Multifunctional donepezil analogues as cholinesterase and BACE1 inhibitors

molecules

Article

Multifunctional Donepezil Analogues as

Cholinesterase and BACE1 Inhibitors

Keith D. Green, Marina Y. Fosso and Sylvie Garneau-Tsodikova *

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington,

KY 40536-0596, USA; kgr234@uky.edu (K.D.G.); marina.fosso@uky.edu (M.Y.F.)

* Correspondence: sylviegtsodikova@uky.edu

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

Academic Editors: Diego Muñoz-Torrero and Michael Decker

Received: 25 October 2018; Accepted: 7 December 2018; Published: 8 December 2018

ꢀꢁꢂꢃꢄꢅꢆ

Abstract: A series of 22 donepezil analogues were synthesized through alkylation/benzylation

and compared to donepezil and its 6-O-desmethyl adduct. All the compounds were found to be

potent inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), two enzymes

responsible for the hydrolysis of the neurotransmitter acetylcholine in Alzheimer’s disease patient

brains. Many of them displayed lower inhibitory concentrations of EeAChE (IC₅₀= 0.016

±

0.001 µM

to 0.23 0.03 M) and EfBChE (IC₅₀= 0.11 0.01 M to 1.3 0.2 M) than donepezil. One of the

±

µ

±

µ

±

µ

better compounds was tested against HsAChE and was found to be even more active than donepezil

and inhibited HsAChE better than EeAChE. The analogues with the aromatic substituents were

generally more potent than the ones with aliphatic substituents. Five of the analogues also inhibited

the action of β-secretase (BACE1) enzyme.

Keywords: Alzheimer’s disease; acetylcholinesterase; butyrylcholinesterase; β-secretase; inhibitors

1. Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by memory loss

and cognitive deﬁcits. It is the most common form of dementia among older adults and the sixth

leading cause of death in the United States [

that there have been more than 2 million deaths associated to AD and other dementias in 2016, and this

number has doubled since 2000 [ ]. In the United States alone, more than 5 million people are currently

1]. In 2018, the World Health Organization (WHO) reported

2

living with AD, and this number is expected to triple by 2050. Unfortunately, there is currently no cure

for AD, which contributes to the deadly nature of this disease.

Despite all the research efforts invested, the speciﬁc cause(s) of AD remain(s) unclear [

molecular mechanisms of AD have been proposed, including the -amyloid cascade, oxidative stress,

metal imbalance, and cholinergic hypothesis [ ]. The latter appears to be the most efﬁcient therapeutic

3]. Several

β

4

avenue in providing temporary relief of AD symptoms. Indeed, ﬁve drugs have been approved by

the United States Food and Drug Administration (FDA) for the symptomatic treatment of AD, four of

which are acetylcholinesterase (AChE) inhibitors: rivastigmine, galantamine, donepezil, and tacrine.

These drugs prevent the action of cholinesterases (ChEs), which are responsible for the hydrolysis

of the neurotransmitter acetylcholine (ACh), thereby increasing the levels of ACh in the brain and

improving the cholinergic functions in AD patients [5,6]. In addition to AChE, another type of enzyme

involved in the hydrolysis of the neurotransmitter ACh is butyrylcholinesterase (BChE). The activity

and expression of BChE have been suggested to increase throughout the progression of AD, indicating

that BChE may play an important role in the late stage of AD [7]. Therefore, inhibition of AChE and

BChE remains a potential therapeutic target for AD treatment. However, targeting ChEs alone is

deﬁnitely not sufﬁcient.

Molecules 2018, 23, 3252; doi:10.3390/molecules23123252

ww w .mdpi.com/journal/molecules

Molecules 2018, 23, 3252

2 of 22

Another hallmark of AD pathology is the accumulation of amyloid-

These plaques are composed of A peptides that result from the cleavage of the transmembrane

amyloid precursor protein (APP) by secretases to form A monomers that will aggregate to toxic

ﬁbrils [ ]. -secretase (BACE1) is an aspartyl protease that cleaves APP near the membrane surface,

β (Aβ) plaques on the brain [8].

β

9

β

and it has been targeted for the development of potential therapies against AD [10].

Due to the multifactorial nature of AD, the development of compounds that could target different

pathological features of the disease appears to be a viable research avenue. We previously reported

on the synthesis and biological evaluation of a number of multifunctional molecules derived from

tacrine and chalcones that are capable of targeting ChEs and A

commonly prescribed medication for AD [ ], and several other studies have focused on this drug to

generate multifunctional compounds targeting various hallmarks of AD, including BACE1 [16 19],

β [11–15]. Since donepezil is the most

4

–

we decided to generate multi-targeted analogues derived from donepezil that would inhibit ChEs and

β-secretase.

2. Results and Discussion

2.1. Chemistry

The synthetic route utilized for the synthesis of donepezil analogues is outlined in Scheme 1.

Starting from ferulic acid (

presence of methanesulfonic acid (MsOH) produced ketone

the ketone with the aldehyde through aldol condensation were met with little success. To overcome

this shortcoming, the free hydroxyl group in compound was protected with a TBDMS group to yield

the corresponding ketone with 90% yield. This was then successfully condensed with the aldehyde

in the presence of KOH, to yield the -unsaturated ketone with 65% yield. Selective reduction of

the double bond in the presence of a ketone and a benzyl group was achieved through a controlled

poisoning of the palladium catalyst with thioanisole to give the 6-O-desmethyl donepezil adduct

1

), hydrogenation in the presence of Pd/C, followed by cyclization in the

3

with 67% yield [20 21]. Attempts to react

,

3

5

3

4

5

α

,

β

6

7

with 94% yield. The latter bears a free hydroxyl group that was reacted with the corresponding alkyl

or benzyl halides to yield 22 donepezil analogues (8a–v) with 32–95% yields.

MeO

HO

CO₂H

MeO

HO

CO₂H

MeO

HO

Pd/C, H₂

MsOH

EtOAc, rt

(quant.)

120 °C

(67%)

O

1

2

3

TBDMSCl, DMAP,

Et₃N, CH₂Cl₂, rt

(90%)

OHC

N

MeO

HO

MeO

5

TBDMSO

O

KOH, EtOH, 65 °C

(65%)

4

N

6

Pd/C, H₂, THF,

thioanisole, rt

(94%)

MeO

HO

MeO

RX (X = Cl or Br)

RO

K₂CO₃,

DMF, rt

(32-95%)

O

N

7

8

R =

Y

n

Z

a: Y = NH₂, n = 2 (65%)

b:Y = NH₂, n = 1 (93%)

c: Y = Me, n = 1 (95%)

d:Y = Cl, n = 1 (85%)

e: Y = Br, n = 1 (54%)

f: Y = Br, n = 3 (93%)

g:Y = Br, n = 11 (32%)

h: Z = H (82%)

i: Z = 4-Me (58%)

j: Z = 4-OMe (36%) r: Z = 2,5-diF (85%)

k: Z = 4-NO₂(65%) s: Z = 2,6-diF (93%)

l: Z = 4-Br (84%)

m: Z = 4-F (86%)

n: Z = 3-F (88%)

o: Z = 2-F (86%)

p: Z = 2-CF₃(90%)

q: Z = 2,4-diF (85%)

t: Z = 4-Br-2-F (91%)

u: Z = 2,4,6-triF (93%)

v: Z = 2,3,4,5,6-pentaF (81%)

Scheme 1. Synthetic scheme for the preparation of compounds 8a–v.

Molecules 2018, 23, 3252

3 of 22

2.2. Cholinesterase Inhibition

To evaluate the potential cholinesterase (ChE) inhibitory activity of donepezil, 6-O-desmethyl

donepezil , and the 22 newly synthesized donepezil analogues 8a , their IC₅₀values were determined

7

–v

against AChE from Electrophorus electricus (EeAChE) (Table 1 and Figures S82–S83) and EfBChE

from equine serum (Equus ferus) (Table 1 and Figures S84–S85) using the well-established Ellman

method [22].

Table 1. Inhibition (IC₅₀values in µM, with standard error) of the activity of EeAChE (from Electrophorus

electricus) and BChE (from Equus ferus) by donepezil and its analogues 7 and 8a–v, and the selectivity

index (SI) for each inhibitor based on IC₅₀values.

Cpd

EeAChE

EfBChE

2.0 ± 0.1

SI

17

Donepezil HCl

0.12 ± 0.01

0.41 ± 0.05

0.054 ± 0.003

0.021 ± 0.003

0.14 ± 0.02

0.059 ± 0.004

0.044 ± 0.003

0.061 ± 0.007

0.79 ± 0.28

0.13 ± 0.01

0.23 ± 0.03

0.13 ± 0.01

0.13 ± 0.02

0.071 ± 0.015

0.081 ± 0.005

0.16 ± 0.02

0.12 ± 0.02

0.032 ± 0.010

0.11 ± 0.01

0.090 ± 0.009

0.016 ± 0.001

0.054 ± 0.007

0.027 ± 0.004

0.17 ± 0.02

7

8a

8b

8c

8d

8e

8f

4.3 ± 0.4

11

0.57 ± 0.04

0.48 ± 0.03

2.1 ± 0.3

1.3 ± 0.1

1.3 ± 0.2

5.2 ± 1.6

0.70 ± 0.05

1.0 ± 0.2

0.67 ± 0.17

0.46 ± 0.06

0.72 ± 0.10

0.57 ± 0.10

0.96 ± 0.15

0.76 ± 0.12

0.25 ± 0.08

0.48 ± 0.08

0.60 ± 0.15

0.44 ± 0.05

0.37 ± 0.05

0.20 ± 0.03

0.11 ± 0.01

11

23

15

22

30

21

8g

8h

8i

6.6

5.4

4.3

5.2

3.5

10

8j

8k

8l

8m

8n

8o

8p

8q

8r

7.0

6.0

6.3

7.8

4.4

6.7

28

8s

8t

6.9

7.4

0.69

8u

8v

2.2.1. AChE Inhibition

When comparing the 6-O-desmethyl donepezil

(R = Me; IC₅₀= 0.12 0.01 M) and its analogues 8a

groups; IC₅₀= 0.016 0.001 M to 0.23 0.03 M), it becomes evident that 6-O-alkylation/benzylation

enhances EeAChE inhibition, with the only exception being 8g (IC₅₀= 0.79 0.28 M), which bears a

7

(R = H; IC₅₀= 0.41

±

0.05 µM) with donepezil

±

µ

–

f

and 8h (R = various alkyl and benzylic

–v

±

µ

±

µ

±

µ

hydrophobic 1-bromododecyl group. Other analogues were equal to or even better than donepezil at

inhibiting the action of EeAChE in vitro. With similar IC₅₀values, compound 8c (R = n-propyl;

IC₅₀= 0.14 ± 0.02 µM) was as potent as donepezil (R = Me; IC₅₀= 0.12

±

0.01

µ

M). However,

substituting the terminal methyl in the R group of compound 8c (R = n-propyl; IC₅₀= 0.14

±

0.02

Molecules 2018, 23, 3252

4 of 22

µ

M) by a terminal amine in compound 8b (R = H₂NCH₂CH₂; IC₅₀= 0.021

±

0.003

µ

M) drastically

increased the potency. Indeed, a 6-fold reduction of the IC₅₀value of donepezil was observed.

The amine group may form hydrogen bonds with Tyr70, Asp72, and Gln74 residues near the PAS [11].

Cancellation of this hydrogen bonding by replacing the terminal amine in the R group of compound 8b

(R = H₂NCH₂CH₂; IC₅₀= 0.021

±

0.003

µ

M) with a chlorine atom in 8d (R = ClCH₂CH₂; IC₅₀= 0.059

±

0.004 M) or a bromine atom in 8e (R = BrCH₂CH₂; IC₅₀= 0.044

µ

±

0.003 M) resulted in an increase in

µ

the IC₅₀values, which only represented a 2- or 3-fold enhanced potency when compared to donepezil,

respectively. Elongation of the R group also appeared to worsen the IC₅₀values. Indeed, adding an

extra methylene to compound 8b (R = H₂NCH₂CH₂; IC₅₀= 0.021

(R = H₂NCH₂CH₂CH₂; IC₅₀= 0.054 0.003 M), while the addition of two methylene groups to

compound 8e (R = BrCH₂CH₂; IC₅₀= 0.044 0.003 M) gives compound 8f (R = BrCH₂CH₂CH₂CH₂;

IC₅₀= 0.061 0.007 M). Nevertheless, all these analogues remained better inhibitors of EeAChE

than donepezil.

