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KaoHsiung Medical School KaoHsiung, Taiwan December 14, 2009. The Importance of Fluorine in the Design of BACE Inhibitors. James R. McCarthy. 14,000,000. 12,000,000. 10,000,000. 8,000,000. NUMBER OF VICTIMS. 6,000,000. 4,000,000. 2,000,000. 0. 1900. 1910. 1920. 1930. 1940.

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slide1

KaoHsiung Medical School

KaoHsiung, Taiwan

December 14, 2009

The Importance of Fluorine in the Design of BACE Inhibitors

James R. McCarthy

slide2

14,000,000

12,000,000

10,000,000

8,000,000

NUMBER OF VICTIMS

6,000,000

4,000,000

2,000,000

0

1900

1910

1920

1930

1940

1950

1960

1970

1980

1990

2000

2010

2020

2030

2040

2050

YEARS

Age 65-74 Years

Age 75-84 Years

Age 85+ Years

Prevalence of Alzheimer’s Disease (AD)(By decades in U.S.A. from 1900-2050)

slide3

Contrast Between the Healthy Brain and

the Advanced AD Brain

Ventricle

Hippocampus

slide4

Two main pathological hallmarks of AD:

neuritic plaques and neurofibrillary tangles

Neurtic plaques

Neurofibrillary tangles

Largely Ab peptide

Hyperphosphorylated tau

More specific to AD

Found in many neurological disorders

slide5

BACE (b-SECRETASE) HYPOTHESIS

Blocking the first cleavage of APP by b-secretase will limit the substrate for g-secretase and diminish Ab peptide production.

sAPPb

Amyloid Precursor Protein

KPI OX

NH2+

NH2+

b-amyloid (Ab38-49)

(b-secretase)

First step

+

CLEARANCE

Diffuse

Primitive (Immature)

g-secretase

COO-

COO-

Classic (Typical)

C99 (b-CTF)

Compact (Burnt-out)

-

Lilly Confidential

BACE

Clark and Trojanowski, 2000, Neurodegenerative Dementias, p151.

slide6

Common isosteres that mimic the tetrahedral intermediate of amide bond hydrolysis as catalyzed by aspartyl proteases (TSAs):

N-terminal

Central core

C-terminal

P1’

P2

P1

P3

P2’

Ethanolamines

Hydroxyethylenes

BACE (b-APP Cleaving Enzyme) is a membrane

bound aspartyl protease

Aspartyl protease mechanism of action

slide7

BACE1, an Aspartyl Protease, co-crystallizes with a substrate mimetic inhibitor OM99-2

(Tang, Hong et. al., Science, 2000, 290, 150-153)

The active site of BACE is defined by OM99-2, containing the hydroxyethylene core (Leu*Ala)

Ki= 1.6 nM

slide9

Hippocampus

Vehicle

0.5

treated

0.4

67%

67%

60%

60%

0.3

***

***

Hippocampus Ab (x-40) (ng/gm)

**

**

0.2

0.1

0.0

mdr_KO PO

mdr_KO SQ

A Novel Sulfone That Is Efficacious in Pgp Transporter Deficient Mice

mcaFRET IC50 = 0.4 nM

Ab IC50 = 1 nM

Mouse microsome metabolism = 90%

Rat microsome metabolisn = 87%

Caco2 = 3%

slide11

Physical properties of BACE inhibitors are key for brain penetration

For a reasonable chance of compound crossing Blood-Brain Barrier (BBB), following physical properties are important:

  • cLog P > 2 and < 5
  • PSA < 85 Å2
  • MW < 500
  • Caco-2 > 200 nM/sec
  • cpKa < 8

Van der Waterbeemd, H.; Camenisch, G.; Folkers, G.; Chretien, J.R.; Raevsky, O.A. Estimation of blood-brain barrier crossing of drugs

Using molecular size and shape and H-bonding descriptors. J. Drug Targeting, 1998, 6(2), 151-165

Kelder, J.; Grootenhuis, P.D.J.; Bayada, D.M.; Delbressine, L.P.C.; Ploemen, J.-P. Polar molecular surface as a dominating determinant

For oral absorption and brain penetration of drugs. Pharm. Res., 1999, 16(10), 1514-1519

slide12

Designing Efficacious BACE Inhibitors

  • Which pockets are important to occupy?
  • Utilize directed SAR to define binding opportunities.
slide13

Strategy: Design BACE inhibitors with

clogP > 2 and < 5 PSA < 85

MW < 500 cpKa < 8

  • Replace polar N-terminal groups with smaller groups
  • Retain potency through novel C-terminal rings to increase
  • rigidity

R = Me

BACE1 IC50 = 80 mM

BACE1 IC50 > 100 mM

BACE1 IC50=2 nM

slide14

Difluorophenyl at P1 is 5 times more potent vs. phenyl

Increase in potency consistent with van der Waals

Forces between the fluorines and the surface of

the protein. (Will be illustrated with a crystal structure)

slide15

BACE1 IC50 = 20 nM

9.2 Hz

9.5Hz

1.8Hz

8

9

7

6

unsubstituted

9.2 Hz

6.5Hz

3.05 Hz

7

9

8

6

Extreme coupling constants for compound 1 indicate a more rigid solution structure

