DATE – OCTOBER 23 rd, 2009 PRESENTED BY – AKUL MEHTA VIRGINIA COMMONWEALTH UNIVERSITY SCHOOL OF...

DIRECT RENIN INHIBITORS AS ANTIHYPERTENSIVE DRUGS

DATE – OCTOBER 23rd, 2009

PRESENTED BY – AKUL MEHTA

VIRGINIA COMMONWEALTH UNIVERSITYSCHOOL OF PHARMACYDEPARTMENT OF MEDICINAL CHEMISTRY

www.pharmaxchange.info

http://www.pharmaxchange.info/

WHAT IS HYPERTENSION?

Normal Blood Pressure = 120/80 Systolic = 90 – 119 mm Hg Diastolic = 60 – 79 mm Hg

Hypertension = High Blood Pressure

Known risk factor of several Cardiovascular Disorders

2Chobanian, A. V. et al. Hypertension 2003, 42, 1206-1252



3

RENIN ANGIOTENSIN SYSTEM

AngII

ACE

AngI = Angiotensin I ACE = Angiotensin converting enzyme AngII = Angiotensin II

Image adapted from - Li, Y. C. Curr. Opin. Invest. Drugs 2007, 8, 750-757.

Angiotensinogen AngI

Renin

Rate DeterminingStep INCREASE IN BLOOD

PRESSURE



4

CURRENT DRUGS TARGETING THE RENIN-ANGIOTENSIN SYSTEM FOR HYPERTENSION

IS THERE REALLY A NEED FOR ANOTHER ANTIHYPERTENSIVE DRUG?

•Angiotensin Converting Enzyme Inhibitors

•Angiotensin II Receptor Blockers

•Aldosterone Antagonists



5

NEED FOR ANOTHER ANTIHYPERTENSIVE

Li, Y. C. Curr. Opin. Invest. Drugs 2007, 8, 750-757.Image from - http://blog.wineenthusiast.com/wp-content/uploads/2008/12/balance.jpg

Hypertension needs modulation



6

NEED FOR ANOTHER ANTIHYPERTENSIVE

Li, Y. C. Curr. Opin. Invest. Drugs 2007, 8, 750-757.

Angiotensinogen AngI AngII Ang II RECEPTORSAT1

Renin

Rate DeterminingStep

ACE

Ang II RECEPTORSAT2

ChymaseAT1 RECEPTOR BLOCKER (ARB)

ACE INHIBITOR

INVOLVED INBRADYKININMETABOLISM

Limitations of current drugs targeting the system



7

NEED FOR ANOTHER ANTIHYPERTENSIVE Hypertension 1° risk factor in cardiovascular

diseases

Worldwide prevalance of ≈ 1 billion people (almost 1/6th of the human population)

United States of America > 60 million people

< 30% of patients achieve treatment goals

Yokokawa, F. et al. Expert Opin. Ther. Patents 2008, 18(6), 581-602.

IS THERE REALLY A NEED FOR ANOTHER ANTIHYPERTENSIVEDRUG?YES THERE IS DEFINITELY A NEED



8

CURRENT INTEREST IN INHIBITION OF RENIN ANGIOTENSIN SYSTEM

1985-1989 1990-1994 1995-1999 2000-2004 2005-20080

2040

6080

100120

140160

138 135

12 12

75

Patent Applications from 1985-2008 for Renin Inhibitors

Yokokawa, F. et al. Expert Opin. Ther. Patents 2008, 18(6), 581-602.



9

INTRODUCING RENIN

History

Structure

Function

Mechanism

PDB ID – 2v0z



10

RENIN - HISTORY 1898- Tigerstedt and

Bergman

Kidney extracts produce pressor effects- coined the term ‘renin’

Phillips, M. I. News Physiol. Sci. 1999, 14, 271-274



RENIN - HISTORY 1934- Harry Goldblatt

Induced experimental hypertension in dogs

1st to prove that renin system blockade would reduce hypertension

11Basso, N. et al. Hypertension 2001, 38, 1246-1249.



12

RENIN - HISTORY 1970- Tadashi Inigami

Isolated pure renin from hog kidney

This was followed by isolation from rat and human kidney.