Replacing the alkyl group in donepezil (R = Me; IC₅₀= 0.12

in compound 8h (R = Bn; IC₅₀= 0.13 0.01 M) did not affect the IC₅₀value. Likewise, additional

substitutions at the para-position of the benzyl group resulted in IC₅₀values that were similar to

that of donepezil. Indeed, compounds 8i (R = 4-MeBn; IC₅₀= 0.23 0.03 M), 8j (R = 4-OMeBn;

IC₅₀= 0.13 ± 0.01 µM), 8k (R = 4-NO₂Bn; IC₅₀= 0.13 0.02 M), 8l (R = 4-BrBn; IC₅₀= 0.071

0.015 M), and 8m (R = 4-FBn; IC₅₀= 0.081 0.005 M) displayed IC₅₀values that were still within

1- to 2-fold of that of donepezil (R = Me; IC₅₀= 0.12 0.01 M). Similarly, when the ﬂuoro group

was moved from the para-position in compound 8m (R = 4-FBn; IC₅₀= 0.081 0.005 M) to the

meta- or ortho-positions in compounds 8n (R = 3-FBn; IC₅₀= 0.16 0.02 M) and 8o (R = 2-FBn;

IC₅₀= 0.12 ± 0.02 µM), respectively, the potency of these analogues was comparable to donepezil

(R = Me; IC₅₀= 0.12 0.01 M). However, replacing the ﬂuoro group in 8o (R = 2-FBn; IC₅₀= 0.12 0.02

M) by a CF₃group in 8p (R = 2-CF₃Bn; IC₅₀= 0.032 0.010 M) improved the IC₅₀by 4-fold. This may

±

0.003 µM) gives compound 8a

±

µ

±

µ

±

µ

±

0.01 µM) by an aromatic group

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

suggest that enhanced electron-withdrawing effect on the aromatic ring may improve the potency of

the analogue. Attempts to spread out the electron-withdrawing effect throughout the aromatic ring led

to compounds 8q (R = 2,4-diF-Bn; IC₅₀= 0.11

8s (R = 2,6-diF-Bn; IC₅₀= 0.016 0.001

with two electron-withdrawing groups, and compounds 8u (R = 2,4,6-triF-Bn; IC₅₀= 0.027

±

0.01

M), and 8t (R = 4-Br-2-F-Bn; IC₅₀= 0.054 ± 0.007 µM),

0.004

µ

M), 8r (R = 2,5-diF-Bn; IC₅₀= 0.090 ± 0.009 µM)

,

±

µ

±

µM) and 8v (R = 2,3,4,5-pentaF-Bn; IC₅₀= 0.17 ± 0.02 µM), with three and ﬁve electron-withdrawing

groups, respectively. It thus appears that both ortho-positions on the benzyl group are very sensitive

to the presence of electron-withdrawing groups, since 8s (R = 2,6-diF-Bn; IC₅₀= 0.016

±

0.001

µM)

and 8u (R = 2,4,6-triF-Bn; IC₅₀= 0.027

±

0.004 µM) were eight and ﬁve times more potent than

donepezil, respectively.

In order to conﬁrm that the data obtained with EeAChE would also apply to HsAChE (from Homo

sapiens), we tested donepezil along with a compound that displayed better inhibition than donepezil,

8t. We found that both donepezil and compound 8t inhibited HsAChE better than the EeAChE (Table 2).

Compound 8t (R = 4-Br-2-F-Bn; IC₅₀= 0.0018

donepezil (R = Me; IC₅₀= 0.032 0.011 M). In the case of EeAChE, compound 8t (R = 4-Br-2-F-Bn;

IC₅₀= 0.054 0.007 M) had an IC₅₀value that was 2.2-fold better than donepezil (R = Me; IC₅₀= 0.12

±

0.0006 µM) inhibited HsAChE 18-fold better than

±

µ

±

µ

± 0.01 µM). These data would suggest that our inhibitors are well suited for working with HsAChE.

Table 2. Inhibition (IC₅₀values in µM) of the activity of HsAChE by donepezil and its analogue 8t.

Cpd

Donepezil HCl

8t

IC₅₀(µM)

0.032 ± 0.011

0.0018 ± 0.0006

SI ^a

3.8

30

a

Selectivity index of EeAChE versus HsAChE, based on IC₅₀values.

Molecules 2018, 23, 3252

5 of 22

2.2.2. EfBChE Inhibition

As expected, donepezil analogues 8a

–v

were less effective against EfBChE than EeAChE. Indeed,

donepezil is highly selective for EeAChE over EfBChE [23], and as a result, it is expected for its

analogues to behave similarly. However, when compared to donepezil (R = Me; IC₅₀= 2.0 0.1 M),

all but compounds 8c (R = CH₃CH₂CH_2;IC₅₀= 2.1 0.3 M) and 8g (IC₅₀= 5.2 1.6 M) appeared to

be more effective at inhibiting the action of BChE. The presence of a terminal amine in compounds 8a

(R = H₂NCH₂CH₂CH₂; IC₅₀= 0.57 0.04

M) and 8b (R = H₂NCH₂CH₂; IC₅₀= 0.48 ± 0.03 µM) still

drastically increased their potency by 4-fold when compared to donepezil. Substitution of the amine

group in 8b (R = H₂NCH₂CH₂; IC₅₀= 0.48 0.03 M) with a chlorine atom in 8d (R = ClCH₂CH₂;

IC₅₀= 1.3 0.1 M) or a bromine atom in 8e (R = BrCH₂CH₂; IC₅₀= 1.3 0.2 M) resulted

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

again in an increase in the IC₅₀values, which only represented a 2-fold enhanced potency when

compared to donepezil. Elongation of the R group did not have much effect, as the IC₅₀value of 8f

(R = BrCH₂CH₂CH₂CH₂; IC₅₀= 1.3 ± 0.2 µM) still remained within 2-fold that of donepezil.

A greater improvement of the IC₅₀values was more noticeable when the alkyl group in donepezil

(R = Me; IC₅₀= 2.0

0.05 M) was 3-fold more potent than donepezil. Substitutions at the para-position of the benzyl

group also contributed to reducing the IC₅₀values. Indeed, compounds 8i (R = 4-Me-Bn; IC₅₀= 1.0

0.2 M), 8j (R = 4-OMe-Bn; IC₅₀= 0.67 0.17 M), 8k (R = 4-NO₂Bn; IC₅₀= 0.46 0.06 M),

8l (R = 4-Br-Bn; IC₅₀= 0.72 0.10 M), and 8m (R = 4-F-Bn; IC₅₀= 0.57 0.10 M) displayed

inhibitory efﬁcacies of 2- to 4-fold better than donepezil (R = Me; IC₅₀= 2.0 0.1 M). Similarly,

when the ﬂuoro group was moved from the para-position in compound 8m (R = 4-F-Bn; IC₅₀= 0.57

0.10 M) to the meta- or ortho-positions in compounds 8n (R = 3-F-Bn; IC₅₀= 0.96 0.15 M) and

8o (R = 2-F-Bn; IC₅₀= 0.76 0.12 M), respectively, the potency of these analogues was reduced

by 1- to 2-fold. Replacing the ﬂuoro group in 8o (R = 2-F-Bn; IC₅₀= 0.76 0.12 M) by a CF₃

group in 8p (R = 2-CF₃Bn; IC₅₀= 0.25 0.08 M) once again improved the IC₅₀by 8-fold. This is in

±

0.1

µM) was replaced by an aromatic group. Compound 8h (R = Bn; IC₅₀= 0.70

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

agreement with the trend observed in EeAChE inhibition. Indeed, the additional electron-withdrawing

effect on the aromatic ring still appeared to increase the potency of the analogue. Compounds 8q

(R = 2,4-diF-Bn; IC₅₀= 0.48

IC₅₀= 0.44 0.05 M), and 8t (R = 4-Br-2-F-Bn; IC₅₀= 0.37

groups, and compounds 8u (R = 2,4,6-triF-Bn; IC₅₀= 0.20

IC₅₀= 0.11 ± 0.01 µM), with three and ﬁve electron-withdrawing groups, respectively, were all better

EfBChE inhibitors than donepezil (R = Me; IC₅₀= 2.0 0.1 M). Compound 8q (R = 2,4-diF-Bn;

IC₅₀= 0.48 0.08 M) was 4-fold better than donepezil, while 8r (R = 2,5-diF-Bn; IC₅₀= 0.60 0.15

M) and 8s (R = 2,6-diF-Bn; IC₅₀= 0.44 0.05 M) were 3- and 5-fold better, respectively. Replacing

the ﬂuorine atom at the para-position in 8q (R = 2,4-diF-Bn; IC₅₀= 0.48 0.08 M) with a bromine

atom in 8t (R = 4-Br-2-F-Bn; IC₅₀= 0.37 0.05 M) did not impart a noticeable change. Compounds

8u (R = 2,4,6-triF-Bn; IC₅₀= 0.20 0.03 M) and 8v (R = 2,3,4,5-pentaF-Bn; IC₅₀= 0.11 0.01 M),

±

0.08

µ

M), 8r (R = 2,5-diF-Bn; IC₅₀= 0.60

0.05 M), with two electron-withdrawing

0.03 M) and 8v (R = 2,3,4,5-pentaF-Bn;

±

0.15 µM), 8s (R = 2,6-diF-Bn;

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

±

µ

with three and ﬁve electron-withdrawing groups, respectively, were 10- and 18-fold better than

donepezil. The active site gorge of BChE is less constrained than that of AChE, allowing BChE to

better accommodate bulky inhibitors [24]. This supports our observations that additional substitution

on the aromatic ring increased the potency of the donepezil analogues against EfBChE more than

against EeAChE.

We also calculated the selectivity index (SI) to understand the utility of the compounds. For all

but one compound, 8v, EeAChE was inhibited 3.5- to 30-fold better than EfBChE. Interestingly,

compound 8v was 1.5-fold more selective for EfBChE. Clearly the donepezil analogues are better

suited for inhibiting EeAChE. We also looked at the selectivity of the inhibitors for EeAChE versus

HsAChE. We observed that donepezil was 3-fold more selective for HsAChE. Perhaps more interesting,

compound 8t was 30-fold more selective for HsAChE over EeAChE.

Molecules 2018, 23, 3252

6 of 22

2.3. BACE1 Inhibition

It has previously been reported that donepezil has some BACE1 inhibitory activity [19]. Keeping

this in mind we decided to test these compounds for BACE1 inhibitory activity (Table 3). Unlike with

AChE and BChE, in general, donepezil analogues 8a–v were not better than the parent donepezil

at inhibiting the action of BACE1 in vitro, with the exception of 8c

the low micromolar range. Indeed, 8c (R = CH₃CH₂CH_2;IC₅₀= 6.1

,

±

8e

0.1

M), and 8l (R = 4-Br-Bn; IC₅₀= 3.4

M) were within 5-fold of the IC₅₀values of donepezil. This suggests that our analogues are more

,

8f, and 8l, which were in

µM), 8e (R = BrCH₂CH₂;

IC₅₀= 7.9

0.1

±

0.9

µM), 8f (R = BrCH₂CH₂CH₂CH₂; IC₅₀= 7.9

±

2.4

µ

±

µ

selective in targeting the ChEs, but they do still target BACE1. As a control for the BACE1 inhibition

assays, we used BACE inhibitor IV. Our inhibitors were poorer inhibitors than BACE inhibitor IV

(IC₅₀= 0.63

±

0.18 nM). While BACE inhibitor IV is better, it was designed to be very speciﬁc for

that one target. However, with an illness such as Alzheimer’s disease, which has many facets and

contributing factors, having multifunctional inhibitors that display activity against BACE1 and ChEs

is beneﬁcial.

Table 3. Inhibition (IC50 values in

µM, unless otherwise noted) of the activity of BACE1 by donepezil

and its analogues 7, and 8a–v. BACE inhibitor IV was used as a control.

Cpd

IC₅₀(µM)

1.5 ± 0.3

–

Donepezil HCl

7

8a

8b

8c

8d

8e

8f

95 ± 12

~100

6.1 ± 0.1

~100

7.9 ± 0.9

7.9 ± 2.4

–

8g

8h

8i

58 ± 1

58 ± 2

–

8j

8k

8l

–

3.4 ± 0.1

~100

8m

8n

8o

8p

8q

8r

12 ± 3

21 ± 4

34 ± 9

37 ± 5

~100

8s

8t

30 ± 8

169 ± 2

91 ± 14

29 ± 6

8u

8v

BACE1 inhibitor IV 0.63

±

0.18 nM

Molecules 2018, 23, 3252

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2.4. BACE1 Modeling

To aid in the understanding of donepezil and its analogues’ inhibitory activity of BACE1, we used

SwissDock to perform some modeling studies. Figure 1 shows the crystal structure (PDB# 4FM7 [25],

with a published inhibitor of BACE1 (published IC₅₀value = 0.1 µM). This inhibitor shares the

vicinyl dioxygen-substituted phenyl ring found in donepezil. Based on the results of the modeling,

the aromatic ring of donepezil aligns with that of the inhibitor originally co-crystallized with BACE1

(Figure 1A,B). When looking at the docking of donepezil (Figure 1C), it is apparent that is binds in a

similar location to the reported co-crystallized inhibitor (Figure 1 B), albeit not as tightly as apparent by

the IC₅₀values, which are 10-fold different. When examining the docking of compound 8l (Figure 1D),

it is slightly twisted, likely due to the bulky 4-bromobenzyl substitution. This slight torsion could

explain the roughly doubled IC₅₀value of compound 8l when compared to that of donepezil. Based on

the modeling, there is also room for more optimization at this location, reasoning that modiﬁcations of

donepezil have the potential to yield better inhibitors than the parent compound if modiﬁed correctly.

A

B

C

D

Figure 1. Molecular docking showing the overlay of donepezil (green) and compound 8l (navy blue)

with the known BACE1 inhibitor (gray) crystallized with BACE1 (PDB# 4FM7 [25]), shown as ribbons.

Panel

A shows the three compounds in the active site of BACE1. Panels B–D show the zoomed-in view

of the known inhibitor (B), donepezil (C), and compound 8l (D).

