From fragment screening to in vivo efficacy: Optimization of a series of 2-aminoquinolines as potent inhibitors of beta-site amyloid precursor protein cleaving enzyme 1 (bace1)

ARTICLE

pubs.acs.org/jmc

From Fragment Screening to In Vivo Efficacy: Optimization of a

Series of 2-Aminoquinolines as Potent Inhibitors of Beta-Site

Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1)^†

Yuan Cheng,*^,‡Ted C. Judd,*^,‡Michael D. Bartberger,^§James Brown,^‡Kui Chen,^{^}Robert T. Fremeau, Jr.,^||

Dean Hickman,^#Stephen A. Hitchcock,³Brad Jordan,^§Vivian Li,^§Patricia Lopez,^‡Steven W. Louie,^#

Yi Luo,^||Klaus Michelsen,^§Thomas Nixey,^‡Timothy S. Powers,^‡Claire Rattan,^||E. Allen Sickmier,^§

David J. St. Jean, Jr.,^‡Robert C. Wahl,^{^}Paul H. Wen,^||and Stephen Wood^||

^‡Chemistry Research and Discovery, ^§Department of Molecular Structure, Department of Neuroscience, ^{^}Department

of HTS and Molecular Pharmacology, ^#Department of Pharmacokinetics and Drug Metabolism, Amgen Inc.,

One Amgen Center Drive, Thousand Oaks, California 91320, United States

S Supporting Information

b

ABSTRACT: Using fragment-based screening of a focused

fragment library, 2-aminoquinoline 1 was identiﬁed as an initial

hit for BACE1. Further SAR development was supported by

X-ray structures of BACE1 cocrystallized with various ligands

and molecular modeling studies to expedite the discovery of

potent compounds. These strategies enabled us to integrate the

C-3 side chain on 2-aminoquinoline 1 extending deep into the

P2⁰binding pocket of BACE1 and enhancing the ligand’s potency. We were able to improve the BACE1 potency to subnanomolar

range, over 10⁶-fold more potent than the initial hit (900 μM). Further elaboration of the physical properties of the lead compounds

to those more consistent with good bloodꢀbrain barrier permeability led to inhibitors with greatly improved cellular activity and

permeability. Compound 59 showed an IC₅₀value of 11 nM on BACE1 and cellular activity of 80 nM. This compound was advanced

into rat pharmacokinetic and pharmacodynamic studies and demonstrated signiﬁcant reduction of Aβ levels in cerebrospinal ﬂuid

(CSF).

’ INTRODUCTION

ﬁrst to generate the amino terminus of Aβ, producing soluble

amyloid precursor protein-β (sAPPβ) and a membrane bound

C-terminal fragment called C99. γ-Secretase then cleaves

C99 to release the mature Aβ peptide. Inhibition of β-secretase

decreases the production of all forms of Aβ, including the most

Alzheimer’s disease (AD) is a progressive neurological disease

of the brain that leads to the irreversible loss of neurons and

dementia. The clinical hallmarks of AD include progressive

impairment in memory, judgment, decision making, orientation

to physical surroundings, and language. Current standards of care

including the acetylcholinesterase inhibitors and NMDA recep-

tor antagonists provide temporary symptomatic relief but do not

alter the underlying disease pathophysiology. A therapeutic

treatment that directly modiﬁes the progression of the disease

remains one of the largest unmet medical needs.

The pathological hallmarks of AD include the deposition of

amyloid β (Aβ) in the neural parenchyma and the formation of

neuronal tangles. The study of disease genes that are associated

with familial cases of AD has implicated the accumulation of the

Aβ peptide as a key early initiating event in the pathogenesis of

this disorder.¹The genetic ﬁndings form the basis for the amyloid

hypothesis of AD, which suggests that accumulation of the Aβ

peptide in toxic protein aggregates due to an imbalance between

production and clearance triggers numerous pathophysiological

changes that ultimately lead to synaptic failure, neurodegenera-

tion, and cognitive dysfunction.²Aβ is produced by the sequen-

tial endoproteolytic cleavage of amyloid precursor protein (APP)

by two proteases, the β- and γ-secretases.^3,4β-Secretase cuts APP

5

pathogenic species, Aβ₄₂indicating that β-secretase is a prime

therapeutic target for the development of disease-modifying

therapies for AD.⁶

In 1999, β-secretase was identiﬁed as the type 1 transmem-

brane aspartyl protease BACE1 that is related to the pepsin and

retroviral aspartic protease families.³The highest levels of BACE1

ꢀ/ꢀ

expression are found in neurons in the brain.³BACE1

mice

are viable, fertil_ꢀe,_/a_ꢀnd do not produce cerebral Aβ.⁷Furthermore,

when BACE1

mice are crossed with APP transgenic mice

that overexpress mutant APP, the deﬁciency in BACE1 activity

rescues the amyloid pathology and synaptic and behavioral

dysfunction observed in the APP transgenic mice.⁸These results

indicate that BACE1 is essential for producing amyloidogenic Aβ

peptides in the mammalian brain.

BACE1 is located in the central nervous system (CNS) and

characterized by a large binding site to recognize 6ꢀ8 amino acid

Received: May 3, 2011

Published: June 27, 2011

r

2011 American Chemical Society

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residues of APP. There are signiﬁcant challenges in designing

low-molecular-weight, brain penetrant, potent BACE1 inhibitors.⁹

Traditional high throughput screening (HTS) methods have met

with very limited success, and most lead compounds have been

derivedbyrationalstructure-based designofpeptidomimetics.^10ꢀ13

The majority of these compounds possess a secondary

alcohol as a transition state mimetic that displaces an

active-site water molecule and forms hydrogen bonding

interactions with the catalytic aspartates. Although some of

the initial peptidomimetic inhibitors were very potent in

vitro, these inhibitors tended to have multiple hydrogen bond

donors and acceptors along with relatively high molecular

weight (MW > 500). These characteristics present challenges

to further development of these compounds into CNS

penetrable BACE1 inhibitors devoid of P-glycoprotein

(Pgp) mediated eﬄux liabilities.^14,15As such, a majority of

these peptidomimetic inhibitors were not able to demon-

strate signiﬁcant CNS Aβ lowering in rodent pharmacody-

namic (PD) models. In recent years, there has been a growing

interest from a number of groups focused on developing

nonpeptidomimetic BACE1 inhibitors.^15ꢀ18However, only a

few compounds in this class are known in the literature to

show both good in vitro potency (in the low nanomolar

range) and also demonstrate signiﬁcant Aβ reduction in CNS.

Therefore, identiﬁcation of nonpeptidic small molecule

BACE1 inhibitors that are both highly potent and CNS

penetrable remains a major challenge.

Our eﬀorts implementing fragment-based screening to iden-

tify potent BACE1 inhibitors have led to a class of aminoquino-

lines that are highly potent and brain penetrable. Starting with a

focused fragment library that was screened using surface plasmon

resonance (SPR) based technology,³²one of the initial fragment

hits, 2-aminoquinoline 1, displayed potency in the millimolar

range with excellent LE (Figure 1). 2-Aminoquinoline 1 has been

reported in the literature as a weak inhibitor of BACE1 along with

its cocrystal structure revealing its mode of binding.^30,31On the

basis of the high ligand eﬃciency of 1 and the existence of

structural information regarding its binding, this fragment was

selected as one of the more promising starting points for further

optimization.

Development of 1 was by supported by X-ray determination of

key BACE1 cocrystal structures along the optimization pathway

in conjunction with modeling studies, expediting the discovery of

compounds with increasingly greater aﬃnity. Close attention was

paid to the physical properties of the lead compounds, giving rise

to inhibitors with favorable cellular activity and permeability. The

optimized aminoquinoline BACE1 inhibitor 59 was evaluated in

rat pharmacodynamic studies and demonstrated signiﬁcant CSF

Aβ reduction. The evolution of the structureꢀactivity relation-

ship, as well as optimization of physical properties conducive to

bloodꢀbrain barrier penetration, will be the central focus of this

report.

’ CHEMISTRY

In an eﬀort to identify nonpeptidic leads with the potential for

desired CNS penetration, we employed a fragment-based screen-

ing approach. Fragment-based screening has become a valuable

alternative to traditional HTS screening, oﬀering the possibility

of identifying novel leads with much lower molecular weight than

those generated from HTS.^19ꢀ22Fragment-based hits are typi-

cally small (MW 150ꢀ250) and have weak aﬃnity (K_d∼

100ꢀ2000 μM) but high ligand eﬃciency (LE).^22ꢀ25Optimiza-

tion of fragment hits guided by LE and physical properties can

provide opportunities to design lead compounds with the desired

drug-like properties. However, fragment-based screening can be

challenging in practice as robust detection techniques are neces-

sary to detect low aﬃnity compounds. Ideally, having more than

one screening method is desirable in order to conﬁrm the on-

mechanism activity of fragment hits and minimize the number of

compounds that give false positive results. In practice, access to

cocrystal structures of the target and fragment hits is also crucial

for further conﬁrmation and optimizations of the hits. Because

these requirements were able to be satisﬁed with the BACE1

enzyme target, the fragment-based screening approach appeared

to be an appealing strategy.

The synthesis of bromo-substituted 2-aminoquinolineanalogues

as shown in Table 1 is summarized in Scheme 1. Commercially

available bromo-substituted quinolin-2(1H)-ones (2) was reﬂuxed

with phosphorus oxychloride to give 2-chloroquinoline 3. The

chlorine displacement with 4-methoxybenzylamine followed by

removal of the 4-methoxylbenzyl group using triﬂuoroacetic acid

aﬀorded bromo-substituted 2-aminoquinolines 28ꢀ31.

6-Substituted 2-aminoquinoline analogues 32ꢀ38 shown in

Table 2 were prepared from Suzuki coupling of boronic acids

with 6-bromo-2-aminoquinoline 4 (Scheme 2). 6-(2-(3,3-Di-

methylbut-1-ynyl)phenyl)quinolin-2-amine 39 was prepared

from 6-aminoquinoline 5 by converting the amino group to

Scheme 1. Synthesis of Bromo-Substituted 2-Aminoquino-

line Derivatives^a

^aReagents and conditions: (a) phosphorus oxychloride, reﬂux; (b)

4-methoxybenzylamine, N-methylpyrrolidinone, 140 ꢀC; (c) triﬂuoroa-

cetic acid, 60 ꢀC.

There have been reports in recent years demonstrating the

success of fragment-based screening approach on BACE1^17,26ꢀ31

and several fragment hits inhibiting BACE1 with good LE having

been identiﬁed. However, few of these inhibitors developed from

fragment-based screening were reported to reduce Aβ peptide

production in the CNS.

Scheme 2. Synthesis of 6-Substituted 2-Aminoquinoline

Derivatives^a

^aReagents and conditions: (a) potassium carbonate, PS (polystyrene)-

triphenylphosphine palladium(0), dimethoxyethane/ethanol, micro-

wave, 140 ꢀC, 10 min.

Figure 1. 2-Aminoquinoline as an initial fragment hit capable of

inhibiting BACE.

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Scheme 3. Synthesis of 6-(2-(3,3-Dimethylbut-1-ynyl)phenyl)quinolin-2-amine^a

^aReagents and conditions: (a) NaNO₂, HCl, NaI, ethyl acetate; (b) 2-bromophenylboronic acid, potassium carbonate, tetrakistriphenylphosphine

palladium(0), ethanol, reﬂux; (c) m-chloroperoxybenzoic acid, chloroform, reﬂux; (d) triﬂuoromethylbenzene, tert-butylamine, p-toluenesulfonic

anhydride, 25 ꢀC; (e) triﬂuoroacetic acid, 80 ꢀC; (f) triethylamine, copper(I) iodide, dichlorobis(triphenylphosphine)palladium(II), 4,4-dimethylpent-

2-yne, dimethylformamide, 140 ꢀC.

Scheme 4. Synthesis of 3-Substituted 2-Aminoquinoline Derivatives^a

^aReagents and conditions: (a) methyl-(triphenylphosphoranylidene)-acetate, THF, 60 ꢀC; (b) lithium hydroxide, methanol/H₂O, 50 ꢀC; (c) thionyl

chloride, 80 ꢀC; (d) N,N-diisopropylethylamine, R₁R₂NH, dichloromethane, 25 ꢀC; (e) H₂, Pd/C, methanol; (f) m-chloroperoxybenzoic acid,

chloroform, reﬂux; (g) triﬂuoromethylbenzene, tert-butylamine, p-toluenesulfonic anhydride, 25 ꢀC; (h) triﬂuoroacetic acid, 80 ꢀC.

Scheme 5. Synthesis of 6-Aryl C-3 Substituted 2-Aminoquinoline Derivatives^a

^aReagents and conditions: (a) lithium aluminum hydride, THF, ꢀ78 ꢀC; (b) manganese dioxide, dichloromethane; (c) methyl 3,3-dimethoxypro-

pionate, toluenesulfonic acid, toluene, 140 ꢀC; (d) diisobutylaluminum hydride, dichloromethane, ꢀ78 ꢀC; (e) DessꢀMartin periodinane,

dichloromethane; (f) methyl (triphenylphosphoranylidene) acetate, THF, 70 ꢀC; (g) o-tolylboronic acid, potassium acetate, bis(4-(di-tert-

butylphosphino)-N,N-dimethylbenzenamine) dichloropalladium(II), water, ethanol, reﬂux; (h) H₂, Pd/C, ethanol; (i) LiOH, methanol, 50 ꢀC; (j)

thionyl chloride, 80 ꢀC, then R₁NH₂, or R₁R₂NH, N,N-diisopropylethylamine, dichloromethane; (k) m-chloroperoxybenzoic acid, chloroform, reﬂux;

(l) triﬂuoromethylbenzene, tert-butylamine, p-toluenesulfonic anhydride, 25 ꢀC; (m) triﬂuoroacetic acid, 80 ꢀC.

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Scheme 6. Synthesis of r-Substituted Amide Intermediates^a

^aReagents and conditions: (a) potassium tert-butoxide, THF, 25 ꢀC.

Scheme 7. Synthesis of 6-o-Tolyl 2-Aminoquinoline Amide Derivatives^a

^aReagents and conditions: (a) phosphorus oxychloride, DMF, 75 ꢀC; (b) 4-methoxybenzylamine, NMP, 130 ꢀC or tert-butylamine, ethanol, 125 ꢀC; (c)

ethyl 2-(diethoxyphosphoryl)propanoate, lithium chloride, acetonitrile, 25 ꢀC; (d) o-tolylboronic acid, potassium acetate, bis(4-(di-tert-

butylphosphino)-N,N-dimethylbenzenamine) dichloropalladium(II), water, ethanol, reﬂux; (e) H₂, Pt/C, ethanol; (f) LiOH, methanol/THF; (g)

O-(benzotriazol-1-yl)-N,N,N⁰,N⁰-tetramethyluronium tetraﬂuoroborate, R₂NH₂, N,N-diisopropylethylamine, NMP; (h) triﬂuoroacetic acid, 80 ꢀC.

Scheme 8. Synthesis of 6-Substituted 2-Aminoquinoline 3,3-Dimethylbutanyl Amide Derivatives^a

^aReagents and conditions: (a) LiOH, methanol/THF; (b) H₂, Pt/C, ethanol. (b) O-(benzotriazol-1-yl)-N,N,N⁰,N⁰-tetramethyluronium tetraﬂuoroborate, 3,3-

dimethylbutanamine, N,N-diisopropylethylamine, NMP; (c) H₂, Pt/C, ethanol; (d) R₂-boronic acid, potassium acetate, bis(4-(di-tert-butylphosphino)-N,N-

dimethylbenzenamine) dichloropalladium(II), water, ethanol, reﬂux; (e) triﬂuoroacetic acid, 80 ꢀC; (f) dichloro(1,1⁰-bis(diphenylphosphino)ferrocene)

palladium(II), potassium acetate, 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane, dioxane, 85 ꢀC; (g) R₃Br, potassium

acetate, bis(4-(di-tert-butylphosphino)-N,N-dimethylbenzenamine) dichloropalladium(II), water, ethanol, reﬂux.

the iodide followed by Suzuki coupling with 2-bromophenyl-

boronic acid to give 6-(2-bromophenyl)quinoline 6 (Scheme 3).

The 2-amino group was installed by formation of the quinoline

N-oxide 7 by oxidation with m-chloroperoxybenzoic acid and

then treatment with toluenesulfonic anhydride and tert-butyl-

amine in triﬂuoromethylbenzene to aﬀord the corresponding

the tert-butylamine derivative 8. Removal of the tert-butyl group

under acidic conditions gave the free 2-amino analogue 6-(2-

bromophenyl)quinolin-2-amine 9. Palladium catalyzed coupling

of 9 with 4,4-dimethylpent-2-yne aﬀorded 6-(2-(3,3-dimethyl-

but-1-ynyl)phenyl)quinolin-2-amine 39.