Currently working Vanderbilt University School of Medicine



13

RENIN - STRUCTUREHighly Species Specific

340 amino acids

Two beta pleated sheets make two lobes

Active site between the two lobes

Aspartates for the active site provided by each lobe – i.e. Asp 32 and Asp. 215

Eder, J. et al. Current Pharmaceutical Design, 2007, 13, 271-285.PDB ID- 2v0z

ASP32

ASP215



14

RENIN - FUNCTION

Renin cleaves peptide bondbetween Leu10-Val11

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu10 + Val11-Ile-His-Asn---

Angiotensin I

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu10--Val11-Ile-His-Asn---Angiotensinogen (480 amino acid long)

Angiotensinogen AngI

Renin

Rate DeterminingStep



15

RENIN - MECHANISM

Asp

O

O

H

Asp

O-

O

HO

H

NH

HN

O

OP1

P1'

ENZYMEACTIVE SITE

Asp

O-

O Asp

O

O

NH

HN

O

OP1

P1'O

H H

H

Asp

OH

O Asp

O-

O

NH

HN

O

OP1

P1'

OH+ H

Eder, J. et al. Current Pharmaceutical Design, 2007, 13, 271-285.



HOW DOES ONE MEASURE RENIN INHIBITION?

In Vitro Human renin is

incubated with inhibitor

Angiotensinogen is added.

Angiotensin I produced is measured by radioimmunoassay

16

In Vivo Animal testing was

done on sodium depleted marmosets

Now even transgenic rats are used

Changes in blood pressure and heart rate is measured telemetrically.

Image from - http://www.bukisa.com/articles/50597_marmoset-monkeys-and-their-habitats



17

INHIBITORS OF RENIN

OHN

H

OHN

O

NH

HN

O

HN

N

O

O

Cl CH3

Cl

OH

H2NHN

O

NH2

O

O

O

O

HN

O

O

Cl

N

HN

O

O

N

HN

O

O

O

O



18

INHIBITORS OF RENIN

Early Inhibitors of Renin Antibodies

Transition State Analogs Peptide Mimetic Inhibitors Non-Peptide Inhibitors

▪ Recently developed Inhibitors



19

INHIBITORS OF RENIN






20

EARLY INHIBITORS OF RENIN

Monoclonal Antibodies Against Renin

Excellent tools to study enzyme and its hypertensive effects

However – ▪ parenteral administration ▪ immunogenecity

therefore less application in medicine.

Galen, F. X. J. Clin. Invest. 1984, 74, 723-735.



21

INHIBITORS OF RENIN






22

SCHECHTER AND BERGER NOMENCLATURE FOR PROTEASES

Mitti, P. R. Curr. Opin. Struct. Biol. 2006, 16, 769-775.



23

THEORY OF TRANSITION STATE ANALOGS

SubstrateProduct

TransitionState

Enzyme Active Site

Schramm, V. L. Annu. Rev. Biochem. 1998, 67, 693-720.

Reaction Co-ordinate

En

erg

y

I

Inhibitor withstructure similar to transition statewill have highaffinity.



24

PEPTIDE MIMETIC INHIBITORS

Modification of scissile bond with statine and its variants

Normal Substrate: AngiotensinogenAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn-]

Statine Analog:Boc-Phe-His-Stat-Leu-Phe-NH2

IC50 – 190nM Normal Peptide

Boger, J. et al. J. Med. Chem. 1985, 28, 1779-1790.

NH

HN

R1 H

H OH

HH

O

O

R2H

NH

HN

O

OR1

H

H

R2



25

PEPTIDE MIMETIC INHIBITORS

Statine Analog:Boc-Phe-His-Stat-Leu-Phe-NH2

ACHPA Analog:Boc-Phe-His-ACHPA-Leu-Phe-NH2

IC50 – 49nM

O

NH

OHHN

(3S,4S)-4-amino-3-hydroxy-5-cyclohexylpentanoic acid

O

R2H

O

NH

R1 H

NH

HN

R1 H

H OH

HH

O

O

R2H



26

NON-PEPTIDE INHIBITORS OF RENIN STORY OF ALISKIREN

(DEVELOPED BY NOVARTIS)

DEVELOPMENT OF PIPERIDINE CLASS OF RENIN INHIBITORS (DEVELOPED BY HOFFMAN LA ROCHE AND OTHERS)

OH

H2NHN

O

O

O

O

O

NH2

HN

O

O

Cl



27

NON-PEPTIDE INHIBITORS OF RENIN

STORY OF ALISKIREN

OH

H2NHN

O

O

O

O

O

NH2



28

STORY OF ALISKIRENBoc-Phe-His-ACHPA-Leu-Phe-NH2

OHHN

HN

O

NH

O

N

HN

O

SOO

CGP38560

IC50 = 1nMThe Renin-Angiotensin System; Robertson, J. I. S., Nicholls, M. G., Eds.; Mosby: London, 1993.