3. Materials and Methods

3.1. General Information

All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), Alfa Aesar (Ward Hill,

MA, USA), and AK scientiﬁc (Union, CA, USA), and used without further puriﬁcation. Chemical

reactions were monitored by thin layer chromatography (TLC) using Merck (Darmstadt, Germany),

Silica gel 60 F₂₅₀plates. Visualization was achieved using UV (model UVGL-58, UVP, Upland, CA,

USA) light and a ceric molybdate stain (5 g (NH₄)₂Ce(NO₃)₆, 120 g (NH₄)₆Mo₇O₂₄4H₂O, 80 mL

H₂SO₄, 720 mL H₂O). ¹H and ¹³C-NMR spectra were recorded at 400 and 100 MHz, respectively, on a

Varian 400 MHz spectrometer (Varian, Palo Alto, CA, USA), using the indicated deuterated solvents.

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Chemical shifts (δ) are given in parts per million (ppm). Coupling constants (J) are given in Hertz (Hz),

and conventional abbreviations used for signal shape are as follows: s = singlet; d = doublet; t = triplet;

m = multiplet; dd = doublet of doublets; ddd = doublet of doublet of doublets; br s = broad singlet;

dt = doublet of triplets. High-resolution mass spectra were recorded on an AB SCIEX Triple TOF 5600

System (AB SCIEX, Framingham, MA, USA). The purity of the compound was further conﬁrmed to

be

≥

95% by RP-HPLC (model 1260 Inﬁnity, Agilent, Santa Clara, CA, USA) by using the following

= 254 nm; column = Vydac 201SP^TMC18, 250

4.6 mm, 90A 5 m;

method: Flow rate = 0.5 mL/min;

λ

×

µ

eluents: A = H₂O + 0.1% TFA, B = MeCN; gradient proﬁle: starting from 5% B, increasing from 5% to

100% B over 17 min, holding at 100% for 5 min, decreasing from 100% to 5% over 3 min. Prior to each

injection, the HPLC column was equilibrated for 5 min with 5% B.

3.2. Synthesis of Compounds 2–8v

3.2.1. 3-(4-Hydroxy-3-methoxyphenyl)propanoic acid (2)

A catalytic amount of 10% Pd/C (0.43 g) was added to a solution of ferulic acid (1, 6.0 g, 30.9 mmol)

in degassed EtOAc (100 mL). The reaction ﬂask was then sealed with a rubber septum and freed

of air. The reaction mixture was stirred at room temperature (RT) overnight under H₂atmosphere.

Upon completion, the reaction mixture was ﬁltered through a bed of celite, and concentrated to

1

afford the known compound

which matches the literature [26], Figure S1)

aromatic), 6.69 (s, 1H, aromatic), 6.68 (d, J = 7.6 Hz, 1H, aromatic), 5.60 (very br s, 1H, O

2

[

26] (6.1 g, quant.) as an off-white solid: H-NMR (400 MHz, CDCl₃,

δ

10.50 (very br s, 1H, CO₂

H

), 6.82 (d, J = 7.6 Hz, 1H,

), 3.85 (s, 3H,

H

PhOCH₃), 2.87 (t, J = 7.2 Hz, 2H, PhCH₂CH₂CO₂H), 2.64 (t, J = 7.2 Hz, 2H, PhCH₂CH₂CO₂H).

3.2.2. 6-Hydroxy-5-methoxy-2,3-dihydroinden-1-one (3)

A solution of compound

2 (6.3 g, 32.1 mmol) in methanesulfonic acid (50 mL) was reﬂuxed at

120 ^◦C for 1 h. After cooling to RT, the reaction mixture was poured into ice-water, stirred for 5 min,

and ﬁltered to afford a crude dark brown solid, which was recrystallized from EtOH to afford the

1

known compound

3

[

20] (3.8 g, 67%) as a yellow solid: H-NMR (400 MHz, (CD₃)₂SO, which matches

the lit. [20], Figure S2)

δ

9.38 (s, 1H, OH), 7.03 (s, 1H, aromatic), 6.89 (s, 1H, aromatic), 3.83 (s, 3H,

OCH₃), 2.92 (t, J = 5.6 Hz, 2H, CH₂CH₂C=O), 2.49 (t, J = 5.6 Hz, 2H, CH₂CH₂C=O).

3.2.3. 6-[tert-Butyl(dimethyl)silyl]oxy-5-methoxy-2,3-dihydroinden-1-one (4)

TBDMSCl (3.2 g, 21.3 mmol) was added to a solution of compound

3 (1.9 g, 10.7 mmol),

DMAP (0.5 g, 4.3 mmol) and Et₃N (3.0 mL, 21.3 mmol) in freshly distilled CH₂Cl₂(100 mL). The reaction

mixture was stirred at RT overnight before being quenched with H₂O (100 mL). The organic layer

was separated, washed with H₂O (2

×

100 mL) and brine (100 mL), dried over anhydrous Mg₂SO₄,

ﬁltered, and concentrated under reduced pressure to afford a crude dark brown solid, which was

puriﬁed by ﬂash column chromatography (SiO₂gel, pure hexanes to hexanes:EtOAc/3:1, R_f0.44 in

Hexanes:EtOAc/3:1) to yield a brown solid, which was further triturated in hexanes to give compound

4

(2.8 g, 90%) as a white solid: ¹H-NMR (400 MHz, CDCl₃, Figure S3)

aromatic), 3.87 (s, 3H, PhOC ₃), 3.02 (app. t, J = 5.6 Hz, 2H, C

CH₂CH₂C=O), 0.98 (s, 9H, SiC(CH₃)₃), 0.14 (s, 6H, Si(CH₃)₂); ¹³C-NMR (100 MHz, CDCl₃, Figure S4)

205.7 (C=O), 157.5 (C), 150.9 (C), 145.2 (C), 130.0 (C), 114.1 (CH), 107.8 (CH), 55.6 (CH₃), 36.6 (CH₂),

δ

7.17 (s, 1H, aromatic), 6.84 (s, 1H,

H

H₂CH₂C=O), 2.64 (app. t, J = 5.6 Hz, 2H,

δ

25.62 (CH₃, three carbons), 25.56 (CH₂), 18.4 (C),

[M + H]⁺293.1567; found 293.1563.

−

4.7 (CH₃, two carbons); m/z calcd. for C₁₆H₂₅O₃Si⁺

3.2.4. (E)-2-[(1-Benzylpiperidin-4-yl)methylene]-6-hydroxy-5-methoxy-2,3-dihydroinden-1-one (6)

To a solution of compound 4 (1.00 g, 3.42 mmol) and N-benzylpiperidine-4-carboxaldehyde (5,

0.68 mL, 3.42 mmol) in EtOH (10 mL) was added KOH (0.5 g), and the mixture was reﬂuxed at 65 ^◦C.

After 1 h, the reaction was analyzed by TLC (CH₂Cl₂:MeOH/19:1, R_f0.30 in CH₂Cl₂:MeOH/19:1).

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The reaction mixture was concentrated under reduced pressure to give a crude yellow solid, which was

re-dissolved in H₂O (10 mL). 1 N aqueous HCl was then slowly added until pH 5 to yield a yellow

precipitate, which was recrystallized in MeCN to afford compound

¹H-NMR (400 MHz, CDCl₃, Figure S5)

7.32–7.24 (m, 6H, aromatic), 6.87 (s, 1H, aromatic), 6.63 (d,

J = 10.0 Hz, 1H, C=C ), 5.70 (br s, 1H, O ), 3.98 (s, 3H, OCH₃), 3.56 (s, 2H), 3.51 (s, 2H), 2.91 (d,

J = 11.6 Hz, 2H), 2.30 (m, 1H), 2.04 (t, J = 11.6 Hz, 2H), 1.70–1.60 (m, 4H); 13C-NMR (100 MHz, CDCl₃,

Figure S6) 192.6 (C=O), 152.6 (C), 145.8 (C), 143.4 (C), 139.9 (C), 138.2 (C), 135.5 (CH), 132.5 (CH),

6 (0.81 g, 65%) as a yellow solid:

δ

H

δ

129.2 (CH, two carbons), 128.2 (CH, two carbons), 127.0 (C), 108.7 (CH), 106.8 (CH), 63.5 (CH₂),

56.2 (CH₃), 53.1 (CH₂, two carbons), 37.2 (CH₂), 31.2 (CH₂, two carbons), 29.5 (CH); m/z calcd. for

C₂₃H₂₆NO₃⁺[M + H]⁺364.1907; found 364.1909.

3.2.5. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-hydroxy-5-methoxy-2,3-dihydroinden-1-one (7)

To a solution of compound

(wet support, Sigma 520829-10G, 10 mg). The reaction ﬂask was then sealed with a rubber septum

and freed of air. Thioanisole (14.2 L of a stock solution comprising

10⁻⁷mL, obtained using 5

14.2 L of thioanisole in 50 mL of anhydrous THF) was added, and the reaction mixture was stirred

at RT overnight under H₂atmosphere. Upon completion, the reaction mixture was ﬁltered through

a bed of celite, and concentrated to yield the known compound (96 mg, 94%) as a yellow solid:

¹H-NMR (400 MHz, CDCl₃, Figure S7)

7.30–7.20 (m, 6H, aromatic), 6.82 (s, 1H, aromatic), 3.96 (s,

6 (101 mg, 0.28 mmol) in degassed THF (2.5 mL), 10% Pd/C was added

×

µ

7

δ

3H, OCH₃), 3.49 (s, 2H, NCH₂Ph), 3.20 (dd, J₁= 18.0 Hz, J₂= 7.6 Hz, 1H), 2.87 (m, 2H), 2.66 (dt,

J₁= 13.6 Hz, J₂= 3.6 Hz, 2H), 1.98–1.82 (m, 3H), 1.72–1.63 (m, 2H), 1.48 (m, 1H), 1.39–1.24 (m, 3H);

13C-NMR (100 MHz, CDCl₃, Figure S8) δ 207.8 (C=O), 152.9 (C), 147.6 (C), 145.8 (C), 138.3 (C), 130.0 (C),

129.3 (CH, two carbons), 128.1 (CH, two carbons), 126.9 (CH), 108.1 (CH), 106.9 (CH), 63.4 (CH₂),

56.2 (CH₃), 53.7 (CH₂, two carbons), 45.3 (CH), 38.7 (CH₂), 34.4 (CH₂), 33.4 (CH₂), 32.9 (CH₂), 31.7 (CH);

+

m/z calcd. for C₂₃H₂₈NO₃[M + H]⁺366.2064; found 366.2065. The purity of the compound was

further conﬁrmed by RP-HPLC: R_t= 17.17 min (96%; Figure S9).

3.2.6. tert-Butyl N-(3-chloropropyl)carbamate (Boc-protected 3-chloropropylamine).

A solution of NaHCO₃(5.9 g, 70.8 mmol) in H₂O (15 mL) was slowly added to a mixture of

3-chloropropylamine hydrochloride (1.0 g, 7.69 mmol), Boc₂O (3.0 g, 13.8 mmol) and 1,4-dioxane

◦

(10 mL). The resulting mixture was stirred at 60 C for 3 h. The reaction mixture was then diluted with

H₂O, and extracted with EtOAc (3

×

). The combined organic layers were washed with H₂O (3 ) and

×

brine (3 ), dried over anhydrous MgSO₄, ﬁltered, and concentrated under reduced pressure. The crude

×

product obtained was puriﬁed by column chromatography (SiO₂gel, hexanes:EtOAc/5:1; R_f0.31 in

hexanes:EtOAc/5:1) to yield the known compound tert-butyl N-(3-chloropropyl)carbamate [27] (0.55 g,

1

36%) as a colorless oil: H-NMR (400 MHz, CDCl₃, which matches the lit. [27], Figure S10)

δ 4.65

(br s, 1H, N

H), 3.56 (t, J = 6.4 Hz, 2H, ClCH₂CH₂), 3.26 (q, J = 6.4 Hz, 2H, CH₂CH₂NHBoc), 1.94 (p,

J = 6.4 Hz, 2H, CH₂CH₂CH₂), 1.42 (s, 9H, C(CH₃)₃).

3.2.7. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(3-tert-butyl-N-propylcarbamate)oxy]-5-methoxy-2,3-

dihydroinden-1-one (Boc-protected compound 8a)

A solution of compound

1.18 mmol), Cs₂CO₃(575 mg, 1.76 mmol), and TBAI (109 mg, 0.29 mmol) in anhydrous DMF (5 mL) was

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc (3 ).

