Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A β-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer's Disease

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Drug Annotation

pubs.acs.org/jmc

Discovery of the 3‑Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative

Verubecestat (MK-8931)−A β‑Site Amyloid Precursor Protein

Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer’s

Disease

Jack D. Scott,*^,†Sarah W. Li,^†Andrew P. J. Brunskill,^⊥Xia Chen,^‡,■Kathleen Cox,^§Jared N. Cumming,^†

Mark Forman,^ΔEric J. Gilbert,^†Robert A. Hodgson,^‡,□Lynn A. Hyde,^‡Qin Jiang,^∇Ulrich Iserloh,^†,●

Irina Kazakevich,^∥Reshma Kuvelkar,^‡Hong Mei,^§John Meredith,^¶Jeﬀrey Misiaszek,^†,○Peter Orth,^#

Lana M. Rossiter,^∇Meagan Slater,^∇,▲Julie Stone,^§Corey O. Strickland,^#Johannes H. Voigt,^#,◊

Ganfeng Wang,^§Hongwu Wang,^#Yusheng Wu,^†,⧫William J. Greenlee,^†,%Eric M. Parker,^‡

Matthew E. Kennedy,^‡and Andrew W. Stamford*^,†

Departments of ^†Discovery Chemistry, ^‡Neuroscience, ^§Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, ^ΔTranslational

#

⊥

∥

Medicine, Structural Chemistry, Molecular and Materials Characterization, Pharmaceutical Sciences and Clinical Supply, and

^¶Toxicological Sciences, MRL, Merck & Co. Inc., 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United States

^∇Albany Molecular Research Inc., 26 Corporate Circle, Albany, New York 12203, United States

S

* Supporting Information

ABSTRACT: Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-

thiadiazinane 1,1-dioxide derivative, is a high-aﬃnity β-site amyloid precursor protein

cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation

for the treatment of mild to moderate and prodromal Alzheimer’s disease. Although not

selective over the closely related aspartyl protease BACE2, verubecestat has high

selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and

profoundly lowers CSF and brain Aβ levels in rats and nonhuman primates and CSF Aβ

levels in humans. In this annotation, we describe the discovery of 3, including design,

validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as

aspects of its preclinical and Phase 1 clinical characterization.

precursor protein (APP), ﬁrst by β-site amyloid precursor

protein cleaving enzyme 1 (BACE1) followed by cleavage of

the resulting C-terminal fragment C99 by γ-secretase. This

cleavage sequence results in production of a family of Aβ

peptides, of which Aβ40 is the most abundant isoform and

Aβ42 is more highly prone to aggregate into neurotoxic,

oligomeric species.⁴According to the amyloid hypothesis,

aberrant production and/or accumulation of Aβ peptides,

principally Aβ42, over a period of decades is causative of the

underlying disease pathogenesis that ultimately leads to

neuronal cell death.^4,5In addition to the invariant presence of

amyloid plaques in the brains of AD patients, the amyloid

hypothesis is underpinned by several other lines of evidence.

First, many distinct neurodegenerative diseases are associated

with the invariant presence of abnormal protein aggregates

analogous to amyloid plaques. Second, low levels of Aβ42 in

the CSF are a reasonably good diagnostic/prognostic

biomarker for AD. Finally, and most signiﬁcantly, early onset

autosomal dominant familial AD is associated with mutations in

INTRODUCTION AND BIOLOGICAL RATIONALE

■

Alzheimer’s disease (AD) is a devastating, progressive neuro-

degenerative disease that is associated with up to 80% of the

estimated 47 million cases of dementia worldwide and is a

leading cause of death in the United States.^1,2As the elderly

population increases, the worldwide incidence of dementia is

expected to nearly triple to approximately 132 million by 2050,

creating an unsustainable socioeconomic burden.¹Currently

available therapies, which include acetylcholinesterase inhibitors

and the N-methyl-^D-aspartate receptor antagonist memantine,

produce modest and transient improvement in cognitive

function but do not alter the progression of AD.³Treatments

that delay or halt disease progression by targeting the

underlying causes of AD would have lasting impacts on patient

function and quality of life and would address an urgent unmet

medical need.

Two histopathological hallmarks are invariably observed in

the brains of AD patients, namely, extracellular amyloid plaques

composed primarily of β-amyloid (Aβ) peptides and intra-

neuronal neuroﬁbrillary tangles composed primarily of

aggregates of abnormally phosphorylated tau protein. Aβ

peptides are formed by two sequential cleavages of the amyloid

Received: February 26, 2016

A

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Drug Annotation

Figure 1. Structures of isothiourea NMR fragment screening hits and clinical compound 3.

Figure 2. Iminoheterocyclic BACE1 inhibitors developed from isothiourea NMR fragment screening hits 1 and 2.

APP and the presenilin proteins (which are components of the

γ-secretase enzyme), and all of these mutations share the

common phenotype of increasing total Aβ levels or the relative

proportion of Aβ42.⁶Given this multifaceted evidence

supporting the role of Aβ peptides in AD progression,

substantial eﬀorts have been invested in the development of

amyloid-lowering therapies as a disease-modifying approach to

AD treatment.⁷Prominent among these has been inhibition of

BACE1 to reduce or prevent production of the Aβ peptides.

This approach has been further supported by the recent ﬁnding

that a rare mutation (A673T) near the BACE1 cleavage site in

APP reduces Aβ peptide production and is associated with

reduced risk of developing AD and improved cognitive function

in the elderly.⁸

BACE1 is a membrane-bound aspartyl protease expressed

primarily in the central nervous system (CNS), is the sole

enzymatic activity responsible for the initial β-site APP

cleavage, and is required for Aβ peptide production in vivo.⁹

In the brain, BACE1 is expressed mainly in neurons and cleaves

APP predominantly in the endosomal compartments where the

acidic pH is near the optimum for its enzymatic activity (pH

5).¹⁰Since the characterization of BACE1 more than 15 years

ago,¹¹there have been intensive eﬀorts to overcome the

challenges of identifying small molecule inhibitors that can

penetrate the CNS and inhibit the formation of centrally

derived Aβ peptides.¹²These eﬀorts have been driven by

evidence that BACE1 inhibition, in comparison with γ-secretase

inhibition and antiamyloid immunotherapy, may be an

inherently safer amyloid-lowering approach,⁷a notion that

has been informed by an evolving understanding of BACE1

biology. In this regard, Bace1 knockout mice have been

reported to have a number of subtle phenotypes, including

reduction of central and peripheral nerve myelination, and

several putative BACE1 substrates other than APP have

recently been proposed.^10,13,14However, many of these

phenotypes and substrates remain to be independently

conﬁrmed, have little if any functional consequence, are not

recapitulated by pharmacological inhibition of BACE, or may

be mitigated through partial BACE1 inhibition.^8,10,13−15

In this annotation, we describe the design and character-

ization of a novel iminothiadiazinane dioxide class of BACE1

inhibitors, originally derived from fragment screening hits 1 and

2 (Figure 1),¹⁶and key aspects of the extensive SAR explored

around this core that culminated in the discovery of the highly

optimized inhibitor verubecestat 3 (MK-8931, N-[3-[(5R)-3-

amino-5,6-dihydro-2,5-dimethyl-1,1-dioxido-2H-1,2,4-thiadia-

zin-5-yl]-4-ﬂuorophenyl]-5-ﬂuoro-2-pyridinecarboxamide).¹⁷

Compound 3 is currently in Phase 3 clinical trials with the

potential to robustly test BACE1 inhibition as a disease-

modifying treatment for AD.

B

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Figure 3. (A) BACE1 cocrystal with isothiourea hit 2. (B) BACE1 cocrystal with designed iminohydantoin fragment 4. (C) BACE1 cocrystal with

lead iminohydantoin 5. The ﬂap residues of the enzyme have been removed for clarity. The surface of the two catalytic aspartic acids is colored

salmon.

BACKGROUND

COMPOUND DESIGN AND OPTIMIZATION

■

As a continuation of our eﬀorts to discover BACE1 inhibitors

with pharmacodynamic, pharmacokinetic, and safety proﬁles

suitable for long-term clinical evaluation, we explored a series of

iminothiadiazinane dioxides represented by 9 (Figure 2). We

were attracted to the novel intellectual property space oﬀered

by the iminothiadiazinane dioxide motif as we anticipated

increased competition in the iminoheterocyclic class of BACE1

inhibitors following the disclosure of cyclic acylguanidines in

2005−2006 by us and others.²³

We have previously described the discovery of a series of

iminoheterocyclic BACE1 inhibitors (Figure 2) originating

from the isothiourea fragment screening hit 2 (Figure 1).^16,18

The isothiourea functionality of 2 engaged in hydrogen bond

donor−acceptor interactions with the catalytic dyad of BACE1

(Asp³²and Asp²²⁸), representing an aspartyl protease binding

motif that was unprecedented at the time of its discovery

(Figure 3A). Conceptually, the novel iminoheterocyclic class of

aspartyl protease inhibitors was designed by replacing the

isothiourea catalytic dyad binding motif with an iminoheter-

ocyclic core embodied by iminohydantoin 4 (Figure 2). An X-

ray cocrystal structure of BACE1 complexed with designed

fragment 4 (Figure 3B) conﬁrmed that the weakly basic

amidine moiety engaged in a hydrogen bond donor−acceptor

network with the catalytic dyad.¹⁸Through application of

structure-based design with focus on balancing potency and

lipophilicity, 4 was evolved into the highly ligand eﬃcient

diphenyl iminohydantoin lead compound 5 (Figure 3C).¹⁸

Iminohydantoin 5 embodies the minimum BACE1 pharmaco-

phore of a conformationally restricted benzyl amidine that

projects the phenyl group into the hydrophobic S1 pocket.¹⁹

Eﬃcient occupancy of S3 and the S3 subpocket (S3^sp) through

structure-guided multiparameter optimization of this orally

bioavailable, brain-penetrant lead resulted in the identiﬁcation

of iminohydantoin 6,²⁰which showed signiﬁcant reduction of

the pharmacodynamic biomarker, CSF Aβ40, after a single oral

dose of 30 mg/kg to rats (Figure 2).

Subsequently, we disclosed a series of core-modiﬁed

iminopyrimidinone BACE1 inhibitors with more potent

pharmacodynamic activity exempliﬁed by the high aﬃnity and

highly ligand eﬃcient inhibitor 7.²¹Following oral admin-

istration to rats, compound 7 exhibited robust, dose-dependent

reductions of Aβ40 in CSF and cortex with ED₅₀values of 4

and 6 mg/kg, respectively.²¹In an alternative display, an

approach using structure-based design to build into a water-

ﬁlled pocket of the enzyme near S2′ led to the discovery of the

novel pyrrolidine-fused iminopyrimidinone analogue 8 that also

showed robust CNS pharmacodynamic activity in rats with

CSF and cortex Aβ40 oral ED₅₀values of 5 and 13 mg/kg,

respectively.²²Although the discovery of the optimized

inhibitors 6−8 represented signiﬁcant advances, they did not

meet criteria for long-term AD clinical trials, and further

optimization eﬀorts focused on addressing deﬁciencies in the

proﬁles of these molecules.

Modeling simulations of 9 suggested that replacement of the

planar carbonyl group of 7 with the tetrahedral sulfonyl moiety

would not signiﬁcantly alter the conformational landscape; the

required pseudoaxial orientation of the biaryl motif correspond-

ing to the bound conformation of 9 was calculated to be close

to the lowest energy conformation (within 0.5 kcal/mol of the

energy minimum). In a model of 9 docked with the cocrystal

structure of 7 and BACE1 (Figure 4A), the predicted binding

modes of the iminothiadiazinane dioxide core and the S1−S3

biaryl motif of 9 were virtually superimposable with the crystal

structure of 7. In comparison to the iminopyrimidinone core,

the slightly increased volume of the iminothiadiazinane ring

and the steric demand of the sulfonyl group did not result in

additional close contacts with the enzyme. The quaternary C5

methyl group of 9 projects toward the protein surface, and

based on SAR developed in the iminopyrimidinone series²¹and

the similar conformations of the iminopyrimidinone and

iminothiadiazinane dioxide rings, groups larger than methyl at

this position were unlikely to be tolerated due to steric clashes

with the enzyme. With respect to calculated physicochemical

properties, replacement of the carbonyl with sulfonyl was

predicted to modestly increase PSA and decrease lipophilicity,

remaining compatible with CNS penetration.

In the event, iminothiadiazinane dioxide 9 (K_i= 2.4 nM) was

a potent BACE1 inhibitor with a K_ivalue similar to that of

iminopyrimidinone 7 (Table 1). The X-ray crystal structure of

9 was almost superimposable with the bound structure of 7

(Figure 4B), consistent with the initial docking study. Although

iminothiadiazinane dioxide 9 was equipotent to 7 in the

puriﬁed enzyme assay, the compounds were diﬀerentiated on

the basis of whole cell and in vivo activity and physical

properties (Table 1). In comparison with 7, iminothiadiazinane

dioxide 9 was less potent in the cell-based assay with a more

pronounced shift (23-fold) between the enzymatic K_iand cell

IC₅₀values despite its higher Caco-2 permeability. This may be

a consequence of the reduced basicity of iminothiadiazinane

dioxide 9 relative to its iminopyrimidinone counterpart 7

(Table 1) and is in agreement with literature reports and our

C

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Although the pharmacodynamic activities of analogues 7 and

9 were considered favorable for advancement, these com-

pounds were not developed further. Metabolite proﬁling

following oral administration of tritiated 9 to rats revealed

biliary excretion of metabolites corresponding to direct

glutathione addition and glutathione adducts derived from

oxidative metabolism. Although not deﬁnitive, mass spectros-

copy implicated the propynylpyridine moiety of 9 as the most

likely site of glutathione addition. Additionally, the modest

cathepsin D (CatD) selectivity of 7 and 9 (21- and 47-fold,

respectively, based on enzyme K_ivalues) was considered to be

inadequate and a major limitation to their progression. CatD,

an aspartyl protease widely expressed in central and peripheral

tissues, plays an essential role in lysosome function. Genetic

deletion of CatD in mice results in neuronal lipopigment

accumulation, neurodegeneration, blindness, and postnatal

lethality. These phenotypes resemble neuronal ceroid lip-

ofuscinosces, a group of rare human neurodegenerative

disorders resulting from genetic disruption of lysosome

function.²⁸

Subsequent literature reports of the BACE1 inhibitors

LY2811376 (10) and AMG-8718 (11) have substantiated

toxicity concerns related to CatD inhibition (Figure 5).^29,30In a

Figure 4. (A) Overlay of docked model of 9 (aqua) and the X-ray

cocrystal structure of 7 (yellow) with BACE1. (B) X-ray cocrystal

structures of 9 (purple; PDB: 5HTZ) and 7 (yellow) with BACE1.