O

NH

OHHN

(3S,4S)-4-amino-3-hydroxy-5-cyclohexylpentanoic acid

O

R2H

O

NH

R1 H



29

STORY OF ALISKIREN

OHHN

HN

O

NH

O

N

HN

O

SOO

S3 S1

CGP38560

S3 S1

IC50 = 1nM

Goshke, R. et al. Bioorg. Med. Chem. Lett. 1997, 7, 2735-2740.

OH

H2NHN

O

R2R1R3

R4

OHHN

HN

O

NH

O

N

HN

O

SOO



30

STORY OF ALISKIREN

OH

H2NHN

O

R2R1R3

R4

S3S1

-phenyl substituent equipotent to -t-butyl

-OCH2COOCH3 > -OCH2CONH2 > -OCH2COOH

-isopropyl was found optimumIncrease or decrease in substituent size caused decrease in activity

R-epimer showed significant drop in activity

Can be changed to methyl



31

OH

H2NHN

OO

O

H2N

STORY OF ALISKIRENOH

H2NHN

O

R2R1R3

R4

Most IC50 in μM range

IC50 = 20nM

OH

H2NHN

OO

O

H2N



32

STORY OF ALISKIRENOH

H2NHN

OO

O

H2N

OH

H2NHN

O

R1

O

R3

R2 R4

To improve physicochemical properties



33

STORY OF ALISKIREN

R1

-t-butyl

-O-CH3

OH

H2NHN

O

R1

O

R3

R2 R4

When R2 = -CH2CONH2

R3= -MeR4= -n-butyl

Goshke, R. et al. J. Med. Chem. 2007, 50, 4818-4831.



34

OH

H2NHN

O

O

O

R3

R2 R4

STORY OF ALISKIREN

R2

(R3= Me, R4= n-butyl)IC50 Human Renin

Purified (nM)IC50 Plasma Renin (nM)

-CH2CONHMe 11 38

-CH2CH2OMe 4 32

-CH2CH2CH2OMe 1 1

-CH2CH2CH2OEt 3 20

-CH2CH2CH2OH 6 36

-CH2CH2CH2CH2OMe 2 22

-(CH2)4CH3 4 70



35

STORY OF ALISKIREN

R3

R4=n-butylIC50 Human Renin

Purified (nM)IC50 Plasma Renin (nM)

-CH3 1 3

-isopropyl 0.7 0.9

OH

H2NHN

O

O

O

R3

R4

O

Maibaum, J. et. al. J. Med. Chem. 2007, 50, 4832-4844.



36

STORY OF ALISKIREN

R4 IC50 Human Renin Purified (nM)

IC50 Plasma Renin (nM)

-CH2CONH2 3 10

-CHMeCONH2 4 12

-(CH2)2CONHMe 3 7

0.6 0.6

-CH2CH2CH2-morpholine 3 3

OH

H2NHN

O

O

O

R4

O

CH2

H3C CH3

O

NH2



37

STORY OF ALISKIREN

OH

H2NHN

O

O

O

O

O

NH2

Aliskiren

Green= CGP38560Purple= Aliskiren

Wood, J. M. et al. Biochem. Biophys. Res. Comm. 2003, 308, 698-705.PDB ID – 2v0z



38

SUMMARY OF ALISKIREN

OHHN

HN

O

NH

O

N

HN

O

SOO

CGP38560

OH

H2NHN

O

O

O

O

O

NH2

Aliskiren



39

NON-PEPTIDE INHIBITORS OF RENIN-

PIPERIDINE CLASS OF INHIBITORS

HN

O

O

Cl



40

Screening of the Roche Compound Library

IC50 = 50 μMHighly selective forrenin.

HN

O

O

Cl

Vieira, E. et al. Bioorg. Med. Chem. Lett. 1999, 9, 1397-1402.