The combined organic layers were washed with H₂O (3 ) and brine (3 ), dried over anhydrous MgSO₄,

7 (215 mg, 0.59 mmol), tert-butyl N-(3-chloropropyl)carbamate (228 mg,

×

filtered, and concentrated under reduced pressure. The crude product obtained was purified by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.55 in CH₂Cl₂:MeOH/9:1) to yield

2-[(1-benzylpiperidin-4-yl)methyl]-6-[(3-tert-butyl-N-propylcarbamate)oxy]-5-methoxy-2,3-dihydroinden-

1-one (276 mg, 90%) as a white foam: ¹H-NMR (400 MHz, CDCl₃, Figure S11)

7.11 (s, 1H, aromatic), 6.82 (s, 1H, aromatic), 5.52 (very br t, 1H, N

δ

7.30–7.20 (m, 5H, aromatic),

), 4.08 (t, J = 5.6 Hz, 2H, OCH₂CH₂),

H

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3.93 (s, 3H, OCH₃), 3.49 (s, 2H, NCH₂Ph), 3.34 (q, J = 5.2 Hz, 2H, CH₂CH₂NHBoc), 3.20 (dd, J₁= 17.6 Hz,

J₂= 8.0 Hz, 1H), 2.88 (m, 2H), 2.67 (m, 2H), 2.02–1.85 (m, 5H), 1.72–1.62 (m, 2H), 1.44 (s, 9H, C(CH₃)₃),

1.36–1.02 (m, 4H); 13C-NMR (100 MHz, CDCl₃, Figure S12)

δ 207.7 (C=O), 156.0 (CH), 155.6 (C=O),

148.9 (C), 148.4 (C), 138.3 (C), 129.2 (CH, two carbons), 128.1 (CH, three carbons), 126.9 (C), 107.4 (CH),

105.3 (CH), 78.9 (C), 68.3 (CH₂), 63.4 (CH₂), 56.0 (CH₃), 53.74 (CH₂), 53.72 (CH₂), 45.4 (CH), 39.1 (CH₂),

38.7 (CH₂), 34.4 (CH₂), 33.4 (CH₂), 33.0 (CH₂), 31.7 (CH), 29.0 (CH₂), 28.5 (CH₃, three carbons); m/z calcd.

for C₃₁H₄₃N₂O₅⁺[M + H]⁺523.3166; found 523.3131.

3.2.8. 6-[(3-Aminopropyl)oxy]-2-[(1-benzylpiperidin-4-yl)methyl]-5-methoxy-2,3-dihydroinden-1-

one (8a)

A solution of the 2-[(1-benzylpiperidin-4-yl)methyl]-6-[(3-tert-butyl-N-propylcarbamate)oxy]-5-

methoxy-2,3-dihydroinden-1-one (100 mg, 0.19 mmol) in CH₂Cl₂(2 mL) was treated with TFA (1 mL)

and allowed to stir at RT for 5 min. The reaction was then quenched by addition of saturated aqueous

NaHCO₃and the resulting mixture was extracted with CH₂Cl₂(3 ). The combined organic layers

×

were washed with brine, dried over anhydrous MgSO₄, ﬁltered, and concentrated under reduced

pressure. The crude product obtained was puriﬁed by column chromatography (SiO₂gel, pure CH₂Cl₂

to CH₂Cl₂:MeOH/19:1; R_f0.12 in CH₂Cl₂:MeOH/9:1) to yield compound 8a (53 mg, 65%) as a white

1

solid: H-NMR (400 MHz, CDCl₃, Figure S13)

δ 7.28–7.18 (m, 5H, aromatic), 7.13 (s, 1H, aromatic),

6.81 (s, 1H, aromatic), 4.09 (t, J = 6.4 Hz, 2H, OCH₂CH₂), 3.90 (s, 3H, OCH₃), 3.47 (s, 2H, NCH₂Ph),

3.19 (dd, J₁= 17.6 Hz, J₂= 8.0 Hz, 1H), 2.84–2.90 (m, 4H), 2.67 (m, 2H), 1.99–1.84 (m, 7H), 1.72–1.58

(m, 2H), 1.45 (m, 1H), 1.38–1.23 (m, 3H); 13C-NMR (100 MHz, CDCl₃, Figure S14)

δ 207.8 (C=O),

155.7 (C), 148.73 (C), 148.66 (C), 138.4 (C), 129.2 (CH, three carbons), 128.1 (CH, two carbons), 126.9 (C),

107.5 (CH), 105.6 (CH), 67.4 (CH₂), 63.4 (CH₂), 56.2 (CH₃), 53.76 (CH₂), 53.74 (CH₂), 45.4 (CH),

39.4 (CH₂), 38.7 (CH₂), 34.4 (CH₂), 33.3 (CH₂), 33.0 (CH₂), 32.5 (CH₂), 31.8 (CH); m/z calcd. for

+

C₂₆H₃₅N₂O₃[M + H]⁺423.2642; found 423.2656. The purity of the compound was further conﬁrmed

by RP-HPLC: R_t= 15.86 min (96%; Figure S15).

3.2.9. tert-Butyl N-(2-chloroethyl)carbamate.

A solution of NaHCO₃(6.7 g, 79.3 mmol) in H₂O (15 mL) was slowly added to a mixture

of 2-chloroethylamine hydrochloride (1.0 g, 8.6 mmol), Boc₂O (3.4 g, 15.5 mmol) and 1,4-dioxane

◦

(10 mL) at 0 C. The resulting mixture was allowed to warm to RT and was stirred overnight.

The reaction mixture was then diluted with H₂O, and extracted with CH₂Cl₂(3

×

). The combined

organic layers were washed with H₂O (3 ) and brine (3 ), dried over anhydrous MgSO₄, ﬁltered,

×

and concentrated under reduced pressure. The crude product obtained was puriﬁed by column

chromatography (SiO₂gel, Hexanes:EtOAc/9:1; R_f0.55 in hexanes:EtOAc/4:1) to yield the known

compound tert-butyl N-(2-chloroethyl)carbamate [28] (1.25 g, 83%) as a colorless oil: ¹H-NMR

(400 MHz, CDCl₃, which matches the lit. [28], Figure S16)

δ 4.94 (br s, 1H, NH), 3.57 (t, J = 6.0

Hz, 2H, ClCH₂CH₂), 3.44 (q, J = 6.0 Hz, 2H, CH₂CH₂NHBoc), 1.47 (s, 9H, C(CH₃)₃).

3.2.10. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(3-tert-butyl-N-ethylcarbamate)oxy]-5-methoxy-2,3-

dihydroinden-1-one (Boc-protected compound 8b).

A solution of compound

(56 mg, 0.15 mmol) in anhydrous DMF (5 mL) was stirred at RT overnight. The reaction mixture

was then diluted with H₂O, and extracted with EtOAc (3 ). The combined organic layers were

washed with H₂O (3 ) and brine (3 ), dried over anhydrous MgSO₄, filtered, and concentrated under

7 (216 mg, 1.20 mmol), Cs₂CO₃(196 mg, 0.60 mmol), and TBAI

×

reduced pressure. The crude product obtained was purified by column chromatography (SiO₂gel,

pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.48 in CH₂Cl₂:MeOH/9:1) to yield 2-[(1-benzylpiperidin-

4-yl)methyl]-6-[(3-tert-butyl-N-ethylcarbamate)oxy]-5-methoxy-2,3-dihydroinden-1-one (93 mg, 61%) as

1

a pale yellow solid: H-NMR (400 MHz, CDCl₃, Figure S17)

δ

7.34–7.20 (m, 5H, aromatic), 7.14 (s, 1H,

aromatic), 6.83 (s, 1H, aromatic), 5.05 (m, 1H, N

H

), 4.05 (br t, 2H, OC ₂CH₂), 3.92 (s, 3H, OC ₃), 3.53 (m,

H

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4H, NCH₂Ph, CH₂CH₂NHBoc), 3.21 (dd, J₁= 17.2 Hz, J₂= 8.4 Hz, 1H), 2.90 (m, 2H), 2.67 (m, 2H), 1.99 (m,

2H), 1.88 (m, 1H), 1.69 (m, 2H), 1.43 (m, 10H), 1.37–1.23 (m, 3H); 13C-NMR (100 MHz, CDCl₃, Figure S18)

δ

207.6 (C=O), 155.8 (C and C=O), 149.2 (C), 148.3 (C), 138.3 (C), 129.3 (CH, two carbons), 128.1 (CH,

three carbons), 126.9 (C), 107.7 (CH), 106.7 (CH), 79.5 (C), 68.8 (CH₂), 63.4 (CH₂), 56.1 (CH₃), 53.7 (CH₂,

two carbons), 45.4 (CH), 39.9 (CH₂), 38.7 (CH₂), 34.4 (CH₂), 33.3 (CH₂), 32.9 (CH₂), 31.7 (CH), 28.4 (CH₃,

three carbons); m/z calcd. for C₃₀H₄₁N₂O₅⁺[M + H]⁺509.3010; found 509.3025.

3.2.11. 6-[(3-Aminoethyl)oxy]-2-[(1-benzylpiperidin-4-yl)methyl]-5-methoxy-2,3-dihydroinden-1-

one (8b)

A solution of 2-[(1-benzylpiperidin-4-yl)methyl]-6-[(3-tert-butyl-N-ethylcarbamate)oxy]-5-

methoxy-2,3-dihydroinden-1-one (83 mg, 0.16 mmol) in CH₂Cl₂(1 mL) was treated with TFA (1 mL)

and allowed to stir at RT. After 1 h, the reaction was analyzed by TLC (CH₂Cl₂:MeOH/9:1, R_f

0.19 in CH₂Cl₂:MeOH/9:1). The reaction was then quenched by addition of saturated aqueous

NaHCO₃and the resulting mixture was extracted with CH₂Cl₂(3 ). The combined organic layers

×

were washed with brine, dried over anhydrous MgSO₄, ﬁltered, and concentrated under reduced

1

pressure to yield compound 8b (62 mg, 93%) as a white solid: H-NMR (400 MHz, CDCl₃, Figure

S19)

δ 7.32–7.20 (m, 5H, aromatic), 7.15 (s, 1H, aromatic), 6.83 (s, 1H, aromatic), 4.03 (t, J = 5.2 Hz,

2H, OCH₂CH₂), 3.92 (s, 3H, OCH₃), 3.57 (s, 2H, NCH₂Ph), 3.21 (dd, J₁= 17.6 Hz, J₂= 8.0 Hz, 1H),

3.11 (t, J = 5.2 Hz, 2H, OCH₂CH₂), 2.95 (m, 2H), 2.67 (m, 2H), 2.03 (m, 2H), 1.89 (m, 1H), 1.71 (m,

2H), 1.52 (m, 2H), 1.44–1.23 (m, 4H); ¹³C-NMR (100 MHz, CDCl₃, Figure S20)

δ 207.7 (C=O), 155.8 (C),

148.9 (C), 148.6 (C), 138.1 (C), 129.3 (CH, four carbons), 129.2 (CH), 128.1 (CH, two carbons), 127.0 (C),

107.6 (CH), 106.0 (CH), 71.1 (CH₂), 63.3 (CH₂), 56.1 (CH₃), 53.68 (CH₂), 53.65 (CH₂), 45.4 (CH),

+

41.2 (CH₂), 38.6 (CH₂), 34.3 (CH₂), 33.3 (CH₂), 32.8 (CH₂), 31.7 (CH); m/z calcd. for C₂₅H₃₃N₂O₃

[M + H]⁺409.2486; found 409.2496. The purity of the compound was further conﬁrmed by RP-HPLC:

R_t= 15.74 min (95%; Figure S21).

3.2.12. 2-[(1-Benzylpiperidin-4-yl)methyl]-5-methoxy-6-propoxy-2,3-dihydroinden-1-one (8c).

A solution of compound

7 (50 mg, 0.14 mmol) and K₂CO₃(95 mg, 0.68 mmol) in anhydrous

DMF (5 mL) was treated with 1-bromopropane (0.06 mL, 0.68 mmol), and the resulting mixture

was stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with

EtOAc (3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3 ), dried over

×

anhydrous MgSO₄, ﬁltered, and concentrated under reduced pressure. The crude product obtained

was puriﬁed by column chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.38 in

CH₂Cl₂:MeOH/19:1) to yield compound 8c (53 mg, 95%) as an off-white solid: H-NMR (400 MHz,

1

CDCl₃, Figure S22) δ 7.32–7.20 (m, 5H, aromatic), 7.13 (s, 1H, aromatic), 6.82 (s, 1H, aromatic), 3.98 (t,

J = 6.8 Hz, 2H, CH₃CH₂CH₂OAr), 3.92 (s, 3H, OCH₃), 3.51 (s, 2H, NCH₂Ph), 3.20 (dd, J₁= 17.6 Hz,

J₂= 8.4 Hz, 1H), 2.90 (m, 2H), 2.66 (dt, J₁= 14.0 Hz, J₂= 3.6 Hz, 2H), 1.98 (m, 2H), 1.90 (m, 1H),

1.85 (sextet, J = 7.2 Hz, 2H, CH₃C

H

₂CH₂OAr), 1.73–1.64 (m, 2H), 1.49 (m, 1H), 1.40–1.24 (m, 3H), 1.02 (t,

207.8 (C=O), 155.8 (C),

J = 7.2 Hz, 3H, CH₃CH₂CH₂OAr); ¹³C-NMR (100 MHz, CDCl₃, Figure S23)

δ

148.9 (C), 148.5 (C), 129.3 (CH, two carbons), 129.2 (C), 128.2 (CH, three carbons), 127.1 (C), 107.5 (CH),

105.5 (CH), 70.5 (CH₂), 63.2 (CH₂), 56.2 (CH₃), 53.7 (CH₂), 53.6 (CH₂), 45.4 (CH), 38.6 (CH₂), 34.3

+

(CH₂), 33.3 (CH₂), 32.7 (CH₂), 31.6 (CH), 22.2 (CH₂), 10.3 (CH₃); m/z calcd. for C₂₆H₃₄NO₃[M + H]⁺

408.2533; found 408.2524. The purity of the compound was further conﬁrmed by RP-HPLC: R_t= 19.30

min (96%; Figure S24).