Figure 5. BACE1 inhibitors with potential CatD-mediated toxicity.

three month rat toxicology study of 10, retinal photoreceptor

degeneration, neuronal degeneration, and lipofuscinosis were

observed, halting clinical evaluation of the compound.²⁹

Although the reported selectivity of 10 for BACE1 over

CatD was 63-fold,²⁹in our hands the selectivity is only 7-fold,

supporting a role of CatD inhibition in the observed toxicity.³¹

Additionally, BACE inhibitor 11 elicited retinal toxicity leading

to thinning of the outer nuclear layer of the retina in a one

month rat toxicity study.³⁰Although the origin of the toxicity

was not established, it was speculated that inhibition of other

proteases including CatD may have contributed to the observed

toxicity.

Given the concerns related to metabolism and CatD

selectivity of 7 and 9, we focused on modiﬁcations to the

biaryl motif with the dual objectives of removal of the

metabolically labile propynylpyridine and improvement of

CatD selectivity. To this end, we had previously disclosed

iminohydantoins 12 and 13 in which the two aryl groups

previous observations that related inhibitors such as 6 (pK_a6.9)

had decreased cellular potency relative to their K_ivalues as a

function of their weaker basicities.^20,21,24,25In an acute rat

pharmacodynamic assay, 9 (10 mg/kg p.o.) aﬀorded robust

reductions of CSF and cortex Aβ40 levels. Although

iminothiadiazinane dioxide 9 displayed weaker cellular potency

than that of iminopyrimidinone 7, it was signiﬁcantly more

potent than 7 in vivo based on comparison of unbound plasma

concentrations that elicited similar central pharmacodynamic

eﬀects (Table 1). The greater in vivo potency of 9 is likely a

consequence of improved permeability and enhanced CNS

penetration imparted by the iminothiadiazinane dioxide core. In

addition to favorable in vivo properties, iminothiadiazinane

analogues had improved hydrolytic stability²⁷(see Supporting

Information) in comparison to the corresponding iminopyr-

imidinones, further validating the iminothiadiazinane dioxide

core as a potentially druggable motif.

Table 1. Proﬁle Comparsion between Iminopyrimidinone 7 and Iminothiadiazinane Dioxide 9

f

BACE1 K_i

cell Aβ40 IC₅₀

Caco-2 P_app[nm s⁻¹

(eﬄux ratio)

]

pK /

^ac

CatD/

Aβ40 reduction CSF/

rat C_u,pl,3h,[nM]

plasma f_urat/

human [%]

a

b

d

e

g

[nM]

PSA

BACE1

cortex [%]

(b/p)

7

9

1.7

2.4

11

55

153 (2.4)

278 (1.6)

7.5/69

6.6/86

21

47

−81/−61

−82/−53

33 (2.9)

8.0 (2.3)

3.6/1.6

1.6/0.7

a

b

BACE1 K_ivalues were determined using puriﬁed human enzyme and are the mean of at least two determinations.²¹IC₅₀values were determined

in HEK293 cells expressing human APP with the Swedish and London familial AD mutations.¹⁸PSA was calculated as described in ref 26. Ratio of

K_ivalues. Percent change relative to vehicle control 3 h after 10 mg/kg p.o. administration to rats. Unbound plasma concentration 3 h after 10 mg/

kg p.o. administration to rats. Ratio of total brain to total plasma compound concentrations.

c

d

e

f

g

D

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binding in the enzyme S1 and S3 pockets are linked by a

aﬃnity. A similar strategy has been employed in the

hydroxyethylamine class of BACE inhibitors.³⁴

carboxamide (Figure 6).³²Although the BACE1 aﬃnity of

Furthermore, in the course of our SAR investigations, we had

observed that BACE1 aﬃnity could be highly dependent on the

iminoheterocyclic core for a given S1−S3 binding motif.²⁰For

example, in the iminohydantoin series, regioisomeric analogues

14 and 15 that bear an S1-binding thienyl group and an

optimized cyclopropyl substituent,²⁰were less potent than the

corresponding S1 phenyl analogue 16 (Figure 8). The same

Figure 6. Iminohydantoin S1−S3 amides.

these unoptimized analogues was relatively weak, they

displayed some intrinsic selectivity over CatD. We were

drawn to the unique binding features of the diaryl amide

binding motif revealed by an X-ray cocrystal structure of (R)-13

(Figure 7A) obtained from soaking the racemate whereby the

Figure 8. Iminohydantoin and iminopyrimidinone biaryl analogues.

rank order of aﬃnity was observed for analogues of 14−16 in

which the cyclopropyl group was replaced by methyl (not

shown). Conversely, in the iminopyrimidinone biaryl series

bearing a methyl group as the optimal quaternary C6

substituent, the thienyl analogues 17 and 18 were more potent

than phenyl analogue 19.²¹Most striking was the signiﬁcant

diﬀerence in K_ibetween the 2,5-thienyl analogues 14 and 17.

Accordingly, we prepared a series of iminohydantoin,

iminopyrimidinone, and iminothiadiazinane dioxide derivatives

bearing a diaryl amide motif to ascertain their BACE1 aﬃnity

and selectivity proﬁles.

Iminopyrimidinone 21 (Table 2) bearing a picolinamide

substituent was an order of magnitude more potent than

similarly substituted iminohydantoin 12 (Figure 6). We were

gratiﬁed to ﬁnd that novel iminothiadiazinane dioxide diaryl

amide 27 also possessed high BACE1 aﬃnity and was

approximately 6-fold more potent than optimized iminohy-

dantoin 20 bearing the matched diaryl amide motif. Strikingly,

selectivity of the diaryl amides 21 and 27 for BACE1 inhibition

relative to CatD was exceedingly high.

In the iminopyrimidinone series, introduction of a

substituent at the pyridyl 5-position resulted in increased

BACE1 aﬃnity compared to that of 21 that lacks substitution at

this position with the methyl, ﬂuoro, chloro, and triﬂuor-

omethyl analogues (22−25) displaying single-digit to sub-

nanomolar K_ivalues while maintaining high selectivity over

CatD. Cell IC₅₀values for these analogues were virtually

unshifted relative to their K_ivalues. Introduction of the

picolinamide scaﬀold also resulted in a signiﬁcantly higher

unbound plasma fraction compared to those of the biaryl

analogues 7 and 9 with rat values in the range of 15−40%.

However, in comparison with 7 and 9, picolinamide

iminopyrimidinone analogues 23−25 displayed relatively

modest reductions of CSF and cortex Aβ40 in the rat

pharmacodynamic assay 3 h after oral administration (10

mg/kg) despite achieving similar unbound plasma concen-

trations. The more modest pharmacodynamic activities of

iminopyrimidinones 23−25 compared to those of 7 and 9 is

consistent with their signiﬁcantly lower P_appvalues and higher

Figure 7. (A) Cocrystal structure of carboxamide (R)-13 highlighting

the hydrogen bond with BACE1 Gly²³⁰(PDB: 5HU0). (B) Overlay of

the cocrystal structures of (R)-13 (yellow) and 9 (purple) with

BACE1.

phenyl and furanyl groups occupied the S1 and S3 pockets,

respectively, and the amide N−H engaged in a hydrogen-

bonding interaction with the carbonyl of Gly²³⁰. Additionally,

the furanyl ring of (R)-13 projected deeper into the S3

subpocket of BACE1 in comparison with the pyridyl ring of 9

(Figure 7B). On the basis of a comparison of the BACE1 and

CatD crystal structures, their S3 subpockets diﬀer in topology

with the CatD S3 subpocket being slightly smaller than that of

BACE1.³³We postulated that diﬀerences in inhibitor binding in

the S3 domains of the respective enzymes contributed to the

modest selectivity of 13 over CatD. Thus, it was of interest to

explore SAR targeting of this region as a means of gaining

improved selectivity over CatD while maintaining high BACE1

E

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Table 2. N-Phenyl Carboxamide Analogues

a

b

BACE1 K_ivalues were determined using puriﬁed human enzyme and are the mean of at least two determinations.²¹IC₅₀values were determined

in HEK293 cells expressing human APP with the Swedish and London familial AD mutations.¹⁸Ratio of mean K_ivalues. Change in Aβ40 versus

vehicle control 3 h after oral administration (10 mg/kg) of compound to rats. Unbound plasma concentration of compound 3 h after oral

administration to rats. Ratio of total brain to total plasma compound concentrations.

c

d

e

f

Caco-2 eﬄux ratios, suggesting that reduced permeability and

Pgp-mediated eﬄux restricts their brain penetration (vide

infra). Methylpyridine 22 showed low plasma levels in the

pharmacodynamic screen, likely as a consequence of its

susceptibility to eﬄux and/or oxidative metabolism; the latter

is consistent with the high rat hepatocyte clearance of 22 (35

μL min⁻¹[10⁶cells]⁻¹) compared to that of analogues 23−25

(≤8 μL min⁻¹[10⁶cells]⁻¹) that lack the pyridyl methyl group.

Similar SAR trends were observed for the novel iminothia-

diazinane dioxide carboxamide analogues 3 and 28−31 with

respect to BACE1 aﬃnity, CatD selectivity, cell-based potency,

and unbound plasma fraction. Comparison of the 6-

ﬂuorophenyl analogues 3 and 31 with the corresponding

desﬂuoro analogues 26 and 27 demonstrates that the 6-ﬂuoro

substituent is beneﬁcial (5-fold) for aﬃnity (Table 2) and is

consistent with the increased aﬃnity imparted by the chloro

F

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a

Table 3. Enzymatic and Cell-Based Activity of 3 at BACE1 and Related Aspartyl Proteases

human enzyme K_i[nM]

HEK293 APP^Swe/Loncell IC₅₀[nM]

BACE1

2.2

BACE2

0.38

CatD

CatE

pepsin

renin

Aβ1−40

Aβ1−42

sAPPβ

≫100,000

33,800

2.1

0.7

4.4

a

K_ivalues were determined using puriﬁed human aspartyl proteases, and IC₅₀values were determined in HEK293 cells expressing human APP with

the Swedish and London familial AD mutations as previously described.^18,21Data shown represent the mean of 3−5 independent experiments

performed in duplicate.

substituent on the thiophene ring of biaryl analogue 7 that we

had reported previously.²¹A major advantage of iminothiadia-

zinane dioxide picolinamides 3 and 29−31 over their

iminopyrimidinone counterparts 22−25 was their signiﬁcantly

higher permeabilities and reduced eﬄux ratios in the Caco-2

assay. Overall permeability values and eﬄux ratios for 3 and

29−31 approached those of the biaryl analogues 7 and 9. This

translated into robust activity in the acute rat pharmocadynamic

screen, where the ﬂuoro- and chloropicolinamides 3 and 30

elicited substantial CSF and cortex Aβ40 lowering that was

greater than that observed for the corresponding iminopyr-

imidinones 23 and 24. Further characterization of iminothia-

diazinane dioxide 3 and corresponding iminopyrimidinone 23

in LLC-PK1 cells transfected with MDR1, an assay that is more

sensitive than the Caco-2 assay for the detection of Pgp

substrates, demonstrated that both compounds are Pgp

substrates. In LLC-MDR1 cells, iminothiadiazinane dioxide 3

(P_app= 29 × 10⁻⁶cm sec⁻¹; eﬄux ratio = 11) had higher

permeability and a lower eﬄux ratio than those of 23 (P_app= 19

× 10⁻⁶cm sec⁻¹; eﬄux ratio > 29), consistent with the trends

observed in the Caco-2 assay. The excellent permeability

conferred by the iminothiadiazinane ring likely accounts for the

robust CNS pharmacodynamic activity of 3 and its analogues

and is suﬃcient to overcome Pgp-mediated eﬄux. The higher

overall permeability observed for the iminothiadiazinane

dioxide picolinamides compared to the corresponding

iminopyrimidinones is ascribed to the weaker basicity of the

iminothiadiazinane dioxide core as reported for structurally

related BACE1 inhibitors.²⁵The lower pK_aof the iminothia-

diazinane core may also contribute to reduced Pgp eﬄux

susceptibility, consistent with reports for structurally unrelated

compounds.³⁵Accordingly, the pK_aof iminothiadiazinane

dioxide 3 was determined to be 7.5, signiﬁcantly lower than

that of the corresponding iminopyrimidinone 23 (8.3).

to its lack of inhibition of CatD in vitro, 3 does not signiﬁcantly

inhibit cathepsin E (CatE), pepsin, or renin. Compound 3 is a

potent inhibitor of the BACE1 homologue BACE2 (K_i= 0.38

nM), which is not unexpected given the similarity of the S1 and

S3 pockets of the two aspartyl proteases.³⁹

The X-ray cocrystal structure of 3 bound to BACE1 shown in

Figure 9A conﬁrms a number of speciﬁc enzyme−inhibitor

Figure 9. (A) Cocrystal structure of 3 and BACE 1 (PDB: 5HU1).

