Andrew Stamford, PhD

Discovery ChemistryMerck Research LaboratoriesMerck & Co., Inc., Kenilworth, NJ USA

Bionomics – Merck3rd Annual Symposium Drug Discovery and Development for Cognition and Alzheimer’s Disease Adelaide, 16 November 2015

Discovery of MK-8931 (verubecestat) as a Disease Modifying Treatmentfor Alzheimer’s disease

Amyloid hypothesis

MRI Image from C. Jack MD Aging

and Dementia Imaging Lab Mayo Clinic, http://mayoresearch.mayo.edu/jack_lab/

decades

Alzheimer’s Disease (AD) – a human health crisisAlzheimer’s Disease (AD) – a human health crisis• Fatal progressive neurodegenerative disease

– Impaired memory, ability to perform activities of daily living.

– Personality and behavioral disturbances.

• >5 million AD patients in the US*; ~24 million worldwide†

– Worldwide costs estimated at US $604 billion in 2010†

• Currently no disease modifying therapy available

– Current treatments are symptomatic and provide modest temporary benefit

• Incidence is growing rapidly as population ages, creating an unsustainable patient, caregiver, and economic burden

*Alzheimer’s Association, 2010 Alzheimer’s Disease Facts and Figures, Alzheimer’s & Dementia, Volume 6 and references therein.

†Alzheimer’s Disease International, World Alzheimer Report 2010, http://www.alz.co.uk/research/world-report

neuron

• Synaptic dysfunction

• Neuronal cell death

• Neuroinflammation

ββββ

A673T Protective

A673V Causative

BACE1

γγγγ

APP sAPPββββ

C99

γγγγ-secretase

Aββββ40Aββββ42

FADmutationspromote

membrane

αααα

N

C

FADmutationspromote

• The first amyloidogenic step in neurons requires BACE1 (APP ββββ-site cleavage)

• Mutations that increase Aββββ42 production or Aββββ42/40 ratio lead to early-onset AD (FAD)

• APP mutation A673T is associated with a reduced risk of developing AD and results in ~40%

inhibition of BACE1 mediated APP processing in cells

• Inhibition of Aββββ formation should slow or halt the progression of cell death and cognitive decline

Amyloid hypothesis

Jonsson, et al., Nature, 2012, 488, 96-99Karran et al., Nat. Rev. Drug. Disc., 2011, 10, 698

BACE1 characteristics and discovery challenges

Asp32

Asp228

Flap

OM99-2

A. Ghosh et al., JACS, 2000, 122, 3522J. Tang et al., Science, 2000, 290, 150

BACE1 – OM99-2

BACE1 Ki = 1.6 nM

• Membrane bound aspartyl protease intra-cellularly localized in neurons

• Active site within endosomes (pH ~5)

• Inhibitors must be brain penetrant and cell permeable

• Classical aspartyl protease inhibitor designs are Pgp substrates and poorly brain penetrant

S1 S2′

S2

S3

S4

S3′S1′

S4′

5

• Expression systems developed for large scale production of soluble BACE1 (10 g)

– 15N-BACE1 for NMR fragment screen

– Crystalline BACE1 for X-ray crystallography

• 10K member fragment library (< 300 Da) screened vs. 15N-BACE1

– HSQC NMR as mixtures of 12 @ 500 µM

• Weak binders detected by NMR chemical shift perturbations

• Confirmation of hits as singles by NMR

BACE1 NMR fragment screening

• Opportunity to discover novel chemotypes

KD(NMR): 550 µµµµM 290 µµµµM 100 µµµµM 3700 µµµµM

Isothiourea fragment optimization

Asp32

Asp228

8.60 8.40 1H (ppm)

13

5.5

13

2.1

15N

(p

pm

)

Asp228

Asp32

Ch

em

ica

l S

hift

[p

pm

]

8.04 7.84 1H (ppm)

11

3.2

1

11

.8

1

5N

(p

pm

)

Gly13

(S3)

Gly74

(Flap)

Black: BACEGreen: BACE + 12 compound mixture

KD(NMR) = 550 µµµµMIle118

Gly34

Gly230

Black: BACEMagenta: BACE + isothiourea

KD(NMR) = 15 µµµµM

NMR screening204 isothioureaanalogs

1 2

6

Optimized isothiourea fragment:

x-ray co-crystal structure

FlapS1

S3

Y.-S. Wang et al., J. Med. Chem., 2010, 53, 942

2Kd (NMR) = 15 µM

BACE1 IC50 >200 µM

2.8Å2.9Å

2.9Å

2.7Å

2.7Å

Asp228

Asp32

• Isothiourea – BACE1 co-crystals used to generate all subsequent co-crystal structures

• Turnaround time <1 week

7

Design of a novel iminohydantoin scaffold

acylguanidinepKa: ~8

CreatininepKa: 5

2pKa: ~10

• Minimize H-bond donors• Tune basicity• Access sub-pockets adjacent to catalytic dyad

N3 substitutedpKa: ~ 7

51

3

8

model

Iminohydantoin scaffold validation!