3.2.13. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(chloroethyl)oxy]-5-methoxy-2,3-dihydroinden-1-one (8d

)

A solution of compound (50 mg, 0.14 mmol) and K₂CO₃(189 mg, 1.37 mmol) in anhydrous

7

DMF (5 mL) was treated with 1,2-dichloroethane (0.11 mL, 1.37 mmol), and the resulting mixture

was stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with

Molecules 2018, 23, 3252

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CH₂Cl₂(3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3

×

), dried over

anhydrous MgSO₄, ﬁltered, and concentrated under reduced pressure. The crude product obtained

was puriﬁed by column chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.38 in

CH₂Cl₂:MeOH/19:1) to yield compound 8d (50 mg, 85%) as a brown oil: H-NMR (400 MHz, CDCl₃,

1

Figure S25)

2H), 3.91 (s, 3H, OC

1H), 2.88 (m, 2H), 2.66 (dt, J₁= 14.4 Hz, J₂= 2.8 Hz, 2H), 1.99–1.93 (m, 2H), 1.92–1.85 (m, 1H), 1.72–1.63

(m, 2H), 1.47 (m, 1H), 1.40–1.23 (m, 3H); ¹³C-NMR (100 MHz, CDCl₃, Figure S26)

207.6 (C=O),

δ

7.30–7.20 (m, 5H, aromatic), 7.14 (s, 1H, aromatic), 6.84 (s, 1H, aromatic), 4.25 (t, J = 6.0 Hz,

H

₃), 3.82 (t, J = 6.0 Hz, 2H), 3.49 (s, 2H, NC ₂Ph), 3.20 (dd, J₁= 17.6 Hz, J₂= 8.4 Hz,

H

δ

155.9 (C), 149.5 (C), 148.0 (C), 138.1 (C), 129.3 (CH, two carbons), 129.2 (CH), 128.1 (CH, two carbons),

127.0 (C), 107.9 (CH), 106.6 (CH), 69.0 (CH₂), 63.3 (CH₂), 56.2 (CH₃), 53.68 (CH₂), 53.65 (CH₂), 45.4 (CH),

+

41.4 (CH₂), 38.6 (CH₂), 34.3 (CH₂), 33.3 (CH₂), 32.8 (CH₂), 31.7 (CH); m/z calcd. for C₂₅H₃₁ClNO₃

[M + H]⁺428.1987; found 428.1984. The purity of the compound was further conﬁrmed by RP-HPLC:

R_t= 18.90 min (96%; Figure S27).

3.2.14. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(bromoethyl)oxy]-5-methoxy-2,3-dihydroinden-1-

one (8e)

A solution of compound 7 (100 mg, 0.27 mmol) and K₂CO₃(380 mg, 2.74 mmol) in anhydrous

DMF (5 mL) was treated with 1,2-dibromoethane (0.24 mL, 2.74 mmol) and the resulting mixture

was stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with

CH₂Cl₂(3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3 ), dried over

×

anhydrous MgSO₄, ﬁltered, and concentrated under reduced pressure. The crude product obtained

was puriﬁed by column chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.49 in

CH₂Cl₂:MeOH/19:1) to yield compound 8e (70 mg, 54%) as an off-white solid: H-NMR (400 MHz,

1

CDCl₃, Figure S28)

J = 6.4 Hz, 2H), 3.93 (s, 3H, OC

J₂= 8.0 Hz, 1H), 2.88 (m, 2H), 2.67 (dt, J₁= 14.0 Hz, J₂= 2.8 Hz, 2H), 1.98–1.86 (m, 3H), 1.72–1.62 (m,

2H), 1.47 (m, 1H), 1.40–1.24 (m, 3H); ¹³C-NMR (100 MHz, CDCl₃, Figure S29)

207.6 (C=O), 155.9 (C),

δ

7.30-7.20 (m, 5H, aromatic), 7.15 (s, 1H, aromatic), 6.85 (s, 1H, aromatic), 4.32 (t,

H

₃), 3.65 (t, J = 6.4 Hz, 2H), 3.49 (s, 2H, NC ₂Ph), 3.21 (dd, J₁= 17.6 Hz

H

,

δ

149.5 (C), 147.8 (C), 138.4 (C), 129.2 (CH, 3 carbons), 128.1 (CH, 2 carbons), 126.9 (C), 108.0 (CH),

106.6 (CH), 68.8 (CH₂), 63.4 (CH₂), 56.3 (CH₃), 53.75 (CH₂), 53.72 (CH₂), 45.4 (CH), 38.7 (CH₂),

+

34.4 (CH₂), 33.4 (CH₂), 33.0 (CH₂), 31.8 (CH), 28.4 (CH₂); m/z calcd. for C₂₅H₃₁BrNO₃[M + H]⁺

472.1482; found 472.1477. The purity of the compound was further conﬁrmed by RP-HPLC: R_t= 19.10

min (97%; Figure S30).

3.2.15. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(bromobutyl)oxy]-5-methoxy-2,3-dihydroinden-1-one (8f

)

A solution of compound (50 mg, 0.14 mmol) and K₂CO₃(189 mg, 1.37 mmol) in anhydrous

7

DMF (5 mL) was treated with 1,4-dibromobutane (0.16 mL, 1.37 mmol), and the resulting mixture

was stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with

CH₂Cl₂(3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3 ), dried over

×

anhydrous MgSO₄, ﬁltered, and concentrated under reduced pressure. The crude product obtained

was puriﬁed by column chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.38 in

CH₂Cl₂:MeOH/19:1) to yield compound 8f (63 mg, 93%) as an off-white solid: H-NMR (400 MHz,

1

CDCl₃, Figure S31)

J = 6.4 Hz, 2H), 3.92 (s, 3H, OC

J₂= 8.0 Hz, 1H), 2.90 (m, 2H), 2.67 (m, 2H), 2.10–1.93 (m, 6H), 1.92–1.85 (m, 1H), 1.73–1.64 (m, 2H),

1.48 (m, 1H), 1.40–1.23 (m, 3H); ¹³C-NMR (100 MHz, CDCl₃, Figure S32)

207.8 (C=O), 155.8 (C),

δ

7.32–7.22 (m, 5H, aromatic), 7.13 (s, 1H, aromatic), 6.83 (s, 1H, aromatic), 4.04 (t,

H

₃), 3.52 (s, 2H, NC ₂Ph), 3.48 (t, J = 6.4 Hz, 2H), 3.20 (dd, J₁= 17.6 Hz,

H

δ

148.8 (C), 148.6 (C), 129.4 (CH, two carbons), 129.2 (C), 128.2 (CH, three carbons), 127.2 (C), 107.5 (CH),

105.6 (CH), 68.0 (CH₂), 63.2 (CH₂), 56.2 (CH₃), 53.6 (CH₂, two carbons), 45.3 (CH), 38.6 (CH₂),

34.2 (CH₂), 33.3 (CH₂, two carbons), 32.6 (CH₂), 31.5 (CH), 29.4 (CH₂), 27.6 (CH₂); m/z calcd. for

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C₂₇H₃₅BrNO₃⁺[M + H]⁺500.1795; found 500.1794. The purity of the compound was further conﬁrmed

by RP-HPLC: R_t= 20.22 min (95%; Figure S33).

3.2.16. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(bromododecyl)oxy]-5-methoxy-2,3-dihydroinden-1-

one (8g)

A solution of compound 7 (100 mg, 0.27 mmol) and K₂CO₃(380 mg, 2.74 mmol) in anhydrous

DMF (5 mL) was treated with 1,12-dibromododecane (900 mg, 2.74 mmol) and the resulting mixture

was stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with

CH₂Cl₂(3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3 ), dried over

×

anhydrous MgSO₄, ﬁltered, and concentrated under reduced pressure. The crude product obtained

was puriﬁed by column chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.49 in

CH₂Cl₂:MeOH/19:1) to yield compound 8g (53 mg, 32%) as an off-white solid: H-NMR (400 MHz,

1

CDCl₃, Figure S34)

J = 6.8 Hz, 2H), 3.92 (s, 3H, OC

J₂= 8.0 Hz, 1H), 2.91 (m, 2H), 2.66 (dt, J₁= 14.0 Hz, J₂= 3.6 Hz, 2H), 1.99 (m, 2H), 1.92–1.78 (m, 5H),

1.78–1.62 (m, 3H), 1.44–1.38 (m, 6H), 1.36–1.22 (m, 13H); ¹³C-NMR (100 MHz, CDCl₃, Figure S35)

δ

7.32–7.22 (m, 5H, aromatic), 7.13 (s, 1H, aromatic), 6.82 (s, 1H, aromatic), 3.99 (t,

H

₃), 3.53 (s, 2H, NC ₂Ph), 3.38 (t, J = 6.8 Hz, 2H), 3.20 (dd, J₁= 17.6 Hz,

H

δ

207.8 (C=O), 155.8 (C), 148.9 (C), 148.5 (C), 129.3 (CH, two carbons), 129.2 (C), 128.2 (CH, three carbons),

127.1 (C), 107.4 (CH), 105.5 (CH), 69.1 (CH₂), 63.2 (CH₂), 56.2 (CH₃), 53.6 (CH₂, two carbons),

45.4 (CH), 38.6 (CH₂), 34.3 (CH₂), 34.1 (CH₂), 33.3 (CH₂), 32.8 (CH₂), 32.6 (CH₂), 31.6 (CH), 29.5 (CH₂,

two carbons), 29.4 (CH₂), 29.3 (CH₂), 28.9 (CH₂), 28.7 (CH₂), 28.1 (CH₂), 26.9 (CH₂), 25.9 (CH₂);

+

m/z calcd. for C₃₅H₅₁BrNO₃[M + H]⁺612.3047; found 612.3045. The purity of the compound was

further conﬁrmed by RP-HPLC: R_t= 25.00 min (96%; Figure S36).

3.2.17. 6-[(Benzyl)oxy-2-[(1-benzylpiperidin-4-yl)methyl]-5-methoxy-2,3-dihydroinden-1-one (8h)

A solution of compound

(5 mL) was treated with benzyl bromide (20

at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc (3

The combined organic layers were washed with H₂O (3 ) and brine (3 ), dried over anhydrous

7

(50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

L, 0.16 mmol), and the resulting mixture was stirred

).

µ

×

MgSO₄, and ﬁltered. After standing at RT overnight, white solids precipitated out, which were

ﬁltered off. The ﬁltrate was further concentrated under reduced pressure, and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.37 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8h (51 mg, 82%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S37)

δ

7.42 (d, J = 7.6 Hz, 2H, aromatic), 7.35 (t, J = 7.6 Hz, 2H, aromatic), 7.31–7.28 (m, 5H, aromatic),

7.24 (s, 1H, aromatic), 7.19 (s, 1H, aromatic), 6.85 (s, 1H, aromatic), 5.13 (s, 2H, OCH₂Ph), 3.93 (s,

3H, OCH₃), 3.51 (s, 2H, NCH₂Ph), 3.20 (dd, J₁= 17.6 Hz, J₂= 8.4 Hz, 1H), 2.89 (m, 2H), 2.63 (dt,

J₁= 14.0 Hz

,

J₂= 4.0 Hz, 2H), 1.97 (m, 2H), 1.88 (m, 1H), 1.73–1.64 (m, 2H), 1.48 (m, 1H), 1.40–1.23 (m,

207.6 (C=O), 156.0 (C), 148.9 (C), 148.5 (C), 136.3 (C),

3H); ¹³C-NMR (100 MHz, CDCl₃, Figure S38)

δ

129.4 (CH, two carbons), 129.2 (C), 128.6 (CH, two carbons), 128.2 (CH, two carbons), 128.0 (CH,

two carbons), 127.4 (CH, two carbons), 127.1 (C), 107.6 (CH), 106.4 (CH), 70.8 (CH₂), 63.2 (CH₂),

56.2 (CH₃), 53.6 (CH₂, two carbons), 45.3 (CH), 38.6 (CH₂), 34.3 (CH₂), 33.4 (CH₂), 32.7 (CH₂), 31.6 (CH);

+

m/z calcd. for C₃₀H₃₄NO₃[M + H]⁺456.2533; found 456.2528. The purity of the compound was

further conﬁrmed by RP-HPLC: R_t= 20.05 min (96%; Figure S39).