The synthesis of 3-substituted 2-aminoquinoline analogues in

Table 3 started with commercial available quinoline-3-aldehyde

10, which was converted to methyl 3-(quinolin-3-yl)acrylate by

Wittig oleﬁnation with methyl-(triphenylphosphoranylidene)-

acetate (Scheme 4). Hydrolysis of the ester group gave the acid

11 and amide formation followed by hydrogenation of the oleﬁn

provided the amide 12. The 2-amino group was installed as

previously described in the preparation of 39 in Scheme 3 to give

the 2-amino analogues 40 and 44.

Scheme 5 describes the synthesis of 6-o-tolyl 3-substituted

2-aminoquinoline analogues that are shown in Table 3. The

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methyl ester of 13 was converted to the aldehyde 14 by reduction

with lithium aluminum hydride followed by oxidation with manga-

nese dioxide. Heating of 14 with methyl 3,3-dimethoxypropionate

and catalytic toluenesulfonic acid in toluene aﬀorded methyl

6-bromoquinoline-3-carboxylate 15. The ester group of 15 was

converted to the aldehyde 16, which underwent a Witting

oleﬁnation with methyl (triphenylphosphoranylidene) acetate

to give 17. Suzuki coupling with o-tolylboronic acid, followed by

hydrogenation of the oleﬁn, provided 18. Hydrolysis of the

methyl ester and amide coupling with various amines aﬀorded

19. The 2-amino group was installed as described in Scheme 3 to

give compound 41ꢀ43.

Figure 2. The binding mode of amidine-like groups with catalytic

aspartates in the active site of BACE.

The compounds with substitution at the R-position of the

amide in Table 4 were prepared in an analogous manner as

described in Scheme 6, except the C-3 linker was installed with

the aldehyde 16 by HornerꢀWadsworthꢀEmmons reaction

with various substituted phosphonate esters to give 20. 45ꢀ50

were prepared by the same procedures (17 to 41ꢀ43) as

described in Scheme 5.

Alternatively, R-methyl amide analogues as shown in Scheme 7

couldbe synthesized fromthe3-aldehyde quinoline22 prepared in

one step via Vilsmeier reaction using a modiﬁed procedure from

the literature³³and functionalization at the 2-position. Com-

pounds in Table 5 were prepared using this method. Thus N-

(4-bromophenyl)acetamide 21 was treated with phosphorus

oxychloride and DMF to give 2-chloroquinoline aldehyde 22.

The chlorine atom was displaced by 4-methoxybenzylamine or

tert-butylamine to aﬀord 23. HornerꢀWadsworthꢀEmmons

oleﬁnation on aldehyde 23 with ethyl 2-(diethoxyphosphoryl)-

propanoate gave the common intermediate ester 24, which was

further modiﬁed by both amide replacement of the ester and

6-bromo substitution to access the ﬁnal compounds. Suzuki

coupling between the o-tolylboronic acid, followed by hydrolysis

of the ester group and hydrogenation of the oleﬁn, aﬀorded the

acid 25. At this stage, the (R)-enantiomer could be isolated by

chiral HPLC separation. The acid 25 was coupled with various

amines under the standard amide coupling conditions followed by

removal of the tert-butyl or 4-methoxybenzyl group of the

2-aminoquinoline to give 51ꢀ57.

Figure 3. Co-crystal structure of 32 in BACE1 (white) superimposed

with the 2-aminoquinoline co-crystal structure 2OHL (1, green.) Of

note for 32 are the conformational change of ﬂap Tyr71 and CꢀH

π

3 3 3

interactions with Tyr71 and Phe108. Distances are reported in Å.

bidentate-type interactions afforded by aminoheterocycles and

amidine-like moieties such as depicted in Figure 2.^30,31

About 4000 compounds were chosen for screening. SPR

technology was used to screen the fragment library rather than

a ﬂuorescence-based enzymatic assay because ﬂuorescence-based

detection is well-known to be susceptible to compound inter-

ference at high concentrations (>100 μM). SPR technology has

been shown to be able to monitor and evaluate the relatively

weak interactions with the target protein and allows for determi-

nation of binding constants >100 μM.^29c,32After screening the

fragment library, 106 fragment hits were identiﬁed with potency

<10 mM by the SPR assay. These compounds were next tested in

an ¹⁹F-NMR-based functional assay which directly measured

inhibition of BACE1 activity using a ¹⁹F-labeled substrate pep-

tide. Eight compounds were conﬁrmed with potency less than

1 mM. These fragment hits (<1 mM) were further triaged based

on LE, cocrystallization with the BACE1 protein, and initial SAR

studies. Among these hits, 2-aminoquinoline 1 was characterized

as a weak inhibitor with potency at 900 μM but excellent ligand

eﬃciency at 0.38. These results are in the same range as reported

previously by Astex,^30,31who have also disclosed the binding

modes for a number of aminoheterocyclic BACE1 inhibitors,

including parent 2-aminoquinoline 1 with BACE1 (PDB ac-

The 6-substituted compounds in Table 6 were prepared from

the intermediate 24 as shown in Scheme 8. Hydrolysis of the

ester, then amide coupling with 3,3-dimethylbutanamine and

hydrogenation of the oleﬁn, provided 26. Suzuki coupling of 26

with various boronic acids and removal of the tert-butyl or

4-methoxybenzyl group of the 2-aminoquinoline gave 60ꢀ62.

Alternatively, the bromide of 30 could be converted to the boronic

ester 27 via palladium cross-coupling with bis-pinacol borane.

Suzuki coupling of 27 with aryl halides, followed by removal of

the 2-amino protecting group, aﬀorded 58 and 59.

’ RESULTS AND DISCUSSION

Fragment Screening and Subsequent SAR Development

Using Protein X-ray Crystallography. The fragment library was

designed to contain small, soluble, low-molecular-weight com-

pounds that were selected by applying physicochemical filters,

such as polar surface area (PSA) < 30 Å², the number of

hydrogen bonding donors e2, and molecular weight <300.

The library also included small heterocyclic compounds posses-

sing functional groups which could form one or more polar or

charged interactions with the two catalytic Asp residues in

the BACE active site. Of particular interest were the known

cession code: 2OHL). In this structure, a CꢀH

π interac-

3 3 3

tion (3.9ꢀ4.5 Å) is observed between the Tyr71 residue on the

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Table 1. Activity of 2-Aminoquinoline and Bromo-Substituted 2-Aminoquinolines

^aLigand eﬃciency is deﬁned as free energy of binding per heavy atom and calculated based on K_dfrom SPR (LE = ΔG/N_{non-hydrogen atoms}).

Table 2. Activity of 6-Substituted 2-Aminoquinolines

which complements the hydrogen bonding interactions be-

tween the basic N1 and the 2-amino group with aspartic acid

residues 32 and 228, respectively (Figure 2). On the basis of the

observed tight interaction between the carbon atom at the

6-position and the π face of Tyr71 in the binding of 1 with

BACE, as well as its perpendicular trajectory with respect to the

location of the BACE aﬃnity pockets (e.g., P1, P1⁰, P2⁰), it

would appear that substitution in the 6-position may not be

well-tolerated as an access point to these various BACE sub-

sites, which has been noted by others.³⁰Nonetheless, we

decided to examine the eﬀect of adding a hydrophobic group

at the various positions of the 2-aminoquinoline core using a

bromine atom as shown in Table 1. Encouragingly, it was found

that all of the bromo-substituted 2-aminoquinolines (28ꢀ31)

displayed improved potency about 2ꢀ4 fold, with 29, 30, and

31 demonstrating higher ligand eﬃciency (0.41, 0.41, and 0.42

respectively) than parent 2-aminoquinoline 1. Notably, the

improved potency observed for 30 was striking in light of the

existing cocrystal structure of 1. We therefore decided to further

investigated substitution at the 6-position with aryl groups

(Table 2). Attachment of a thiophene (32) or phenyl (33)

group at the 6-position resulted in a greater than 20-fold

improvement in binding aﬃnity. Compound 32 was cocrystal-

lized with BACE enzyme and the superimposition of the

cocrystal structures of 32 and the literature structure of 1 is

shown in Figure 3^a.

Although both the protein backbone and the majority of the

side chains in the region of the active site remain highly

conserved, of note was the signiﬁcant conformational change

of Tyr71. This conformational change moves the Tyr71 side

chain from the rear of the aminquinoline core to a position above

^aLigand eﬃciency is calculated based on K_dfrom SPR.

it, resulting in CꢀH π contacts between the face of the Tyr71

3 3 3

ﬂap region of BACE1 and the 6-position of the 2-aminoquino-

line core. This interaction is likely a signiﬁcant driver of potency

side chain and the 4- and 5- positions of the inhibitor. Moreover, the

thiophene substituent at the 6-position engages in a CꢀH

π

3 3 3

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Table 3. Activity of 3-Substituted and 3,6-Substituted 2-Aminoquinolines

^aIC₅₀values are the means of at least two experiments. ^bND = not determined. ^cLigand eﬃciency is calculated based on K_dfrom SPR. ^dLigand eﬃciency

is calculated based on IC₅₀from the FRET assay.

Table 4. r-Substituted Amide Aminoquinolines

Figure 4. Co-crystal structure of 39 (light-blue with surface shown) in

BACE1 (green with beige surface; ﬂap residues shown in magenta)

depicting occupancy of the P1 subsite by the tert-butyl alkyne of 39.

Distances are reported in Å.

phenyl ring (37) was not tolerated. Compound 38, with a

cyclohexyl group at the 6-position showed a 13-fold loss in

^aIC₅₀values are the means of at least two experiments. ^bRacemic.

potency, underlining the importance of the edgeꢀface interac-

tion between Phe108 and the face of the 6-aryl group as a

signiﬁcant source of binding aﬃnity.

interaction with the edge of Phe108. The overall binding motif is

reminiscent of that of a dihydroquinazoline class of BACE1

inhibitors previously reported in the literature.³⁰

Substitution at both ortho (34 and 35) and meta (36) position

of the C-6 aryl ring enhanced the potency by about 2-fold while

retaining good ligand eﬃciency, whereas para substitution of the

The ortho position of the 6-phenyl group appeared to furnish

an access point into the P1 aﬃnity pocket. Analogues such as the

tert-butyl alkyne derivative 39 were synthesized to explore this

area, with the predicted binding in the P1 veriﬁed by crystal-

lography (Figure 4). Although 39 displayed a 3.7-fold increase in

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Table 5. 3-Substituted Amide 2-Aminoquinolines

^aIC₅₀values are the means of at least two experiments. ^bRacemic. ^c(R)-isomer. ^d(R)-isomer at the pyran ring determined by X-ray crystal structure.

^eEﬄux ratio in LLC-PK cells transfected with rat MDR1. ^fcLogP values are calculated by Daylight Toolkit 4.81.

spacer of seemingly ideal length and possessing a minimal

number of rotatable bonds.

The P1 pocket is comprised in part by aromatic residues

Phe108 and Trp115, and thus aromatic rather than purely

aliphatic hydrophobic substitution in P1 might be expected to

furnish some additional potency. However, it was projected that

such potency gains in P1 might not be commensurate with the

molecular weight and hydrophobicity expense necessary to

access it. We therefore sought to identify other opportunities

to access the various aﬃnity pockets of BACE1 from other

positions on the 2-aminoquinoline core, which might increase

binding aﬃnity in a more ligand eﬃcient manner.

Akin to a previously reported class of dihydroquinazoline

inhibitors¹⁸found to access the P1 aﬃnity site, amide substitution

at the 3-position of the aminoquinoline core was pursued.

Compound 40 was prepared and cocrystallized with the BACE1

enzyme (Table 3). The crystal structure demonstrated that the

side chain conformation at the 3-position was similar to that

observed in the literature, with preferential occupation of the P1

pocket by the cyclohexyl substituent of the N,N-disubstituted

amide (Figure 5). In the absence of a 6-substituent, the conformation

of overhead Tyr71 in the structure of 40 reverted to that

observed in the structure of the parent aminoquinoline, re-

Figure 5. Co-crystal structure of 40 (light-blue) in BACE1 (green with

beige surface; ﬂap residues in transparent magenta) depicting occupancy

of the P1 subsite by the N-cyclohexyl substituent.

potency compared to the 6-phenyl substituted parent compound

33, and modest 2-fold improvement over the much smaller

methyl and chloro-substituted analogues 34 and 35, it rendered

an overall decrease in LE compared to the latter two analogues.

This decrease in LE occurred despite the introduction of the

highly shape-complementary tert-butyl hydrophobic group in the

roughly spherical P1 aﬃnity pocket along with the utilization of a

engaging the 5- and 6-positions in a CꢀH π interaction

3 3 3

(cf. 4- and 5-position in 7, Figure 3).

The potency of 40 via SPR measurement was 38.4 μM, 23-fold

more potent than parent 2-aminoquinoline 1. Addition of the

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Table 6. C-6 Substituted 3,3-Dimethylbutyl Amide 2-Aminoquinolines

^aIC₅₀values are the means of at least two experiments. ^bRacemic. ^c(R)-isomer. ^dEﬄux ratio in LLC-PK cells transfected with rat MDR1. ^ecLogP values

are calculated by Daylight Toolkit 4.81.

(1 and 34), their LE were signiﬁcantly less than that of both 34

and parent 2-aminoquinoline 1 (0.26 vs 0.38 and 0.24 vs 0.38,

respectively). This suggested that occupancy of P1 pocket via a

tethered, N,N-disubstituted amide side chain might not lead to

high-eﬃciency binding to the BACE1 protein. Theobserved gains

in potency were, in our view, insuﬃcient to oﬀset the increase in

molecular weight necessary for P1 occupancy.

However, removal of the N-methyl group on the pendant

amide function, coupled with an o-tolyl substituent at the 6-posi-

tion (42), resulted in an astonishing increase in potency, with an

IC₅₀value of 0.074 μM. This represented a 123-fold improve-

ment over 34 and approaching 10500-fold over the initial

fragment hit 1. The cocrystal structure of 42 with the BACE

enzyme revealed that the 3-amide chain was no longer oriented

toward the P1 side of the active site. Instead, the N-cyclohexyl

occupies the P2⁰region, with the NH of the pendant amide

forming a hydrogen bonding interaction with the carbonyl

Figure 6. Co-crystal structure of 42 (white) in BACE1 (cutaway surface

oxygen of Gly34 on the underside of the active site (Figure 6).

view), depicting P2⁰subsite binding of the N-cyclohexyl moiety. Flap

This contact is augmented by an interaction between the

residues (surface not shown) in transparent magenta, active site residues

amide π face and the edge of nearby Tyr198. The o-methyl

in green, and BACE1 active site surface colored by cavity depth.

group of the 6-tolyl function is oriented toward the P1 site. In

Occupation of P1 by a glycerol molecule (from cryoprotectant in

addition, the structure also revealed the P2⁰site was not fully

crystallography sample) is depicted in orange.

occupied by the cyclohexyl amide group, indicating that an

amide group with longer alkyl chain might extend further into

optimal 6-tolyl group gave rise to 41 (ca. 4 μM), 9-fold more

potent than 40 and 2-fold improved over 34. At this point, the

observed potencies were entering a range (<10 μM) where it was

possible to accurately compare and verify SPR measurements via

our standard enzymatic assay, which were found to be consistent

for 41 (4.2 and 5.2 μM, respectively).

the P2⁰pocket, allowing for higher binding aﬃnity. In light of

this observation, we added a simple methylene linker and

prepared cyclohexylmethyl amide analogue 43. Indeed, 43

exhibited an IC₅₀value of 34 nM in the enzymatic assay, a

2-fold improvement over 42.

The presence of the 6-tolyl substitution appears to direct the

3-amide group away from the nonprime region and into the P1⁰/

P2⁰aﬃnity sites, likely due in part to the presence of the o-methyl

Although the potency of 40 and 41 were improved versus their

respective parent compounds which lacked the 3-side chain

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Figure 8. Co-crystal structure of 50 (cyan) in BACE1 (cutaway surface

in beige; active site residues in green, ﬂap residues in magenta) depicting

occupancy of P1⁰and P2⁰.