KD (NMR): 200 µµµµMBACE1 Ki: >500 µµµµMLE: 0.30

Predicted binding mode

KD (NMR): 120 µµµµMBACE1 Ki: ~300 µµµµMLE: 0.27

Unexpected binding mode

Asp228

Asp32

X-rayAsp228

Asp32

X-ray

X BACE1 IC50

73 µM

72 µM

73 µM

Enabling chemistry: biochemically active compounds

~600 compounds

X BACE1 IC50

7 µM

15 µM

9 µM

Optimization: affinity

S1

S2′

S3

BACE1 Ki: 73 µM

occupancy: S1, S3

Thr232

2.71Å

2.86Å

S1

S3 S2′

BACE1 Ki: 71 nM

occupancy: S1, S3, S2’

Z. Zhu et al., J. Med. Chem., 2010, 53, 951

Optimization: lead-like properties

Z. Zhu et al., J. Med. Chem., 2010, 53, 951

truncation reoptimization

BACE1 Ki nM 71 860 3,700

MW (LE) 536 (0.25) 319 (0.36) 265 (0.37)

Cell Aβ40 IC50 nM 700 2,600 13,000

CatD / BACE1 <1 1 >20

ClogP 7.5 5.4 1.9

Rat AUC0-6h

10 mpk PO µµµµM.h<1 4.8

brain/plasma 1.6

S3

S1

S2′

S1S3 S2′

S3sp

13

BACE1 Ki (LE) 3.7 µµµµM (0.37)cell Aββββ40 IC50 13 µµµµMCatD/BACE1 >20Plasma EC50,U

for rat CSF Aββββ40 reduction

1.7 nM (0.48)10 nM2115 nM

Optimization: drug-like properties

• Core ring expansion• Maximize structural diversity

MBI-1

5.0 nM (0.38)13 nM43030 nM

MBI-4

Measurement of Aββββ40 in rat cerebral spinal fluid (CSF):

• Key in vivo assay for differentiating compounds.

• High-throughput and used early in ROP to drive SAR development.

• Translational endpoint; human CSF Aβ is a key biomarker for AD clinical program

A. Stamford et al., ACS Med. Chem. Lett. 2012, 3, 897; M. Mandal et al., J. Med. Chem. 2012, 55, 9331

Beyond iminopyrimidones: iminothiadiazine core

PSA (ClogP) 69 (3.2) 86 (2.7)

MBI-1Model

• Iminothiadiazine analog of MBI-1

– favorable calculated properties

• Modeling suggested an excellent overlay of the two cores with

– no unfavorable contacts due to sp3

sulfonyl vs. sp2 acyl

– axial disposition of biaryl motif

– favorable trajectory in S1-S3 despite difference in shape of the core. S1S3

S3sp

• Iminothiadiazine analog of MBI-1

– favorable calculated properties

• Modeling suggested an excellent overlay of the two cores with

– no unfavorable contacts due to sp3

sulfonyl vs. sp2 acyl

– axial disposition of biaryl motif

– favorable trajectory in S1-S3 despite difference in shape of the core.

PSA (ClogP) 69 (3.2) 86 (2.7)

BACE1 Ki nM 1.7 nM (LE = 0.48) 2.4 nM (LE = 0.45)

MBI-1Model

X-ray

S1S3

S3sp

Beyond iminopyrimidones: iminothiadiazine core

Profile of iminothiadiazine biaryl analog vs. MBI-1

• Profile of thiadiazine analog compared favorably to MBI-1

– increased cell shift, likely driven by lower pKa.

– higher permeability than MBI-1; robust lowering of central Aβ40 in rat.