3.2.18. 2-[(1-Benzylpiperidin-4-yl)methyl]-5-methoxy-6-[(4-methylbenzyl)oxy-2,3-dihydroinden-1-

one (8i)

A solution of compound

7 (50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

(5 mL) was treated with 4-methylbenzyl bromide (30 mg, 0.16 mmol), and the resulting mixture was

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with CH₂Cl₂

(3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3

×

), dried over anhydrous

MgSO₄, and ﬁltered. After standing at RT overnight, the white solids precipitated out, which were

Molecules 2018, 23, 3252

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ﬁltered off. The ﬁltrate was further concentrated under reduced pressure, and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.35 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8i (37 mg, 58%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S40)

δ 7.31

(d, J = 8.0 Hz, 2H, aromatic), 7.31–7.29 (m, 4H, aromatic), 7.24 (s, 1H, aromatic), 7.18 (s, 1H, aromatic),

7.15 (d, J = 8.0 Hz, 2H, aromatic), 6.83 (s, 1H, aromatic), 5.09 (s, 2H, OCH₂Ph), 3.92 (s, 3H, OCH₃),

3.51 (s, 2H, NC

Hz, 2H), 2.32 (s, 3H, C

(m, 3H); ¹³C-NMR (100 MHz, CDCl₃, Figure S41)

H

₂Ph), 3.20 (dd, J₁= 17.6 Hz, J₂= 8.4 Hz, 1H), 2.89 (m, 2H), 2.63 (dt, J₁= 13.6 Hz, J₂= 3.6

₃Ph), 1.97 (m, 2H), 1.91–1.84 (m, 1H), 1.72–1.64 (m, 2H), 1.47 (m, 1H), 1.40–1.23

207.6 (C=O), 156.0 (C), 148.9 (C), 148.5 (C), 137.8 (C),

H

δ

133.3 (C), 129.30 (CH, two carbons), 129.25 (CH, two carbons), 129.17 (C), 128.2 (CH, three carbons),

127.5 (CH, 2 carbons), 127.0 (C), 107.6 (CH), 106.4 (CH), 70.7 (CH₂), 63.3 (CH₂), 56.2 (CH₃), 53.7 (CH₂,

2 carbons), 45.4 (CH), 38.7 (CH₂), 34.3 (CH₂), 33.4 (CH₂), 32.8 (CH₂), 31.7 (CH), 21.2 (CH₃); m/z calcd. for

+

C₃₁H₃₆NO₃[M + H]⁺470.2690; found 470.2681. The purity of the compound was further conﬁrmed

by RP-HPLC: R_t= 20.65 min (95%; Figure S42).

3.2.19. 2-[(1-Benzylpiperidin-4-yl)methyl]-5-methoxy-6-[(4-methoxybenzyl)oxy-2,3-dihydroinden-1-

one (8j)

A solution of compound 7 (50 mg, 0.14 mmol) and K₂CO₃(189 mg, 1.37 mmol) in anhydrous DMF

(5 mL) was treated with 4-methoxybenzyl chloride (0.19 mL, 1.37 mmol), and the resulting mixture

was stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with

CH₂Cl₂(3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3 ), dried over

×

anhydrous MgSO₄, ﬁltered, and concentrated under reduced pressure. The crude product obtained

was puriﬁed by column chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.30 in

CH₂Cl₂:MeOH/19:1) to yield compound 8j (24 mg, 36%) as an off-white solid: H-NMR (400 MHz,

1

CDCl₃, Figure S43)

aromatic), 6.87 (d, J = 8.8 Hz, 2H, aromatic), 6.83 (s, 1H, aromatic), 5.05 (s, 2H, OCH₂Ph), 3.91 (s, 3H,

OC ₃), 3.78 (s, 3H, OC ₃), 3.56 (s, 2H, NC ₂Ph), 3.20 (dd, J₁= 17.6 Hz, J₂= 8.4 Hz, 1H), 2.95 (m, 2H),

2.65 (dt, J₁= 13.6 Hz, J₂= 3.6 Hz, 2H), 2.08–1.98 (m, 2H), 1.92–1.82 (m, 1H), 1.76–1.64 (m, 2H), 1.53 (m,

1H), 1.40–1.23 (m, 3H); ¹³C-NMR (100 MHz, CDCl₃, Figure S44)

207.6 (C=O), 159.5 (C), 156.0 (C),

δ 7.34 (d, J = 8.8 Hz, 2H, aromatic), 7.33–7.26 (m, 5H, aromatic), 7.19 (s, 1H,

H

δ

148.9 (C), 148.5 (C), 137.2 (C), 129.5 (CH, two carbons), 129.2 (CH, two carbons), 129.1 (C), 128.4 (CH),

128.2 (CH, two carbons), 127.3 (C), 114.0 (CH, 2 carbons), 107.6 (CH), 106.5 (CH), 70.6 (CH₂), 63.1 (CH₂),

56.2 (CH₃), 55.3 (CH₃), 53.6 (CH₂), 53.5 (CH₂), 45.3 (CH), 38.6 (CH₂), 34.1 (CH₂), 33.4 (CH₂), 32.4 (CH₂),

31.4 (CH); m/z calcd. for C₃₁H₃₆NO₄[M + H]⁺486.2639; found 486.2635. The purity of the compound

+

was further conﬁrmed by RP-HPLC: R_t= 19.93 min (95%; Figure S45).

3.2.20. 2-[(1-Benzylpiperidin-4-yl)methyl]-5-methoxy-6-[(4-nitrobenzyl)oxy-2,3-dihydroinden-1-

one (8k).

A solution of compound

7 (50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

(5 mL) was treated with 4-nitrobenzyl bromide (35 mg, 0.16 mmol), and the resulting mixture was

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3 ), dried over anhydrous

×

MgSO₄, and ﬁltered. After standing at RT overnight, white solids precipitated out, which were

ﬁltered off. The ﬁltrate was further concentrated under reduced pressure and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.37 in CH₂Cl₂:MeOH/19:1)

1

to yield compound 8k (44 mg, 65%) as a brown foam: H-NMR (400 MHz, CDCl₃, Figure S46)

δ

8.22 (d, J = 8.4 Hz, 2H, aromatic), 7.60 (d, J = 8.4 Hz, 2H, aromatic), 7.31–7.28 (m, 4H, aromatic),

7.24 (s, 1H, aromatic), 7.14 (s, 1H, aromatic), 6.88 (s, 1H, aromatic), 5.23 (s, 2H, OCH₂Ph), 3.97 (s,

3H, OCH₃), 3.50 (s, 2H, NCH₂Ph), 3.22 (dd, J₁= 17.6 Hz, J₂= 8.0 Hz, 1H), 2.89 (m, 2H), 2.68 (m,

2H), 1.96 (m, 2H), 1.87 (m, 1H), 1.72–1.63 (m, 2H), 1.47 (m, 1H), 1.36–1.23 (m, 3H); ¹³C-NMR (100

MHz, CDCl₃, Figure S47)

δ 207.5 (C=O), 155.9 (C), 149.5 (C), 147.8 (C), 147.6 (C), 143.8 (C), 129.3 (CH),

129.2 (CH), 128.2 (CH, two carbons), 127.5 (CH, three carbons), 127.0 (C), 123.9 (CH, two carbons +

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C), 107.9 (CH), 106.6 (CH), 69.5 (CH₂), 63.3 (CH₂), 56.3 (CH₃), 53.7 (CH₂, two carbons), 45.4 (CH),

+

38.6 (CH₂), 34.3 (CH₂), 33.4 (CH₂), 32.8 (CH₂), 31.6 (CH); m/z calcd. for C₃₀H₃₃N₂O₅[M + H]⁺

501.2384; found 501.2385. The purity of the compound was further conﬁrmed by RP-HPLC: R_t= 19.98

min (96%; Figure S48).

3.2.21. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(4-bromobenzyl)oxy-5-methoxy-2,3-dihydroinden-1-

one (8l)

A solution of compound

7 (50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

(5 mL) was treated with 4-bromobenzyl bromide (41 mg, 0.16 mmol) and the resulting mixture was

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3 ), dried over anhydrous

×

MgSO₄, and ﬁltered. After standing at RT overnight, white solids precipitated out, which were

ﬁltered off. The ﬁltrate was further concentrated under reduced pressure and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.37 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8l (61 mg, 84%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S49)

δ

7.47 (d, J = 8.4 Hz, 2H, aromatic), 7.30 (d, J = 8.4 Hz, 2H, aromatic), 7.31–7.28 (m, 4H, aromatic),

7.24 (s, 1H, aromatic), 7.14 (s, 1H, aromatic), 6.85 (s, 1H, aromatic), 5.07 (s, 2H, OCH₂Ph), 3.94 (s,

3H, OCH₃), 3.51 (s, 2H, NCH₂Ph), 3.21 (dd, J₁= 17.6 Hz, J₂= 8.4 Hz, 1H), 2.89 (m, 2H), 2.67 (dt,

J₁= 14.4 Hz, J₂= 4.0 Hz, 2H), 1.96 (m, 2H), 1.87 (m, 1H), 1.72–1.63 (m, 2H), 1.47 (m, 1H), 1.40–1.23 (m,

3H); ¹³C-NMR (100 MHz, CDCl₃, Figure S50)

δ 207.6 (C=O), 155.9 (C), 149.1 (C), 148.2 (C), 135.4 (C),

131.7 (CH, two carbons + C), 129.4 (CH), 129.2 (CH), 129.0 (CH, three carbons), 128.2 (CH, two carbons),

127.1 (C), 122.0 (C), 107.7 (CH), 106.5 (CH), 70.1 (CH₂), 63.2 (CH₂), 56.2 (CH₃), 53.6 (CH₂, two carbons),

45.3 (CH), 38.6 (CH₂), 34.2 (CH₂), 33.4 (CH₂), 32.7 (CH₂), 31.6 (CH); m/z calcd. for C₃₀H₃₃BrNO₃⁺[M

+ H]⁺534.1638; found 534.1650. The purity of the compound was further conﬁrmed by RP-HPLC:

R_t= 21.05 min (96%; Figure S51).

3.2.22. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(4-ﬂuorobenzyl)oxy-5-methoxy-2,3-dihydroinden-1-

one (8m)

A solution of compound

(5 mL) was treated with 4-ﬂuorobenzyl bromide (20

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3 ). The combined organic layers were washed with H₂O (3 ) and brine (3 ), dried over anhydrous

7

(50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

µL, 0.16 mmol), and the resulting mixture was

×

MgSO₄, and ﬁltered. The ﬁltrate was concentrated under reduced pressure and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.31 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8m (56 mg, 86%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S52)

δ 7.40

(dd, J₁= 8.8 Hz, J₂= 5.6 Hz, 2H, aromatic), 7.30 (m, 4H, aromatic), 7.24 (s, 1H, aromatic), 7.18 (s, 1H,

aromatic), 7.04 (t, J = 8.8 Hz, 2H, aromatic), 6.85 (s, 1H, aromatic), 5.08 (s, 2H, OCH₂Ph), 3.93 (s, 3H,

OCH₃), 3.51 (s, 2H, NCH₂Ph), 3.21 (dd, J₁= 17.6 Hz, J₂= 8.4 Hz, 1H), 2.90 (m, 2H), 2.67 (dt, J₁= 14.0

Hz, J₂= 3.6 Hz, 2H), 1.98 (m, 2H), 1.88 (m, 1H), 1.69 (m, 2H), 1.49 (m, 1H), 1.36–1.23 (m, 3H); ¹³C-NMR

(100 MHz, CDCl₃, Figure S53)

δ

207.6 (C=O), 163.8–161.3 (d, ¹J_C-F= 245.2 Hz, C, one carbon), 155.9 (C),

149.1 (C), 148.3 (C), 138.1 (C), 132.14–132.11 (d, ⁴J_C-F= 3.8 Hz, C, one carbon), 129.4–129.27 (d, ³J_C-F

=

8.4 Hz, CH, two carbons), 129.28 (CH, two carbons), 129.2 (CH), 128.2 (CH, two carbons), 127.0 (C),

2

115.6–115.4 (d, J_C-F= 21.2 Hz, CH, two carbons), 107.7 (CH), 106.4 (CH), 70.1 (CH₂), 63.3 (CH₂),

56.2 (CH₃), 53.70 (CH₂), 53.68 (CH₂), 45.4 (CH), 38.7 (CH₂), 34.3 (CH₂), 33.3 (CH₂), 32.8 (CH₂),

31.7 (CH); m/z calcd. C₃₀H₃₃FNO₃⁺[M + H]⁺474.2439; found 474.2442. The purity of the compound

was further conﬁrmed by RP-HPLC: R_t= 20.14 min (96%; Figure S54).

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3.2.23. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(3-ﬂuorobenzyl)oxy-5-methoxy-2,3-dihydroinden-1-

one (8n)

A solution of compound

(5 mL) was treated with 3-ﬂuorobenzyl bromide (20

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3 ). The combined organic layers were washed with H₂O (3 ) and brine (3 ), dried over anhydrous

7

(50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

µL, 0.16 mmol), and the resulting mixture was

×

MgSO₄, and ﬁltered. After standing at RT overnight, the white solids precipitated out, which were

ﬁltered off. The ﬁltrate was further concentrated under reduced pressure, and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.31 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8n (57 mg, 88%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S55)

δ

7.34–7.26 (m, 5H, aromatic), 7.24 (s, 1H, aromatic), 7.18–7.12 (m, 3H, aromatic), 6.98 (td, J₁= 8.4 Hz,

J₂= 2.4 Hz, 1H, aromatic), 6.86 (s, 1H, aromatic), 5.12 (s, 2H, OCH₂Ph), 3.95 (s, 3H, OCH₃), 3.54 (s,

2H, NCH₂Ph), 3.21 (dd, J₁= 17.6 Hz, J₂= 8.0 Hz, 1H), 2.92 (m, 2H), 2.67 (dt, J₁= 14.4 Hz, J₂= 4.4 Hz,

2H), 2.0 (m, 2H), 1.87 (m, 1H), 1.73–1.66 (m, 2H), 1.50 (m, 1H), 1.40-1.26 (m, 3H); ¹³C-NMR (100 MHz,

CDCl₃, Figure S56) δ

207.6 (C=O), 164.2–161.8 (d, ¹J_C-F= 245.2 Hz, C, 1 carbon), 155.9 (C), 149.2 (C),

148.2 (C), 139.0–138.9 (d, ³J_C-F= 7.6 Hz, C, one carbon), 137.4 (C), 130.2–130.1 (d, ³J_C-F= 7.6 Hz, CH,

one carbon), 129.4 (CH, two carbons), 129.1 (CH), 128.2 (CH, two carbons), 127.2 (C), 122.7–122.6 (d,

⁴J_C-F= 3.1 Hz, CH, one carbon), 115.0–114.8 (d, ²J_C-F= 20.5 Hz, CH, one carbon), 114.2–114.0 (d, ²J_C-F

=

22.0 Hz, CH, one carbon), 107.7 (CH), 106.4 (CH), 69.9 (CH₂), 63.1 (CH₂), 56.2 (CH₃), 53.6 (CH₂), 53.5

+

(CH₂), 45.3 (CH), 38.6 (CH₂), 34.2 (CH₂), 33.4 (CH₂), 32.5 (CH₂), 31.5 (CH); m/z calcd. for C₃₀H₃₃FNO₃

[M + H]⁺474.2439; found 474.2426. The purity of the compound was further conﬁrmed by RP-HPLC:

R_t= 20.20 min (96%; Figure S57).