(B) X-ray structure of 3. (C) Cocrystal structure of 3 and BACE1

overlaid with CatD X-ray structure (PDB: 1LYB). CatD residues

Phe¹³¹and Asp³²³are highlighted in blue.

interactions. The two H-bond donors of the iminothiadiazinane

dioxide core are hydrogen-bonded to the Asp³²and Asp²²⁸

active site residues, and the phenyl group projects pseudoaxially

from the thiadiazine core to occupy the hydrophobic S1 pocket

deﬁned by Leu³⁰, Phe¹⁰⁸, Trp¹¹⁵, and Ile¹¹⁸. The amide exists in

the energetically preferred E-conﬁguration, and the amide

hydrogen is within H-bonding distance of the Gly²³⁰carbonyl

oxygen. The two aryl rings are essentially planar with the

ﬂuoropyridine substituent positioned between the two loops

comprised of Ser¹⁰, Gly¹¹and Gln¹²and Gly²³⁰, Thr²³¹and

Thr²³²that deﬁne the S3 pocket. The pyridine ﬂuoro

substituent extends into the S3 subpocket and is in close

proximity to the Ala³³⁵methyl side chain (not shown).

The small molecule X-ray crystal structure of 3 (Figure 9B)

recapitulates the key conformational features of the verubece-

stat bioactive conformation deﬁned by the BACE1-bound

inhibitor structure (Figure 9A). These include the conforma-

tion of the iminothiadiazinane dioxide ring and the pseudoaxial

disposition of the ﬂuorophenyl substituent, the E amide

orientation, and the position of the pyridine nitrogen to form

an internal H-bond to the amide hydrogen. A conformational

search on 3 conﬁrmed that the crystal structures of 3

correspond to low energy conformations with the pseudoaxial

conformation of the core phenyl substituent enforced by the

quaternary methyl group and the internal hydrogen bond

locking the conformation of the picolinamide, eﬀectively

limiting the number of rotatable bonds to two.

Comparison of 5-chloropyridine derivative 30 (previously

reported as MBi-3)³⁶with corresponding benzamide 32

highlights the profoundly beneﬁcial eﬀects of the pyridine

nitrogen with respect to BACE1 aﬃnity and eﬄux suscepti-

bility. The reduced permeability and higher eﬄux ratio of the

benzamide 32 can be attributed to the lack of an internal H-

bond to the amide hydrogen, eﬀectively unmasking the amide

H-bond donor. The loss of aﬃnity is likely a consequence of

the diﬀerence in conformational bias of the benzamide relative

to the picolinamide, which in the case of the benzamide 32

disfavors the near-planar bioactive conformation of the

picolinamide motif (vide infra).³⁷

PRECLINICAL CHARACTERIZATION

■

5-Fluoropyridine analogue 3 was selected for detailed character-

ization with key in vitro potency and selectivity data detailed in

Table 3.³⁸In a cell-based assay, 3 potently inhibits both Aβ40

and Aβ42 formation with similar IC₅₀values. A similar cell-

based IC₅₀value is also observed for inhibition of sAPPβ, one

of the direct products of BACE1 cleavage of APP. In addition

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Insight into the lack of aﬃnity of 3 for CatD is provided by

comparison of the S1−S3 pockets of CatD and BACE1 (Figure

9C). Binding of 3 to CatD should be disfavored by a close

contact of the amide carbonyl of 3 with the S1 Phe¹³¹residue.

Moreover, S3 and S3^spof CatD are smaller than the

corresponding BACE1 pockets with Ala¹³(S3) and Asp³²³

(S3^sp) of CatD unable to accommodate the steric demand of

the ﬂuoropyridyl group of 3.³³Rotation of the pyridyl group to

alleviate steric clashes within S3 of CatD would be disfavored

due to disruption of the energetically preferred planar

picolinamide conformation of the inhibitor.

There is a notable discrepancy between the robust CNS

activity of 3 (vide infra) and the compound’s high calculated

polar surface area (115 Å²),²⁶which is beyond the upper limit

of 90 Å²suggested for CNS active drugs.⁴⁰The internal H-

bond between the pyridine nitrogen and the amide hydrogen

eﬀectively masks the polarity of these groups,⁴¹reducing the

solvent accessible surface area and hence the dehydration

penalty necessary to achieve eﬃcient cell membrane perme-

ability and blood-brain barrier permeation. The high perme-

ability of 3 is suﬃcient to mitigate the eﬀects of Pgp-mediated

eﬄux. The importance of the internal hydrogen bond to the

CNS drug-like properties of the series is reinforced by the

observation that its removal through replacement of the pyridyl

with phenyl reduces permeability and increases Pgp eﬄux

susceptibility (cf. 30 and 32 in Table 2).

attribute this delay in response of CSF Aβ levels to the kinetics

of CSF Aβ turnover and to the time required for transit of

newly synthesized Aβ from the brain parenchyma to the CSF.

This is consistent with results from a separate experiment in

which a 10 mg/kg oral dose of 3 to cynomolgus monkeys

aﬀorded greater reduction of brain Aβ levels (72%) compared

to CSF Aβ levels (60%) 4 h postdose.³⁸Of note, in the cisterna

magna-cannulated monkey study, CSF levels of 3 were

signiﬁcantly lower than unbound plasma levels with C_CSF

/

C_u,plasmaranging from 0.1 to 0.6. These ratios increased over

time and were consistent between the two dose levels. The

observation that CSF levels of 3 deviate from the unbound

plasma concentration is consistent with susceptibility of 3 to

Pgp-mediated eﬄux. Despite the susceptibility to transport by

Pgp, these acute pharmacodynamic studies demonstrated that 3

achieves central BACE1 inhibition that translates to robust

brain Aβ reduction.³⁸

The pharmacokinetic properties of 3 were evaluated in

Sprague−Dawley (SD) rats, cynomolgus monkeys, and beagle

dogs (Table 4).³⁸Compound 3 exhibited good to excellent oral

bioavailability, low to moderate clearance, and moderate to high

volume of distribution. The compound had a high plasma

unbound fraction across preclinical species and a comparable

human plasma unbound fraction (45%). Fixed exponent

allometric scaling of the rat, dog, and monkey PK parameters

predicted a human plasma t_1/2of 11 h for 3, consistent with QD

dosing in the clinic.⁴²Human dose predictions employed an

E_maxconcentration−response model and predicted a human

QD dose of 35 mg to achieve a steady-state AUC_24hAβ40

reduction of 75% based on the monkey brain and CSF Aβ40

pharmacodynamic proﬁle.³⁸The predicted unbound plasma

C_maxof 3 required to achieve this eﬀect was 82 nM. The

crystalline free base of 3 has excellent aqueous solubility (1.6

mM), was selected as the development form, and was predicted

to be a BCS class 1 substance at projected clinical doses.

Metabolism of 3 was evaluated in human hepatocytes

resulting in identiﬁcation of two metabolites: N-2 desmethyl

metabolite 33 and aniline metabolite 34 (Figure 11). Each of

these metabolites had BACE1 inhibitory activity that was >100-

fold less than that of 3. In vivo, 33 and 34 were observed as

circulating metabolites following oral administration of 3 to rats

and monkeys. Aniline 34 was not mutagenic in a 5-strain Ames

test.

The pharmacokinetic-pharmacodynamic proﬁle of 3 was

more fully characterized in rats and cynomolgus monkeys. In

rats, acute oral administration of 3 dose-dependently reduced

CSF and cortex Aβ40 with ED₅₀values of 5 and 8 mg/kg,

respectively, corresponding to unbound plasma EC₅₀values of

48 and 81 nM, respectively. Plasma levels of 3 increased in a

dose-proportional manner, whereas increases in CSF and total

brain exposure were less than dose proportional.³⁸

Compound 3 was also evaluated in a time course study in

cisterna magna-cannulated cynomolgus monkeys at oral doses

of 3 and 10 mg/kg (Figure 10). CSF Aβ40 levels were

Many BACE inhibitors including 3 display a basic moiety

situated in proximity to a hydrophobic group, a pharmacophore

that is also recognized by the I_KRchannel encoded by the

human Ether-A-Go-Go gene (hERG).⁴³Inhibition of the hERG

channel is implicated in QT prolongation, which can lead to

cardiac arrhythmia and sudden death.⁴⁴In vitro, 3 was

determined to have an IC₅₀value for inhibition of the hERG

channel of 2.2 μM, 27-fold higher than the projected human

unbound C_maxof 82 nM. In a single-dose cardiovascular study

in telemetered cynomolgus monkeys, 3 had no eﬀect on the

QT interval at an unbound plasma concentration of 1.5 μM,

which is approximately 18-fold higher than the predicted

Figure 10. Time course of changes in CSF Aβ40 and concentrations

of 3 (3 mg/kg dose) in plasma (unbound) and CSF in cynomolgus

monkeys following single oral doses of 3.

human unbound C_max

.

Compound 3 does not signiﬁcantly inhibit human CYP

isoforms 1A2, 2C9, 2C19, 2D6, and 3A4 (all IC₅₀> 40 μM),

indicating that the compound is unlikely to be a perpetrator of

CYP-mediated drug−drug interactions. Additionally, 3 at

concentrations up to 15 μM did not induce CYP 3A4 or 1A2

expression in human hepatocytes. To further examine the

potential of 3 for liver enzyme induction, the compound was

monitored via sequential CSF sampling for 24 h after

administration of 3. Profound, sustained reduction of CSF

Aβ40 levels was observed at each dose. Peak eﬀects of 3 on

CSF Aβ lowering (72 and 81% reduction at 3 and 10 mg/kg,

respectively) were observed 12 h after dosing, which was

signiﬁcantly delayed from the T_maxof 2 and 6 h for

concentrations of 3 in plasma and CSF, respectively. We

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Table 4. Pharmacokinetic Parameters of 3 in SD Rat, Beagle Dog, and Cynomolgus Monkey

a

b

IV administration

Cl [mL min⁻¹kg⁻¹

oral administration (3 mg/kg)

dose [mg kg⁻¹

]

V_dss[L kg⁻¹

]

t_1/2[h]

AUC [μM h]

C_max[μM]

F [%]

plasma f_u[%]

rat

3

1

46

21

5.4

7.5

2.7

1.9

4.9

1.1

5.7

42

0.27

0.60

1.6

45

95

37

50

36

monkey

dog

4.3

9.7

100

a

b

Dosed as a solution of the HCl salt in 20% hydroxypropyl-β-cyclodextrin. Dosed as a solution of the HCl salt in 0.4% hydroxypropyl

methylcellulose.

unexpected diﬀerence in properties manifested by alteration

of the iminoheterocycle core.

In chronic toxicology studies in rats (doses up to 75 mg kg⁻¹

day⁻¹for 6 months) and monkeys (doses up to 100 mg kg⁻¹

day⁻¹for 9 months), 3 did not elicit neurodegeneration or

lipofuscinosis in the CNS consistent with its high selectivity for

BACE1 over CatD (>45,000-fold), nor did it alter the

histomorphological proﬁle of sciatic or CNS nerve myelina-

tion.³⁸As mentioned previously, 3 is a potent inhibitor of

BACE2. Although knowledge of the speciﬁc functions of

BACE2 is evolving, recent reports have demonstrated that

BACE2 processes the melanosome structural protein PMEL17,

which is required for proper pigmentation and is consistent

with the lighter coat color observed in Bace2 knockout mice.⁴⁵

In addition, it has been postulated that BACE2 plays a role in

the function of pancreatic β cells through processing of the

transmembrane protein Tmem27. Bace2 knockout mice display

improved glucose homeostasis, and Bace2 inhibition has been

reported to produce beneﬁcial eﬀects on glucose homeostasis

Figure 11. Metabolites of 3.

administered orally at a dose of 30 mg/kg BID for 5 days to

rats. This subchronic administration of 3 caused a modest (1.4-

fold) induction of CYP 3A1 activity but did not signiﬁcantly

alter the expression of CYPs 1A1, 1A2, 2B, 3A2, or 4A. There

was also no eﬀect of subchronic administration of 3 on liver

weights. In contrast, subchronic administration of the

iminopyrimidinone congener 23 (30 mg/kg BID, 5 days)

caused signiﬁcant induction of CYPs 1A, 2B, and 3A1, liver

weight gain, and autoinduction as evidenced by a 3-fold

decrease in plasma levels on day 5, thus revealing an

a

Scheme 1. Synthesis of Iminothiadiazinane Dioxide 9

a

Reagents and conditions: (a) (R)-2-methylpropane-2-sulﬁnamide, Ti(OEt)₄, THF, reﬂux, 61%; (b) MsCl, Et₃N, CH₂Cl₂, rt, 90%; (c) n-BuLi, THF

−78 °C; 36, THF, −78 °C, 75%; (d) 4 N HCl in dioxane, MeOH, CH₂Cl₂, rt; (e) TFA, 1,3-dimethoxybenzene, CH₂Cl₂, rt. 99% (crude, 2 steps); (f)

benzoylisothiocyanate, CH₂Cl₂, rt; (g) NaOMe, MeOH, rt; (h) MeI, EtOH, reﬂux, 81% (3 steps); (i) di-tert-butyldicarbonate, Et₃N, CH₂Cl₂, rt,

72%; (j) NBS, DMF, 50 °C, 63%; (k) (5-(prop-1-ynyl)pyridin-3-yl)boronic acid, 2 M Na₂CO₃, PdCl₂(dppf)·CH₂Cl₂, 1,4-dioxane, 65 °C; di-tert-

butyldicarbonate, Et₃N, CH₂Cl₂; (l) CF₃CO₂H, CH₂Cl₂, rt, 21% (3 steps).