BACE1 Ki nM (LE) 2 nM (0.48) 2 nM (0.45)

PSA (ClogP) 69 (3.2) 86 (2.7)

Aβ40 IC50 nM (pKa) 11 (7.5) 55 (6.6)

CatD / BACE1 21x 47x

Caco-2 Papp (ER) 153 nm·s-1 (2.4) 278 nm·s-1 (1.6)

Rat Aβ40 10 mpk PO, CSF (ctx)Pl3h,u nM (B/P)

-81% (-61%)30 (2.9)

-82% (-53%)7 (2.3)

MBI-1

Re-investigating P1-P3 substituents: Diaryl amides

• Investigation of new P1-P3 biaryl modifications driven by

– susceptibility of propynyl pyridine to glutathione addition in vivo

– need for improved selectivity over CatD

• structural differences between CatD vs. BACE1 in S3sp may contribute to modest selectivity

• P1-P3 diaryl amides investigated following from previous efforts in the iminohydantoin series

– distal aryl ring in the iminohydantoin diaryl amide bound deeper in S3sp than in the simple biaryls

N

HN

HN

OHN

O

O

BACE1 Kicell Aββββ40 IC50

CatD / B1

2 nM55 nM47X

600 nMn.d.

~100X S1S3

S3sp

Diaryl amides: Iminopyrimidinone vs. iminothiadiazine

BACE1 Ki nM (LE) 4 (0.42) 2 (0.43)

Aβ40 IC50 nM 5 2

CatD / BACE1 >10,000X >10,000X

PSA, ClogP, pKa 98, 2.8, 8.3 115, 1.4, 7.5

• P1-P3 Biaryl amide analogs with both iminopyrimidinone and iminothiadiazinecores showed excellent BACE1 potency with no shift in the cell-based assay.

• Unprecedented selectivity over CatD compared to earlier biaryl analogs.

Diaryl amides: Iminopyrimidinone vs. iminothiadiazine

BACE1 Ki nM (LE) 4 (0.42) 2 (0.43)

Aβ40 IC50 nM 5 2

CatD / BACE1 >10,000X >10,000X

PSA, ClogP, pKa 98, 2.8, 8.3 115, 1.4, 7.5

Rat Aβ40 10 mpk PO, CSF (ctx)Pl3h,u nM (B/P)

-53% (-11%)22 (0.4)

-85% (-53%)36 (0.6)

Caco-2 Papp nm·s-1 (ER) 22 (11) 128 (2.4)

• Cores differentiated significantly on ability of analogs to lower central Aβ40 in rat in alignment with differences in permeability and susceptibility to Pgp efflux.

– Unanticipated at design stage given PSA and ClogP values.

– May be driven by differences in pKa.

Diaryl amides: Iminopyrimidinone vs. iminothiadiazine

BACE1 Ki nM (LE) 4 (0.42) 2 (0.43)

Aβ40 IC50 nM 5 2

CatD / BACE1 >10,000X >10,000X

PSA, ClogP, pKa 98, 2.8, 8.3 115, 1.4, 7.5

Rat Aβ40 10 mpk PO, CSF (ctx)Pl3h,u nM (B/P)

-53% (-11%)22 (0.4)

-85% (-53%)36 (0.6)

Caco-2 Papp nm·s-1 (ER) 22 (11) 128 (2.4)

Rat 5-day 30 mpk BIDCYP induction

Liver weight gain

1A1, 2B, 3A1yes

nonenone

• Core also impacted induction of CYP isozymes upon sub-chronic dosing in rat.

MK-8931: Structure

MK-8931verubecestat

BACE1 Ki nM (LE) 4 (0.42) 2 (0.43)

Aβ40 IC50 nM 5 2

CatD / BACE1 >10,000X >10,000X

PSA, ClogP, pKa 98, 2.8, 8.3 115, 1.4, 7.5

Rat Aβ40 10 mpk PO, CSF (ctx)Pl3h,u nM (B/P)

-53% (-11%)22 (0.4)

-85% (-53%)36 (0.6)

Caco-2 Papp nm·s-1 (ER) 22 (11) 128 (2.4)

Rat 5-day 30 mpk BIDCYP induction

Liver weight gain

1A1, 2B, 3A1yes

nonenone

MK-8931: Profile and binding

HN

S

N

NH

F

HN

O

NF

O

O

• Amide NH is within H-bonding distance of Gly230.

• Only 2 rotatable bonds – amide may provide pre-organization through restricted rotation.

• Position of pyridyl nitrogen may serve to mask impact of amide NH through an intramolecular H-bond.