3.2.24. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(2-ﬂuorobenzyl)oxy-5-methoxy-2,3-dihydroinden-1-

one (8o)

A solution of compound

(5 mL) was treated with 2-ﬂuorobenzyl bromide (20

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3 ). The combined organic layers were washed with H₂O (3 ) and brine (3 ), dried over anhydrous

7

(50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

µL, 0.16 mmol), and the resulting mixture was

×

MgSO₄, and ﬁltered. After standing at RT overnight, white solids precipitated out, which were

ﬁltered off. The ﬁltrate was further concentrated under reduced pressure and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.31 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8o (56 mg, 86%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S58)

δ

7.49 (t, J = 7.6 Hz, 1H, aromatic), 7.31–7.25 (m, 5H, aromatic), 7.24 (s, 1H, aromatic), 7.23 (d, J = 7.6

Hz, 1H, aromatic), 7.12 (t, J = 7.6 Hz, 1H, aromatic), 7.06 (t, J = 8.4 Hz, 1H, aromatic), 6.85 (s, 1H,

aromatic), 5.18 (s, 2H, OCH₂Ph), 3.93 (s, 3H, OCH₃), 3.52 (s, 2H, NCH₂Ph), 3.21 (dd, J₁= 17.6 Hz,

J₂= 8.0 Hz, 1H), 2.90 (m, 2H), 2.67 (m, 2H), 1.98 (m, 2H), 1.88 (m, 1H), 1.73–1.65 (m, 2H), 1.49 (m,

1H), 1.36–1.23 (m, 3H); ¹³C-NMR (100 MHz, CDCl₃, Figure S59) 207.6 (C=O), 161.7–159.2 (d, ¹J_C-F

δ

= 245.9 Hz, C, one carbon), 156.0 (C), 149.2 (C), 148.3 (C), 129.8 (CH), 129.7 (CH), 129.57-129.53 (d,

³J_C-F= 3.8 Hz, CH, one carbon), 129.3 (C, two carbons), 129.2 (CH), 128.2 (CH, two carbons), 127.1 (C),

124.23–124.20 (d, ³J_C-F= 3.8 Hz, CH, one carbon), 123.66–123.51 (d, ²J_C-F= 14.4 Hz, CH, one carbon),

115.5–115.3 (d, ²J_C-F= 20.5 Hz, CH, one carbon), 107.7 (CH), 106.6 (CH), 64.80–64.75 (d, ³J_C-F= 4.5 Hz,

CH₂, one carbon), 63.2 (CH₂), 56.2 (CH₃), 53.6 (CH₂, two carbons), 45.4 (CH), 38.6 (CH₂), 34.3 (CH₂),

+

33.4 (CH₂), 32.7 (CH₂), 31.6 (CH); m/z calcd. for C₃₀H₃₃FNO₃[M + H]⁺474.2439; found 474.2429.

The purity of the compound was further conﬁrmed by RP-HPLC: R_t= 20.06 min (96%; Figure S60).

3.2.25. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(2-triﬂuoromethylbenzyl)oxy-5-methoxy-2,3-

dihydroinden-1-one (8p)

A solution of compound

(5 mL) was treated with 2-triﬂuoromethylbenzyl bromide (25

7

(50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

L, 0.16 mmol) and the resulting mixture

µ

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was stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3 ). The combined organic layers were washed with H₂O (3 ) and brine (3 ), dried over anhydrous

×

MgSO₄, and ﬁltered. The ﬁltrate was concentrated under reduced pressure, and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.41 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8p (65 mg, 90%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S61)

δ 7.75

(d, J = 7.6 Hz, 1H, aromatic), 7.67 (d, J = 8.0 Hz, 1H, aromatic), 7.54 (t, J = 8.0 Hz, 1H, aromatic), 7.39 (t,

J = 7.6 Hz, 1H, aromatic), 7.31–7.27 (m, 4H, aromatic), 7.24 (m, 1H, aromatic), 7.18 (s, 1H, aromatic),

6.88 (s, 1H, aromatic), 5.31 (s, 2H, OCH₂Ph), 3.96 (s, 3H, OCH₃), 3.51 (s, 2H, NCH₂Ph), 3.22 (dd,

J₁= 17.6 Hz, J₂= 8.0 Hz, 1H), 2.90 (m, 2H), 2.68 (dt, J₁= 14.4 Hz, J₂= 3.2 Hz, 2H), 1.97 (m, 2H), 1.88 (m,

1H), 1.69 (m, 2H), 1.50 (m, 1H), 1.40–1.24 (m, 3H); 13C-NMR (100 MHz, CDCl₃, Figure S62)

δ 207.6,

156.0, 149.4, 148.2, 138.1, 135.07, 135.06, 132.1, 129.3 (two carbons), 129.2, 128.4, 128.2 (two carbons),

127.8, 127.5, 127.2, 127.0, 126.01, 125.95, 125.90, 125.8, 125.6, 122.9, 107.8, 106.6, 67.04, 67.01, 63.3,

+

56.2, 53.71. 53.68, 45.4, 38.7, 34.3, 33.4, 32.8, 31.7; m/z calcd. for C₃₁H₃₃F₃NO₃[M + H]⁺524.2407;

found 524.2401. The purity of the compound was further conﬁrmed by RP-HPLC: R_t= 20.90 min (96%;

Figure S63).

3.2.26. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(2,4-diﬂuorobenzyl)oxy-5-methoxy-2,3-dihydroinden-1-

one (8q)

A solution of compound

(5 mL) was treated with 2,4-diﬂuorobenzyl bromide (21

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3 ). The combined organic layers were washed with H₂O (3 ) and brine (3 ), dried over anhydrous

7

(50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

µL, 0.16 mmol) and the resulting mixture was

×

MgSO₄, and ﬁltered. The ﬁltrate was concentrated under reduced pressure, and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.17 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8q (57 mg, 85%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S64)

δ

7.46 (dd, J₁= 14.8 Hz, J₂= 8.4 Hz, 1H, aromatic), 7.31–7.27 (m, 4H, aromatic), 7.24 (s, 1H, aromatic),

7.22 (s, 1H, aromatic), 6.88–6.79 (m, 3H, aromatic), 5.12 (s, 2H, OC ₂Ph), 3.93 (s, 3H, OC ₃), 3.52 (s, 2H,

H

NCH₂Ph), 3.22 (dd, J₁= 17.6 Hz, J₂= 8.0 Hz, 1H), 2.90 (m, 2H), 2.67 (dt, J₁= 14.4 Hz, J₂= 3.2 Hz, 2H),

1.98 (m, 2H), 1.89 (m, 1H), 1.69 (m, 2H), 1.49 (m, 1H), 1.40–1.26 (m, 3H); 13C-NMR (100 MHz, CDCl₃,

Figure S65)

δ 207.6, 164.2, 164.0, 161.9, 161.8, 161.7, 161.6, 159.4, 159.3, 156.0, 149.3, 148.2, 137.9, 130.81,

130.75, 130.72, 130.66, 129.3 (two carbons), 129.2, 128.2 (two carbons), 127.0, 119.7, 119.6, 119.54, 119.50,

111.55, 111.51, 111.34, 111.30, 107.7, 106.6, 104.2, 103.9, 103.7, 64.34, 64.30, 63.3, 56.2, 53.7, 53.6, 45.4, 38.6,

34.3, 33.4, 32.8, 31.6; m/z calcd. for C₃₀H₃₂F₂NO₃⁺[M + H]⁺492.2345; found 492.2353. The purity of

the compound was further conﬁrmed by RP-HPLC: R_t= 20.25 min (95%; Figure S66).

3.2.27. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(2,5-diﬂuorobenzyl)oxy-5-methoxy-2,3-dihydroinden-1-

one (8r)

A solution of compound

(5 mL) was treated with 2,5-diﬂuorobenzyl bromide (21

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3 ). The combined organic layers were washed with H₂O (3 ) and brine (3 ), dried over anhydrous

7

(50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

µL, 0.16 mmol), and the resulting mixture was

×

MgSO₄, and ﬁltered. The ﬁltrate was concentrated under reduced pressure and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.17 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8r (67 mg, 85%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S67)

δ

7.32–7.28 (m, 4H, aromatic), 7.24 (m, 2H, aromatic), 7.19 (s, 1H, aromatic), 7.02 (td, J₁= 8.8 Hz, J₂= 4.0

Hz, 1H, aromatic), 6.97–6.91 (m, 1H, aromatic), 6.87 (s, 1H, aromatic), 5.16 (s, 2H, OC ₂Ph), 3.95 (s, 3H,

H

OCH₃), 3.54 (s, 2H, NCH₂Ph), 3.22 (dd, J₁= 17.6 Hz, J₂= 8.0 Hz, 1H), 2.92 (m, 2H), 2.68 (dt, J₁= 14.0

Hz, J₂= 3.6 Hz, 2H), 2.05 (m, 2H), 1.88 (m, 1H), 1.70 (m, 2H), 1.51 (m, 1H), 1.40–1.24 (m, 3H); 13C-NMR

(100 MHz, CDCl₃, Figure S68)

δ 207.5, 160.02, 160.00, 157.62, 157.60, 157.21, 157.19, 155.9, 154.79, 154.77,

149.4, 148.0, 137.7, 129.4 (two carbons), 129.2, 128.2 (two carbons), 127.1, 125.6, 125.5, 125.4, 125.3, 116.6,

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116.5, 116.3, 116.2, 116.0, 115.9, 115.8, 115.7, 115.6, 115.43, 115.38, 107.8, 106.5, 64.2, 64.1, 63.2, 56.2, 53.60,

+

53.57, 45.3, 38.6, 34.2, 33.4, 32.6, 31.5; m/z calcd. for C₃₀H₃₂F₂NO₃[M + H]⁺492.2345; found 492.2350.

Purity of the compound was further conﬁrmed by RP-HPLC: R_t= 20.28 min (96%; Figure S69).

3.2.28. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(2,6-diﬂuorobenzyl)oxy-5-methoxy-2,3-dihydroinden-1-

one (8s)

A solution of compound

7 (50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

(5 mL) was treated with 2,6-diﬂuorobenzyl bromide (34 mg, 0.16 mmol), and the resulting mixture was

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3 ), dried over anhydrous

×

MgSO₄, and ﬁltered. The ﬁltrate was concentrated under reduced pressure and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.34 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8s (62 mg, 93%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S70)

δ

7.32–7.27 (m, 6H, aromatic), 7.24 (m, 1H, aromatic), 6.90 (t, J = 8.0 Hz, 2H, aromatic), 6.84 (s, 1H,

aromatic), 5.14 (s, 2H, OCH₂Ph), 3.88 (s, 3H, OCH₃), 3.53 (s, 2H, NCH₂Ph), 3.22 (dd, J₁= 17.6 Hz,

J₂= 8.0 Hz, 1H), 2.91 (m, 2H), 2.68 (dt, J₁= 14.4 Hz, J₂= 4.4 Hz, 2H), 2.00 (m, 2H), 1.89 (m, 1H), 1.70 (m,

2H), 1.50 (m, 1H), 1.40–1.24 (m, 3H); 13C-NMR (100 MHz, CDCl₃, Figure S71)

δ 207.6, 163.3, 163.2,

160.8, 160.7, 156.2, 149.5, 148.4, 137.7, 131.0, 130.9, 130.8, 129.4 (two carbons), 129.2, 128.2 (two carbons),

127.1, 112.4, 112.2, 112.0, 111.6, 111.5, 111.4, 111.3, 107.8, 107.3, 63.2, 59.19, 59.15, 59.11, 56.2, 53.61,

+

53.58, 45.4, 38.6, 34.2, 33.4, 32.7, 31.6; m/z calcd. for C₃₀H₃₂F₂NO₃[M + H]⁺492.2345; found 492.2352.

The purity of the compound was further conﬁrmed by RP-HPLC: R_t= 19.99 min (96%; Figure S72).