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a

Scheme 2. Synthesis of Iminopyrimidinone Amides 21−25

a

Reagents and conditions: (a) (R)-2-methylpropane-2-sulﬁnamide, Ti(OEt)₄, THF, reﬂux, 64%; (b) LDA, methyl acetate, ClTi(OⁱPr)₃, THF, −78

i

°C; 45, THF, −78 °C, 75%; (c) HCl, 1,4-dioxane, rt; (d) EDCI, N-[(methylamino)thioxomethyl]-t-butylcarbamate, Pr₂NEt, THF, rt, 49% (2

steps); (e) H₂, Pd/C, MeOH, rt, 94%; (f) RCO₂H, BOPCl, pyr, rt; (g) CF₃CO₂H, CH₂Cl₂, rt, 23−89% (2 steps).

a

Scheme 3. Synthesis of Iminothiadiazinane Dioxide Amides 3 and 28−32

a

Reagents and conditions: (a) n-BuLi, THF, −78 °C, 45; THF, −78 °C, 56%; (b) 4 N HCl in 1,4-dioxane, MeOH, CH₂Cl₂, rt; (c) TFA, 1,3-

dimethoxybenzene, CH₂Cl₂, rt, 90% (2 steps); (d) 5 M BrCN in MeCN, n-BuOH, reﬂux; (e) di-tert-butyldicarbonate, Et₃N, DCM, rt, 59% (2

steps); (f) H₂, Pd/C, EtOH, THF, rt, 90%; (g) RCO₂H, BOPCl, pyr, rt; (h) CF₃CO₂H, CH₂Cl₂, rt, 35−89% (2 steps).

and increased pancreatic β-cell mass in ob/ob mice.⁴⁶Fur

hypopigmentation was observed in mice and rabbits treated

with 3; however, in the aforementioned monkey chronic

toxicology study, there was no evidence of fur hypopigmenta-

tion or changes in pigmentation of any tissue. In addition, no

changes in blood glucose levels were observed in chronic rat

and monkey studies.³⁸Overall, the relatively benign phenotypes

of Bace knockout mice, current understanding of the role of

BACE2 processing of its endogenous substrates, and the

outcome of preclinical toxicology studies has mitigated

concerns related to the lack of selectivity of 3 over BACE2.

sAPPβ. Mean reduction of each CSF analyte was >80% at the

60 mg dose. Notably, there were no reports of changes in skin

or hair pigmentation as a potential consequence of BACE2

inhibition in any of the Phase 1 studies; however, treatment

time of longer duration would likely be required for

pigmentation changes to manifest. Compound 3 is currently

under evaluation in Phase 3 clinical trials in prodromal and mild

to moderate AD patients at doses of 12 and 40 mg. These doses

were selected on the basis of simulations of the Phase 1

pharmacodynamic data, which predicted that 95% of patients

will achieve CSF Aβ peptide reductions of >50 and >75% at the

12 and 40 mg doses, respectively.³⁸

CLINICAL EVALUATION

■

CHEMISTRY

The safety, pharmacokinetics, and pharmacodynamics of 3 were

assessed in Phase 1 clinical trials in healthy volunteers and mild-

to-moderate AD patients.³⁸In healthy volunteers, single oral

doses of 3 up to 550 mg were safe and well tolerated. Plasma

and CSF levels of 3 increased in a dose-proportional manner,

and the terminal plasma half-life was 14−24 h. Compound 3

elicited dose-dependent reductions of CSF Aβ40, Aβ42, and

sAPPβ with >90% reduction of CSF Aβ40 achieved at the 550

mg dose. Additionally, no QTc prolongation was observed in

healthy volunteers administered daily oral doses of 3 at or

below the supratherapeutic dose of 250 mg for 14 days. In a

Phase 1b study of 3 in patients with mild to moderate AD, oral

doses of 12, 40, and 60 mg once per day for 7 days resulted in

sustained dose-dependent reductions of CSF Aβ40, Aβ42, and

■

Synthesis of iminothiadiazinane dioxide 9 (Scheme 1) began

with treatment of (R)-tert-butylsulﬁnyl ketimine 36, readily

available from ketone 35, with the conjugate base of

sulfonamide 38 made from commercial 1-(4-methoxyphenyl)-

N-methylmethanamine 37. This process generated the

protected β-amino sulfonamide 39 in 75% yield and high

diastereomeric ratio (dr 98:2).^47,48The tert-butylsulﬁnamide

group was cleaved upon treatment of adduct 39 with hydrogen

chloride, and the p-methoxy benzyl (PMB) group was

subsequently removed using TFA to aﬀord β-amino

sulfonamide 40. The iminothiadiazinane dioxide core was

then formed in high yield using a three step protocol.⁴⁹First,

treatment of amine 40 with benzoylisothiocyanate was followed

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by cleavage of the benzoyl group to provide an intermediate

thiourea. Activation of the thiourea with methyl iodide induced

an intramolecular ring closure to provide iminothiadiazinane

dioxide 41. Protection of the guanidine with a Boc group

provided 42, which was followed by regioselective bromination

of the thiophene ring to aﬀord key intermediate 43. A Suzuki

coupling between 43 and (5-(prop-1-ynyl)pyridin-3-yl)boronic

acid and subsequent Boc deprotection provided biaryl

iminothiadiazinane dioxide 9.

Scheme 2 describes the preparation of the iminopyrimidi-

none amides 21−25. The iminopyrimidinone core was

prepared in a similar fashion to our previously described

chemistry.²¹Ketimine 45, derived from methyl ketone 44, was

subjected to an asymmetric aldol addition with the enolate of

methyl acetate providing the β-amino ester 46. Sulﬁnamide

cleavage under acidic conditions was followed by addition of

the resultant amine to N-[(methylamino)thioxomethyl]-t-

butylcarbamate. Subsequent ring closure aﬀorded the imino-

pyrimidinone core, which was Boc protected to provide 47.

Nitro reduction was followed by amide coupling with a set of

pyridine carboxylic acids and Boc deprotection to provide the

iminopyrimidinone carboxamide analogues 21−25.

The iminothiadiazinane dioxide amides 3 and 28−32 were

prepared using a modiﬁed route (Scheme 3) compared to that

described for the preparation of 9. The addition of the lithium

anion of sulfonamide 38 to ketimine 45 aﬀorded amino

sulfonamide 49 in 64% yield in a diastereomeric ratio of

93.5:6.5 favoring the desired R,R diastereomer. The minor

diastereomer was easily removed by silica gel column

chromatography. After acidic cleavage of the sulﬁnamide and

PMB groups to aﬀord the β-amino sulfonamide derivative 50,

the iminothiadiazinane dioxide ring was formed in a single step

using cyanogen bromide in moderate yield. The previously

described three step protocol employing benzylisothiocyanate

aﬀorded the desired thiadiazinane as well; however, the yields

were poor, and the reproducibility was inconsistent due to

multiple side products formed via aromatic nucleophilic

substitution on the nitro-activated ﬂuorobenzene moiety.

After thiadiazininane ring formation, the guanidine was Boc-

protected to aﬀord 51, which was followed by nitro reduction

to aﬀord aniline 52. Finally, amide formation and deprotection

aﬀorded the heterocyclic iminothiadiazinane dioxide amides 3⁵⁰

and 28−32.

preclinical species, which translated to profound reductions of

CSF Aβ peptides in healthy volunteers and AD patients.

Compound 3 is a ﬁrst-in-class BACE1 inhibitor that has

advanced to Phase 3 trials in mild to moderate AD and

prodromal AD and presents a unique opportunity to provide a

robust test of the amyloid hypothesis.

EXPERIMENTAL SECTION

■

Synthetic Materials and Methods. Reagents and solvents were

obtained from commercial sources and used without further

1

puriﬁcation. H NMR spectra (400 and 500 MHz) were collected

on Varian spectrometers. Chemical shifts are reported in ppm relative

to the residual solvent peak in the indicated solvent, and for ¹H NMR

spectra, multiplicities, coupling constants in hertz, and numbers of

protons are indicated parenthetically. Optical rotations were

determined using a PerkinElmer model 341 polarimeter, and substrate

concentration c is reported in g/100 mL. Purities of all reported

compounds were greater than 95% based on HPLC chromatograms

obtained on an Agilent 1100 LCMS system.

(R)-N-(1-(3-Chlorothiophen-2-yl)ethylidene)-2-methylpro-

pane-2-sulﬁnamide (36). To a solution of 1-(3-chlorothiophen-2-

yl)ethanone (35) (10 g, 62 mmol) in THF (40 mL) were added (R)-

2-methylpropane-2-sulﬁnamide (7.9 g, 65 mmol) followed by

titanium(IV)ethoxide (13 g, 69 mmol). The resultant mixture was

heated to reﬂux and stirred for 16 h. After that time, the mixture was

cooled to room temperature and poured into ice water, and the

mixture was ﬁltered. The ﬁlter cake was washed with CH₂Cl₂, and the

combined ﬁltrates were then extracted with CH₂Cl₂. The combined

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated in

vacuo. The crude residue was puriﬁed by ﬂash chromatography (SiO₂;

gradient elution 0−35% EtOAc in hexanes) to aﬀord 36 (10 g, 61%

yield) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J = 5.6

Hz, 1H), 6.96 (d, J = 5.6 Hz, 1H), 2.88 (s, 3H), 1.30 (s, 9H).

N-(4-Methoxybenzyl)-N-methylmethanesulfonamide (38).

To a solution of 1-(4-methoxyphenyl)-N-methylmethanamine (37)

(10.1 g, 66.8 mmol) in CH₂Cl₂(100 mL) at 0 °C were added Et₃N

(12.1 mL, 86.4 mmol) followed by the dropwise addition of

methanesulfonyl chloride (5.95 mL, 76.8 mmol). The mixture was

stirred at 0 °C for 30 min and then allowed to warm to room

temperature with stirring for an additional 16 h. After that time, the

mixture was diluted with CH₂Cl₂and washed with 1 N HCl(aq) and

sat. NaHCO₃(aq). The organic layer was then dried over Na₂SO₄,

ﬁltered, and concentrated in vacuo. The crude residue was puriﬁed by

ﬂash chromatography (SiO₂; gradient elution 0−50% EtOAc in

1

hexanes) to aﬀord 38 (13.8 g, 90% yield) as an oﬀ white solid. H

NMR (400 MHz, CDCl₃) δ 7.27 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 8.0

Hz, 2H), 4.25 (s, 3H), 3.81 (s, 2H), 2.81 (s, 3H), 2.75 (s, 3H). ESI MS

m/z 252.0 [M + Na]⁺.

CONCLUSIONS

■

(S)-2-(3-Chlorothiophen-2-yl)-2-((R)-1,1-dimethylethylsulﬁ-

namido)-N-(4-methoxybenzyl)-N-methylpropane-1-sulfona-

mide (39). To a ﬂame-dried round-bottom ﬂask containing a solution

of 38 (7.8 g, 34 mmol) in THF (75 mL) at −78 °C under an

atmosphere of nitrogen was added dropwise a solution of n-BuLi (1.6

M in hexanes, 21 mL, 34 mmol), and the mixture was stirred for 30

min. After that time, the solution was transferred by cannula into a

precooled solution (−78 °C) of ketimine 36 (4.0 g, 15 mmol) in THF

(75 mL). The resultant mixture was stirred at −78 °C for 5 h. After

that time, the mixture was quenched with water and allowed to warm

to room temperature. The mixture was then extracted with EtOAc

(3×). The combined organic layers were dried over Na₂SO₄, ﬁltered,

and concentrated in vacuo. The crude residue was puriﬁed by ﬂash

chromatography (SiO₂; gradient elution 0−70% EtOAc in hexanes) to

In this work, we have established the novel iminothiadiazinane

dioxide core as a viable BACE1 catalytic dyad binding motif and

have applied this innovative core to develop novel BACE1

inhibitors with favorable CNS drug-like properties. These

eﬀorts have culminated in the discovery of compound 3, which

incorporates key structural features that are critical for ﬁne-

tuning its properties into ranges that provide robust central

pharmacodynamic activity and minimize undesirable oﬀ-target

activities. Eﬄux susceptibility and permeability of 3 are

favorably modulated by the reduced basicity of the

iminothiadiazinane dioxide core relative to the iminopyrimidi-

none core and by the presence of an intramolecular hydrogen

bond between the pyridine and amide hydrogen. The

importance of the N-phenylpicolinamide S1−S3 binding

motif is underscored by the high BACE1 aﬃnity and CatD

selectivity that this structural feature imparts when combined

with the iminothiadiazinane dioxide core. Oral administration

of 3 elicits robust reduction of centrally derived Aβ40 in

1

aﬀord 39 (5.6 g, 75% yield). H NMR (400 MHz, CDCl₃) δ 7.24−

7.18 (m, 3H), 6.96 (d, J = 6.0 Hz, 1H), 6.85 (d, J = 8.8 Hz, 2H), 6.36

(s, 1H), 4.21 (d, J = 14.0 Hz, 1H), 4.17 (s, 2H), 3.79 (s, 3H), 3.68 (d, J

= 14.0 Hz, 1H), 2.71 (s, 3H), 1.99 (s, 3H), 1.34 (s, 9H). ESI MS m/z

493.08 [M + H]⁺.

(S)-2-Amino-2-(3-chlorothiophen-2-yl)-N-methylpropane-1-

sulfonamide (40). To a solution of 39 (5.6 g, 11 mmol) in a mixture

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Drug Annotation

of CH₂Cl₂:MeOH (3:1, 50 mL) at room temperature was added a

solution of HCl (4 N in dioxane, 17 mL, 68 mmol). The mixture was

stirred at room temperature for 1 h. After that time, the mixture was

concentrated in vacuo, and the resultant residue was treated with

toluene (100 mL) and concentrated in vacuo again. To the obtained

residue were added CH₂Cl₂(50 mL) followed by TFA (26 mL, 341

mmol) and 1,3-dimethoxybenzene (10 mL, 80 mmol). The reaction

mixture was stirred at room temperature for 16 h. After that time, the

volatiles were removed in vacuo, and 1 M HCl(aq) was added to the

mixture, which was then extracted with Et₂O (3×). The aqueous layer

was then adjusted to pH ∼10 with the addition of sat. Na₂CO₃(aq).

The aqueous layer was then extracted with CH₂Cl₂(3×). The

combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo to aﬀord 40 (3.0 g, 99% crude yield), which was

carried on without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ

7.15 (d, J = 5.2 Hz, 1H), 6.90 (d, J = 5.2 Hz, 1H), 4.36 (br s, 1H), 3.94

(d, J = 14.4 Hz, 1H), 3.45 (d, J = 14.4 Hz, 1H), 2.66 (d, J = 5.2 Hz,

3H), 2.49 (br s, 2H), 1.76 (s, 3H). ESI MS m/z 269.2 [M + H]⁺.