BACE1 Ki nM 2

BACE2 Ki nM 0.4

Selectivity vs. CatD >45,000X

CatE >45,000X

Pepsin >45,000X

Renin 15,000X

Cell Aββββ40 IC50 nM 2

Caco-2 Papp nm·s-1

(ER)128(2.4)

LLC-PK1 Papp 10-6cm·s-1

(MDR1 ER)29

(11)

D. Millan et al., Med. Chem. Comm. 2011, 2, 669a.

S1S3

S3sp

Gly230

MK-8931: Rat Aβ40 pharmacodynamic activity

• Excellent lowering of central Aβ in rat with single PO doses.

• CSF ED50 = 5 mg/kgEC50,u in plasma = 48 nM

• Cortex ED50 = 8 mg/kgEC50,u in plasma = 78 nM

• Rat plasma unbound fraction = 37%

BACE1 Ki nM 2

BACE2 Ki nM 0.4

Selectivity vs. CatD >45,000X

CatE >45,000X

Pepsin >45,000X

Renin 15,000X

Cell Aββββ40 IC50 nM 2

Caco-2 Papp nm·s-1

(ER)128(2.4)

LLC-PK1 Papp 10-6cm·s-1

(MDR1 ER)29

(11)

0

20

40

60

80

100

CSFCortex

1 10 100

SCH 900931 mg/kg, po

Aββ ββ

40(%

Veh

icle

)

HN

S

N

NH

F

HN

O

NF

O

O

MK-8931

CSF Aββββ40 Time Course

MK-8931: Monkey Aβ40 pharmacodynamic activity

Control CSF Cortex0

20

40

60

80

100

-60%-72%

10 mg/kg PO 4h

A40 % of Control

CSF / Cortex Aββββ40

• MK-8931 produced a profound and sustained reduction of CSF Aβ40 over 24hin cisterna magna cannulated Cynomolgus monkeys following a single PO dose.

• Cortical Aβ40 was also dramatically reduced by a single PO dose of MK-8931.

– Supports a direct link between CSF and cortical Aβ and mimics the Phase 1 clinical design.

– Mitigated concerns about Pgp limiting the ability of MK-8931 to achieve sufficient brain BACE1 occupancy required to effect reduction of Aβ.

MK-8931: ADME profile and human dose projection

• Good to excellent pharmacokinetic profiles in rat, dog, and monkey.

• Low human in vitro hepatocyte clearance (<1 µL/min/106 cells).

• High free fraction in pre-clinical species (36-50%) and human (45%).

• Low probability for drug-drug interactions

– No inhibition, time-dependent inhibition, or induction of major CYP isoforms.

• BCS Class 1 molecule with properties consistent with QD dosing.

– Human dose projections made based on allometry from PK parameters across species in combination with PK/PD from monkeys.

– Projected dose for 75% reduction of CSF Aβ40 AUC0-24h,ss: 35 mg

Bioavailability 45 – 100%

t1/2 2 – 10 h

Vd,ss 2.7 – 7.5 L/kg

B/P (rat, monkey) 0.4 – 1.3

MK-8931 Dramatically reduces CSF Aβ in AD patients

� Extensive Phase 1 program in both healthy volunteers and Alzheimer’s patients to thoroughly define CSF Aβ PK/PD and safety profile.

� Similar levels of CSF Aβ reduction in both populations.

� Single and multiple doses generally well-tolerated.

Placebo (N=6 Planned)MK-8931 12 mg (N=8 Planned)MK-8931 40 mg (N=8 Planned)MK-8931 60 mg (N=8 Planned) � Dose-dependent reduction in

CSF Aβ40 (lumbar catheterization) with a mean reduction from baseline up to 90%.

� Similar reduction in CSF Aβ42.

� Dose proportional increase in plasma and CSF exposures.

� CSF/PlasmaU ~ 0.24.

MK-8931: Phase 2/3 Clinical program

• EPOCH: Phase 2/3 safety and efficacy trial in mild-to-moderate AD– NCT01739348 (clinicaltrials.gov)

– December 2012: initiated Phase 2 (safety cohort)

• n = 400; placebo, 12, 40 and 60 mg, QD

– December 2013: initiated dosing for Phase 3 (main cohort)

• n = 1560; placebo, 12 and 40 mg, QD

– 18 Month dosing duration

• APECS: Phase 3 safety and efficacy trial in prodromal AD– NCT01953601 (clinicaltrials.gov)

– December 2013: trial initiated

• n = 1500; placebo, 12 and 40 mg, QD

– 24 Months dosing duration

• From Phase 1 data, simulation of Aββββ reduction distributions predicts that >95% of patients will have:

– 50-75% Aβ reduction with 12 mg QD

– >75% Aβ reduction with 40 mg QD

Summary and outlook•28

• Building on an iminoheterocycle platform developedfrom fragment-based screening, we have designed a novel aspartyl protease binding motif, the iminothiadiazine.