Figure 7. Co-crystal structure of 44 (cyan and yellow) in BACE1

(cutaway surface in beige; active site residues in green, ﬂap residues in

magenta) depicting dual binding conformations occupying both the P1

and P2⁰aﬃnity pockets. Superimposed (based on crystal structure

alignment) is 42 from Figure 6 (transparent white). Distances are

reported in Å.

analogue 43. The (R)-isomer 46 was preferred to the (S)-isomer

47 (2.5 nM vs 970 nM in the FRET assay). Larger

R-amide groups 48, 49, and 50 (all racemic), however, afforded

less potent analogues versus R-methyl analogue 45 (racemic),

suggesting that the (R)-isomer of R-methyl substituent was

optimal. Successful cocrystallization of R-propyl analogue 50 as

its racemic mixture confirmed the occupation of the P1⁰and P2⁰

subsites by the (R)-enantiomer (Figure 8).

group oriented toward P1. Interestingly, compound 44, the

direct des-tolyl analogue of 43, cocrystallizes in BACE1 in both

prime- and nonprime-site binding conformations, as seen clearly

from the electron density (Figure 7). In each of the two, nearly

Cs-symmetric binding conformations, the amide NH from the

inhibitor engages the CdO of a glycine residue on the underside

of the active site (Gly34 and Gly230), orienting the cyclohex-

ylmethyl substituent into both the P1 and P2⁰subsites. For

6-tolyl analogue 43, nonprime-site binding of this hydrophobic

group would likely not be possible due to an intramolecular steric

clash (ca. 2.4ꢀ2.8 Å) illustrated by the alignment of the crystal-

lographic poses of 44 and 42 (Figure 7).

With two hydrogen bond donors and molecular weight below

400, the lead compound 42 displayed good BACE1 potency with

high LE at 0.34. This compound was evaluated in the cell-based

Aβ production assay (Aβ40) and showed moderate activity with

an IC₅₀= 0.71 μM. Additionally, we employed an in vitro

permeability assay using LLC-PK cell line transfected with human

or rat Pgp to estimate passive bloodꢀbrain barrier permeability

and eﬄux susceptibility.³⁴For CNS penetrable compounds, the

targeted in vitro passive permeability is >100 nm/s with an eﬄux

ratio (ER) less than 3.^9a,35The LLC-PK assay showed that 42 had

modest permeability (66 nm/s) and low ER at 0.6. Overall, the

results indicated that compound 42 represented a promising

proﬁle as a lead compound for further development into CNS

penetrable BACE1 inhibitors. We therefore carried out SAR

studies on this compound, particularly focused on improving cell

potency and permeability.

Optimization of 2-Aminoquinoline Lead Compound 42:

Conformational Stabilization and P1⁰Access. Analysis of the

binding of 42 with BACE1 protein revealed that an R-alkyl

substituent in the (R)-configuration (anti with respect to the

quinoline core) should enhance binding affinity via enrichment

of the binding (amide-gauche) conformation by destabilization of

other rotamers about the C_benzylꢀC_Rσ bond. Such a substituent

would also be ideally poised to occupy the previously empty

P1⁰pocket (Figure 6). To investigate this strategy, a series of

R-substituted amide analogues of 43 were prepared (Table 4). A

simple R-methyl group (45) showed single-digit nanomolar po-

tency at 3.7 nM, which is nearly a 10-fold increase over des-methyl

Having secured the optimal R-amide substitution, attention

shifted to exploring the P2⁰region. This region of the molecule

appeared to be fairly accommodating, accepting alkyl groups as

well as saturated and unsaturated ring systems. The results for the

amide analogues are provided in Table 5. Compound 51 was

designed to have a longer alkyl chain, and the 3,3-dimethylbu-

tanyl group can position deeper into the P2⁰pocket. This

compound achieved subnanomolar BACE1 potency at 0.65

nM in the enzymatic assay. The benzyl group of 52 was also

tolerated and showed potency at 20 nM in the enzymatic assay.

However, both compound 51 and 52 showed large cell shifts

(380-fold and 260-fold, respectively) and modest permeability

(65 and 65 nm/s, respectively), most likely due to the high cLogP

(5.6 and 5.5, respectively) of these two compounds. In an

attempt to improve permeability and reduce cell shift, polarity

was introduced through heteroaryl derivatives such as pyridin-3-

ylmethanamide 53, thiazol-5-ylmethanamide 54, and pyrazin-2-

ylmethanamide 55, each having lower cLogP at 3.5, 3.3, and 2.5,

respectively. Although these heteroaryl analogues showed com-

parable or slightly decreased enzyme activity (53 and 54 are

racemic) as compared to the benzylamide 52, the above-men-

tioned analogues demonstrated much improved cell shift versus

52 (6.4ꢀ14-fold vs 260-fold) as well as improved permeability

(160, 120, and 200 nm/s respectively). Unfortunately, these

heteroaryl compounds all showed elevated ER in both rat and

human transfected Pgp cell lines (ER data in rat shown in

Table 5). We also introduced a heteroatom to saturated ring

systems (56 and 57). 56 containing a tetrahydropyran group that

had cLogP of 3.2, displayed good potency in the enzymatic assay

at 10 nM and cell potency of 0.17 μM with relatively low cell shift

(17-fold). This compound also had good permeability (120 nm/

s), but the ER was high at 25. To modulate the polarity and

reduce Pgp-mediated eﬄux, a gem-dimethyl group was intro-

duced next to the oxygen atom on the pyran ring in 57. This

compound possessed cLogP of 3.6 and demonstrated excellent

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Figure 10. Aβ40 reduction of 59 in CSF observed in the rat PD model.

A known γ-secretase inhibitor, (S)-2,3-dimethyl-N-((S)-1-(((S)-5-

methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)amino)-1-oxo-

propan-2-yl)butanamide, was used as a positive control.³⁶

Compound 59 had desired overall properties in cell potency,

permeability, and ER. Dosed at 60 mg/kg subcutaneously,

compound 59 was evaluated in the rat PD model for lowering

of Aβ40/42 measured in the CSF. Evaluated at the 2 h time

point, 59 showed a signiﬁcant 42% reduction of CSF Aβ40/42

formation after a single dose (p < 0.0001) (Figure 10). This result

demonstrated that with desired properties, the 2-aminoquinoline

BACE1 inhibitor 59 was able to aﬀord good CNS penetration

and produce signiﬁcant in vivo eﬃcacy in Aβ reduction. Un-

fortunately, compound 59 displayed high human and rat micro-

somal clearance. Eﬀorts to improve metabolic stability of this

class of compounds will be the focus of future eﬀorts.

Figure 9. Co-crystal structure of 57 (white) in BACE1 (cutaway surface

view), depicting P1⁰and P2⁰subsite binding of the R-methyl and N-

dimethylpyranyl groups, respectively. Flap residues (surface not shown)

in transparent magenta, active site residues in green, and BACE1 active

site surface colored by cavity depth. Occupation of P1 by a glycerol molecule

(from cryoprotectant in crystallography sample) is depicted in orange.

potency in the enzymatic assay at 0.7 nM and good cell potency

at 18 nM. The permeability was moderate at 89 nm/s, and the ER

was notably decreased compared to 56 (5.8 vs 25). These results

and subsequent investigation on the relationship of cLogP with

permeability and cell shift of aminoquinoline compounds sug-

gested that it is essential to design compounds with cLogP values

below 5 in order to achieve good permeability as well as low cell

shift. Compound 57 was crystallized in BACE1 (Figure 9) as its

single (R) enantiomer at the P1⁰occupying, R-methyl position.

The single conﬁguration at the pyran substituent, while not

conﬁrmed via synthesis, was determined from the electron

density to be (R).

In the eﬀort to optimize the physical properties, a number of

polar substitution groups at 6-position were also investigated.

The 3,3-dimethylbutanyl amide group that oﬀered the best

potency was used at the P2⁰region. The SAR of 6-substitutions

revealed that the pyridin-2-yl group is tolerated (58ꢀ61,

Table 6). The cell shift was only 2-fold with the 3-methyl

pyridine-2-yl analogue 58, having an IC₅₀of 16 nM in the

enzymatic assay and 35 nM in the cell assay. This compound

also had good permeability at 150 nm/s, however, the ER was

7.8. The 3-chloro pyridin-2-yl analogue 59 exhibited potency of

11 nM in the enzymatic assay and 80 nM in the cell assay. The

permeability was 120 nm/s with a desirable ER of 3.1. The

3-methoxy and 3-cyano pyridine-2-yl analogues 60 and 61

showed less potency in the enzymatic and the cell assay, but

both of these compounds had good permeability (130 and

150 nm/s, respectively). The o-methylketone phenyl analogue

62 also showed good potency in the enzymatic assay (15 nM) as

well as in the cell assay (110 nM), demonstrating good perme-

ability (120 nm/s) and low ER at 1.8. The cLogP is lower than 5

for all of these compounds (58ꢀ62) that exhibited low cell shift

(<10) and good permeability (>100 nm/s). This result indicated

that we could improve cell potency as well as achieve good

permeability by modiﬁcation of the physical properties.

’ CONCLUSION

Using SPR to screen a focused fragment library, 2-aminoqui-

noline 1 was identiﬁed as an initial fragment hit that displayed

potency in the millimolar range. A rapid optimization of the

2-aminoquinoline hit guided by LE and supported by X-ray

crystal structures led to the identiﬁcation of a lead compound 42

with a potency of 74 nM for BACE1, reﬂecting a 12000-fold

increase in potency over the starting fragment hit while preser-

ving ligand eﬃciency. Further SAR investigation delivered potent

BACE inhibitors in the subnanomolar range with over 10⁶-fold

increase in potency. Subsequently, modiﬁcation of the physical

properties of these 2-aminoquinoline analogues led to further

improvements including decreasing the enzyme/cell shift,

improving permeability, and lowering ER, culminating in potent

BACE1 inhibitors with desirable properties for CNS penetration.

Compound 59 exempliﬁed these improvements displaying sig-

niﬁcant Aβ reduction in CSF in a rat PD model.

’ EXPERIMENTAL SECTION

SPR Assay. Dissociation constant (K_d) measurements were per-

formed on Biacore S51 and T100 instruments (GE Healthcare). BACE1

protein and inhibitors for Biacore measurements were generated in-

house; all other reagents were purchased from GE Healthcare or Sigma-

Aldrich. Glycosylated BACE1 was reacted with sodium periodate to

oxidize cis-diols groups on sugar chains to aldehydes. The oxidized

BACE was immobilized at high density (10000ꢀ12000 RU) onto CM5

chips using aldehyde coupling chemistry and resulted in surface activities

close to 100% based on binding of a reference inhibitor, N-((2S,3S,5R)-

6-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-3-hydroxy-5-methyl-6-

oxo-1-phenylhexan-2-yl)-3-(2-oxopyrrolidin-1-yl)benzamide. The im-

mobilization running buffer consisted of 10 mM HEPES pH 7.4 with

To elucidate the value of these aminoquinolines in reducing

Aβ in vivo, we chose 59 for further investigation in a rat model.

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150 mM NaCl, and immobilization steps consisted of a 3ꢀ4 min

EDC/NHS activation step[200 mM 1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride, 50 mM N-hydroxysuccinimide], 7 min

5 mM carbohydrizide in water, 7 min 1 M ethanolamine hydrochloride

pH 8.5, 10 min 20 μg/mL oxidized BACE in 10 mM sodium acetate pH

4.0, 7 min 100 mM sodium cyanoborohydride in 100 mM sodium

acetate pH 4.0, 3 ꢁ 30 s 50 mM glycine pH 9.5, and 3 ꢁ 30s 1 M NaCl in

100 mM NaHCO₃pH 9.5.

For K_dmeasurements the buﬀer was replaced with 50 mM sodium

acetate pH 5.0, 150 mM NaCl, 0.005 (v/v) Tween20, and 5% (v/v)

DMSO. Compound stocks prepared in DMSO (typically 20 mM) were

serially diluted in running buﬀer and injected over the immobilized

BACE1. Association and dissociation time were typically set to 45 s and

90 s, respectively. The runs were performed at 25 ꢀC with a ﬂow rate

from 10 to 90 μL/min and data collection rate of 10 Hz.

The data was processed and analyzed using Scrubber-2 analysis

software (BioLogic Software, Campbell, Australia). The sample re-

sponse observed on the reference spot was subtracted from the sample

response with immobilized BACE1 to correct for systematic noise and

baseline drift. Data was solvent corrected and the response from blank

injections was used to double-reference the binding data. The data was

molecular weight normalized and K_dvalues established using simple

steady analysis with one global R_max(or individual R_maxin cases were

complete saturation was achieved).

sandwich ELISA to detect Aβ40 was performed in 96-well microtiter

plates, which were precoated with goat antirabbit IgG (Pierce).

The capture and detection antibody pair that was used to detect Aβ40

from cell supernatants consists of affinity purified pAβ40 (Invitrogen)

and biotinylated 6E10 (Covance), respectively. Conditioned media was

incubated with capture antibody overnight at 4 ꢀC, followed by washing.

The detecting antibody incubation was for 3 h at 4 ꢀC, again followed by

the wash steps as described previously. The plate was developed using

Delfia reagents (Streptavidin-Europium and Enhancement solution

(Perkin-Elmer)) and time-resolved fluorescence was measured on an

EnVision multilabel plate reader (Perkin-Elmer).

Permeability Assay. The wild-type cell line LLC-PK1 (porcine

renal epithelial cells, WT-LLC-PK1) was purchased from American

Type Culture Collection (ATCC, Manassass, VA). Transfections of

WT-LLC-PK1 cells with human MDR1 gene (hMDR1-LLC-PK1) and

rat mdr1a gene (rMdr1a-LLC-PK1) were generated. Cells were grown

in Medium 199 supplemented with 10% fetal bovine serum.³⁹Cells were

seeded onto matrigel-coated transwell filter membranes at a density of

90000 cells/well. Media change was performed on day 3. Compound

incubations were performed 5ꢀ6 days post seeding. All cultures were

incubated at 37 ꢀC in a humidified (95% relative humidity) atmosphere

of 5% CO₂/95% air.

Prior to the transport experiment,⁴⁰culture medium was aspirated

from both apical and basolateral wells, and cells were rinsed with warmed

(37 ꢀC) Hank’s balanced salt solution supplemented with 10 mM Hepes

at pH 7.4 (HHBSS, Invitrogen, Grand Island, NY). HHBSS was

removed from wells prior to dosing with test drugs at 5 μM in transport

buﬀer (HHBSS containing 0.1% bovine serum albumin). Then 150 μL

of transport buﬀer were added to receiver chambers prior to dosing in

triplicate to apical or basolateral chambers. The dosed transwell plates

containing the cell monolayers were incubated for 2 h at 37 ꢀC on a

shaking platform. At the end of the incubation period, 100 μL samples

were collected from receiver reservoirs and analyzed by LC-MS/MS on

an API4000 (Applied Biosystem, Foster City, CA) triple quadruple mass

spectrometer interfaced with turbo IonSpray operated in positive mode

using Analyst 1.4.2 software.

BACE1 ¹⁹F NMR Enzymatic Assay. An NMR-based enzymatic

assay was designed using a ¹⁹F-labeled BACE1 substrate peptide with the

sequence EVNLDAEF(CF₃). BACE1 cleaves this peptide between the L

and D residues, which resulted in distinct ¹⁹F NMR signals for the

substrate and product. Integration of the signal area yielded concentra-

tions of each component and allowed calculation of K_i. The assay was

conducted in 96-well plate format using 200 nM of BACE1 reacted

with 100 μM of the substrate peptide. Compounds of interest were

added at various concentrations and were followed with a blank DMSO

control well as well as a control inhibitor with a K_iof 400 nM. The

experiment was conducted in 25 mM sodium acetate, pH 5.0, and

allowed to react for a period of 20 min, at which time the reaction was

quenched by the addition of 300 μL of 8 M urea (20% D₂O for NMR

frequency lock). All experiments were conducted on a Bruker Avance III

500 MHz NMR spectrometer with an SEF cryoprobe for ¹⁹F direct

detection.

BACE1 Enzymatic Assay. BACE1 enzymatic activity was deter-

mined by the enhancement of fluorescence intensity upon enzymatic

cleavage of the fluorescence resonance energy transfer substrate. The

BACE recognition and cleavage sequence of the substrate is derived

from the reported literature,³⁷and the fluorophore and quencher dyes

are attached to side chain of Lys residues at the termini of the substrate

peptide. The human recombinant BACE1³⁸assay was performed in

50 mM acetate, pH 4.5/8% DMSO/100 μM Genepol/0.002% Brij-35.