(S)-5-(3-Chlorothiophen-2-yl)-3-imino-2,5-dimethyl-1,2,4-

thiadiazinane 1,1-dioxide (41). To a solution of 40 (3.0 g, 11

mmol) in CH₂Cl₂(50 mL) was added benzoylisothiocyanate (1.8 mL,

13 mmol), and the mixture was stirred at room temperature overnight.

After that time, the volatiles were removed in vacuo. To the residue

was added MeOH (50 mL) followed by a solution of sodium

methoxide (25% in MeOH, 6.4 mL, 28 mmol). The mixture was

stirred at room temperature for 45 min after which the solvent was

removed in vacuo. The residue was partitioned between CH₂Cl₂and

water, and the aqueous layer was adjusted to pH ∼8 with the addition

of 1 N HCl(aq) and sat. NaHCO₃(aq). The aqueous layer was then

extracted with CH₂Cl₂(3×). The combined organic layers were dried

over Na₂SO₄, ﬁltered, and concentrated in vacuo to aﬀord the crude

intermediate thiourea. To the thiourea were added EtOH (50 mL)

followed by methyl iodide (0.84 mL, 13 mmol), and the mixture was

heated to reﬂux with stirring for 8 h. The mixture was then cooled to

room temperature, and the solvent was removed in vacuo. The residue

was partitioned between water and CH₂Cl₂. The aqueous layer was

basiﬁed (pH >9) with sat. Na₂CO₃(aq). The layers were separated,

and the aqueous layer was extracted with CH₂Cl₂(2×). The combined

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated in

vacuo. The crude residue was puriﬁed by ﬂash chromatography [SiO₂;

gradient elution 0−5% MeOH (with 10% concn NH₄OH) in CH₂Cl₂]

(S)-5-(3-Chloro-5-(5-(prop-1-yn-1-yl)pyridin-3-yl)thiophen-

2-yl)-3-imino-2,5-dimethyl-1,2,4-thiadiazinane 1,1-Dioxide (9).

To a vial containing 43 (360 mg, 0.76 mmol) were added [5-(prop-1-

yn-1-yl)pyridin-3-yl]boronic acid (210 mg, 1.3 mmol) followed by

PdCl₂(dppf)·CH₂Cl₂(93 mg, 0.11 mmol). The vial was transferred

into a glovebox under an atmosphere of nitrogen. To the vial were

added t-BuOH (6 mL) followed by an aqueous solution of K₂CO₃

(2M, 1.1 mL, 2.2 mmol), and the mixture was stirred at 65 °C

overnight. After that time, the mixture was cooled to room

temperature and ﬁltered. To the ﬁltrate was added water, and the

mixture was extracted with CH₂Cl₂(3×). The combined organic layers

were dried over Na₂SO₄, ﬁltered, and concentrated in vacuo. To the

residue were added CH₂Cl₂(6 mL) followed by diisopropylethylamine

(0.27 mL, 1.5 mmol) and di-tert-butyldicarbonate (0.18 mL, 0.76

mmol). The mixture was stirred at room temperature for 24 h. The

reaction was quenched with water, and the aqueous layer was extracted

with CH₂Cl₂(3×). The combined organic layers were dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo. The residue was puriﬁed

by ﬂash chromatography (SiO₂; gradient elution 0−40% EtOAc in

hexanes) to aﬀord the intermediate Boc carbamate (100 mg, 26%

yield) as a white foam. To a solution of this intemediate in CH₂Cl₂(1

mL) was added TFA (1 mL). The resultant solution was stirred at

room temperature for 2 h. After that time, the soluton was

concentrated in vacuo. The crude residue was puriﬁed by HPLC

(C₁₈; gradient elution 10−45% MeCN:water with 0.1% TFA) to aﬀord

9 (80 mg, 82% yield) as the TFA salt. ¹H NMR (500 MHz, CDCl₃) δ

8.80 (d, J = 2.2 Hz, 1H), 8.61 (d, J = 2.0 Hz, 1H), 7.34 (s, 1H), 4.71

(d, J = 14.2 Hz, 1H), 4.55 (br s, 3 H), 3.76 (d, J = 14.2 Hz, 1H), 3.41

(s, 3H), 2.15 (s, 3H), 2.14 (s, 3H). ESI MS m/z 409.1 [M + H]⁺.

20

[α]_D−43.1° (c 0.515, CH₃OH).

(R)-N-(1-(2-Fluoro-5-nitrophenyl)ethylidene)-2-methylpro-

pane-2-sulﬁnamide (45). Ketimine 45 (37.4 g, 64% yield) was

1

prepared from 44 following the procedure used to prepare 36. H

NMR (400 MHz, CDCl₃) δ 8.54 (dd, J = 6.3, 2.9 Hz, 1H), 8.30 (dd, J

= 9.1, 3.5 Hz, 1H), 7.28 (t, J = 9.5 Hz, 1H), 2.79 (d, J = 3.5 Hz, 3H),

1.32 (s, 9H). ESI MS m/z 287.1 [M + H]⁺.

(R)-2-((R)-1,1-Dimethylethylsulﬁnamido)-2-(2-ﬂuoro-5-nitro-

phenyl)-N-(4-methoxybenzyl)-N-methylpropane-1-sulfona-

mide (49). To a ﬂame-dried round-bottom ﬂask containing a solution

of 38 (60.0 g, 262 mmol) in THF (750 mL) at −75 °C under an

atmosphere of nitrogen was added over 10 min a solution of n-BuLi

(1.6 M in hexanes, 165 mL, 262 mmol), and the mixture was stirred

for 30 min. After that time, to the mixture was added a solution of

ketimine 45 (50.0 g, 175 mmol) in THF (250 mL). The resultant

mixture was stirred at −75 °C for 1 h. After that time, the mixture was

slowly warmed to rt over 1 h, and then the reaction was quenched with

water and brine. The resultant mixture was then extracted with EtOAc

(3×). The combined organic layers were washed with brine, dried over

Na₂SO₄and MgSO₄, ﬁltered, and concentrated in vacuo. The crude

residue (HPLC; dr 93.5:6.5) was puriﬁed by ﬂash chromatography

(SiO₂; gradient elution 0−70% EtOAc in hexanes) to aﬀord 49 (50.8

g, 56% yield) as a single diastereomer. ¹H NMR (400 MHz, CDCl₃) δ

8.55 (m, 1H), 8.22 (m, 1H), 7.18−7.24 (m, 4 H), 6.84 (dd, J = 8.4, 1.2

Hz, 1H), 5.99 (s, 1H), 4.21 (d, J = 14.8 Hz, 2H), 3.86 (d, J = 14.8 Hz,

2H), 3.78 (s, 3H), 2.71 (s, 3H), 1.61 (s, 3H), 1.40 (s, 9H). ESI MS m/

z 538.2 [M + Na]⁺.

1

to aﬀord 41 (2.6 g, 81% yield) as an oﬀ white solid. H NMR (400

MHz, DMSO-d₆) δ 7.38 (d, J = 5.2 Hz, 1H), 6.93 (d, J = 5.2 Hz, 1H),

6.06 (br s, 2H), 3.91 (s, 2H), 3.04 (s, 3H), 1.62 (s, 3H). ESI MS m/z

294.2 [M + H]⁺.

(S)-tert-Butyl (5-(3-Chlorothiophen-2-yl)-2,5-dimethyl-1,1-

dioxido-1,2,4-thiadiazinan-3-ylidene)carbamate (42). To a

solution of 41 (9.6 g, 33 mmol) in THF (100 mL) was added

triethylamine (5.5 mL, 39 mmol) and di-tert-butyldicarbonate (8.6 g,

39 mmol), and the resultant mixture was stirred at room temperature

for 16 h. After that time, the volatiles were removed in vacuo, and the

residue was puriﬁed by ﬂash chromatography (SiO₂; gradient elution

0−20% EtOAc in hexanes) to aﬀord 42 (9.3 g, 72%) as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 10.71 (s, 1H), 7.21 (d, J = 6.0 Hz,

1H), 6.96 (d, J = 6.0 Hz, 1H), 4.43 (d, J = 14.0 Hz, 1H), 3.64 (d, J =

14.0 Hz, 1H), 3.28 (s, 3H), 2.02 (s, 3H), 1.52 (s, 9H). ESI MS m/z

394.0 [M + H]⁺.

(R)-2-Amino-2-(2-ﬂuoro-5-nitrophenyl)-N-methylpropane-1-

sulfonamide (50). Amine 50 (15.5 g, 90% yield) was prepared from

(S)-tert-Butyl (5-(5-Bromo-3-chlorothiophen-2-yl)-2,5-di-

methyl-1,1-dioxido-1,2,4-thiadiazinan-3-ylidene)carbamate

(43). To a solution of 42 (2.2 g, 5.6 mmol) in DMF (60 mL) was

added NBS (2.7 g, 15 mmol). The mixture was heated to 50 °C with

stirring for 8 h then allowed to cool to rt. The reaction mixture was

quenched with sat. Na₂S₂O₅(aq), partioned between water and EtOAc,

and the aqueous layer was extracted with EtOAc (3×). The combined

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated in

vacuo. The residue was puriﬁed by ﬂash chromatography (SiO₂;

gradient elution 0−17% EtOAc in hexanes) to aﬀord 43 (1.7 g, 63%

yield). ¹H NMR (400 MHz, CDCl₃) δ 10.78 (br s, 1H), 7.26 (s, 1H),

4.48 (d, J = 14.0 Hz, 1H), 3.58 (d, J = 14.0 Hz, 1H), 3.28 (s, 3H), 2.97

(s, 3H), 1.53 (s, 9H). ESI MS m/z 495.8 [M + Na]⁺.

1

49 following the procedure used to prepare 40. H NMR (400 MHz,

CDCl₃) δ 8.60 (dd, J = 7.2, 2.4 Hz, 1H), 8.18 (ddd, J = 7.2, 3.6, 3.6 Hz,

1H), 7.17 (dd, J = 10.4, 9.4 Hz, 1H), 4.33 (br s, 1H), 3.85 (d, J = 16.0

Hz, 1H), 3.41 (d, J = 16.0 Hz, 1H), 2.67 (s, 3H), 2.28 (br s, 2H), 1.62

(s, 3H). ESI MS m/z 292.2 [M + H]⁺.

(R)-tert-Butyl (5-(2-Fluoro-5-nitrophenyl)-2,5-dimethyl-1,1-

dioxido-1,2,4-thiadiazinan-3-ylidene)carbamate (51). To a

slurry of 50 (26.2 g, 89.9 mmol) in 1-butanol (300 mL) was added

a solution of cyanogen bromide (5 M in MeCN, 19.8 mL, 98.9 mmol),

and the mixture was heated to reﬂux with stirring for 4 h. After that

time, the mixture was cooled to room temperature and concentrated in

vacuo to approximately 1/3 of the original volume. To the mixture was

L

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Journal of Medicinal Chemistry

Drug Annotation

then added Et₂O (300 mL), and the resultant precipitate was removed

by ﬁltration. The precipitate was partitioned between EtOAc and sat.

Na₂CO₃(aq), and the mixture was extracted with EtOAc (3×). The

combined organic layers were washed with brine, dried over Na₂SO₄,

ﬁltered, and concentrated in vacuo. To the residue was added CH₂Cl₂

(300 mL) followed by triethylamine (16.6 mL, 120 mmol) and di-tert-

butyldicarbonate (22.4 g, 102 mmol). The resultant mixture was

stirred at rt for 16 h. The mixture was then diluted with CH₂Cl₂and

washed with Na₂CO₃(aq). The layers were separated, and the aqueous

layer was extracted with CH₂Cl₂(2×). The combined organic layers

were dried over Na₂SO₄, ﬁltered, and concentrated in vacuo. The

crude residue was puriﬁed by ﬂash chromatography (SiO₂; gradient

elution 0−40% EtOAc in hexanes) to aﬀord 51 (22.3 g, 59% yield) as

oxazolidinyl)phosphinic chloride (4.54 g, 17.8 mmol) followed by

pyridine (8 mL). The resultant mixture was stirred at rt for 1 h. The

mixture was then partitioned between water and EtOAc and ﬁltered,

and the layers were separated. The aqueous layer was extracted with

EtOAc (2×). The combined organic layers were dried over Na₂SO₄,

ﬁltered, and concentrated in vacuo. The crude residue was puriﬁed by

ﬂash chromatography (SiO₂; gradient elution 0−35% EtOAc in

hexanes) to aﬀord the intermediate amide (3.50 g, 87% yield). To a

ﬂask containing the amide were added CH₂Cl₂(30 mL) followed by

TFA (10 mL). The resultant solution was stirred at rt for 2 h. After

that time, to the solution was added Et₂O (50 mL), and the mixture

was poured into hexanes (400 mL). The resultant precipitate was

isolated by ﬁltration and dried under high vacuum. The obtained solid

was partitioned between EtOAc and sat. Na₂CO₃(aq). The layers were

separated, and the aqueous layer was extracted with EtOAc (2×). The

combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo to aﬀord 3 (2.70 g, 89% yield) as a white solid.

¹H NMR (400 MHz, DMSO-d₆) δ 10.57 (s, 1H), 8.73 (d, J = 2.8 Hz,

1H), 8.22 (dd, J = 8.8, 4.8 Hz, 1H), 8.03−7.95 (m, 2H), 7.79 (m, 1H),

7.14 (dd, J = 11.6, 8.8 Hz, 1H), 6.03 (br s, 2H), 3.78 (s, 1H), 3.34 (s,

1

a light tan solid. H NMR (400 MHz, CDCl₃) δ 10.82 (s, 1H), 8.26

(m, 2H), 7.28 (m, 1H), 4.29 (d, J = 14.4 Hz, 1H), 3.68 (d, J = 14.4 Hz,

1H), 3.23 (s, 3H), 1.90 (s, 3H), 1.56 (s, 9H). ESI MS m/z 439.0 [M +

Na]⁺.