7

8

1

BACE1 IC50 = 926 nM

9

2

slide16

Cyclization Strategies

BACE1 IC50 = 20 nM

• 5 and 6-membered rings exhibit optimal binding to BACE1

(by DOCKing experiments)

BACE1 IC50 = 0.35nM

Ab IC50 = 5.7nM

slide17

Elimination of polar group at P2 & decrease in MW have a small impact on BBB penetration

P2

P2

P2’

P3

P1

P3

P1

P1

2

3

1

BACE1 IC50 = 0.4 nM

Ab IC50 = 5.7 nM

B/P = 0.02

MW = 553

PSA = 132

BACE1 IC50 = 0.5 nM

Ab IC50 = 1.5 nM

B/P = 0.02

MW = 580

PSA = 127

BACE1 IC50 =80000 nM

B/P = 0.06

MW = 312

PSA = 61

Hypothesis: Basicity of nitrogen on compound 3

(cpKa = 9.5) hinders blood brain barrier penetration

slide18

Optimization strategy

  • Di-axial groups are required to fill S1’ and S2’ pockets
  • Addition of hydrophobic P1’ & P2’ groups led to the design of smaller less polar inhibitors

S1’ pocket

BACE1 IC50 = 0.35 nM

S2’ pocket

slide19

Introduce P2’ group to improve potency

Both forms exist in solution

slide21

Synthesis of 3-Alkyl-2-Alkoxy morpholine BACE inhibitors

X-ray crystal structure unequivocally established the stereochemistry at all three chiral centers

slide24

BACE IC50 = 1320 nM

BACE IC50 = 222 nM

BACE IC50 = 306 nM

B/P 0.07

(Brain to Plasma exposure ratio)

B/P 0.42

B/P 0.48

Progression of the SAR: P2’/P1’ on morpholine

2434074

BACE IC50 = 77 nM

Ab IC50 = 91 nM

MW =414

BACE IC50 = 2800 nM

Ab IC50 = 2160 nM

B/P 0.38

Morpholine ring decreased pKa & provided anomeric effect

Introduction of P1’ methyl increased potency

slide25

P1’ axial

P2’

cLogP = 2.4

PSA = 70 A2

MW = 414

cpKa = 7

Caco-2 perm ~ 300 nM/sec

Anomeric effect favors axial

P2’ O-neopentyl

2434074 exhibits desired physical properties for a CNS drug

o

slide26

Pharmacokinetics of 2434074 in wild type mice shows minimal exposure after oral administration (10 mg/kg)

s.c

2434074

slide27

rat

A

P+O

B

O-dealkylated

B

A

control

parent

Rat hepatocyte metabolism for 2434074

  • Metabolism predominantly on P2’ neopentyl group
slide28

Approach to decrease metabolism at P2’

  • Mouse hepatic microsomal experiments show that most metabolism
  • is concentrated on the P2’ position

Introduction of fluorine at P2’

slide29

Fluorinated alcohols designed for attachment at

the P2’ site on the morpholine inhibitors

slide30

Routes to Fluoro-Neopentyl Alcohols Represented by the

Syntheses of 3-Fluoro-2-fluoromethyl-2-methyl-propan-1-ol (3)

slide33

Approach to decrease metabolism at P2’

2434074

The bis (difluoroMe) analog of 2434074 maintains potency with decreased metabolism

slide34

Approach to decrease metabolism at P2’

  • Introduction of oxygen or fluorine on cyclic P2´substituents lead to decreased metabolism but also decreased activity
slide35

S2’

Iterative Docking and X-ray Studies for introduction of fluorine on P2’ Cyclohexyl

  • SAR showed polar groups not tolerated in S2’ pocket
  • Docking suggested 3-cyclohexyl position may allow addition of metabolic stabilizing difluoro substitution
  • Subsequent x-ray structure (yellow) verified this finding

{

slide36

Significant difference in activity of the two enantiomers

mcaFRET IC50 = 45 nM

Ab IC50 = 67 nM

Rat surrogate metabolism: 49%

PSA = 72.9

cLogP = 2.6

MW = 475

Caco-2 = 24.2

mcaFRET IC50 = 280 nM

Ab IC50 = 473 nM

Rat surrogate metabolism: 50%

slide39

Summary

  • Structure-based drug design was utilized to obtain drugable BACE
  • inhibitors
  • The importance of fluorine in the design of the BACE inhibitors was
  • shown to be two-fold:
  • - Fluorines substituted on the meta-positions of P1 phenyl group resulted
  • in an increase in potency via van der Waals forces between the surface
  • of the protein and the fluorines.
  • - The addition of the aliphatic fluorines on the 3-position of the P2’
  • cyclohexyl group resulted in decreased metabolism. The crystal
  • structure shows that the fluorines are situated at the edge of the
  • pocket and are not interfering with interactions of the cyclohexyl group
  • with amino acids in the S2’ pocket.
slide40

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