In doseꢀresponse IC₅₀assays, 10 point 1:3 serial dilutions of compound

in DMSO were preincubated with the enzyme for 60 min at room

temperature. Subsequently, the substrate was added to initiate the

reaction. After 60 min at room temperature, the reaction was stopped

by addition of 0.1 M Tris base to raise the pH above the enzyme active

range, and the increase of fluorescence intensity was measured on Safire

II microplate reader (Tecan, M€annedorf, Switzerland).

The apparent permeability coeﬃcient (P_app) of all tested agents was

estimated from the slope of a plot of cumulative amount of the agent

versus time based on the following equation:

P_app¼ ðdQ=dtÞ=ðA C₀Þ

3

where dQ/dt is the penetration rate of the agent (ng/s), A is the surface

area of the cell layer on the Transwell (0.11 cm²), and C₀is the initial

concentration of the test compound (ng/mL). Eﬄux ratio (ER) was

calculated from the basolateral-to-apical permeability divided by the

apical-to-basolateral permeability: ER = P_appB > A/P_appA > B.

X-ray Crystal Structure. Recombinant human BACE1 residues

14ꢀ453 was overexpressed in bacteria as inclusion bodies and refolded

using a procedure described by Patel et al.⁴¹Crystals were grown in

similar conditions described in Patel et al.⁴¹BACE1 protein was

concentrated to 8 mg/mL in a buffer containing 20 mM Tris (pH

8.2), 150 mM NaCl, and 1 mM DTT. DMSO (3% v/v) was added to the

protein immediately prior to crystallization. Apo crystals of BACE1 were

grown at 20 ꢀC using the hanging drop method/vapor diffusion method,

the drops containing 1 μL of BACE1 solution and 1 μL of reservoir

solution. The reservoir solution consisted of 20% (w/v) PEG 5000

monomethylethyl ether (MME), 200 mM sodium citrate (pH 6.6), and

200 mM sodium iodide. Apo BACE1 crystal was soaked overnight in a

5.0 mM compound solution with 33% (w/v) PEG 5000 monomethyl-

ethyl ether (MME), 110 mM sodium citrate, 220 mM sodium iodide,

and 10% DMSO (from compound dilution). The following day, the

crystal was briefly transferred to a cryoprotectant (5.0 mM compound

with 33% (w/v) PEG 5000 monomethylethyl ether (MME), 110 mM

sodium citrate, 220 mM sodium iodide, 10% DMSO and 22% glycerol)

Cell-Based Assay. Human embryonic kidney cells (HEK293)

stably expressing APP_SWwere plated at a density of 100K cells/well in

96-well plates (Costar). The cells were cultivated for 6 h at 37 ꢀC and 5%

CO₂in DMEM supplemented with 10% FBS. Cells were incubated

overnight with test compounds at concentrations ranging from 0.0005 to

10 μM. Following incubation with the test compounds the conditioned

media was collected and the Aβ40 levels were determined using a

sandwich ELISA. The IC₅₀was calculated from the percent of control

Aβ40 as a function of the concentration of the test compound. The

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and flash frozen in liquid nitrogen. Data was collected at Advanced

Light Source Beamline 5.0.2 (Lawrence Berkeley National Laboratory,

Berkeley, CA) at 100 K. Data was processed and scaled using

HKL2000.⁴²The crystals belong to the space group P6₁22 with approxi-

mate unit cell dimensions of a = b = 103 Å, c = 169 Å. Molecular replace-

ment was performed using Phaser,⁴³using an apo BACE-1 structure

(PDB entry 1w50) as a search model. The structure was refined using

Refmac 5,⁴⁴and the model with ligand was built with Coot.⁴⁵

to cool to room temperature and then evaporated to dryness under

reduced pressure. Then ice water was added to the resulting residue

followed by dichloromethane (80 mL). This mixture was stirred, and

potassium carbonate was added carefully until the mixture had been

neutralized to pH 7. The organic phase was separated and dried with

magnesium sulfate, filtered, and concentrated to dryness under reduced

pressure. This crude material was dissolved in a mixture of dry ethanol

(12 mL) and toluene (4 mL) with heating and was transferred to a

microwave vessel. 4-Methoxybenzylamine (1.6 mL, 12 mmol) was

added, and the vial was sealed and heated to 155 ꢀC for 45 min. Then

the reaction was evaporated to dryness under reduced pressure and

purified by column chromatography on silica gel (eluting with a gradient

of 20ꢀ100% ethyl acetate in hexane) to give N-(4-methoxybenzyl)-8-

bromoquinolin-2-amine. This material was dissolved in trifluoroacetic

acid (20 mL) and heated to 60 ꢀC for 90 min and then evaporated to

dryness under reduced pressure. The residue was purified by column

chromatography on silica gel (eluding with a gradient of 50ꢀ100% ethyl

acetate in hexane) to give the 8-bromoquinolin-2-amine (0.102 g, 3.2%

yield over 3 steps) as a brown solid. ¹H NMR (400 MHz, methanol-d₄) δ

8.37 (d, J = 9.4 Hz, 1H), 8.09 (dd, J = 7.8, 1.2 Hz, 1H), 7.93 (dd, J = 7.9,

1.1 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.16 (d, J = 9.4 Hz, 1H), 4.8 (br s,

2H). MS (ESI, positive ion) m/z: 223 (M + 1).

Pharmacodynamic Assay. Male SpragueꢀDawley rats (175ꢀ

200 g) were purchased from Harlan and were maintained on a 12 h light/

dark cycle with unrestricted access to food and water until use. Rats were

dosed subcutaneously with 59 at 20 and 60 mg/kg in 15% HPBCD,

pH 4. Rats were euthanized with CO₂inhalation for 2 min, and cisterna

magna was quickly exposed by removing the skin and muscle above it.

CSF (50ꢀ100 μL) was collected with a 30 gauge needle through the

dura membrane covering the cisterna magna. Blood was withdrawn

by cardiac puncture and plasma obtained by centrifugation for drug

exposures. Brains were removed and, along with the CSF, immediately

frozen on dry ice and stored at ꢀ80 ꢀC until use. The frozen brains were

subsequently homogenized in 10 volumes of (w/v) of 0.5% Triton

X-100 in TBS with protease inhibitors. The homogenates were cen-

trifuged at 100000 rpm for 30 min at 4 ꢀC. The supernatants were

analyzed for Aβ40 levels by immunoassay as follows: Meso Scale 96-well

avidin plates were coated with Biotin-4G8 (Covance) and detected with

ruthenium-labeled Fab specific for Aβ40. Plates were read in MSD

Sector6000 imager according to manufacturer’s recommended protocol

(Meso Scale Discovery, Inc.). Aβ40 concentrations were plotted

using Graphpad Prism and analyzed by one-way ANOVA followed

by Dunnett’s multiple comparison analysis to compare drug-treated

animals to vehicle-treated controls.

7-Bromoquinolin-2-amine (29). Using 2-aminoquinolin-7-ol

1

and following the procedure used to prepare 28 gave 29. H NMR

(400 MHz, chloroform-d) δ 7.81ꢀ7.88 (m, 2H), 7.47 (d, J = 8.4 Hz,

1H), 7.35 (dd, J = 8.5, 1.9 Hz, 1H), 6.72 (d, J = 8.8 Hz, 1H, 5.27 (br s,

2H). MS (ESI, positive ion) m/z: 223 (M + 1).

6-Bromoquinolin-2-amine (30). Using 2-aminoquinolin-6-ol

1

and following the procedure used to prepare 28 gave 30. H NMR

(400 MHz, chloroform-d) δ 7.72ꢀ7.78 (m, 2H), 7.60 (dd, J = 8.8, 2.5

Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H), 5.1 (br s, 2H).

MS (ESI, positive ion) m/z: 223 (M + 1).

Chemistry. Unless otherwise noted, all materials were obtained

from commercial suppliers and used without further purification.

Anhydrous solvents were obtained from Aldrich or EM Science and

used directly. All reactions involving air- or moisture-sensitive reagents

were performed under a nitrogen or argon atmosphere. All microwave

assisted reactions were conducted with a Smith synthesizer from

Personal Chemistry, Uppsala, Sweden. Silica gel chromatography was

performed using either glass columns packed with silica gel (230ꢀ400

mesh, EMD Chemicals, Gibbstown, NJ) or prepacked silica gel car-

5-Bromoquinolin-2-amine (31). Using 2-aminoquinolin-5-ol

1

and following the procedure used to prepare 28 gave 31. H NMR

(400 MHz, chloroform-d) δ 8.28 (d, J = 9.0 Hz, 1H), 7.63 (d, J = 8.1 Hz,

1H), 7.53 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 6.80 (d, J = 9.0 Hz,

1H), 5.14 (br s, 2H). MS (ESI, positive ion) m/z: 223 (M + 1).

6-(Thiophen-3-yl)quinolin-2-amine (32). A mixture of thio-

phen-3-ylboronic acid (0.25 mmol, 32 mg), 6-bromo-2-aminoquinoline

(30, 50 mg, 0.22 mmol), K₂CO₃(3 M aq, 100 μL, 0.3 mmol), PS-PPh₃-

Pd (0.11 mmol/g, 2.2 μmol, 20 mg), and dimethoxyethane/EtOH

(50%, 1 mL) was heated in microwave at 140 ꢀC for 10 min. The

resulting mixture was cooled, filtered, and purified by HPLC (10ꢀ60%

CH₃CN/water modified with 0.1% TFA) to give 6-(thiophen-3-

yl)quinolin-2-amine (0.012 g, 24% yield). ¹H NMR (400 MHz, MeOH)

δ 8.39 (d, J = 9.5 Hz, 1H), 8.18 (d, J = 2.0 Hz, 1H), 8.14 (dd, J = 8.8,

2.0 Hz, 1H), 7.80ꢀ7.84 (m, 1H), 7.70 (d, J = 8.8 Hz, 1H), 1H,

7.55ꢀ7.61 (m, 2H), 7.09 (d, J = 9.5 Hz, 1H). MS (ESI, positive ion)

m/z: 227 (M + 1).

1

tridges (Biotage or ISCO). H NMR spectra were determined with a

Bruker 300 MHz or DRX 400 MHz spectrometer. Chemical shifts are

reported in parts per million (ppm, δ units) downfield from tetra-

methylsilane. All tested compounds were purified to g95% purity as

determined by reverse phase HPLC. HPLC analysis was obtained on

Agilent 1100, using one or two of the following three methods. HPLC

method A (3.6 min LC-MS run): Zorbax analytical C18 column (50 mm

ꢁ 3 mm, 3.5 μm, 40 ꢀC); mobile phase, A = 0.1% TFA in water, B = 0.1%

TFA in acetonitrile; gradient, 0.0ꢀ3.6 min, 5ꢀ95% B; flow rate =

1.5 mL/min; 254 nm; 0 min post time; 1.0 μL injection. HPLC method

B (10 min LC-MS run): YMC ODS-AM (100 mm ꢁ 2.1 mm, 5 μm,

40 ꢀC); mobile phase, A = 0.1% HCOOH in water, B = 0.1% HCOOH

in acetonitrile; gradient, 0.0ꢀ0.5 min, 10% B; 0.5ꢀ7.0, 10ꢀ100% B;

7.0ꢀ10 min, 100% B; flow rate = 0.5 mL/min; 254 nm; 1.5 min post

time; 3.0 μL injection. HPLC method C (5 min LC-MS Run):

Phenomenex Synergi MAX-RP (50 mm ꢁ 2.0 mm, 4.0 mm, 40 ꢀC);

mobile phase, A = 0.1%TFA in water, B = 0.1% TFA in acetonitrile;

gradient, 0.0ꢀ0.2 min, 10% B, 0.2ꢀ3.0 min, 10ꢀ100% B, 3.0ꢀ4.5 min

100% B, 4.5ꢀ5.0 min, 100ꢀ10% B; flow rate = 0.8 mL/min; 254 nm; 1.5

min post time; 3.0 μL injection. Low-resolution mass spectral (MS) data

were determined on a Perkin-Elmer-SCIEX API 165 mass spectrometer

using ES ionization modes (positive or negative).

6-Phenylquinolin-2-amine (33). Using phenylboronic acid and

1

following the procedure used to prepare 32 gave 33. H NMR (400

MHz, methanol-d₄) δ 8.41 (d, J = 9.2 Hz, 1H), 8.14 (d, J = 2.0 Hz, 1H),

8.09 (dd, J = 8.7, 2.0 Hz, 1H), 7.69ꢀ7.78 (m, 3H), 7.48ꢀ7.55 (m, 2H),

7.38ꢀ7.46 (m, 1H), 7.10 (d, J = 9.2 Hz, 1H). MS (ESI, positive ion)

m/z: 221 (M + 1).

6-o-Tolylquinolin-2-amine (34). Using o-tolylboronic acid and

following theprocedure used to prepare 32 gave 34. ¹H NMR(400 MHz,

methanol-d₄) δ 8.38 (d, J = 9.2 Hz, 1H), 7.82ꢀ7.87 (m, 1H), 7.75ꢀ7.81

(m, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.22ꢀ7.36 (m, 4H), 7.10 (d, J = 9.2 Hz,

1H), 2.28 (s, 3H). MS (ESI, positive ion) m/z: 235 (M + 1).

6-(2-Chlorophenyl)quinolin-2-amine (35). Using 2-chloro-

phenylboronic acid and following the procedure used to prepare 32 gave

35. ¹H NMR (400 MHz, methanol-d₄) δ 8.40 (d, J = 9.4 Hz, 1H), 7.96

(d, J = 2.0 Hz, 1H), 7.89 (dd, J = 8.6, 2.0 Hz, 1H), 7.75 (d, J = 8.6 Hz),

8-Bromoquinolin-2-amine (28). 8-Bromoquinolin-2-ol (3.16 g,

14 mmol) was mixed with phosphorus oxychloride (40 mL, 429 mmol)

and heated to reflux in a 140 ꢀC oil bath for 2 h. The mixture was allowed

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1H, 7.55ꢀ7.61 (m, 1H), 7.40ꢀ7.50 (m, 3H), 7.12 (d, J = 9.4 Hz, 1H).

MS (ESI, positive ion) m/z: 255, 257 (M + 1).

(30 mL) and heated to reﬂux. After 10 min, the reaction mixture was

worked up with 1N sodium hydroxide (60 mL) and extracted with

dichloromethane. The organic layer was dried with magnesium sulfate,

ﬁltered, and concentrated. The crude material was suspended in triﬂuo-

romethylbenzene (20 mL) and treated with tert-butylamine (1.0 mL,

9.51 mmol). Chloroform (3 mL) was added, followed by p-toluenesul-

fonic anhydride (0.58 g, 1.78 mmol, added in small portions). The

reaction mixture was stirred for 10 min and then was worked up with

sodium hydroxide (1N, 50 mL) and water (50 mL) and extracted with

dichloromethane. The organic layer was separated, dried with magne-

sium sulfate, ﬁltered, and concentrated. The crude material was puriﬁed

with column chromatography on silica gel (eluting with a gradient of

0ꢀ100% dichloromethane in hexane) to give 6-(2-bromophenyl)-N-

tert-butylquinolin-2-amine (0.31 g, 64% yield) as a colorless oil.

6-(2-Bromophenyl)-N-tert-butylquinolin-2-amine (0.31 g, 0.87 mmol)

was dissolved in triﬂuoroacetic acid (40 mL) and heated to reﬂux for

90 min. The solvent was removed, and the residue was redissolved

in dichloromethane and washed with 1N NaOH, water, and brine. The

organic layer was separated, dried on sodium sulfate, ﬁltered, and

concentrated. Thecrudematerialwaspuriﬁedbycolumnchromatography

on silica gel (eluting with a gradient of 30ꢀ100% ethyl acetate in hexane)

to give 6-(2-bromophenyl)quinolin-2-amine (0.26 g, 99% yield) as a

white solid.

6-(2-Bromophenyl)quinolin-2-amine (0.24 g, 0.82 mmol) was dis-

solved in triethylamine (0.57 mL, 4.08 mmol) and dry DMF (1.5 mL)

and transferred to a microwave vessel. Copper(I) iodide (7.8 mg, 0.041

mmol), dichlorobis(triphenylphosphine)palladium(II) (0.029 g, 0.041

mmol), and 3,3-dimethyl-1-butyne (0.15 mL, 1.22 mmol) were added,

and the reaction was heated to 140 ꢀC for 20 min. The crude mixture was

partitioned between water (60 mL) and ethyl acetate (100 mL). The

organic layer was dried with magnesium sulfate, ﬁltered, and concen-

trated. The crude material was puriﬁed by column chromatography on

silica gel (eluting with a gradient of 0ꢀ100% dichloromethane in ethyl

acetate) to give enriched material, which was further puriﬁed using

reverse phase HPLC to give 6-(2-(3,3-dimethylbut-1-ynyl)phe-

nyl)quinolin-2-amine (0.065 g, 26% yield). ¹H NMR (400 MHz,

chloroform-d) δ 7.87 (m, 3H), 7.68 (d, J = 8.4 Hz, 1H), 7.51 (dd, J =

7.6, 1.2 Hz, 1H) 7.44 (dd, J = 7.6, 1.2 Hz, 1H), 7.34 (td, J = 7.4 (ꢁ2), 1.6

Hz, 1H), 7.26 (ddd, J = 8.0, 7.0, 1.4 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H),

4.87 (br s, 2H), 1.15 (s, 9H). MS (ESI, positive ion) m/z: 301 (M + 1).