(R)-tert-Butyl (5-(5-Amino-2-ﬂuorophenyl)-2,5-dimethyl-1,1-

dioxido-1,2,4-thiadiazinan-3-ylidene)carbamate (52). To a

hydrogenation vessel containing degassed solution 51 (7.3 g,18

mmol) in 1:1 EtOH:THF (130 mL) was added 10% Pd/C (1.1 g, 0.52

mmol, 50 wt %/wt H₂O). The vessel was sealed, evacuated, and

backﬁlled with nitrogen (3×) and then evacuated again and backﬁlled

with hydrogen (3×). The vessel was pressurized to 55 psi with

hydrogen, and the mixture was shaken at room temperature for 4 h.

The mixture was then purged with nitrogen, and the catalyst was

removed by ﬁltration. The ﬁlter cake was washed with 1:1 EtOH:THF,

and the ﬁltrate was concentrated in vacuo. The crude residue was

puriﬁed by ﬂash chromatography (SiO₂; gradient elution 0−45%

EtOAc in hexanes) to aﬀord 52 (6.1 g, 90% yield) as a white solid. ¹H

NMR (400 MHz, CDCl₃) δ 10.48 (br s, 1H), 6.87 (ddd, J = 12.0, 8.4,

1.6 Hz, 1H), 6.60−6.50 (m, 2H), 5.28 (s, 2H), 4.25 (d, J = 14.0 Hz,

1H), 3.63 (d, J = 14.0 Hz, 1H), 3.22 (s, 3H), 1.83 (s, 3H), 1.53 (s,

9H). ESI MS m/z 387.0 [M + H]⁺.

General Procedure A for the Preparation of Compounds

28−32. To a ﬂask containing 52 (100 mg, 0.26 mmol) were added the

requisite carboxylic acid (0.34 mmol) and bis(2-oxo-3-oxazolidinyl)-

phosphinic chloride (106 mg, 0.42 mmol) followed by pyridine (1.2

mL). The resultant mixture was stirred at rt for 16 h. After that time,

the mixture was partitioned between water and EtOAc. The layers

were separated, and the aqueous layer was extracted with EtOAc. The

combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated in vacuo. The crude residue was puriﬁed by ﬂash

chromatography (SiO₂; gradient elution 0−60% EtOAc in hexanes) to

aﬀord the intermediate amide. To a ﬂask containing the amide were

added CH₂Cl₂(3 mL) followed by TFA (1 mL). The resultant

solution was stirred at rt for 2 h. After that time, the solution was

concentrated in vacuo and dried further under high vacuum to aﬀord

the title compound as the triﬂuoroacetate salt.

20

1H), 3.05 (s, 3H), 1.61 (s, 3H). ESI MS m/z 410.2 [M + H]⁺. [α]_D

37.2° (c 0.367, CH₃OH). To generate its hydrochloride salt, 3

prepared above was added to CH₂Cl₂(50 mL) followed by a solution

of HCl (2 N in Et₂O, 3.6 mL, 7.2 mmol), and the mixture was

concentrated in vacuo. The product was slurried in distilled water (50

mL) and lyophilized to aﬀord the HCl monohydrate salt of 3 (2.58 g,

1

79% yield, 3 steps) as a white solid. H NMR (400 MHz, CD₃OD) δ

8.59 (d, J = 2.8 Hz, 1H), 8.26 (dd, J = 8.8, 4.8 Hz, 1H), 8.02 (dd, J =

7.6, 2.8 Hz, 1H), 7.82−7.75 (m, 2H), 7.22 (dd, J = 12.0, 8.8 Hz, 1H),

4.49 (dd, J = 14.4, 0.8 Hz, 1H), 4.30 (d, J = 14.4 Hz, 1H), 3.30 (s, 3H),

1.96 (s, 3H). ESI MS m/z 410.2 [M + H]⁺. Anal. Calcd

(C₁₇H₂₀ClF₂N₅O₄S): C, 44.02; H, 4.35; N, 15.10; Cl, 7.64; S, 6.91.

Found: C, 43.77; H, 4.32; N, 14.81; Cl, 7.84; S, 7.04.

(R)-5-Chloro-N-(4-ﬂuoro-3-(3-imino-2,5-dimethyl-1,1-dioxi-

do-1,2,4-thiadiazinan-5-yl)phenyl)picolinamide (30). Com-

pound 30 (89% yield) was prepared following general procedure A.

¹H NMR (400 MHz, CDCl₃) δ 11.28 (br s, 1H), 9.75 (s, 1H), 8.90 (br

s, 2H), 8.40 (d, J = 2.4 Hz, 1H), 7.96−7.90 (m, 2H), 7.66 (dd, J = 8.8,

2.4 Hz, 1H), 7.46 (ddd, J = 6.4, 2.8, 2.4 Hz, 1H), 7.11 (dd, J = 11.6, 8.8

Hz, 1H), 4.51 (dd, J = 14.8, 2.8 Hz, 1H), 3.81 (d, J = 14.8 Hz, 1H),

3.34 (s, 3H), 1.95 (s, 3H). ESI MS m/z 426.2 [M + H]⁺.

(R)-N-(4-Fluoro-3-(3-imino-2,5-dimethyl-1,1-dioxido-1,2,4-

thiadiazinan-5-yl)phenyl)-5-(triﬂuoromethyl)picolinamide

(31). Compound 31 (41% yield) was prepared following general

1

procedure A. H NMR (400 MHz, CDCl₃) δ 12.00 (br s, 1H), 10.82

(br s, 1H), 9.62 (s, 1H), 8.71 (s, 1H), 8.08 (dd, J = 6.8, 1.6 Hz, 1H),

7.95 (d, J = 8.0 Hz, 1H) 7.82 (d, J = 8.0 Hz, 1H), 7.20 (m, 1H), 7.06

(dd, J = 11.6, 8.8 Hz, 1H), 4.60 (dd, J = 14.4, 2.8 Hz, 1H), 3.80 (d, J =

14.4 Hz, 1H), 3.42 (s, 3H), 1.96 (s, 3H). ESI MS m/z 460.0 [M + H]⁺.

(R)-4-Chloro-(4-ﬂuoro-3-(3-imino-2,5-dimethyl-1,1-dioxido-

1,2,4-thiadiazinan-5-yl)phenyl)benzamide (32). Compound 32

(R)-N-(4-Fluoro-3-(3-imino-2,5-dimethyl-1,1-dioxido-1,2,4-

thiadiazinan-5-yl)phenyl)picolinamide (28). Compound 28 (78%

1

(56% yield) was prepared following general procedure A. H NMR

1

yield) was prepared following general procedure A. H NMR (400

(400 MHz, CDCl₃) δ 10.91 (s, 1H), 9.09 (s, 1H), 8.37 (br s, 2H), 7.75

(m, 1H), 7.65 (m 3H), 7.25 (d, J = 8.0 Hz, 2H) 7.07 (dd, J = 11.6, 8.8

Hz, 1H), 4.34 (d, J = 12.4 Hz, 1H), 3.73 (d, J = 12.4 Hz, 1H), 3.03 (s,

3H), 1.83 (s, 3H). ESI MS m/z 425.2 [M + H]⁺.

MHz, CDCl₃) δ 11.16 (br s, 1H), 10.09 (s, 1H), 9.50 (br s, 2H), 8.53

(d, J = 4.8 Hz, 1H), 8.10 (d, J = 7.6 Hz, 1H), 7.92 (dd, J = 7.6, 2.8 Hz,

1H), 7.83 (ddd, J = 7.6, 1.6, 1.6 Hz, 1H), 7.59 (ddd, J = 8.8, 2.8, 2.8

Hz, 1H), 7.45 (ddd, J = 7.6, 4.8, 1.6 Hz, 1H), 7.12 (dd, J = 12.0, 8.8

Hz, 1H), 4.45 (dd, J = 14.8, 2.4 Hz, 1H), 3.82 (d, J = 14.8 Hz, 1H),

3.31 (s, 3H), 1.95 (s, 3H). ESI MS m/z 392.2 [M + H]⁺.

(R)-N-(4-Fluoro-3-(3-imino-2,5-dimethyl-1,1-dioxido-1,2,4-

thiadiazinan-5-yl)phenyl)-5-methylpicolinamide (29). Com-

pound 29 (35% yield) was prepared following general procedure A.

¹H NMR (400 MHz, CDCl₃) δ 9.91 (br s, 1H), 8.31 (s, 1H), 8.05 (d, J

= 8.0 Hz, 1H), 7.91 (dd, J = 7.2, 2.8 Hz, 1H), 7.64 (m, 2H), 7.03 (dd, J

= 11.6, 8.8 Hz, 1H), 3.95 (br s, 2H), 3.90 (d, J = 14.0 Hz, 1H), 3.68 (d,

J = 14.0 Hz, 1H), 3.20 (s, 3H), 2.38 (s, 3H), 1.76 (s, 3H). ESI MS m/z

406.2 [M + H]⁺.

N-[3-[(5R)-3-Amino-5,6-dihydro-2,5-dimethyl-1,1-dioxido-

2H-1,2,4-thiadiazin-5-yl]-4-ﬂuorophenyl]-5-ﬂuoro-2-pyridine-

carboxamide (3). To a ﬂask containing 52 (3.00 g, 7.76 mmol) were

added 5-ﬂuoropicolinic acid (1.64 g, 11.6 mmol) and bis(2-oxo-3-

ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b00307.

Synthetic methods and characterization data for

compounds ³H-9, 12−16, and 20−27, molecular

modeling details, crystallographic information, and

methods for in vitro, in vivo, pharmacodynamic,

hydrolytic stability, and pharmacokinetic assays (PDF)

K_iand IC₅₀values for the compounds (CSV)

M

DOI: 10.1021/acs.jmedchem.6b00307

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Drug Annotation

therapy with acetylcholinesterase inhibitors and memantine. Expert

Opin. Drug Saf. 2014, 13, 759−774.

Accession Codes

3: 5HU1; 9: 5HTZ; and 13: 5HU0.

(4) Haass, C.; Selkoe, D. J. Soluble protein oligomers in

neurodegeneration: Lesson from the Alzheimer’s amyloid β-peptide.

Nat. Rev. Mol. Cell Biol. 2007, 8, 101−112.

AUTHOR INFORMATION

Corresponding Authors

*Phone: 908-740-4729. E-mail: jack.scott@merck.com.

*Phone: 973-868-2088. E-mail: andy.stamford1@gmail.com.

■

(5) (a) Hardy, J.; Selkoe, D. J. The amyloid hypothesis of Alzheimer’s

disease: Progress and problems on the road to therapeutics. Science

2002, 297, 353−356. (b) Karran, E.; Mercken, M.; De Strooper, B.

The amyloid cascade hypothesis for Alzheimer’s disease: an appraisal

for the development of therapeutics. Nat. Rev. Drug Discovery 2011, 10,

698−712.

(6) Tanzi, R. E. The genetics of Alzheimer disease. Cold Spring

Harbor Perspect. Med. 2012, 2, 1−10.

(7) Citron, M. Alzheimer’s disease: strategies for disease

modification. Nat. Rev. Drug Discovery 2010, 9, 387−398.

(8) Jonsson, T.; Atwal, J. K.; Steinberg, S.; Snaedal, J.; Jonsson, P. V.;

Bjornsson, S.; Stefansson, H.; Sulem, P.; Gudbjartsson, D.; Maloney,

J.; Hoyte, K.; Gustafson, A.; Liu, Y.; Lu, Y.; Bhangale, T.; Graham, R.

Present Addresses

^■(X.C.): Tandem Laboratories, Ridgeﬁeld, Connecticut, USA

^●(U.I.): Big Boost Marketing, Hoboken, New Jersey, USA

^□(R.A.H.): Charles River Laboratories, Boston, Massachusetts,

USA

^○(J.M.): Metrohm, New Jersey, USA

^▲(M.S.): Momentive Performance Materials, Waterford, New

York, USA

^◊(J. H.V.): Gilead Sciences, Foster City, California, USA

^⧫(Y.W.): Tetranov International, Inc., New Brunswick, New

Jersey, USA

R.; Huttenlocher, J.; Bjornsdottir, G.; Andreassen, O. A.; Jonsson, E.

̈

G.; Palotie, A.; Behrens, T. W.; Magnusson, O. T.; Kong, A.;

Thorsteinsdottir, U.; Watts, R. J.; Stefansson, K. A mutation in APP

protects against Alzheimer’s disease and age-related cognitive decline.

Nature 2012, 488, 96−99.

^%(W.J.G.): MedChem Discovery Consulting, Teaneck, New

Jersey, USA

Notes

(9) Roberds, S. L.; Anderson, J.; Basi, G.; Bienkowski, M. J.;

Branstetter, D. G.; Chen, K. S.; Freedman, S. B.; Frigon, N. L.; Games,

D.; Hu, K.; Johnson-Wood, K.; Kappenman, K. E.; Kawabe, T. T.;

Kola, I.; Kuehn, R.; Lee, M.; Liu, W.; Motter, R.; Nichols, N. F.;

Power, M.; Robertson, D. W.; Schenk, D.; Schoor, M.; Shopp, G. M.;

Shuck, M. E.; Sinha, S.; Svensson, K. A.; Tatsuno, G.; Tintrup, H.;

Wijsman, J.; Wright, S.; McConlogue, L. BACE knockout mice are

healthy despite lacking the primary beta-secretase activity in brain:

implications for Alzheimer’s disease therapeutics. Hum. Mol. Genet.

2001, 10, 1317−1324.

(10) Vassar, R.; Kuhn, P.-H.; Haass, C.; Kennedy, M. E.; Rajendran,

L.; Wong, P. C.; Lichtenthaler, S. F. Function, therapeutic potential

and cell biology of BACE proteases: current status and future

prospects. J. Neurochem. 2014, 130, 4−28.