41

X-Ray Structure Interpretation

HN

O

O

Cl

Asp32-COOH HOOC-Asp215

S4

S1 S3




42


HN

O

R2

R1

R1

R1 R2 IC50 (nM)

H 26

H

5.8x102

H

2.6x103

H 41

H 53

H 8

H 1.5

OCH3

0.060

NH

OO

N

O

OO

O

OO

NO

NO

OO

OO

O

OO

O

OO

S4

S1 S3



43

X-Ray Analysis

Lifts a whole flap region fromThr72 to Ser81


HN

O

OCH3

OCH3

O

O

O

S3sp

S1 S3

HN

O

O

Cl


S4

S1 S3




44

S3sp

HN

R1

Y Z

O O

R2

Guller, R. et al. Bioorg. Med. Chem. Lett. 1999, 9, 1403-1408.

Y Z R1 R2 IC50 (nM)Rec.

Renin

IC50 (nM)

Plasma renin

CH CH

H

5.1 3.3x102

CH CH OCH3

0.67 37

N CH

H

5.9x102 7.1x103

N

N

H 8.2x102 8.2x103

O*

O*

HN

O*

HN

O*


S1 S3

* Indicates point of attachmentto piperidine ring



45Marki, H. P. et al. Il Farmaco 2001, 56, 21-27.

PIPERIDINE CLASS OF INHIBITORSHN

O

ONR

O

O

R IC50 (nM) Rec. Renin

IC50 (nM) Plasma Renin

-CH2-CH2-OH 0.30 18

-CH2-CH2-NH-CO-CH3 0.039 0.60

-CH2-CH2-CH2-NH2 8.0 n.d

-CH2-CH2-CH2-NH-CO-CH3 2.5 n.d

S3sp

S1 S3

Story for these molecules in literature ends here



46

PIPERIDINE CLASS OF INHIBITORS-KETOPIPERAZINES

Holsworth, D. D. et al. Bioorg. Med. Chem. 2005, 13, 2657-2664.

N

HN

O

O

O

O

ON

HN

O

O

O

O

HN

O

O

O

O

IC50 = 2nM IC50 = 180nM IC50 = 54nM

SAR studies



47


N

HN

O O

O

OR

R IC50(nM)

1700

1020

440

0.50

*

N*

NH

ON

NH

O

N

HN

O O

O

OO

* Indicates point of attachment to O.

CH2

*

CH2

*

S3sp

S1S3

Found to inhibit CYP3A4



48

PIPERIDINE CLASS OF INHIBITORS-KETOPYRAZINES

Holsworth, D. D. et al. Bioorg. Med. Chem. Lett. 2006, 16, 2500-2504.

N

HN

O O

O

OO

N

R

R

IC50 RENIN (nM) 0.29 4.0

IC50 CYP3A4 (nM) 82 18

*NH

O

*N

O

R

IC50 RENIN (nM) 606 >10,000 0.42

IC50 CYP3A4 (nM) 42 3790 n.d

N*

O

NO

*O

*O

O

* Indicates point of attachment to N of tetrahydroquinoline

S3sp

S1 S3* Indicates point of attachment to N of tetrahydroquinoline



49


Holsworth, D. D. et al. Bioorg. Med. Chem. Lett. 2006, 16, 2500-2504.

N

HN

O O

O

OO

N

R

R

IC50 RENIN (nM) 9.0 550

IC50 CYP3A4 (nM) n.d 54

R

IC50 RENIN (nM) 3.2 37 450

IC50 CYP3A4 (nM) 12 455 10,000

*O

O

*O

O

* O * OH* OH

O

* Indicates point of attachment to N of tetrahydroquinoline

S3sp

S1 S3* Indicates point of attachment to N of tetrahydroquinoline



50


Holsworth, D. D. et al. Bioorg. Med. Chem. Lett. 2006, 16, 2500-2504.PDB ID – 2fs4

Tyr 14 Tyr14

H2O

Crystal Structure

N

O

H

Schematic View



R

IC50 RENIN (nM) 0.18 364 1,200

IC50 CYP3A4 (nM) 14 25,000 15,000

51


N

HN

O O

O

RO

R

IC50 RENIN (nM) 7 0.68

IC50 CYP3A4 (nM) 35 n.d

N*O

O

O

O

N*O

O

O

O

N*S

O

O

O

N*O O

HO

O

O

N*S O

HO

O

* Indicates point of attachment to C of Methyl

S3sp

S1

S3



52

SUMMARY OF PIPERIDINE CLASS

HN

O

ON

O

O

NH

O

HN

O

O

Cl

HN

O

O N

O

O

O

O



53

RECENTLY DEVELOPED INHIBITORS OF RENIN 3,9-diazobicyclo [3.3.1] nonene derivatives

Bezencon, O. et al. J. Med. Chem. 2009, 52, 3689-3702.