3-(2-Aminoquinolin-3-yl)-N-cyclohexyl-N-methylpropana-

mide (40). Quinoline-3-carboxaldehyde (1.0 g, 6.36 mmol) was dis-

solved in dry tetrahydrofuran (30 mL) and treated with methyl

(triphenylphosphoranylidene) acetate (2.13 g, 6.36 mmol). The reaction

was heated to 60 ꢀC for 90 min. Then the solvent was removed under

reduced pressure. The crude material was purified by column chroma-

tography on silica gel (eluting with a gradient of 0ꢀ30% ethyl acetate in

dichloromethane) to give the methyl 3-(quinolin-3-yl)acrylate as a white

solid. This material was dissolved in methanol (60 mL) and treated with a

solution of lithium hydroxide monohydrate (1.33 g, 31.8 mmol) in water

(30 mL). The solution was heated to 50 ꢀC and stirred for 40 min. The

reaction mixture was neutralized with 5N hydrochloric acid (6.5 mL), and

the solvent was removed to dryness under reduced pressure. The crude

material was dried under high vacuum with heat to remove remaining

residual water. The crude 3-(quinolin-3-yl)acrylic acid was suspended in

thionyl chloride (85 mL, 1.17 mol) and heated to 80 ꢀC for 90 min. The

reaction mixture was evaporated to dryness under reduced pressure and

further dried under high vacuum. Dichloromethane (80 mL) was added,

and a solution of diisopropylethylamine (3.5 mL, 20 mmol) and N-

methylcyclohexylamine (1.0 mL, 7.8 mmol) in dichloromethane (80 mL)

was added. The suspension was stirred for 10 min. Then water (100 mL)

and ethyl acetate (100 mL) were added. The organic layer was separated,

dried on sodium sulfate, filtered, and concentrated. The crude material

was purified by column chromatography on silica gel (eluting with a

6-(3-Chlorophenyl)quinolin-2-amine (36). Using 3-chloro-

phenylboronic acid and following the procedure used to prepare 32 gave

36. ¹H NMR(400 MHz, methanol-d₄) δ 8.43 (d, J = 9.4 Hz, 1H), 8.19 (d,

J = 2.0 Hz, 1H), 8.10 (dd, J = 8.7, 2.0 Hz, 1H), 7.74ꢀ7.79 (m, 2H), 7.69

(dt, J = 7.7, 1.4Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.43ꢀ7.47 (m, 1H), 7.12

(d, J = 9.4 Hz, 1H). MS (ESI, positive ion) m/z: 255, 257 (M + 1).

6-(4-Chlorophenyl)quinolin-2-amine (37). Using 4-chloro-

phenylboronic acid and following the procedure used to prepare 32

gave 37. ¹H NMR (400 MHz, methanol-d₄) δ 8.02 (d, J = 8.8 Hz, 1H),

7.91 (d, J = 2.0 Hz, 1H), 7.83 (dd, J = 8.8, 2.0 Hz, 1H), 7.69ꢀ7.73 (m,

2H), 7.62 (d, J = 8.8 Hz, 1H), 7.44ꢀ7.49 (m, 2H), 6.87 (d, J = 8.8 Hz,

1H). MS (ESI, positive ion) m/z: 255, 257 (M + 1).

6-Cyclohexylquinolin-2-amine (38). 6-Bromo-N-tert-butylqui-

nolin-2-amine (1.21 g, 4.3 mmol, prepared as described in 28 but using

2-aminoquinolin-6-ol and tert-butylamine) and 1,1⁰-bis(diphenylphos-

phino)ferrocene-palladium(II)dichloride dichloromethane adduct (0.18 g,

0.22 mmol), was dissolved indry tetrahydrofuran (50mL) under nitrogen

and cooledin an ice bath. Cyclohexylzinc bromide (0.5 M solutionin THF,

11 mL, 5.4 mmol) was added. The reaction mixture was heated to 60 ꢀC

for 40 min. Then the solution was evaporated to dryness under reduced

pressure. The crude material was purified by column chromatography on

silica gel (eluting with a gradient of 0ꢀ50% ethyl acetate in hexane) to give

N-tert-butyl-6-cyclohexylquinolin-2-amine (0.28 g, 23% yield).

N-tert-Butyl-6-cyclohexylquinolin-2-amine (0.28 g, 0.99 mmol) was

dissolved in triﬂuoroacetic acid (30 mL) and heated to reﬂux. After

20 min, the solvent was evaporated to dryness under reduced pressure.

The crude material was redissolved in dichloromethane (50 mL) and

washed with saturated aqueous sodium bicarbonate (20 mL). The

organic layer was separated, dried on sodium sulfate, ﬁltered, and

concentrated. The crude product was puriﬁed by column chromatogra-

phy on silica gel (eluting with a gradient of 0ꢀ10% methanol in

dichloromethane) to give 6-cyclohexylquinolin-2-amine (0.049 g, 22%

yield). ¹H NMR (400 MHz, chloroform-d) δ 7.86 (d, J = 8.8 Hz, 1H),

7.62 (d, J = 8.6 Hz, 1H), 7.47 (dd, J = 8.6, 2.0 Hz, 1H), 7.42 (d, J = 2.0 Hz,

1H), 6.70 (d, J = 8.8 Hz, 1H), 5.15 (br s, 2H), 2.60 (m, 1H), 1.7ꢀ2.0

(m, 5 H), 1.2ꢀ1.6 (m, 5H). MS (ESI, positive ion) m/z: 227 (M + 1).

6-(2-(3,3-Dimethylbut-1-ynyl)phenyl)quinolin-2-amine

(39). 6-Aminoquinoline (5.37 g, 37.2 mmol) was dissolved in 5N HCl

(30 mL) and cooled in an ice bath. A solution of sodium nitrite (2.57 g,

37.2 mmol) in water (20 mL) was added slowly, and the mixture was

stirred for 15 min. Ethyl acetate (200 mL) was added, followed by slow

addition of a solution of sodium iodide (5.58 g, 37.2 mmol) in water

(20 mL). After the reaction mixture was stirred for 10 min, the organic

layer was separated and washed sequentially with water (200 mL),

saturated sodium bicarbonate solution (100 mL), and finally sodium

sulfite solution (100 mL). Then it was dried with magnesium sulfate,

filtered, and concentrated. The crude material was purified by column

chromatography on silica gel eluted with dichloromethane in hexane to

give 6-iodoquinoline (4.65 g, 49.0% yield) as a yellow solid.

6-Iodoquinoline (0.39 g, 1.53 mmol), potassium carbonate (0.42 g,

3.05 mmol), 2-bromophenylboronic acid (0.32 g, 1.60 mmol), tetrakis-

triphenylphosphine palladium(0) (0.088 g, 0.076 mmol), and ethanol

(10 mL) were combined and heated to reﬂux under nitrogen for 2.5 h.

The reaction mixture was concentrated to dryness. Then the crude

material was dissolved in dichloromethane (40 mL) and washed with

water (40 mL). The organic layer was separated, dried with magnesium

sulfate, ﬁltered, and concentrated. The crude material was puriﬁed by

columnchromatographyonsilica gel (eluting with a gradient of 50ꢀ100%

dichloromethane in hexane) to give 6-(2-bromophenyl)quinoline (0.39 g,

90% yield).

6-(2-Bromophenyl)quinoline (0.39 g, 1.37 mmol) and m-chloroper-

oxybenzoic acid (0.81 g, 3.53 mmol) were dissolved in chloroform

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ARTICLE

gradiet of 0ꢀ100% ethyl acetate in dichloromethane) to give N-cyclohex-

yl-N-methyl-3-(quinolin-3-yl)acrylamide (1.38 g, 72% yield over 3 steps).

N-Cyclohexyl-N-methyl-3-(quinolin-3-yl)acrylamide (0.29 g, 0.98

mmol) was dissolved in methanol (20 mL) and treated with palladium

on carbon (0.052 g, 0.049 mmol). The reaction mixture was stirred

under a hydrogen balloon for 3 h then ﬁltered through a pad of Celite,

and the ﬁltrate was evaporated to dryness under reduced pressure. The

crude material was dissolved in chloroform (20 mL) and treated with m-

chloroperoxybenzoic acid (0.45 g, 1.95 mmol). The solution was heated

to reﬂux for 60 min. 1N Sodium hydroxide (50 mL) and dichloro-

methane (50 mL) were added. The organic layer was separated, dried

with magnesium sulfate, ﬁltered, and concentrated. The crude material

was dissolved in a mixture of triﬂuoromethylbenzene (20 mL) and tert-

butylamine (0.80 mL, 7.61 mmol) prior to addition of p-toluenesulfonic

anhydride (0.45 g, 1.36 mmol). The reaction mixture was stirred for

15 min. Then water (20 mL), 1N sodium hydroxide (30 mL), and

dichloromethane (60 mL) were added. The organic layer was separated

and washed with additional 1N sodium hydroxide (50 mL), then

dried with magnesium sulfate, ﬁltered, and concentrated. The crude

material was puriﬁed by column chromatography on silica gel (eluting

with a gradient of 0ꢀ100% ethyl acetate in hexane) to give 3-(2-(tert-

butylamino)quinolin-3-yl)-N-cyclohexyl-N-methylpropanamide (0.16 g,

45% yield).

3-(2-(tert-Butylamino)-6-o-tolylquinolin-3-yl)-N-cyclohexyl-N-

methylpropanamide (0.090 g, 0.24 mmol) was dissolved in triﬂuoroac-

teic acid (40 mL) and heated to reﬂux for 30 min. The solvent was

removed under reduced pressure. The crude material was added

dichloromethane (15 mL) and saturated sodium bicarbonate aqueous

solution (20 mL). The organic layer was separated, dried on sodium

sulfate, ﬁltered, and concentrated. The crude material was puriﬁed by

column chromatography on silica gel (eluting with a gradient of 0ꢀ10%

methanol in dichloromethane) to give 3-(2-amino-6-o-tolylquinolin-3-

yl)-N-cyclohexyl-N-methylpropanamide (0.025 g, 33% yield). ¹H NMR

(400 MHz, chloroform-d) δ 8.70 (br s, 2H), 7.86 (d, J = 3.1 Hz, 1H),

7.76 (d, J = 8.4 Hz, 1H), 7.59 (m, 2H), 7.33 (t, J = 7.5 Hz, 1H),

3.05ꢀ3.15 (m, 2H), 2.82 (s, 3H, 3:2 mix of rotomers), 2.74 (t, J = 6.3 Hz,

1H), 2.69 (t, J = 6.3 Hz, 1H) 1.00ꢀ1.89 (m, 11H). MS (ESI, positive

ion) m/z: 312 (M + 1).

3-(2-Amino-6-o-tolylquinolin-3-yl)-N-cyclohexyl-N-methyl-

propanamide (41). 2-Amino-5-bromobenzaldehyde (2.6 g, 13.0 mmol)

was dissolved in toluene (100 mL) and treated with methyl 3,3-dimethoxy-

propionate (1.84 mL, 13.0 mmol). p-Toluenesulfonic acid monohydrate

(0.25 g, 1.30 mmol) was added, and the solution was heated to reflux with

a DeanꢀStark trap for 90 min. The solvent was removed under reduced

pressure. The crude material was added ethyl acetate (300 mL), water

(100 mL), and saturated sodium bicarbonate aqueous solution (20 mL).

The organic layer was separated, dried with magnesium sulfate, filtered, and

concentrated to give the crude methyl 6-bromoquinoline-3-carboxylate

(3.2 g, 93% yield) as a yellow solid. This material was used without further

purification.

Methyl 6-bromoquinoline-3-carboxylate (0.99 g, 3.71 mmol), potas-

sium carbonate (2.05 g, 14.8 mmol), o-tolylboronic acid (0.96 g, 7.05

mmol), and tetrakis(triphenylphosphine)palladium(0) (0.24 g, 0.21

mmol) were suspended in ethanol (5 mL) and heated in the microwave

to 120 ꢀC for 10 min. The crude mixture was partitioned between water

(100 mL) and ethyl acetate (100 mL). The organic layer was separated,

dried with magnesium sulfate, ﬁltered, and concentrated. The crude

material was puriﬁed by column chromatography on silica gel (eluting

with a gradient of 0ꢀ100% ethyl acetate in hexane) to give methyl 6-o-

tolylquinoline-3-carboxylate. This material was dissolved in dichloro-

methane (40 mL) and cooled in an ice bath under nitrogen. Diisobu-

tylaluminum hydride (1.0 M solution in hexanes, 3.71 mL, 3.71 mmol)

was added, and the solution was stirred for 90 min. Then methanol

(10 mL) and saturated ammonium chloride aqueous solution (10 mL)

were carefully added. The mixture was stirred for 15 min, and then ethyl

acetate (100 mL) and water (100 mL) were added. The organic layer

was separated, dried with magnesium sulfate, ﬁltered, and concentrated.

The crude material was puriﬁed by column chromatography on silica gel

(eluting with a gradient of 0ꢀ10% methanol in dichloromethane) to

give (6-o-tolylquinolin-3-yl)methanol (0.22 g, 24% yield for 2 steps).

(6-o-Tolylquinolin-3-yl)methanol (0.22 g, 0.88 mmol) was dissolved

in dichloromethane (50 mL) and treated with tetrapropylammonium

perruthenate (0.016 g, 0.044 mmol) and 4-methylmorpholine 4-oxide

(0.16 g, 1.32 mmol). The reaction was stirred for 60 min. The crude

mixture was concentrated under reduced pressure to ∼10 mL and

passed through a pad of silica eluting with ethyl acetate to give 6-o-

tolylquinoline-3-carbaldehyde (0.094 g, 44% yield). This material was

dissolved in dry tetrahydrofuran (20 mL) and treated with methyl

(triphenylphosphoranylidene) acetate (0.20 g, 0.60 mmol). The reac-

tion mixture was stirred at room temperature for 20 min. The solvent

was removed, and the crude material was puriﬁed by column chroma-

tography on silica gel (eluting with a gradient of 0ꢀ100% ethyl acetate in

hexane) to give methyl 3-(6-o-tolylquinolin-3-yl)acrylate. This material

was dissolved in methanol (30 mL) and treated with a solution of lithium

hydroxide monohydrate (0.048 g, 1.15 mmol) in water (5 mL). The

solution was stirred for 40 min, and then 5N hydrochloric acid (0.5 mL)

was added. The mixture was evaporated to dryness under reduced

pressure and the crude solid further dried under high vacuum. The crude

material was suspended in excess thionyl chloride (20 mL) and heated to

reﬂux for 30 min. Then the reaction mixture was concentrated to dryness

and further dried under high vacuum. The residue was dissolved in

dichloromethane (20 mL) and treated with N-methylcyclohexylamine

(0.30 mL, 2.27 mmol). After 15 min, the solution was evaporated to

dryness under reduced pressure and the crude material was puriﬁed by

column chromatography on silica gel (eluting with a gradient of

0ꢀ100% ethyl acetate in hexane). The product was further puriﬁed by

reverse phase HPLC to give N-cyclohexyl-N-methyl-3-(6-o-tolylquino-

lin-3-yl)acrylamide (0.15 g, 43% yield).