(11) (a) Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.;

Mendiaz, E. A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.;

Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.; Edenson, S.; Lile, J.;

Jarosinski, M. A.; Biere, A. L.; Curran, E.; Burgess, T.; Louis, J.-C.;

Collins, F.; Treanor, J.; Rogers, G.; Citron, M. β-Secretase cleavage of

Alzheimer’s amyloid precursor protein by the transmembrane aspartic

protease BACE. Science 1999, 286, 735−741. (b) Sinha, S.; Anderson,

J. P.; Barbour, R.; Basi, G. S.; Caccavello, R.; Davis, D.; Doan, M.;

Dovey, H. F.; Frigon, N.; Hong, J.; Jacobson-Croak, K.; Jewett, N.;

Keim, P.; Knops, J.; Lieberburg, I.; Power, M.; Tan, H.; Tatsuno, G.;

Tung, J.; Schenk, D.; Seubert, P.; Suomensaari, S. M.; Wang, S.;

Walker, D.; Zhao, J.; McConlogue, L.; John, V. Purification and

cloning of amyloid precursor protein β-secretase from human brain.

Nature 1999, 402, 537−540. (c) Hussain, I.; Powell, D.; Howlett, D.

R.; Tew, D. G.; Meek, T. D.; Chapman, C.; Gloger, I. S.; Murphy, K.

E.; Southan, C. D.; Ryan, D. M.; Smith, T. S.; Simmons, D. L.; Walsh,

F. S.; Dingwall, C.; Christie, G. Identification of a novel aspartic

protease (Asp 2) as β-Secretase. Mol. Cell. Neurosci. 1999, 14, 419−

427. (d) Yan, R.; Bienkowski, M. J.; Shuck, M. E.; Miao, H.; Tory, M.

C.; Pauley, A. M.; Brashler, J. R.; Stratman, N. C.; Mathews, W. R.;

Buhl, A. E.; Carter, D. B.; Tomasselli, A. G.; Parodi, L. A.; Heinrikson,

R. L.; Gurney, M. E. Membrane-anchored aspartyl protease with

Alzheimer’s disease β-secretase activity. Nature 1999, 402, 533−537.

(e) Lin, X.; Koelsch, G.; Wu, S.; Downs, D.; Dashti, A.; Tang, J.

Human aspartic protease memapsin 2 cleaves the β-secretase site of β-

amyloid precursor protein. Proc. Natl. Acad. Sci. U. S. A. 2000, 97,

1456−1460.

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank Teresa Andreani, Yan Jin, Xian Liang, and Jesse

Wong for their contributions toward compound intermediate

synthesis, David Hesk and Carolee Lavey for radiolabeled

compound synthesis, Ryan Anstatt and Bonnie Werner for

pharmacokinetic and pharmacodynamic studies, and Neil

Johnson for safety analysis. Use of the IMCA-CAT beamline

17-ID at the Advanced Photon Source was supported by the

companies of the Industrial Macromolecular Crystallography

Association through a contract with Hauptman-Woodward

Medical Research Institute. Use of the Advanced Photon

Source was supported by the U.S. Department of Energy,

Oﬃce of Science, Oﬃce of Basic Energy Sciences, under

Contract No. DE-AC02-06CH11357.

ABBREVIATIONS USED

■

AD, Alzheimer’s disease; APP, amyloid precursor protein;

BACE1, β-site APP cleaving enzyme 1; BACE2, β-site APP

cleaving enzyme 2; BCS, biopharmaceutical classiﬁcation

system; Boc, tertiary butoxycarbonyl; CatD, cathepsin D;

CatE, cathepsin E; CNS, central nervous system; CSF,

cerebrospinal ﬂuid; CYP, Cytochrome P₄₅₀; ER, eﬄux ratio;

f_u, unbound fraction; hERG, human ether-a-go-go related gene;

LE, ligand eﬃciency; MDR1, multidrug resistance protein 1;

P

_app, apparent permeability; Pgp, P-glycoprotein; PMB, para-

methoxybenzyl; p.o., per os (dosed by mouth); SAR, structure−

activity relationship; sAPPβ, C-terminally truncated form of

amyloid precursor protein; SD, Sprague−Dawley; TFA,

triﬂuoroacetic acid

REFERENCES

■

(1) (a) Wortmann, M. Dementia: a global health priority −

highlights from an ADI and World Health Organization report.

Alzheimer’s Res. Ther. 2012, 4, 40−43. (b) Alzheimer’s Disease

International. World Alzheimer Report 2015. http://www.alz.co.uk/

research/WorldAlzheimerReport2015.pdf.

(12) (a) Oehlrich, D.; Prokopcova, H.; Gijsen, H. J. M. The

evolution of amidine-based brain penetrant BACE1 inhibitors. Bioorg.

Med. Chem. Lett. 2014, 24, 2033−2045. (b) Yuan, J.; Venkatraman, S.;

Zheng, Y.; McKeever, B. M.; Dillard, L. W.; Singh, S. B. Structure-

based design of β-site APP cleaving enzyme 1 (BACE) inhibitors for

(2) Alzheimer’s Association. 2015 Alzheimer’s disease facts and

ﬁgures. Alzheimers Dement. 2015, 332−384.

(3) Zemek, F.; Drtinova, L.; Nepovimova, E.; Sepsova, V.;

Korabecny, J.; Klimes, J.; Kuca, K. Outcomes of Alzheimer’s disease

N

DOI: 10.1021/acs.jmedchem.6b00307

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Drug Annotation

the treatment of Alzheimer’s disease. disease. J. Med. Chem. 2013, 56,

4156−4180. (c) Probst, G.; Xu, Y. z. Small-molecule BACE1

inhibitors: a patent literature review (2006 - 2011). Expert Opin.

Ther. Pat. 2012, 22, 511−540.

(13) (a) Willem, M.; Garratt, A. N.; Novak, B.; Citron, M.;

Kaufmann, S.; Rittger, A.; DeStrooper, B.; Saftig, P.; Birchmeier, C.;

Haass, C. Control of peripheral nerve myelination by the β-Secretase

BACE1. Science 2006, 314, 664−666. (b) Hu, X.; Hicks, C. W.; He,

W.; Wong, P.; Macklin, W. B.; Trapp, B. D.; Yan, R. Bace1 modulates

myelination in the central and peripheral nervous system. Nat.

Neurosci. 2006, 9, 1520−1525.

(14) Cheret, C.; Willem, M.; Fricker, F. R.; Wende, H.; Wulf-

Goldenberg, A.; Tahirovic, S.; Nave, K.-A.; Saftig, P.; Haass, C.;

Garratt, A. N.; Bennett, D. L.; Birchmeier, C. Bace1 and Neuregulin-1

cooperate to control formation and maintenance of muscle spindles.

EMBO J. 2013, 32, 2015−2028.

Zhang, Q.; Michener, M. S.; Smith, B.; Cox, K.; Grotz, D.; Favreau, L.;

Mitra, K.; Kazakevich, I.; McKittrick, B. A.; Greenlee, W.; Kennedy, M.

E.; Parker, E. M.; Cumming, J. N.; Stamford, A. W. Structure-based

design of an iminoheterocyclic β-site amyloid precursor protein

cleaving enzyme (BACE) inhibitor that lowers central Aβ in

nonhuman primates. J. Med. Chem. 2016, 59, 3231−3248.

(23) (a) Zhu, Z.; McKittrick, B.; Sun, Z.-Y.; Ye, Y. C.; Voigt, J. H.;

Strickland, C.; Smith, E. M.; Stamford, A.; Greenlee, W. J.; Wu, Y.;

Iserloh, U.; Mazzola, R.; Caldwell, J.; Cumming, J.; Wang, L.; Guo, T.;

Le, T. X. H.; Saionz, K. W.; Babu, S. D.; Hunter, R. C. Preparation of

heterocyclic aspartyl protease inhibitors for treating various diseases.

WO2005058311A1, 2005. (b) Malamas, M. S.; Erdei, J. J.; Gunawan, I.

S.; Zhou, P.; Yan, Y.; Quagliato, D. A. Preparation of amino-5,5-

diphenylimidazolone derivatives for inhibition of beta-secretase and

treatment of β-amyloid-related diseases. US20050282825A1, 2005.

(c) Albert, J. S.; Andisik, D.; Arnold, J.; Brown, D.; Callaghan, O.;

Campbell, J.; Carr, R. A. E.; Chessari, G.; Congreve, M. S.; Edwards,

P.; Empﬁeld, J. R.; Frederickson, M.; Koether, G. M.; Krumrine, J.;

Mauger, R.; Murray, C. W.; Patel, S.; Sylvester, M.; Throner, S.

Preparation of substituted 2-aminopyrimidin-4-ones for treating or

preventing Aβ-related pathologies. WO2006041404A1, 2006.

(24) Although we recognized the importance of pK_ato inhibitor

properties, we were unable to make reliable pK_apredictions of the

iminothiadiazinane class of inhibitors using standard ab initio quantum

mechanical calculations performed by commercially available software

packages.

(15) McConlogue, L.; Buttini, M.; Anderson, J. P.; Brigham, E. F.;

Chen, K. S.; Freedman, S. B.; Games, D.; Johnson-Wood, K.; Lee, M.;

Zeller, M.; Liu, W.; Motter, R.; Sinha, S. Partial reduction of BACE1

has dramatic effects on Alzheimer plaque and synaptic pathology in

APP transgenic mice. J. Biol. Chem. 2007, 282, 26326−26334.

(16) Wang, Y.-S.; Strickland, C.; Voigt, J. H.; Kennedy, M. E.; Beyer,

B. M.; Senior, M. M.; Smith, E. M.; Nechuta, T. L.; Madison, V. S.;

Czarniecki, M.; McKittrick, B. A.; Stamford, A. W.; Parker, E. M.;

Hunter, J. C.; Greenlee, W. J.; Wyss, D. F. Application of fragment-

based NMR screening, X-ray crystallography, structure-based design,

and focused chemical library design to identify novel μM leads for the

development of nM BACE-1 (β-site APP cleaving enzyme 1)

inhibitors. J. Med. Chem. 2010, 53, 942−950.

(25) Ginman, T.; Viklund, J.; Malmstrom, J.; Blid, J.; Emond, R.;

̈

Forsblom, R.; Johansson, A.; Kers, A.; Lake, F.; Sehgelmeble, F.;

Sterky, K. J.; Bergh, M.; Lindgren, A.; Johansson, P.; Jeppsson, F.;

(17) The CAS name represents the 3-aminothiadiazine tautomer, and

the structures depicted in the manuscript represent the 3-

iminothiadiazinane tautomer.

Falting, J.; Gravenfors, Y.; Rahm, F. Core refinement toward

̈

permeable β-secretase (BACE-1) inhibitors with low hERG activity.

J. Med. Chem. 2013, 56, 4181−4205.

(18) Zhu, Z.; Sun, Z.-Y.; Ye, Y.; Voigt, J.; Strickland, C.; Smith, E. M.;

Cumming, J.; Wang, L.; Wong, J.; Wang, Y.-S.; Wyss, D. F.; Chen, X.;

Kuvelkar, R.; Kennedy, M. E.; Favreau, L.; Parker, E.; McKittrick, B.

A.; Stamford, A.; Czarniecki, M.; Greenlee, W.; Hunter, J. C. Discovery

of cyclic acylguanidines as highly potent and selective β-site amyloid

cleaving enzyme (BACE) inhibitors: part I-inhibitor design and

validation. J. Med. Chem. 2010, 53, 951−965.

(19) Stamford, A.; Strickland, C. Inhibitors of BACE for treating

Alzheimer’s disease: a fragment-based drug discovery story. Curr. Opin.

Chem. Biol. 2013, 17, 320−328.

(26) The PSA value was calculated using the method described in

Ertl, P.; Rohde, B.; Selzer, P. Fast calculation of molecular polar surface

area as a sum of fragment-based contributions and its application to

the prediction of drug transport properties. J. Med. Chem. 2000, 43,

3714−3717 excluding the contribution from the sulfur atom.

(27) Scott, J. D.; Stamford, A. W.; Gilbert, E. J.; Cumming, J. N.;

Iserloh, U.; Misiaszek, J. A.; Li, G. Iminothiadiazine dioxide

compounds as BACE inhibitors and their preparation, compositions

and use in the treatment of pathologies associated with beta-amyloid

protein. WO2011044181A1, 2011.

(20) Cumming, J. N.; Smith, E. M.; Wang, L.; Misiaszek, J.; Durkin,

J.; Pan, J.; Iserloh, U.; Wu, Y.; Zhu, Z.; Strickland, C.; Voigt, J.; Chen,

X.; Kennedy, M. E.; Kuvelkar, R.; Hyde, L. A.; Cox, K.; Favreau, L.;

Czarniecki, M. F.; Greenlee, W. J.; McKittrick, B. A.; Parker, E. M.;

Stamford, A. W. Structure based design of iminohydantoin BACE1

inhibitors: identification of an orally available, centrally active BACE1

inhibitor. Bioorg. Med. Chem. Lett. 2012, 22, 2444−2449.

(21) Stamford, A. W.; Scott, J. D.; Li, S. W.; Babu, S.; Tadesse, D.;

Hunter, R.; Wu, Y.; Misiaszek, J.; Cumming, J. N.; Gilbert, E. J.;

Huang, C.; McKittrick, B. A.; Hong, L.; Guo, T.; Zhu, Z.; Strickland,

C.; Orth, P.; Voigt, J. H.; Kennedy, M. E.; Chen, X.; Kuvelkar, R.;

Hodgson, R.; Hyde, L. A.; Cox, K.; Favreau, L.; Parker, E. M.;

Greenlee, W. J. Discovery of an orally available, brain penetrant

BACE1 inhibitor that affords robust CNS Aβ reduction. ACS Med.

Chem. Lett. 2012, 3, 897−902.

