabolismApoptosis

Coronary Arterial DiseaseHeart Failure

Venous DiseaseIschemia p p

Immunotherapy Protein degradationONCOLOGY

Arterial HypertensionCARDIOVASCULAR

METABOLISMType II Diabetes Mellitus

Depression / AnxietySchizophrenia

Cognitive ImpairmentNeurodegenerative diseases

(Alzheimer, Parkinson)CNS

RHUMATOLOGYRHUMATOLOGYOsteoporosisOsteoarthritis

Inflammatory & auto-immune diseasesMuscular pathologies

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uscu a pat o og es

Main marketed productsp

CARDIOVASCULARCoversyl Hyperium

Coveram Artex

ONCOLOGYMuphoran

CNSValdoxanSt bl

Preterax ProcoralanFludex Vastarel

METABOLISMDiamicron

Di i MR

StablonTrivastal

Diamicron MRRHUMATOLOGY

Protelos

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