The

N-cyclohexyl-N-methyl-3-(6-o-tolylquinolin-3-yl)acrylamide

(0.1 g, 0.38 mmol) was dissolved in methanol (80 mL) and treated

with palladium on activated carbon (10% by weight, 0.041 g, 0.039

mmol). The solution was stirred under hydrogen for 30 min. Then the

mixture was ﬁltered through a pad of Celite. The ﬁltrate was evaporated

to dryness under reduced pressure. The crude material was puriﬁed by

column chromatography on silica gel (eluting with a gradient of

0ꢀ100% ethyl acetate in dichloromethane) to give N-cyclohexyl-N-

methyl-3-(6-o-tolylquinolin-3-yl)propanamide. This material was dis-

solved in chloroform (20 mL) and treated with m-chloroperoxybenzoic

acid (0.10 g, 0.58 mmol). The solution was heated to reﬂux for 10 min

then diluted with dichloromethane (30 mL) and washed with 1N

sodium hydroxide (70 mL). The organic was dried with magnesium

sulfate and evaporated to dryness under reduced pressure. This material

was combined with triﬂuoromethylbenzene (20 mL) and tert-butyl-

amine (0.20 mL, 1.9 mmol). Then p-toluenesulfonic anhydride (0.15 g,

0.46 mmol) was added in small portions. The mixture was stirred for

20 min. Then water (100 mL), 5N sodium hydroxide (30 mL), and

dichloromethane (70 mL) were added. The organic layer was separated,

dried with magnesium sulfate, ﬁltered, and concentrated. The crude

material was puriﬁed by column chromatography on silica gel (eluting

with a gradient of 0ꢀ100% ethyl acetate in hexane) to give 3-(2-(tert-

butylamino)-6-o-tolylquinolin-3-yl)-N-cyclohexyl-N-methylpropanamide

(0.090 g, 51% yield).

3-(2-(tert-Butylamino)-6-o-tolylquinolin-3-yl)-N-cyclohexyl-N-methyl-

propanamide (0.090 g, 0.20 mmol) was dissolved in triﬂuoroacetic acid

(40 mL) and heated to reﬂux for 20 min. The solvent was removed, and

the residue was taken in dichloromethane (15 mL) and saturated sodium

bicarbonate solution (20 mL). The organic layer was separated, dried

on sodium sulfate, ﬁltered, and concentrated. The crude material was

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puriﬁed by column chromatography on silica gel (eluting with a

gradient of 0ꢀ10% methanol in dichloromethane) to give 3-(2-ami-

no-6-o-tolylquinolin-3-yl)-N-cyclohexyl-N-methylpropanamide (0.025 g,

palladium on carbon (10%, 0.085 g), and stirred under hydrogen for 3 h.

The reaction mixture was ﬁltered through a pad of Celite, and the ﬁltrate

was evaporatedto dryness under reducedpressure. Thecrudeproduct was

dissolved in chloroform (30 mL) and treated with 3-chloroperoxybenzoic

acid (0.17 g, 1.01 mmol). The solution was heated to reﬂux for 60 min.

Then dichloromethane (80 mL) and 1N sodium hydroxide (100 mL)

were added, and the organic layer was separated, dried with magnesium

sulfate, ﬁltered, and concentrated. This material was dissolved in a

mixture of triﬂuoromethylbenzene (10 mL) and tert-butylamine

(0.50 mL, 4.76 mmol) and treated with p-toluenesulfonic anhydride

(0.26 g, 0.81 mmol). The reaction mixture was stirred for 40 min, and

then dichloromethane (70 mL) and 1N sodium hydroxide (50 mL)

were added. The organic layer was separated, dried with magnesium

sulfate, ﬁltered, and concentrated. The crude material was puriﬁed

by column chromatography on silica gel (eluting with a gradient of

0ꢀ30% ethyl acetate in hexane) to give 3-(2-(tert-butylamino)-6-o-

tolylquinolin-3-yl)-N-(cyclohexylmethyl)-2-methylpropanamide (0.14 g,

45% yield).

3-(2-(tert-Butylamino)-6-o-tolylquinolin-3-yl)-N-(cyclohexylmethyl)-

2-methylpropanamide(0.14g, 0.30 mmol) was dissolved in triﬂuoroacetic

acid (20 mL) and heated to reﬂux for 20 min. The solvent was removed,

and the residue was taken in 1N NaOH and extracted with dichloro-

methane. The organic layer was separated, dried on sodium sulfate,

ﬁltered, and concentrated. The crude material was puriﬁed by column

chromatography on silica gel (eluting with a gradient of 0ꢀ10% methanol

in dichloromethane) to give 3-(2-amino-6-o-tolylquinolin-3-yl)-N-

(cyclohexylmethyl)-2-methylpropanamide (0.052 g, 19% yield). ¹H

NMR (400 MHz, methanol-d₄) δ 7.79 (s, 1H), 7.64 (d, J = 8.4 Hz,

1H), 7.53 (m, 2H), 7.43 (s, 1H), 7.27 (m, 4H), 4.24 (br s, 3H), 3.04 (m,

2H), 2.73 (m, 3H), 2.28 (s, 3H), 1.1ꢀ1.6 (m, 9H), 0.6ꢀ1.0 (m, 5H). MS

(ESI, positive ion) m/z: 416 (M + 1).

1

32% yield). H NMR (400 MHz, chloroform-d) δ 7.74 (m, 2H), 7.52

(m, 2H), 7.28(m, 4H), 6.62 (br s, 2H), 3.08 (m, 2H), 2.82 (s, 3H, mix

of rotomers), 2.76 (t, J = 6.5 Hz, 1H), 2.70 (t, J = 6.5 Hz, 1H), 2.28

(s, 3H), 0.9ꢀ1.9 (m, 11H). MS (ESI, positive ion) m/z: 402.2

(M + 1).

3-(2-Amino-6-o-tolylquinolin-3-yl)-N-cyclohexylpropan-

amide (42). Using cyclohexanamine and following the procedure

1

used to prepare 41 gave 42. H NMR (300 MHz, chloroform-d) δ

ppm 0.95ꢀ1.15 (m, 3H) 1.27ꢀ1.41 (m, 2H) 1.52ꢀ1.72 (m, 3H) 1.86

(dd, J = 12.35, 3.43 Hz, 2H) 2.29 (s, 3H) 2.54 (t, J = 6.87 Hz, 2H) 3.01 (t,

J = 6.80 Hz, 2H) 3.68ꢀ3.84 (m, 1H) 5.28ꢀ5.38 (m, 1H) 5.43 (br s, 2H)

7.28 (s, 4 H) 7.46ꢀ7.54 (m, 2H) 7.63ꢀ7.73 (m, 2H). MS (ESI, positive

ion) m/z: 388.2 (M + 1).

3-(2-Amino-6-o-tolylquinolin-3-yl)-N-(cyclohexylmethyl)pro-

panamide (43). Using cyclohexylmethanamine and following the

1

procedure used to prepare 41 gave 43. H NMR (300 MHz, chloro-

form-d) δ ppm 0.69ꢀ0.93 (m, 2H) 0.99ꢀ1.22 (m, 3H) 1.36 (br s, 1H)

1.48ꢀ1.74 (m, 5H) 2.29 (s, 3 H) 2.57 (t, J = 6.80 Hz, 2H) 2.89ꢀ3.16 (m,

4H) 5.62 (br s, 1H) 5.75 (br s, 1H) 7.20ꢀ7.36 (m, 4H) 7.43ꢀ7.56 (m,

2H) 7.61ꢀ7.79 (m, 2H). MS (ESI, positive ion) m/z: 402.2 (M + 1).

3-(2-Aminoquinolin-3-yl)-N-(cyclohexylmethyl)propanamide

(44). Using cyclohexylmethanamine and following the procedure used

1

to prepare 41 gave 44. H NMR (400 MHz, chloroform-d) δ 7.66

(s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 8 Hz, 1H), 7.50 (ddd, J = 8.4,

7.0, 1.5 Hz, 1H), 7.22 (td, J = 7.5, 7.5, 1.1, 1H), 5.61 (br s, 1H), 5.29 (br s,

2H), 3.05 (t, J = 6.4 Hz, 2H), 2.98 (t, J = 6.9 Hz, 2H), 2.54 (t, J = 7.0 Hz,

2H), 1.50ꢀ1.70 (m, 5H), 1.0ꢀ1.45 (m, 4H), 0.7ꢀ0.9 (m, 2H). MS

(ESI, positive ion) m/z: 312 (M + 1).

(R)-3-(2-Amino-6-o-tolylquinolin-3-yl)-N-(cyclohexylme-

thyl)-2-methylpropanamide (46) and (S)-3-(2-Amino-6-o-

tolylquinolin-3-yl)-N-(cyclohexylmethyl)-2-methylpropa-

namide (47). Chiral purificaton of 3-(2-amino-6-o-tolylquinolin-3-yl)-

N-(cyclohexylmethyl)-2-methylpropanamide (45) was performed with

preparative supercritical fluid chromatography (SFC) by Lotus Separa-

tions: Chiralpak IA (3 cm ꢁ 15 cm) column; mobile phase, 20%

methanol (0.2% EDA)/CO₂, 100 bar; flow rate = 80 mL/min; 220 nm;

0.5 mL injection with 25 mg/10 mL in methanol. The resulting 46 and

47 have ee values of >98% and >99%, respectively, according to Lotus.

2-((2-Amino-6-o-tolylquinolin-3-yl)methyl)-N-(cyclohexy-

lmethyl)butanamide (48). Using ethyl 2-(diethoxyphosphoryl)-

butanoate and following the procedure used to prepare 45 gave 48.

¹H NMR (400 MHz, chloroform-d) δ 7.69 (s, 1H), 7.63 (d, J = 8.4 Hz,

1H), 7.47 (m, 2H), 7.26 (m, 4H), 5.68 (br s, 1H), 5.31 (br s, 2H), 3.04

(m, 2H), 2.87 (m, 1H), 2.68 (dd, J = 14.3, 4.3 Hz, 1H), 2.36 (m, 1H),

2.28 (s, 3H), 1.1ꢀ1.9 (m, 9H), 0.85ꢀ1.0 (m, 5H), 0.55ꢀ0.70 (m, 2H).

MS (ESI, positive ion) m/z: 430 (M + 1).

2-((2-Amino-6-o-tolylquinolin-3-yl)methyl)-N-(cyclohexy-

lmethyl)-3-methylbutanamide (49). Using ethyl 2-(diethoxy-

phosphoryl)-3-methylbutanoate and following the procedure used to

prepare 45 gave 49. ¹H NMR (400 MHz, chloroform-d) δ 7.69 (m, 2H),

7.49 (m, 2H), 7.26 (m, 4H), 5.22 (br m, 3H), 3.04 (m, 2H), 2.81 (m,

2H), 2.29 (s, 3H), 2.06 (m, 2H), 0.8ꢀ1.65 (m, 15H), 0.59 (m, 2H). MS

(ESI, positive ion) m/z: 444 (M + 1).

2-((2-Amino-6-o-tolylquinolin-3-yl)methyl)-N-(cyclohexy-

lmethyl)pentanamide (50). Using ethyl 2-(diethoxyphosphoryl)-

pentanoate and following the procedure used to prepare 45 gave 50.

¹H NMR (400 MHz, chloroform-d) δ 7.79 (s, 1H), 7.69 (m, 1H), 7.53

(m, 2H), 7.27 (m, 4H), 6.12 (br s, 2H), 5.89 (br s, 1H), 3.07 (m, 2H),

2.89 (m, 1H), 2.75 (dd, J = 14.5, 4.9 Hz, 1H), 2.52 (m, 1H), 2.28 (s, 3H),

1.78 (m, 1H), 1.1ꢀ1.6 (m, 9H), 0.8ꢀ1.0 (m, 6H), 0.6ꢀ0.8 (m, 2H). MS

(ESI, positive ion) m/z: 444 (M + 1).

3-(2-Amino-6-o-tolylquinolin-3-yl)-N-(cyclohexylmethyl)-

2-methylpropanamide (45). (6-o-Tolylquinolin-3-yl)methanol

(0.32 g, 1.28 mmol, prepared as in 41) was dissolved in toluene

(50 mL) and treated with manganese dioxide (0.33 g, 3.83 mmol).

The mixture was heated to reflux for 80 min. The reaction mixture was

filtered through a pad of Celite into a suspension of triethyl 2-phospho-

nopropionate (0.33 mL, 1.53 mmol) and potassium t-butoxide (0.18 g,

1.59 mmol) in dry tetrahydrofuran (20 mL). The resulting solution was

stirred for 45 min. The solvent was removed, and the crude material was

purified by column chromatography on silica gel (eluting with a gradient

of 0ꢀ100% ethyl acetate in hexane) to give ethyl 2-methyl-3-(6-o-

tolylquinolin-3-yl)acrylate (0.27 g, 64% yield).

Ethyl 2-methyl-3-(6-o-tolylquinolin-3-yl)acrylate (0.27 g, 0.82

mmol) was dissolved in methanol (50 mL) and treated with a solution

of lithium hydroxide monohydrate (0.34 g, 8.21 mmol) in water

(10 mL). The reaction mixture was stirred for 30 min, and then 5N

hydrochloric acid (1.8 mL) was added. The solvent was removed, and

the material was dried on high vacuum. To this material, thionyl chloride

(30 mL, 411 mmol) was added and the mixture was heated to reﬂux for

30 min. The solvent was removed, and the residue was resuspended in

dichloromethane and once again evaporated to dryness under reduced

pressure and further dried under high vacuum. This material was

dissolved in dichloromethane (30 mL) and treated with aminomethyl-

cyclohexane (0.40 mL, 3.08 mmol) and diisopropylethylamine (1.0 mL,

5.74 mmol). The reaction mixture was stirred for 45 min. Water

(100 mL) and ethyl acetate (100 mL) were added, and the organic

layer was separated, dried with magnesium sulfate, ﬁltered, and con-

centrated. The crude material was puriﬁed by column chromatography

on silica gel (eluting with a gradient of 0ꢀ100% ethyl acetate in hexane)

to give the N-(cyclohexylmethyl)-2-methyl-3-(6-o-tolylquinolin-3-

yl)acrylamide (0.29 g, 89% yield over three steps).

N-(Cyclohexylmethyl)-2-methyl-3-(6-o-tolylquinolin-3-yl)acrylamide

(0.29 g, 0.73 mmol) was dissolved in methanol (50 mL), treated with

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(R)-3-(2-Amino-6-o-tolylquinolin-3-yl)-N-(3,3-dimethyl-

butyl)-2-methylpropanamide (51). Using 3,3-dimethylbutan-1-

amine and following the procedure used to prepare 45 afforded

3-(2-Amino-6-o-tolylquinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methyl-

propanamide. Chiral purificaton of the racemic material via preparative

SFC afforded (S)- and (R)-3-(2-amino-6-o-tolylquinolin-3-yl)-N-(3,3-

dimethylbutyl)-2-methylpropanamide: Chiralcel OJ-H (250 mm ꢁ 21

mm, 40 ꢀC, 5 μm) column; mobile phase, 88:12 (A:B), A = liquid CO₂,

B = methanol (0.2% DEA); 100 bar; flow rate = 62 mL/min; 252 nm.

The resulting enantiomers have ee values of 99.98% and >99%,

1N LiOH (10.0 mL, 10.0 mmol) was added to a solution of ethyl

3-(2-(4-methoxybenzylamino)-6-bromoquinolin-3-yl)-2-methylacrylate

(3.6 g, 7.9 mmol) in MeOH (10 mL) and THF (30 mL) at room

temperature. The reaction was stirred 1 h before the organic solvent was

removed and the remaining aqueous was brought to pH 1 with 1N HCl.

The resulting suspension was extracted with a 2:1 chloroform/i-PrOH

mixture. The combined organic layers were washed with brine, dried

over sodium sulfate, and concentrated to aﬀord 3-(2-(4-meth-

oxybenzylamino)-6-bromoquinolin-3-yl)-2-methylacrylic acid, which

was used without further puriﬁcation (2.4 g, 71% yield).

A mixture of 3-(2-(4-methoxybenzylamino)-6-bromoquinolin-3-yl)-

2-methylacrylic acid (0.10 g, 0.23 mmol), 3-(aminomethyl)pyridine

(0.048 mL, 0.47 mmol), and N-ethyl-N-isopropylpropan-2-amine

(0.091 mL, 0.53 mmol) were added followed by addition of

O-(benzotriazol-1-yl)-N,N,N⁰,N⁰-tetramethyluronium tetra ﬂuorobo-

rate (TBTU, 0.098 g, 0.30 mmol) in NMP (2 mL), which was stirred

at room temperature for 1 h. The mixture was quenched with saturated

aqueous NaHCO₃solution and stirred for 15 min. The mixture was

poured into water and extracted with EtOAc. The combined organic

layers were dried over Na₂SO₄, ﬁltered, concentrated, and chromato-

graphed on silica gel using a gradient of 0ꢀ75% ethyl acetate/hexane to

aﬀord 3-(2-(4-methoxybenzylamino)-6-bromoquinolin-3-yl)-2-methyl-

N-(pyridin-3-ylmethyl)acrylamide (0.12 g, 99% yield)

A mixture of 3-(2-(4-methoxybenzylamino)-6-bromoquinolin-3-yl)-

2-methyl-N-(pyridin-3-ylmethyl)acrylamide (0.12 g, 0.23 mmol), bis-

(4-(di-tert-butylphosphino)-N,N-dimethylbenzenamine) dichloropal-

ladium(II) (0.0025 g, 0.0035 mmol), o-tolylboronic acid (0.047 g,

0.35 mmol), and potassium acetate (0.046 g, 0.46 mmol) in EtOH

(10 mL) and water (1 mL) was heated at 85 ꢀC for 3 h. The mixture was

concentrated and chromatographed on silica gel using 0ꢀ75% ethyl

acetate/hexane to aﬀord 3-(2-(4-methoxybenzylamino)-6-o-tolylquino-

lin-3-yl)-2-methyl-N-(pyridin-3-ylmethyl)acrylamide (0.10 g, 82% yield).