(22) (a) Mandal, M.; Zhu, Z.; Cumming, J. N.; Liu, X.; Strickland, C.;

Mazzola, R. D.; Caldwell, J. P.; Leach, P.; Grzelak, M.; Hyde, L.;

Zhang, Q.; Terracina, G.; Zhang, L.; Chen, X.; Kuvelkar, R.; Kennedy,

M. E.; Favreau, L.; Cox, K.; Orth, P.; Buevich, A.; Voigt, J.; Wang, H.;

Kazakevich, I.; McKittrick, B. A.; Greenlee, W.; Parker, E. M.;

Stamford, A. W. Design and validation of bicyclic iminopyrimidinones

as beta amyloid cleaving enzyme-1 (BACE1) inhibitors: conforma-

tional constraint to favor a bioactive conformation. J. Med. Chem.

2012, 55, 9331−9345. (b) Mandal, M.; Wu, Y.; Misiaszek, J.; Li, G.;

Buevich, A.; Caldwell, J. P.; Liu, X.; Mazzola, R. D.; Orth, P.;

Strickland, C.; Voigt, J.; Wang, H.; Zhu, Z.; Chen, X.; Grzelak, M.;

Hyde, L. A.; Kuvelkar, R.; Leach, P. T.; Terracina, G.; Zhang, L.;

(28) (a) Koike, M.; Nakanishi, H.; Saftig, P.; Ezaki, J.; Isahara, K.;

Ohsawa, Y.; Schulz-Schaeffer, W.; Watanabe, T.; Waguri, S.; Kametaka,

S.; Shibata, M.; Yamamoto, K.; Kominami, E.; Peters, C.; von Figura,

K.; Uchiyama, Y. Cathepsin D deficiency induces lysosomal storage

with ceroid lipofuscin in mouse CNS neurons. J. Neurosci. 2000, 20,

6898−6906. (b) Siintola, E.; Partanen, S.; Stromme, P.; Haapanen, A.;

̈

Haltia, M.; Maehlen, J.; Lehesjoki, A.-E.; Tyynela, J. Cathepsin D

̈

deficiency underlies congenital human neuronal ceroid-lipofuscinosis.

Brain 2006, 129, 1438−1445.

(29) May, P. C.; Dean, R. A.; Lowe, S. L.; Martenyi, F.; Sheehan, S.

M.; Boggs, L. N.; Monk, S. A.; Mathes, B. M.; Mergott, D. J.; Watson,

B. M.; Stout, S. L.; Timm, D. E.; LaBell, E. S.; Gonzales, C. R.;

Nakano, M.; Jhee, S. S.; Yen, M.; Ereshefsky, L.; Lindstrom, T. D.;

Calligaro, D. O.; Cocke, P. J.; Hall, D. G.; Friedrich, S.; Citron, M.;

Audia, J. E. Robust central reduction of amyloid-β in humans with an

orally available, non-peptidic β-secretase inhibitor. J. Neurosci. 2011,

31, 16507−16516.

(30) Fielden, M. R.; Werner, J.; Jamison, J. A.; Coppi, A.; Hickman,

D.; Dunn, R. T., II; Trueblood, E.; Zhou, L.; Afshari, C. A.; Lightfoot-

Dunn, R. Retinal toxicity induced by a novel β-secretase inhibitor in

the Sprague-Dawley rat. Toxicol. Pathol. 2015, 43, 581−592.

(31) Independent of our work, selectivity of LY2811376 for BACE1

versus CatD was recently reported to be 6-fold. Butler, C. R.; Brodney,

M. A.; Beck, E. M.; Barreiro, G.; Nolan, C. E.; Pan, F.; Vajdos, F.;

Parris, K.; Varghese, A. H.; Helal, C. J.; Lira, R.; Doran, S. D.; Riddell,

D. R.; Buzon, L. M.; Dutra, J. K.; Martinez-Alsina, L. A.; Ogilvie, K.;

Murray, J. C.; Young, J. M.; Atchison, K.; Robshaw, A.; Gonzales, C.;

O

DOI: 10.1021/acs.jmedchem.6b00307

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Drug Annotation

Wang, J.; Zhang, Y.; O’Neill, B. T. Discovery of a series of efficient,

centrally efficacious BACE1 inhibitors through structure-based drug

design. J. Med. Chem. 2015, 58, 2678−2702.

(32) Zhu, Z.; McKittrick, B. A.; Sun, Z.-Y.; Ye, Y. C.; Voigt, J. H.;

Strickland, C.; Smith, E. M.; Stamford, A.; Greenlee, W. J.; Mazzola,

R.; Caldwell, J.; Cumming, J. N.; Wang, L.; Wu, Y.; Iserloh, U.; Guo,

T.; Le, T. X. H.; Saionz, K. W.; Babu, S. D.; Hunter, R. C.; Morris, M.

L.; Gu, H.; Qian, G.; Tadesse, D. Heterocyclic aspartyl protease

inhibitors. US 20060111370, May 25, 2006.

brain barrier crossing of drugs using molecular size and shape, and H-

bonding descriptors. J. Drug Targeting 1998, 6, 151−165.

(41) Alex, A.; Millan, D. S.; Perez, M.; Wakenhut, F.; Whitlock, G. A.

Intramolecular hydrogen bonding to improve membrane permeability

and absorption in beyond rule of five chemical space. MedChemComm

2011, 2, 669−674.

(42) Tang, H.; Mayersohn, M. A novel model for prediction of

human drug clearance by allometric scaling. Drug Metab. Dispos. 2005,

33, 1297−1303 For 3, the exponent b was set to 0.75 for all three

species used to calculate the projected human dose.

(33) Baldwin, E. T.; Bhat, T. N.; Gulnik, S.; Hosur, M. V.; Sowder, R.

C., II; Cachau, R. E.; Collins, J.; Silva, A. M.; Erickson, J. W. Crystal

structures of native and inhibited forms of human cathepsin D:

implications for lysosomal targeting and drug design. Proc. Natl. Acad.

Sci. U. S. A. 1993, 90, 6796−6800.

(43) Diller, J. D. In silico hERG modelling: challenges and progress.

Curr. Comput.-Aided Drug Des. 2009, 5, 106−121.

(44) Roden, D. M. Drug-induced prolongation of the QT interval. N.

Engl. J. Med. 2004, 350, 1013−1022.

(45) (a) Rochin, L.; Hurbain, I.; Serneels, L.; Fort, C.; Watt, B.;

Leblanc, P.; Marks, M. S.; De Strooper, B.; Raposo, G.; van Niel, G.

BACE2 processes PMEL to form the melanosome amyloid matrix in

pigment cells. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 10658−10663.

(b) Shimshek, D. R.; Jacobson, L. H.; Kolly, C.; Zamurovic, N.;

Balavenkatraman, K. K.; Morawiec, L.; Kreutzer, R.; Schelle, J.; Jucker,

M.; Bertschi, B.; Theil, D.; Heier, A.; Bigot, K.; Beltz, K.; Machauer, R.;

Brzak, I.; Perrot, L.; Neumann, U. Pharmacological BACE1 and

BACE2 inhibition induces hair depigmentation by inhibiting PMEL17

processing in mice. Sci. Rep. 2016, 6, 21917.

(34) Rueeger, H.; Lueoend, R.; Rogel, O.; Rondeau, J.-M.; Mobitz,

̈

H.; Machauer, R.; Jacobson, L.; Staufenbiel, M.; Desrayaud, S.;

Neumann, U. Discovery of cyclic sulfone hydroxyethylamines as

potent and selective β-site APP-cleaving enzyme 1 (BACE1)

inhibitors: structure-based design and in vivo reduction of amyloid

β-peptides. J. Med. Chem. 2012, 55, 3364−3386.

(35) (a) Cox, C. D.; Breslin, M. J.; Whitman, D. B.; Coleman, P. J.;

Garbaccio, R. M.; Fraley, M. E.; Zrada, M. M.; Buser, C. A.; Walsh, E.

S.; Hamilton, K.; Lobell, R. B.; Tao, W.; Abrams, M. T.; South, V. J.;

Huber, H. E.; Kohl, N. E.; Hartman, G. D. Kinesin spindle protein

(KSP) inhibitors. Part V: discovery of 2-propylamino-2,4-diaryl-2,5-

dihydropyrroles as potent, water-soluble KSP inhibitors, and

modulation of their basicity by β-fluorination to overcome cellular

efflux by P-glycoprotein. Bioorg. Med. Chem. Lett. 2007, 17, 2697−

2702. (b) McDonald, I. M.; Mate, R. A.; Zusi, F. C.; Huang, H.; Post-

Munson, D. J.; Ferrante, M. A.; Gallagher, L.; Bertekap, R. L., Jr.;

Knox, R. J.; Robertson, B. J.; Harden, D. G.; Morgan, D. G.; Lodge, N.

J.; Dworetzky, S. I.; Olson, R. E.; Macor, J. E. Discovery of a novel

series of quinolone α7 nicotinic acetylcholine receptor agonists. Bioorg.

Med. Chem. Lett. 2013, 23, 1684−1688. (c) Lerchner, A.; Machauer,

R.; Betschart, C.; Veenstra, S.; Rueeger, H.; McCarthy, C.; Tintelnot-

Blomley, M.; Jaton, A.-L.; Rabe, S.; Desrayaud, S.; Enz, A.; Staufenbiel,

M.; Paganetti, P.; Rondeau, J.-M.; Neumann, U. Macrocyclic BACE-1

inhibitors acutely reduce Aβ in brain after po application. Bioorg. Med.

Chem. Lett. 2010, 20, 603−607.

(36) (a) Hyde, L.; Chen, X.; Stahl, L.; Sondey, M.; Scott, J.;

Cumming, J.; Stamford, A.; Parker, E.; Kennedy, M. Chronic BACE

inhibition dramatically slows the rate of Aβ accumulation and the

development of amyloid plaques in young TgCRND8 mice.

Alzheimer's Dementia 2012, 8, P188. (b) Hyde, L.; Cantu, C.;

Werner, B.; Chen, X.; Sondey, M.; Chen, M.; Weig, B.; LaShomb, A.;

Lu, S.; Hodgson, R.; Scott, J.; Cumming, J.; Stamford, A.; Parker, E.;

Kennedy, M. Chronic BACE inhibition halts further age-related

increases in brain Aβ and amyloid plaques in aged TgCRND8 mice

with established plaques. Alzheimer's Dementia 2012, 8, P188.

(46) Esterhaz

́

y, D.; Stutzer, I.; Wang, H.; Rechsteiner, M. P.;

̈

Beauchamp, J.; Dobeli, H.; Hilpert, H.; Matile, H.; Prummer, M.;

̈

Schmidt, A.; Lieske, N.; Boehm, B.; Marselli, L.; Bosco, D.; Kerr-

Conte, J.; Aebersold, R.; Spinas, G. A.; Moch, H.; Migliorini, C.;

Stoffel, M. BACE2 is a β cell-enriched protease that regulates

pancreatic β cell function and mass. Cell Metab. 2011, 14, 365−377.

́

(47) Velazquez, F.; Arasappan, A.; Chen, K.; Sannigrahi, M.;

Venkatraman, S.; McPhail, A. T.; Chan, T.-M.; Shih, N.-Y.; Njoroge,

F. G. Stereoselective synthesis of β-substituted β-amino sulfones and

sulfonamides via addition of sulfonyl anions to chiral N-sulfinyl imines.

Org. Lett. 2006, 8, 789−792.

(48) Brak, K.; Barrett, K. T.; Ellman, J. A. General one-pot method

for the preparation of N-tert-butanesulfinylamine diastereomer

mixtures as standards for stereoselectivity determinations. J. Org.

Chem. 2009, 74, 3606−3608.

(49) Huang, B.-S.; Chello, P. L.; Yip, L.; Parham, J. C. Synthesis and

properties of the sulfonyl analogues of 4(5)-aminoimidazole-5(4)-

carboxamide, 4(5)-(formylamino)imidazole-5(4)-carboxamide, gua-

nine, and xanthine. J. Med. Chem. 1980, 23, 575−577.

(50) A manufacturing route to verubecestat is described in

Thaisrivongs, D. A.; Miller, S. P.; Molinaro, C.; Chen, Q.; Song, Z.

J.; Tan, L.; Chen, L.; Wenyong Chen, W.; Lekhal, A.; Pulicare, S. K.;

Xu, Y. Synthesis of verubecestat (MK-8931), a BACE1 inhibitor for

the treatment of Alzheimer’s disease. Org. Lett. 2016, DOI: 10.1021/

acs.orglett.6b01793.

(37) Scharfer, C.; Schulz-Gasch, T.; Ehrlich, H.-C.; Guba, W.; Rarey,

̈

M.; Stahl, M. Torsion angle preferences in druglike chemical space: a

comprehensive guide. J. Med. Chem. 2013, 56, 2016−2028.

(38) Kennedy, M. E.; Stamford, A. W.; Chen, X.; Cox, K.; Cumming,

J. N.; Dockendorf, M. F.; Egan, M.; Ereshefsky, L.; Hodgson, R. A.;

Hyde, L. A.; Jhee, S.; Kleijn, H. J.; Kuvelkar, R.; Li, W.; Mattson, B. A.;

Mei, H.; Palcza, J.; Scott, J. D.; Tanen, M.; Troyer, M. D.; Tseng, J. L.;

Stone, J. A.; Parker, E. M.; Forman, M. S. The BACE1 inhibitor

verubecestat (MK-8931) reduces CNS β-amyloid in animal models

and in Alzheimer’s disease patients. Sci. Transl. Med. 2016, 8,

363ra150.

(39) Ostermann, N.; Eder, J.; Eidhoff, U.; Zink, F.; Hassiepen, U.;

Worpenberg, S.; Maibaum, J.; Simic, O.; Hommel, U.; Gerhartz, B.

Crystal structure of human BACE2 in complex with a hydroxyethyl-

amine transition-state inhibitor. J. Mol. Biol. 2006, 355, 249−261.

(40) (a) 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, 1514−1519. (b) Van de Waterbeemd, H.; Camenisch,

G.; Folkers, G.; Chretien, J. R.; Raevsky, O. A. Estimation of blood−

P

DOI: 10.1021/acs.jmedchem.6b00307

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)-A β-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer's Disease

DOI: 10.1021/acs.jmedchem.6b00307

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Full text of DOI:10.1021/acs.jmedchem.6b00307

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