3.2.29. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(4-bromo-2-ﬂuorobenzyl)oxy-5-methoxy-2,3-

dihydroinden-1-one (8t)

A solution of compound

7 (50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

(5 mL) was treated with 4-bromo-2-ﬂuorobenzyl bromide (44 mg, 0.16 mmol), and the resulting mixture

was stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3

×

). The combined organic layers were washed with H₂O (3

×

) and brine (3 ), dried over anhydrous

×

MgSO₄, and ﬁltered. The ﬁltrate was concentrated under reduced pressure and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.56 in CH₂Cl₂:MeOH/19:1) to

1

yield compound 8t (69 mg, 91%) as an off-white solid: H-NMR (400 MHz, CDCl₃, Figure S73)

δ

7.37

(t, J = 7.6 Hz, 1H, aromatic), 7.31–7.26 (m, 6H, aromatic), 7.24 (m, 1H, aromatic), 7.19 (s, 1H, aromatic),

6.86 (s, 1H, aromatic), 5.12 (s, 2H, OCH₂Ph), 3.93 (s, 3H, OCH₃), 3.51 (s, 2H, NCH₂Ph), 3.22 (dd,

J₁= 17.6 Hz, J₂= 8.0 Hz, 1H), 2.90 (m, 2H), 2.68 (dt, J₁= 14.4 Hz, J₂= 3.2 Hz, 2H), 1.98 (m, 2H), 1.88 (m,

1H), 1.69 (m, 2H), 1.48 (m, 1H), 1.40–1.24 (m, 3H); ¹³C-NMR (100 MHz, CDCl₃, Figure S74)

δ 207.5,

161.4, 158.8, 155.9, 149.4, 148.0, 138.1, 130.63, 130.58, 129.3 (two carbons), 129.2, 128.1 (two carbons),

127.64, 127.60, 127.0, 123.0, 122.8, 122.2, 122.1, 119.2, 119.0, 107.8, 106.6, 64.3, 64.2, 63.3, 56.2, 53.70,

53.68, 45.4, 38.6, 34.3, 33.4, 32.9, 31.7; m/z calcd. for C₃₀H₃₂BrFNO₃⁺[M + H]⁺552.1544; found 552.1546.

The purity of the compound was further conﬁrmed by RP-HPLC: R_t= 21.25 min (96%; Figure S75).

3.2.30. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(2,4,6-triﬂuorobenzyl)oxy-5-methoxy-2,3-dihydroinden-

1-one (8u)

A solution of compound

(5 mL) was treated with 2,4,6-triﬂuorobenzyl bromide (22

stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted with EtOAc

(3 ). The combined organic layers were washed with H₂O (3 ) and brine (3 ), dried over anhydrous

7

(50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous DMF

µL, 0.16 mmol) and the resulting mixture was

×

MgSO₄, and ﬁltered. After standing at RT overnight, white solids precipitated out, which were

ﬁltered off. The ﬁltrate was further concentrated under reduced pressure and puriﬁed by column

chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.31 in CH₂Cl₂:MeOH/19:1) to

yield compound 8u (65 mg, 93%) as an off-white solid: ¹H-NMR (400 MHz, CDCl₃, Figure S76)

Molecules 2018, 23, 3252

19 of 22

δ

7.32-7.29 (m, 5H, aromatic), 7.24 (s, 1H, aromatic), 6.84 (s, 1H, aromatic), 6.68 (t, J = 8.4 Hz, 2H,

aromatic), 5.08 (s, 2H, OCH₂Ph), 3.89 (s, 3H, OCH₃), 3.54 (s, 2H, NCH₂Ph), 3.22 (dd, J₁= 17.6 Hz,

J₂= 8.0 Hz, 1H), 2.92 (m, 2H), 2.68 (dt, J₁= 14.4 Hz, J₂= 3.2 Hz, 2H), 2.02 (m, 2H), 1.89 (m, 1H), 1.70 (m,

2H), 1.51 (m, 1H), 1.40–1.26 (m, 3H); 13C-NMR (100 MHz, CDCl₃, Figure S77)

δ 207.6, 164.6, 164.5,

164.3, 163.7, 163.6, 163.5, 163.4, 162.1, 162.0, 161.8, 161.1, 161.04, 161.00, 160.9, 152.2, 149.6, 148.2, 137.7,

129.4 (two carbons), 129.2, 128.2 (two carbons), 127.1, 108.92, 108.87, 108.72, 108.67, 108.53, 108.48,

107.9, 107.4, 100.69, 100.67, 100.6, 100.5, 100.44, 100.42, 100.39, 100.35, 100.2, 100.1, 63.2, 58.80, 58.76,

+

58.7, 56.2, 53.63. 53.59, 45.4, 38.6, 34.2, 33.4, 32.7, 31.6; m/z calcd. for C₃₀H₃₁F₃NO₃[M + H]⁺510.2251;

found 510.2255. The purity of the compound was further conﬁrmed by RP-HPLC: R_t= 20.30 min (95%;

Figure S78).

3.2.31. 2-[(1-Benzylpiperidin-4-yl)methyl]-6-[(2,3,4,5,6-pentaﬂuorobenzyl)oxy-5-methoxy-2,3-

dihydroinden-1-one (8v)

A solution of compound

DMF (5 mL) was treated with 2,3,4,5-pentaﬂuorobenzyl bromide (25

mixture was stirred at RT overnight. The reaction mixture was then diluted with H₂O, and extracted

with EtOAc (3 ). The combined organic layers were washed with H₂O (3 ) and brine (3 ),

7

(50 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol) in anhydrous

µL, 0.16 mmol), and the resulting

×

dried over anhydrous MgSO₄, and ﬁltered. The ﬁltrate was concentrated under reduced pressure

and puriﬁed by column chromatography (SiO₂gel, pure CH₂Cl₂to CH₂Cl₂:MeOH/19:1; R_f0.34 in

1

CH₂Cl₂:MeOH/19:1) to yield compound 8v (61 mg, 81%) as an off-white solid: H-NMR (400 MHz,

CDCl₃, Figure S79)

δ 7.32–7.24 (m, 6H, aromatic), 6.86 (s, 1H, aromatic), 5.13 (s, 2H, OCH₂Ph), 3.90 (s,

3H, OCH₃), 3.53 (s, 2H, NCH₂Ph), 3.23 (dd, J₁= 17.6 Hz, J₂= 8.0 Hz, 1H), 2.91 (m, 2H), 2.69 (dt,

J₁= 13.6 Hz, J₂= 4.0 Hz, 2H), 2.00 (m, 2H), 1.89 (m, 1H), 1.70 (m, 2H), 1.51 (m, 1H), 1.40–1.24 (m, 3H);

13C-NMR (100 MHz, CDCl₃, Figure S80)

δ 207.4, 156.2, 150.1, 147.8, 147.20, 147.16, 147.12, 147.08,

147.04, 147.01, 146.97, 146.93, 144.70, 144.66, 144.62, 144.58, 144.55, 144.51, 144.47, 144.43, 143.23, 143.16,

143.10, 143.05, 142.97, 140.7, 140.61, 140.56, 140.5, 140.4, 138.93, 138.90, 138.8, 138.7, 138.65, 138.60,

138.59, 137.6, 136.42, 136.38, 136.30, 136.26, 136.23, 136.13, 136.10, 129.4 (two carbons), 129.2, 128.5,

128.2 (two carbons), 127.1, 109.91, 109.87, 109.74, 109.70, 109.6, 109.5, 108.0, 107.8, 63.1, 58.7, 56.2, 53.6,

53.5, 45.3, 38.6, 34.2, 33.4, 32.6, 31.5; m/z calcd. for C₃₀H₂₉F₅NO₃⁺[M + H]⁺546.2062; found 546.2058.

The purity of the compound was further conﬁrmed by RP-HPLC: R_t= 20.84 min (95%; Figure S81).

3.3. In Vitro Cholinesterase (ChE) Inhibition Assays

Experiments were performed as previously described [11

(102 pm to 200 M) were dissolved in sodium phosphate buffer ((100

A) and subjected to a 5-fold serial dilution. ChE (either EeAChE or EfBChE) was added to the solution

of inhibitors (50 L, containing 0.08 U/mL ChE (ﬁnal concentration for both EeAChE and EfBChE)

in Buffer A. The mixture of inhibitor and enzyme was incubated for 10 min before initiation with

DTNB (50 L, 0.25 mM ﬁnal concentration) and acylthiocholine (acetylthiocholine for EeAChE and

,13]. Brieﬂy, donepezil analogues

µ

µL), 0.1 M, pH 8.0) (Buffer

µ

butyrylthiochholine for EfBChE) (0.5 mM ﬁnal concentration) in phosphate buffer. The reaction

was monitored at 412 nm taking measurements every 30 s for 10 min using a Spectra Max M5

plate reader (Molecular Devices, San Jose, CA, USA) at 25 ^◦C. Data was corrected with the negative

control (no acylthiocholine), and normalized to the positive control (no inhibitor) using the initial

rates (ﬁrst 5 min). All assays were performed in duplicate or triplicate. HsAChE was treated in

the same manner with the following exceptions: the ﬁnal concentration of HsAChE was 0.16

µ

g/mL

◦

(~0.16 U/mL), and reactions were performed at 37 C. The data was ﬁtted to a sigmoidal curve, and IC₅₀

values were calculated using Sigmaplot 14.0 (Systat Software, San Jose, CA, USA). The IC₅₀curves for

EeAChE and EfBChE inhibition are presented in Table 1 and Figures S82 and S83 (for EeAChE) and

Figures S84 and S85 (for EfBChE). The IC₅₀curves for HsAChE inhibition are presented in Table 2 and

Figure S86.

Molecules 2018, 23, 3252

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3.4. BACE1 Inhibition

Inhibition of BACE1 was tested using the commercial kit (cat CS0010-1KT, Millipore-Sigma,

St. Louis, MO, USA) following the directions accompanying the kit. All compounds were tested in

duplicate at a single concentration (200

showed BACE1 inhibitory activity were then tested in a concentration-dependent manner. Dilutions

were originally performed in DMSO, and 2 L added to the reaction in order to account for any

µM) in order to conﬁrm any activity. All compounds that

µ

moderation of activity from the vehicle. Fluorescent measurements were taken after 2 h. The resulting

rates were normalized to the reaction without inhibitor. In order to get an appropriate sigmoidal ﬁt,

two additional points (400 and 1000

µM) were added to the data when needed. Since the activity of

the enzyme was already negligible at 200

µM, these points aid the sigmoidal nature of the curve ﬁt.

These data are presented in Table 3 and Figure S87.

3.5. Molecular Docking of Donepezil and Compound 8l with BACE1

To further validate the biochemical results obtained against BACE1, we modeled donepezil and

compound 8l using a known crystal structure of BACE1 with an inhibitor, sharing the vicinyl dioxygen

substitution of donepezil as a model (PDB# 4FM7 [25]). Swiss Dock [29,30] was used to identify

the potential binding sites of donepezil or compound 8l with the crystal structure. Once docking

calculations were completed, Chimera [31] was used to compare the potential binding sites with that

of the known inhibitors. The closest alignments were selected, and they are presented in Figure 1.

4. Conclusions

We have synthesized 22 new donepezil analogues, 8a

–

v, and evaluated their biochemical

capabilities, along with that of the parent donepezil and its 6-O-desmethyl adduct

7. Without

exception, these compounds were all able to inhibit the action of EeAChE and EfBChE in the

low-to-sub-micromolar ranges. Compound 8t, one of the better inhibitors of EeAChE and EfBChE

was also a very efﬁcient inhibitor of HsAChE showing the highest preference for this medically

relevant enzyme. Attachment of an alkyl/aromatic group at the 6-O-position of the indanone ring

also seems to enhance their efﬁcacy. While their inhibitory capabilities were greater against EeAChE

than EfBChE, the donepezil analogues 8h

potency when compared to donepezil against EfBChE than EeAChE. The analogues 8a

substituents showed proportional change with respect to donepezil against both EeAChE and EfBChE.

The donepezil analogues 8c 8e 8f, and 8l also displayed potent BACE1 inhibitory activities, and thus

–v

with aromatic substituents displayed a much improved

–g

with alkyl

,

appeared to be multifunctional compounds for the treatment of Alzheimer’s disease.

Supplementary Materials: The Supplementary Materials include ¹H and ¹³C-NMR spectra for the molecules

synthesized, as well as HPLC traces of compounds tested for activity (Figures S1–S81). The IC₅₀curves for

the inhibition of EeAChE, HsAChE, EfBChE, and BACE1 are also provided (Figures S82–S87). The SwissDock

modeling is also provided (Figure S88). These materials are available free of charge via the internet.

Author Contributions: M.Y.F. synthesized all the compounds and conducted the EeAChE and EfBChE inhibition

assays; K.D.G. performed the HsAChE and BACE1 assays; K.D.G., M.Y.F., and S.G.-T. analyzed the data and wrote

the paper.

Funding: This work was supported by startup funds (to S.G.-T.) from the College of Pharmacy at the University

of Kentucky. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource

for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from

NIH P41-GM103311.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Molecules 2018, 23, 3252

21 of 22

Abbreviations

Aβ

amyloid-β

APP

BACE

ChE

amyloid precursor protein

β-secretase

cholinesterase

EeAChE

EfBChE

HsAChE

IC₅₀

acetylcholinesterase (from Electrophorus electricus)

butyrylcholinesterase (from Equus ferus)

acetylcholinesterase (from Homo sapiens)

half maximal inhibitory concentration

potassium hydroxide

KOH

MsOH

TBDMS

methanesulfonic acid

tert-butyldimethylsilyl

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Sample Availability: Samples of the compounds synthesized are available from the authors.

©

2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access

article distributed under the terms and conditions of the Creative Commons Attribution

(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Article Doi

Multifunctional donepezil analogues as cholinesterase and BACE1 inhibitors

DOI: 10.3390/molecules23123252

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