A mixture of 3-(2-(4-methoxybenzylamino)-6-o-tolylquinolin-3-yl)-

2-methyl-N-(pyridin-3-ylmethyl)acrylamide (0.10 g, 0.2 mmol) and

palladium on carbon (0.10 g, 0.9 mmol) in EtOH (20 mL) was stirred

under a hydrogen balloon for 4 h. Then the mixture was ﬁltered through

a pad of Celite, and the ﬁltrate was concentrated. The resulting material

was added triﬂuoroacetic acid (7 mL) and heated at 63 ꢀC for 1 h.

The reaction mixture was concentrated and chromatographed on silica

gel using a gradient of 0ꢀ3% 2 M NH₃in MeOH/dichloromethane

to aﬀord 3-(2-amino-6-o-tolylquinolin-3-yl)-2-methyl-N-(pyridin-3-yl-

methyl)propanamide (0.037 g, 48% yield). ¹H NMR (300 MHz,

chloroform-d) δ 1.31 (d, J = 6.58 Hz, 3H), 2.27 (s, 3H), 2.62ꢀ2.77

(m, 2H), 3.06ꢀ3.18 (m, 1H), 3.47 (s, 1H), 4.20 (dd, J = 1.00 Hz, 1H),

4.40 (dd, J = 14.98, 6.36 Hz, 1H), 5.85 (br s, 1H), 6.84ꢀ6.97 (m, 2H),

7.16ꢀ7.33 (m, 5H), 7.44ꢀ7.56 (m, 2H), 7.65 (d, J = 8.62 Hz, 1H), 7.72

(s, 1H), 8.23 (d, J = 1.75 Hz, 1H), 8.29 (dd, J = 4.82, 1.32 Hz, 1H). MS

(ESI, positive ion) m/z: 411 (M + 1).

3-(2-Amino-6-o-tolylquinolin-3-yl)-2-methyl-N-(thiazol-5-

ylmethyl)propanamide (54). 6-Bromo-2-chloroquinoline-3-carbal-

dehyde (10 g, 37 mmol, prepared as in 53) was dissolved in NMP

200 mL in a 350 mL sealable flask. tert-Butylamine (23 mL, 222 mmol)

was added, and the reaction mixture was sealed and heated at 130 ꢀC for

24 h. After cooling, the mixture was poured into 1N HCl 200 mL and

stirred for 1.5 h. The precipitate was isolated by filtration and washed

with water. The solid was collected and dried on vacuum pump over-

night to give 2-(tert-butylamino)-6-bromoquinoline-3-carbaldehyde

(10.4 g, 92% yield).

1

respectively. H NMR (300 MHz, chloroform-d) δ ppm 0.82 (s, 9H)

1.12ꢀ1.23 (m, 2H) 1.30 (d, J = 6.72 Hz, 3H) 2.30 (s, 3H) 2.48ꢀ2.60

(m, 1H) 2.60ꢀ2.72 (m, 1H) 3.05ꢀ3.23 (m, 3H) 5.16ꢀ5.26 (m, 2H)

5.28ꢀ5.35 (m, 1H) 7.27 (t, J = 1.53 Hz, 4H) 7.47ꢀ7.55 (m, 2H) 7.68 (d,

J = 1.17 Hz, 2H). MS (ESI, positive ion) m/z: 404.1 (M + 1).

(R)-3-(2-Amino-6-o-tolylquinolin-3-yl)-N-benzyl-2-methyl-

propanamide (52). Using benzylamine and following the procedure

used to prepare 45 afforded 3-(2-amino-6-o-tolylquinolin-3-yl)-N-ben-

zyl-2-methylpropanamide. Chiral purification of the racemic material via

preparative SFC: OJH (250 mm ꢁ 21 mm, 5 μm) column; mobile phase

27% methanol (0.2% DEA)/CO₂; flow rate = 65 mL/min; 220 nm; 100

bar. The resulting (S)- and (R)-3-(2-amino-6-o-tolylquinolin-3-yl)-N-

benzyl-2-methylpropanamide have ee values of 100%, respectively.

¹H NMR (300 MHz, chloroform-d) δ ppm 1.35 (d, J = 6.72 Hz, 3H)

2.30 (s, 3H) 2.58ꢀ2.64 (m, 1H) 2.64ꢀ2.76 (m, 1H) 3.06ꢀ3.24 (m,

1H) 4.22ꢀ4.50 (m, 2H) 5.14ꢀ5.27 (m, 2H) 5.61ꢀ5.72 (m, 1H)

6.95ꢀ7.02 (m, 2H) 7.15 (s, 3 H) 7.27ꢀ7.31 (m, 4H) 7.47ꢀ7.50 (m,

1H) 7.50ꢀ7.56 (m, 1H) 7.71 (s, 2H). MS (ESI, positive ion) m/z: 410.0

(M + 1).

3-(2-Amino-6-o-tolylquinolin-3-yl)-2-methyl-N-(pyridin-

3-ylmethyl)propanamide (53). N,N-Dimethylformamide (54 mL,

0.70 mol) was added dropwise (via a syringe pump) to phosphoryl

trichloride (179 mL, 1.96 mol) in a 350 mL sealed tube in an ice bath

under nitrogen. After the addition, the water bath was removed and

N-(4-bromophenyl)acetamide (60 g, 0.28 mol) was added in one

portion and stirred until a homogeneous solution was observed

(approximately 30 min.). The reaction vessel was sealed and heated at

75 ꢀC for 48 h. The reaction was allowed to cool and slowly poured onto

ice (final volume of 2 L) and stirred for 25 min. The solid was filtered and

washed with water until the filtrate was no longer acidic (∼3 L) and the

product was dried in an oven vacuum overnight at 50 ꢀC to afford

6-bromo-2-chloroquinoline-3-carbaldehyde as a light-tan colored solid

(45.7 g, 60% yield).

6-Bromo-2-chloroquinoline-3-carbaldehyde (10 g, 0.37 mol) and

4-methoxybenzylamine (144 mL, 1.11 mol) in EtOH (200 mL) was

heated at 125 ꢀC in a sealed tube for 2.5 h. The reaction mixture was

cooled and poured into 1N HCl (200 mL) and stirred 2 h. The mixture

was extracted with chloroform, and the combined organic layers was

washed with 1N HCl and brine, dried over sodium sulfate, ﬁltered, and

concentrated to aﬀord 2-(4-methoxybenzylamino)-6-bromoquinoline-

3-carbaldehyde (12.4 g, 91% yield).

Lithium chloride (2.41 g, 56.7 mmol) was stirred 4 h in MeCN

(300 mL). To the cloudy solution was added 2-(4-methoxybenzylamino)-

6-bromoquinoline-3-carbaldehyde (10.5 g, 28.4 mmol), ethyl 2-(di-

ethoxyphosphoryl)propanoate (7.4 mL, 34.0 mmol), and 2,3,4,6,7,8,9,

10-octahydropyrimido[1,2-a]azepine (4.3 mL, 28.4 mmol), and the

reaction was stirred 12 h. The reaction was partitioned between 10%

sodium carbonate solution and ethyl acetate. The aqueous layer was

extracted with ethyl acetate, and the combined organic layers were

washed with 10% sodium carbonate aqueous solution, brine, dried over

sodium sulfate, ﬁltered, and concentrated. The crude mixture was puriﬁed

by silica gel chromatography eluted with a gradient of 4:1 hexanes/EtOAc

to aﬀord ethyl 3-(2-(4-methoxybenzylamino)-6-bromoquinolin-3-yl)-2-

methylacrylate (8.59 g, 67% yield).

Lithium chloride (1.4 g, 33 mmol) was stirred overnight in acetonitrile

(150 mL). To the cloudy solution was added 6-bromo-2-(tert-butylami-

no)quinoline-3-carbaldehyde (5.0 g, 16 mmol), ethyl 2-(diethoxy-

phosphoryl)propanoate (4.3 mL, 20 mmol), and 1,8-diazabicyclo-

[5.4.0]undec-7-ene (2.4 mL, 16 mmol). The reaction mixture was stirred

at room temperature for 10 h. To the reaction was added saturated

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sodium bicarbonate solution and extracted with EtOAc three times. The

combined organic layers were washed with brine and dried on sodium

sulfate, ﬁltered, and concentrated. The crude material was puriﬁed by

column chromatography with a gradient of 30ꢀ60% dichloromethane/

hexane to give ethyl 3-(6-bromo-2-(tert-butylamino)quinolin-3-yl)-2-

methylacrylate (5.4 g, 85% yield).

2 h, then water was added and extracted with EtOAc three times. The

combined organic layers were washed with water and brine, dried on

sodium sulfate, ﬁltered, and concentrated. The crude material was

puriﬁed by prep-TLC plate with 70% ethyl acetate/hexane. The puriﬁed

material was treated with 2,2,2-triﬂuoroacetic acid (5 mL) and reﬂuxed

for 2 h. The solvent was removed, and the residue was added dichlo-

romethane and 1N NaOH. The organic layer was separated and dried on

sodium sulfate, ﬁltered, and concentrated. The crude product was

puriﬁed by prep-TLC plate with 5% 2 M NH₃in MeOH/dichloro-

methane to give (R)-3-(2-amino-6-o-tolylquinolin-3-yl)-2-methyl-

N-(pyrazin-2-ylmethyl)propanamide (41 mg, 38% yield). ¹H NMR

(300 MHz, chloroform-d) δ ppm 1.35 (d, J = 6.43 Hz, 3H) 2.30 (s,

3H) 2.54ꢀ2.89 (m, 2H) 2.98ꢀ3.28 (m, 1H) 4.53 (t, J = 4.02 Hz, 2H)

5.15 (br s, 2H) 6.58 (br s, 1H) 7.28 (s, 4H) 7.39ꢀ7.57 (m, 2H)

7.57ꢀ7.78 (m, 2H) 8.29 (s, 1H) 8.39 (s, 1H) 8.51 (s, 1H). MS (ESI,

positive ion) m/z: 412.0 (M + 1).

Ethyl 3-(6-bromo-2-(tert-butylamino)quinolin-3-yl)-2-methylacry-

late (4.43 g, 11 mmol), o-tolylboronic acid (2 g, 17 mmol), bis(4-(di-

tert-butylphosphino)-N,N-dimethylbenzenamine) dichloropalladium-

(II) (0.2 g, 0.2 mmol), potassium acetate (2 g, 23 mmol), and water

(4 mL, 226 mmol) were combined in EtOH 100 mL and heated at 80 ꢀC

for 2 h. Then the solvent was removed, and the crude material was taken

in saturated sodium bicarbonate solution and extracted with EtOAc. The

combined organic layers were washed with brine, dried on sodium

sulfate, ﬁltered, and concentrated. The crude material was puriﬁed by

column chromatography with a gradient of 0ꢀ100% ethyl acetate/

hexane gradient to give ethyl 3-(2-(tert-butylamino)-6-o-tolylquinolin-3-

yl)-2-methylacrylate. This material was dissolved in methanol, and Pd/C

(2.0 g) was added. The reaction mixture was stirred under a hydrogen

balloon overnight. Then the mixture was ﬁltered through a pad of Celite.

The ﬁltrate was collected and concentrated. The crude material was

puriﬁed by column chromatography with 40ꢀ60% dichloromethane/

hexane gradient to give ethyl 3-(2-(tert-butylamino)-6-o-tolylquinolin-3-

yl)-2-methylpropanoate (4.2 g, 92% yield)

Ethyl 3-(2-(tert-butylamino)-6-o-tolylquinolin-3-yl)-2-methylpro-

panoate (4.2 g, 10 mmol) was dissolved in MeOH 150 mL and 1N

sodium hydroxide (42 mL, 42 mmol) was added. The reaction mixture

was reﬂuxed for 2 h and then cooled to room temperature. Then 5N HCl

was added to neutralize the solution to pH 2ꢀ3. The solvent was

removed, and the residue was taken in 1N HCl and extracted with

dichlormethane three times. The combined organic layers were washed

with water and brine, dried on sodium sulfate, ﬁltered, and concentrated

to give 3-(2-(tert-butylamino)-6-o-tolylquinolin-3-yl)-2-methylpropa-

noic acid (3.72 g, 94% yield).

3-(2-(tert-Butylamino)-6-o-tolylquinolin-3-yl)-2-methylpropanoic

acid (0.077 g, 0.2 mmol) was dissolved in dichloromethane 1 mL.

Thiazol-5-ylmethanamine hydrochloride (0.03 g, 0.2 mmol) was added,

followed by addition of N-(3-dimethylaminopropyl)-N⁰-ethylcarbodii-

mide hydrochloride (EDC, 0.05 g, 0.2 mmol). The mixture was stirred at

room temperature for 30 min. The solvent was evaporated, and the

crude material was puriﬁed by column chromatography with a gradient

of 50ꢀ80% ethyl acetate/hexane. The puriﬁed material was treated with

2,2,2-triﬂuoroacetic acid (5 mL) and reﬂuxed for 2 h. The solvent was

evaporated, and the crude material was dissolved in dichloromethane

and washed with 1N NaOH. The organic layer was dried on sodium

sulfate, ﬁltered, and concentrated to give 3-(2-amino-6-o-tolylquinolin-

3-yl)-2-methyl-N-(thiazol-5-ylmethyl)propanamide (0.036 g, 42%

yield). ¹H NMR (300 MHz, chloroform-d) δ ppm 1.29ꢀ1.35 (m,

3 H) 2.30 (s, 3 H) 2.52ꢀ2.77 (m, 2H) 3.12 (s, 1H) 4.54 (d, J = 5.85 Hz,

2H) 5.12 (br s, 2H) 6.00ꢀ6.15 (m, 1H) 7.27ꢀ7.34 (m, 4H) 7.48 (d, J =

1.75 Hz, 1H) 7.49ꢀ7.55 (m, 1H) 7.59 (s, 1H) 7.62ꢀ7.71 (m, 2H) 8.57

(s, 1H). MS (ESI, positive ion) m/z: 417.0 (M + 1).

(R)-3-(2-Amino-6-o-tolylquinolin-3-yl)-2-methyl-N-(pyrazin-

2-ylmethyl)propanamide (55). Chiral purification of 3-(2-(tert-

butylamino)-6-o-tolylquinolin-3-yl)-2-methylpropanoic acid (prepared

as in 54) by preparative SFC: coupling of two Chiralcel OJ-H (21 mm ꢁ

25 cm ꢁ 2, 40 ꢀC) columns; mobile phase, 88:12 (A:B), A = liquid CO₂,

B = methanol (0.2% isopropylamine); 100 bar; flow rate = 70 mL/min.

(R)-3-(2-(tert-Butylamino)-6-o-tolylquinolin-3-yl)-2-methylpropanoic

acid was obtained with 100% ee.

(R)-3-(2-Amino-6-o-tolylquinolin-3-yl)-2-methyl-N-

((tetrahydro-2H-pyran-4-yl)methyl)propanamide (56). Using

(tetrahydro-2H-pyran-4-yl)methanamine and following the procedure

1

used to prepare 55 gave 56. H NMR (400 MHz, chloroform-d) δ

ppm 1.00ꢀ1.17 (m, 3H) 1.23ꢀ1.30 (m, 1H) 1.33 (d, J = 6.65 Hz, 3H)

1.41ꢀ1.51 (m, 1H) 2.30 (s, 3H) 2.54ꢀ2.64 (m, 1H) 2.64ꢀ2.72 (m,

1H) 2.81ꢀ2.92 (m, 1H) 3.01 (m, 1H) 3.05ꢀ3.22 (m, 3H) 3.66ꢀ3.74

(m, 1H) 3.74ꢀ3.83 (m, 1H) 5.18 (br s, 2H) 5.49 (br s, 1H) 7.26ꢀ7.34

(m, 4H) 7.48ꢀ7.54 (m, 2H) 7.65 (d, J = 9.00 Hz, 1H) 7.70 (s, 1H). MS

(ESI, positive ion) m/z: 418.0 (M + 1).