HN

O

ON

O

O

NH

O

HN

O

HN

N

O

Ar1

OAr2

R1

Displaces H2O from active siteaspartates



54

RECENTLY DEVELOPED INHIBITORS OF RENIN 3,9-diazobicyclo [3.3.1] nonene derivatives

Bezencon, O. et al. J. Med. Chem. 2009, 52, 3689-3702.

HN

O

HN

N

O

Ar1

OAr2

R1

HN

O

HN

N

O

O

Cl CH3

Cl

-H, -CH3, -cyclopropane

•short linker preferred•ortho, meta substituentsimproveaffinity.

•ortho di-halo substituents preferred•4-methyl group preferred

IC50 (nM) IC50 (nM)Rec. Renin= 0.20 Plasma Renin= 19



55

RECENTLY DEVELOPED INHIBITORS OF RENIN Alkyl amines

Tice, C. M. et al. Bioorg. Med. Chem. Lett. 2009, 19, 3541-3545.

NHR2

HN

O

NOR1O

X

H

H

S3 S1

S3spHOOC-Asp32

HOOC-Asp215



56

RECENTLY DEVELOPED INHIBITORS OF RENIN Alkyl amines

Human Renin = 0.47 nMPlasma Renin = 13 nMOral Bioavailability = 38%

(in dog)

Tice, C. M. et al. Bioorg. Med. Chem. Lett. 2009, 19, 3541-3545.

NHR2

HN

O

NOR1O

X

H

H-Me> -Et

3-Cl > 3-F > 2-Cl > 2,3-diCl

3o alcohol would interact with Ser 219 γ Oxygen

-Me > -H

NHMe

HN

O

NMeOOH

H

Cl



57

RECENTLY DEVELOPED INHIBITORS OF RENIN Orally bioavailable alkyl amines- crystal structure

Tice, C. M. et al. Bioorg. Med. Chem. Lett. 2009, 19, 3541-3545.PDB ID = 3gw5



58

FUTURE TARGET OF THE RENIN-ANGIOTENSIN

SYSTEM



59

FUTURE TARGET OF RENIN ANGIOTENSIN SYSTEM

Renin Angiotensin

System

Remodeling

Development

Inflammation

Thrombosis

Involvement of Renin-Angiotensin System

Clearly not mediated by Ang II.

Is there a Renin Receptor?

Nguyen, G. et al. Curr. Opin. Nephrol. Hypertens. 2003, 12, 51-55.



Organ of interest mRNA cDNA Cloning

Vector

Put into cellsCells are plated• 1 cell= 1 colony

Cells express cDNA

proteins

Screening of cells against

I125 Renin

Cells with receptors

emit radiation

Isolate the cDNA for receptor

Transfect Human cell lines with

cDNA


60Image adapted from- http://tainano.com/Molecular%20Biology%20Glossary.files/image087.gifNguyen, G. et al. Journ. Clin. Invest. 2002, 109, 1417-1427



61

Functionality of the receptor Effect on Ang I generation in presence of renin


Membrane bound renin 1nM

Free renin 1nM

Membrane bound renin 0.5nM

Free renin 0.5nM

Agen = AngiotensinogenAng I = Angiotensin I



62

Functionality of the receptor Effect on ERK1/ERK2 activation




63

Immunofluorescence studies of receptors on cells of human: Kidney

Placenta


Coronary

Artery



64

Characteristics of renin receptor: 350 amino-acid protein with no homology 45-kDa Multidomain

▪ Hydrophobic amino-terminal domainbinds renin and prorenin activates it

▪ Cytoplasmic tail= 20 amino acids activates ERK1/ERK2 intracellularly

▪ Single transmembrane domain


Campbell, D. J. Hypertension, 2008, 51, 1259-1264.



65

CONCLUSION

PAST PRESENT FUTURE

Over 100 years since discovery of renin

OH

H2NHN

O

NH2

O

O

O

ON

HN

O

O

N

HN

O

O

O

O

OHN

H

OHN

O

NH

HN

O

HN

N

O

O

Cl CH3

Cl



66

ACKNOWLEDGEMENTS

Dr. Umesh Desai

The Desai Group

Department of Medicinal Chemistry at VCU



DATE – OCTOBER 23 rd, 2009 PRESENTED BY – AKUL MEHTA VIRGINIA COMMONWEALTH UNIVERSITY SCHOOL OF...

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