• Combination of the iminothiadiazine core with P1-P3 diaryl amide substitution lead to discovery of MK-8931, with excellent potency against BACE1, very high selectivity over Cathepsin-D, and potent reduction of central Aβ in rat and monkey.

• MK-8931 profoundly reduced CSF Aβ in Phase 1 trials in healthy volunteers and in Alzheimer’s patients. Two long-term Phase 3 outcome trials are currently in progress.

• The pharmacodynamic, pharmacokinetic, and safety profile of MK-8931 enables the most thorough test of the amyloid hypothesis to date with no restriction on the degree of central Aβ lowering attainable in clinical trials.

HN

S

N

NH

F

HN

O

NF

O

O

Summary and outlook (2)•29

• From identification of BACE1 as a target to initiationof first Phase 3 trial with a BACE inhibitor took 14 years.

– 8 years from publication of first iminoheterocycle patent application.

– High tolerance for risk and long time horizon required.

• Very active area of research industry-wide with significant coalescence around benzyl amidine as a minimal pharmacophore.

• Iminoheterocycles in general represent a breakthrough in the ability to target BACE1 and ultimately offer patients hope for development of a disease modifying therapy for AD.

HN

S

N

NH

F

HN

O

NF

O

O

First disclosed

Acknowledgments (1)

Discovery Biology:, Xia Chen, Robert Hodgson, Lynn Hyde, Matt Kennedy, A.

Konovalov, Reshma Kuvelkar, Sherry Lu, Eric Parker, Lixin Song, Giuseppe Terracina, B.

Terry, Lili Zhang, R. Zhang, Qi Zhang, Cinzia Cantu, Zoe Dong, Rob Del Vecchio, Michelle

Sondey, S Villarreal

Medicinal Chemistry: Jared Cumming, Eric Gilbert, Chunli Huang, John Caldwell,

Michale Czarniecki, James Durkin, William Greenlee, Ulrich Iserloh, Guoqing Li, Wei Li,

Rob Mazzola, Brian McKittrick, Jeff Misiaszek, Jianping Pan, Andy Stamford, Jack Scott,

Elizabeth Smith, George Sun, Lingyan Wang, Yusheng Wu, Zhaoning Zhu

Structural Chemistry: Brian Beyer, Alexei Buevich, Peter Orth, Paul Reichert, Mary Senior,

Corey Strickland, Johannes Voigt, H. Wang, Yu-Sen Wang, Daniel Wyss

Drug Metabolism & Pharmacokinetics: Ryan Anstatt, Kathleen Cox, Len Favreau,

Michael Hafey, Yi Han, James Lamca, Rick Morrison, Hong Mei, Diane Rindgen, Gary

Wang

Pharmaceutical Sciences: Irina Kazakevich, Ana Pavlovsky

Safety & Toxicology: Mark Cartwright, John Meredith, Neil Johnson, Rick Briscoe, Kim

Hoagland, Greg Reinhard, Amy Smith, T. Forrest, Marcia Pitzenberger, Sabu Kuruvilla

Acknowledgments (2)

Clinical Neurosciences• Michael Egan• Chris Furtek• David Michelson• Yi Mo

Clinical Pharmacology• Mark Forman• Wen Lin• Jack Tseng• Dennis Wolford

ESD - Statistics• John Palcza• Debbie Panebianco

Clinical Development Lab• Omar Laterza• Michael Tanen• Russell Weiner

Global Clinical Supplies• Irene Michael• Meera Rangarajan

Global Trial Management• Christina Canales

Modeling & Simulation• Huub Kleijn• Otilia Lillin• Julie Stone• Jing Su

Pharmaceutical Sciences• Paul Kokkoris• Mahesh Kulkarni

Chemical Development• Jennifer Albaneze-Walker• Andrew Goodman• Jing Liao• Jamie McCabe Dunn• Michael Williams

PPDM• Freek Eijnthoven• Marissa Dockendorf• Gopal Krishna• Lei Ma• Mark Wirth

PLM• Bach-Yen Nguyen• Pam Rizos• Diane Yen

Regulatory Affairs• Peter Basseches• Sacha Wissink

Toxicology• Britta Mattson• John Meredith