Design and Synthesis of Macrocycles as-Secretase (BACE-1) Inhibitors for the

Treatment of Alzheimer’s Disease

Amyloid precursor protein

Abeta 1-40, 42

Amyloid plaques (extracellular)

Neuron cell death and loss of neurotransmitter function

Alzheimer's disease symptoms

Tau

Tau hyperphosphorylation

Neurofibrillary tangles (intracellular)

Alzheimer’s Disease Pathophysiology

• Alzheimer’s Disease is a progressive, devastating and incurable illness

• Some 4.5 million Americans are affected at a cost of $100 billion a year

Nitasha Manchanda, Alzheimer’s Disease, Decision Resources, June 2007

/ Secretase

(400,000 compound HTS campaign)

NH

HN

NH

NH

HN

NH

OHH2NO

O

O

OH

O

O

O

O

OHONH2

O

O OH

Introduction

OM 99-2 Heptapeptide Ki = 1.6 nM

… to small-molecule BACE inhibitors

• Poor bioavailability• Metabolically unstable

From peptides …

Hong, L. et al. Science, 2000, 290, 5489

N

N N

O

MeNH2

2-Amino-3,4-dihydroquinazolineBACE Ki = 900 nM

O

Macrocylic BACE-1 Inhibitors – Design

Target molecule:

Synthetic strategy:

Bell, Ian M.; et al Journal of Medicinal Chemistry. 2002, 45, 2388-2409

ON

N NH2

Ki = 450 nM

O

N

Ki = 900 nM

N

NO

NH2

N

SAR O

N

N NH2

O

N

O

NO2

NH

F

N

OHO

N

NO

NH2

N

O

NO2

NH

O

N

O

20 Fold better potency

Macrocylic BACE-1 Inhibitors – Initial Synthesis

Poor yield-----Difficult for scaling up -----SAR studies

Baxter, Ellen E.; Huang, Yifang et al. WO 2007/092839

OMe OMe

BocHN

HBTU, DIEA, DMF

H

O

NaBH(OAc)3

ONH

NO2

2. BrCN, CH2Cl2

OH

O

NH2

HBTU,

NH

OH

O

OMe

N

O NHBoc

OMe

N

O NH2 OMe

N

O NH

NO2

F

N

O

ON

N

N

O

NH2

OH

N

O NH

NO2

F

BBr3

K2CO3

DMF

TFA

2. LiAlH4, THF

88% 96%

quantitative 21% 86%

16%

1. H2, Pd/C

30%

Ki = 51 nM

CH2Cl2

DIEA

NO2

F

CH2Cl2

1.

Two Reactive groups

Exploring Alternate Macrocyclization Strategy

Reductive aminationNo literature precedent

N

NO

NH2

N

O

NO2

NH

O

N

O

NO2

OO

N

O

NH2

Macrocyclization via Reductive Amination

NaBH3CN/CH3OH or NaBH4/CH3OH not working.

OH

OH

O

OBn

OH

O

NH2

OBn

NH

O

OBn

NH

LiAlH4

BocHNCO2H

OBn

N

O NHBocOH

N

O NHBoc

O

O

N

O NHBoc

O

NO2

O

N

O NH2

O

NO2

ONH

NO2

N

O

1. BnBr, K2CO3

2. NaOH

96%

1. (COCl)2 DMF(cat)

2.

94%

72%

H2, Pd/C

96%

quantitative

TFA

68%98%

HBTU, DIEA , DMF

F

NO2K2CO3

DMF

H

NaBH(OAc)3

DCMX

THF

Macrocyclization via Reductive Amination-Continued

Imine formation by dehydration

ON

N

N

O

NH2

O

N

O NH2

O

NO2

ONH

NO2

N

O

1. H2, Pd/C

2. BrCN

23%

4 A Molecular Sieves NaBH(OAc)3

78% Ki = 51 nM

DCM

Reflux in benzene with DeanStark trap, NMe4BH(OAc)3

15%

Introducing -branch to the Macrocycles

O

N

NN

NH2O

Me

Ki = 8 nM

ON

N NH2

N O

Baxter, Ellen E.; Huang, Yifang et al. WO 2007/092839

Ki = 5 nM

Synthesis of -Branched Macrocycle

OBn

NH2

OBn

BH3

OBn

HN

BocHNOH

O

O

K2CO3

DMF

NaBH(OAc) 3

OBn

N

ONHBoc

OH

N

ONHBoc

O

N

ONHBoc

NO2

O

O

N

ONH2

NO2

OO

NH

NO2

N O

ON

N NH2

N O

O

71%

H2, Pd/C

4 A Molecular

Sieves

97%

99%

quantitative

93%

78%

1. H2, Pd/C

2. BrCN, CH2Cl2

NaBH(OAc) 3 THF

48%

92%

THF, reflux HBTU, DIEA, DMF

TFA

CH2Cl2

N

NO2

F

CH2Cl2

H

H

H

Major Macrocycles Synthesized-SAR

Position of Amide: important; Chang of functional groups and ring size: No significant impact ----Great potential for imposing desired pharmaceutical properties.

O

N

N

NH2

Ki = 51 nMCellular A IC50 = 516 nM

N

O

O

NN

N

NH2

A (CHO) cellular IC50 = 6.6 nMhERG Ki = 4000 nM

N O

O

N

N

NH2

Ki = 5 nMA (CHO) cellular IC50 = 17 nM

hERG Ki = 19nM

N O

O

N

N

NH2

inactive

N

O

Ki = 31 nMCellular A IC50 = 35 nM

O

N

N

NH2

N O

ON

N

N

O

NH2

Ki = 44 nM

ON

N NH2

N O

Ki = 8 nMCellular IC50 = 81 nMhERG Ki = 1500 nM

ON

N NH2

N O

O

Ki = 17 nMhERG Ki = 3500 nM

ON

N NH2

N O

O

Ki = 22 nMCellular IC50 = 660 nMhERG Ki = 3500 nM

1618

161616

16161617

Macrocycle: In Vivo Testing in Mouse

In vivo efficacy (2 mpk, iv, 2 hr)

Mouse: plasma a -42%, Rat : plasma a -74%,

Plasma A lowing in mice

-22% at 25 mpk, PO/dose, 3 h

O

N

N

NH2

BACE Ki = 5 nMCellular A IC50 = 17 nM

hERG IC50 = 1.9 uMCathepsin D IC50 = 0.78 uM

renin IC50 = 0.34 uM

N O

Acyclic via Macrocyclic compounds

Ki = 5 nMA (CHO) cellular IC 50 = 17 nM hERG Ki = 1900 nM

Plasma A lowing in mice22% at 25 mpk, PO/dose74% at 2 mpk, iv/dose in rat

Ki = 8 nMA (CHO) cellular IC 50 = 44 nM

hERG Ki = 84 nM

No in vivo activity

• Designed and synthesized macrocycles (ki = 5 nM) as BACE-1 enzyme inhibitor Starting from HTS hit (ki = 900 nM).• Developed reductive amination as novel and efficient synthetic approach for cyclizing Macrocycles.

O

N

N

NH2

N O

O

N

NN

NH2O

Me