(R)-3-(2-Amino-6-o-tolylquinolin-3-yl)-N-((R)-2,2-dimethyl-

tetrahydro-2H-pyran-4-yl)-2-methylpropanamide (57).

A

20 mL vial was charged with (R)-3-(2-(tert-butylamino)-6-o-tolylqui-

nolin-3-yl)-2-methylpropanoic acid (0.13 g, 0.33 mmol, prepared as in

55), 3,3-dimethylcyclohexanaminium chloride (0.068 g, 0.42 mmol),

TBTU (0.13 g, 0.42 mmol), and 2 mL of NMP. To the resulting mixture

was added N-ethyl-N-isopropylpropan-2-amine (0.15 mL, 0.83 mmol).

After stirring at room temperature for 2 h, the mixture was diluted with

water and extracted with EtOAc. The combined organic layers were

dried on sodium sulfate, filtered, and concentrated. The resulting two

diastereomers were separated and purified via column chromatography

with a gradient of 0ꢀ50% ethyl acetate/hexane to give (R)-3-(2-amino-

6-o-tolylquinolin-3-yl)-N-((R)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-

2-methylpropanamide and (R)-3-(2-amino-6-o-tolylquinolin-3-yl)-N-

((S)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-2-methylpropanamide.

These two compounds were treated individually with 2,2,2-trifluoroa-

cetic acid (3 mL) and refluxed for 2 h. The solvent was removed and to

the residue was added dichromethane and 1N NaOH. The organic layer

was separated and dried on sodium sulfate, filtered, and concentrated.

The crude product was purified by column chromatography on silica gel

with a gradient of 0ꢀ5% 2 M NH₃in MeOH/dichloromethane to

give (R)-3-(2-amino-6-o-tolylquinolin-3-yl)-N-((S)-2,2-dimethyltetra-

hydro-2H-pyran-4-yl)-2-methylpropanamide and (R)-3-(2-amino-6-

o-tolylquinolin-3-yl)-N-((R)-2,2-dimethyltetrahydro-2H-pyran-4-yl)-2-

methylpropanamide (is consistent with the determined co-crystal

structure). ¹H NMR (400 MHz, methanol-d₄) δ 8.16 (br s, 1H),

7.84ꢀ7.66 (m, 4H), 7.36ꢀ7.16 (m, 4H), 3.71 (br s, 1H), 2.90ꢀ2.62

(m, 2H), 2.26 (m, 3H), 1.84ꢀ1.60 (m, 1H), 1.58ꢀ1.35 (m, 3H),

1.35ꢀ1.17 (m, 5H), 1.06ꢀ0.83 (m, 6H). MS (ESI, positive ion) m/z:

432 (M + 1).

(R)-3-(2-Amino-6-(3-methylpyridin-2-yl)quinolin-3-yl)-

N-(3,3-dimethylbutyl)-2-methylpropanamide (58). TBTU

(1.2 g, 3.7 mmol) was added to 3-(2-(4-methoxybenzylamino)-6-

bromoquinolin-3-yl)-2-methylacrylic acid (1.2 g, 2.8 mmol, prepared

as in 53), 3,3-dimethylbutylamine (1.1 mL, 8.4 mmol), and N,N-

diisopropylethylamine (2.0 mL, 11 mmol) in NMP (10 mL), and the

reaction was stirred for 1 h. The reaction mixture was then poured into a

vigorously stirred solution of saturated aqueous sodium bicarbonate.

(R)-3-(2-(tert-Butylamino)-6-o-tolylquinolin-3-yl)-2-methylpropa-

noic acid (0.10 g, 0.27 mmol) was dissolved in DMF 2 mL. Pyrazin-2-

ylmethanamine (0.029 g, 0.27 mmol) and TBTU (0.10 g, 0.27 mmol)

were added. The reaction mixture was stirred at room temperature for

5853

dx.doi.org/10.1021/jm200544q |J. Med. Chem. 2011, 54, 5836–5857

Journal of Medicinal Chemistry

ARTICLE

After 30 min, ethyl acetate was added. The layers were separated, and the

aqueous layer was extracted with ethyl acetate. The combined organic

layers were washed with water, brine, dried over sodium sulfate, filtered,

and concentrated to afford 3-(6-bromo-2-(4-methoxybenzylamino)quinolin-

3-yl)-N-(3,3-dimethylbutyl)-2-methylacrylamide, which was used with-

out further purification.

1H) 7.16 (dd, J = 4 Hz, J = 8 Hz, 1H) 7.58 (d, J = 8 Hz, 1H) 7.67ꢀ7.71

(m, 3H) 7.73 (s, 1H) 8.52 (d, J = 4 Hz, 1H). MS (ESI, positive ion) m/z:

405 (M + 1).

(R)-3-(2-Amino-6-(3-chloropyridin-2-yl)quinolin-3-yl)-

N-(3,3-dimethylbutyl)-2-methylpropanamide (59). Bis(4-(di-

tert-butylphosphino)-N,N-dimethylbenzenamine) dichloropalladium-

(II) (0.038 g, 0.054 mmol) was added to a degassed solution of

3-(2-(4-methoxybenzylamino)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-

lan-2-yl)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methylacrylamide

(0.60 g, 1.1 mmol, prepared as in 58, potassium acetate (0.211 g, 2.15

mmol), 2-bromo-3-chloropyridine (0.31 g, 1.61 mmol) in EtOH

(12 mL), and water (2 mL). The reaction mixture was refluxed for 12 h.

Then it was cooled and partitioned between dichloromethane and 9:1

saturated ammonium chloride/ammonium hydroxide. The aqueous

layer was extracted with dichloromethane. The combined organic layers

were washed with a 9:1 saturated ammonium chloride/ammonium

hydroxide solution, water, brine, dried over sodium sulfate, filtered,

and concentrated. The crude material was purified by column chroma-

tography eluted with 1.5:1 hexane/ethyl acetate to afford 3-(2-(4-

methoxybenzylamino)-6-(3-chloropyridin-2-yl)quinolin-3-yl)-N-(3,3-

dimethylbutyl)-2-methylacrylamide (0.49 g, 84% yield).

Platinum on carbon (5%), (0.70 g, 0.18 mmol) was added to a

degassed solution of 3-(2-(4-methoxybenzylamino)-6-(3-chloropyridin-

2-yl)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methylacrylamide (0.49 g,

0.9 mmol) in EtOH (10 mL). The ﬂask was degassed with hydrogen

gas and then stirred under a balloon of hydrogen 12 h. The reaction was

ﬁltered through Celite and washed with ethanol and ethyl acetate. The

ﬁltrate was concentrated to aﬀord 3-(2-(4-methoxybenzylamino)-6-(3-

chloropyridin-2-yl)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methylpro-

panamide, which was used directly for the next step without further

puriﬁcation.

TFA (5.0 mL, 65 mmol) wasaddedto 3-(2-(4-methoxybenzylamino)-

6-(3-chloropyridin-2-yl)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methyl-

propanamide (0.30 g, 0.57 mmol), and the reaction was heated to 65 ꢀC.

After 6 h, the reaction was concentrated and was puriﬁed by reverse phase

HPLC to aﬀord 3-(2-amino-6-(3-chloropyridin-2-yl)quinolin-3-yl)-

N-(3,3-dimethylbutyl)-2-methylpropanamide (0.082 g, 21% yield over

two steps).

Chiral puriﬁcaton of the racemic material via preparative SFC aﬀorded

(S)- and (R)-3-(2-amino-6-(3-chloropyridin-2-yl)quinolin-3-yl)-N-(3,3-

dimethylbutyl)-2-methylpropanamide: Chiralpak AD-H (2 cm ꢁ 15 cm,

40 ꢀC) column; mobile phase 20% methanol (0.1% DEA)/CO₂; 100 bar;

ﬂow rate = 70 mL/min, 220 nm. The resulting enantiomers have ee

values of >99%, respectively.

Dichloro(1,1⁰-bis(diphenylphosphino)ferrocene) palladium(II) (0.096 g,

0.12 mmol) was added to a solution of 3-(6-bromo-2-(4-methoxyben-

zylamino)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methylacrylamide

(1.2 g, 2.4 mmol), potassium acetate (0.69 g, 7.1 mmol), and 4,4,5,5-

tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxa-

borolane (0.72 g, 2.8 mmol) in dioxane (20 mL, degassed with

nitrogen). The reaction mixture was heated at 85 ꢀC for 4 h, and then

it was cooled and concentrated. The crude product was diluted with

ethyl acetate and ﬁltered. The ﬁltrate was concentrated to aﬀord

3-(2-(4-methoxybenzylamino)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methylacrylamide, which was

used for the next reaction without further puriﬁcation.

Bis(4-(di-tert-butylphosphino)-N,N-dimethylbenzenamine) dichloropalla-

dium(II) (0.041 g, 0.058 mmol) was added to a degassed solution of

3-(2-(4-methoxybenzylamino)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-

lan-2-yl)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methylacrylamide

(0.60 g, 1.1 mmol), potassium acetate (0.211 g, 2.2 mmol), and

2-bromo-3-methylpyridine (0.18 mL, 1.6 mmol) in EtOH (12 mL)

and water (2 mL). The resulting solution was reﬂuxed for 12 h. Then the

reaction mixture was cooled and partitioned between dichloromethane

and 9:1 saturated ammonium chloride/ammonium hydroxide aqueous

solution. The aqueous layer was extracted with dichloromethane, and

the combined organics were washed with a 9:1 saturated ammonium

chloride/ammonium hydroxide solution, water, brine, dried over so-

dium sulfate, ﬁltered, and concentrated. The crude product was puriﬁed

by silica gel column chromatography eluted with 1:1 ethyl acetate/

hexane to aﬀord 3-(2-(4-methoxybenzylamino)-6-(3-methylpyridin-2-

yl)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methylacrylamide. (0.034 g,

61% yield).

Palladium on carbon (0.71 g, 0.67 mmol) was added to a solution of

3-(2-(4-methoxybenzylamino)-6-(3-methylpyridin-2-yl)quinolin-3-yl)-

N-(3,3-dimethylbutyl)-2-methylacrylamide (0.35 g, 0.67 mmol) in

EtOH (6 mL). The ﬂask was degassed with hydrogen gas and then

stirred under a balloon of hydrogen 12 h. The reaction was ﬁltered

through a pad of Celite and washed with ethanol and ethyl acetate. The

ﬁltrate was concentrated to aﬀord 3-(2-(4-methoxybenzylamino)-6-(3-

methylpyridin-2-yl)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methylpro-

panamide, which was used without further puriﬁcation.

¹H NMR (400 MHz, chloroform-d) δ ppm 0.83 (s, 9H) 1.19ꢀ1.23

(m, 2H) 1.28 (d, J = 8 Hz, 3H) 2.48ꢀ2.55 (m, 1H) 2.63ꢀ2.66 (m, 1H)

3.08ꢀ3.20 (m, 3H) 5.33 (br s, 2H) 5.35ꢀ5.41 (m, 1H) 7.21 (dd, J = 4

Hz, J = 8 Hz, 1H) 7.69 (d, J = 8 Hz, 1H) 7.73 (s, 1H) 7.81 (d, J = 8 Hz,

1H) 7.91 (dd, J = 4 Hz, J = 8 Hz, 1H) 8.00 (s, 1H) 8.61 (d, J = 4 Hz, 1H).

MS (ESI, positive ion) m/z: 425 (M + 1).

3-(2-Amino-6-(3-methoxypyridin-2-yl)quinolin-3-yl)-

N-(3,3-dimethylbutyl)-2-methylpropanamide (60). Using 2-

iodonicotinonitrile and following the procedures used to prepare 58

gave 60. ¹H NMR (300 MHz, chloroform-d) δ ppm 0.83 (s, 9H)

1.08ꢀ1.37 (m, 6H) 2.44ꢀ2.58 (m, 1H) 2.58ꢀ2.73 (m, 1H) 3.15 (br s,

2H) 3.90 (s, 3H) 5.27ꢀ5.49 (m, 1H) 6.08ꢀ6.32 (m, 1H) 7.17ꢀ7.25

(m, 1H) 7.30 (s, 1H) 7.73 (s, 1H) 7.77 (s, 1H) 8.19 (s, 2H) 8.33 (br s,

1H). MS (ESI, positive ion) m/z: 416.0 (M + 1).

3-(2-Amino-6-(3-cyanopyridin-2-yl)quinolin-3-yl)-N-(3,3-

dimethylbutyl)-2-methylpropanamide (61). Using 2-iodo-3-

methoxypyridine and following the procedures used to prepare 58 gave

61. ¹H NMR (300 MHz, chloroform-d) δ ppm 0.84 (s, 9H) 1.11ꢀ1.41

(m, 6H) 2.42ꢀ2.59 (m, 1H) 2.59ꢀ2.74 (m, 1H) 3.18 (d, J = 4.38 Hz,

2H) 5.32ꢀ5.49 (m, 1H) 5.57ꢀ5.85 (m, 1H) 7.31ꢀ7.45 (m, 1H)

TFA(6.0 mL, 78 mmol) was addedto 3-(2-(4-methoxybenzylamino)-

6-(3-methylpyridin-2-yl)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-meth-

ylpropanamide (0.30 g, 0.57 mmol), and the reaction was heated to

65 ꢀC. After 5 h, the reaction was concentrated and the crude material

dissolved in dichloromethane. The organic layer was washed with 1N

NaOH, and the aqueous layer was again extracted with dichloro-

methane. The combined organic layers were washed with brine, dried

over sodium sulfate, and concentrated. The crude product was puriﬁed

by reverse phase HPLC to aﬀord 3-(2-amino-6-(3-methylpyridin-2-

yl)quinolin-3-yl)-N-(3,3-dimethylbutyl)-2-methylpropanamide. (0.20 g,

76% yield over two steps)

Chiral puriﬁcaton of the racemic material via preparative SFC

aﬀorded (S)- and (R)-3-(2-amino-6-(3-methylpyridin-2-yl)quinolin-3-

yl)-N-(3,3-dimethylbutyl)-2-methylpropanamide: Chiralcel OJ-H (4.6 mm ꢁ

15 cm, 40 ꢀC) column; mobile phase 12% 2-propanol (0.2% DEA)/

CO₂; 100 bar; ﬂow rate = 4 mL/min; 252 nm. The resulting enantiomers

have ee values of >99%, respectively.

¹H NMR (400 MHz, chloroform-d) δ ppm 0.81 (s, 9H) 1.17ꢀ1.27

(m, 3H) 1.23 (d, J = 8 Hz, 3H) 2.39 (s, 3H) 2.48ꢀ2.55 (m, 1H)

2.59ꢀ2.64 (m, 1H) 3.04ꢀ3.23 (m, 3H) 5.34 (br s, 2H) 5.62ꢀ5.64 (m,

5854

dx.doi.org/10.1021/jm200544q |J. Med. Chem. 2011, 54, 5836–5857

Journal of Medicinal Chemistry

ARTICLE

7.69ꢀ7.86 (m, 2H) 8.02ꢀ8.15 (m, 2H) 8.23 (s, 1H) 8.82ꢀ8.99 (m,

1H). MS (ESI, positive ion) m/z: 421.0 (M + 1).

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’ ASSOCIATED CONTENT

S

Supporting Information. X-ray crystallographic data

b

(collection details, reﬁnement statistics). This material is avail-

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Accession Codes

^†New protein/ligand coordinates have been deposited in the

PDB with IDs of 3RSV, 3RSX, 3RTH, 3RTM, 3RTN, 3RU1, and

3RVI.

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’ AUTHOR INFORMATION

Corresponding Author

*Phone: 805-447-3543. Fax: 805-480-3016. E-mail: yuanc@

amgen.com (Y.C.) or tjudd@amgen.com (T.C.J.).

Present Addresses

³Envoy Therapeutics, Jupiter, Florida, United States

’ ACKNOWLEDGMENT

We thank Kyung Gahm, Sonia Hua, and Wesley Barnhart for

chiral separation. We also would like to acknowledge the con-

tributions of Dr. Leszek Poppe in the development of the ¹⁹F-

based enzymatic assay described in this paper. The Advanced

Light Source is supported by the U.S. Department of Energy

under contract no. DE-AC02-05CH11231.

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BACE, beta-site APP-Cleaving Enzyme; AD, Alzheimer’s disease;

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From fragment screening to in vivo efficacy: Optimization of a series of 2-aminoquinolines as potent inhibitors of beta-site amyloid precursor protein cleaving enzyme 1 (bace1)

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