Discovery of multifunctional anti-Alzheimer's agents with a unique mechanism of action including inhibition of the enzyme butyrylcholinesterase and γ-aminobutyric acid transporters

European Journal of Medicinal Chemistry 218 (2021) 1 13397

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Discovery of multifunctional anti-Alzheimer’s agents with a unique

mechanism of action including inhibition of the enzyme

butyrylcholinesterase and g-aminobutyric acid transporters

,

1

a

Anna Pasieka ^{a 1}, Dawid Panek ^a, Jakub Jonczyk , Justyna Godyn , Natalia Szałaj ,

ꢀ

ꢀ

Gniewomir Latacz , Julia Tabor , Eva Mezeiova , Fabien Chantegreil , Jose Dias ,

Damijan Knez ^e, Junfeng Lu ^f, Rongbiao Pi ^f, Jan Korabecny ^c, Xavier Brazzolotto ^d,

e

g

a

€

Stanislav Gobec , Georg Hofner , Klaus Wanner , Anna Wie˛ckowska ,

Barbara Malawska ^a

,

*

a

ꢀ

Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688, Krakow, Poland

b

ꢀ

Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688, Krakow, Poland

^cBiomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05, Hradec Kralove, Czech Republic

d

ꢀ

Departement de Toxicologie et Risques Chimiques, Institut de Recherche Biomedicale des Armees, 91223, Bretigny sur Orge, France

^eFaculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000, Ljubljana, Slovenia

^fDepartment of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, China

g

€

Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universitat München, Butenandtstr, 5-13, 81377, Munich, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:

Looking for an effective anti-Alzheimer’s agent is very challenging; however, a multifunctional ligand

strategy may be a promising solution for the treatment of this complex disease. We herein present the

design, synthesis and biological evaluation of novel hydroxyethylamine derivatives displaying unique,

multiple properties that have not been previously reported. The original mechanism of action combines

Received 26 February 2021

Received in revised form

17 March 2021

Accepted 18 March 2021

Available online 31 March 2021

inhibitory activity against disease-modifying targets:

aggregation, along with an effect on targets associated with symptom relief - inhibition of butyr-

ylcholinesterase (BuChE) and -aminobutyric acid transporters (GATs). Among the obtained molecules,

compound 36 exhibited the most balanced and broad activity proﬁle (eeAChE IC₅₀¼ 2.86 M; eqBuChE

IC₅₀¼ 60 nM; hBuChE IC₅₀¼ 20 nM; hBACE1 IC₅₀¼ 5.9 M; inhibition of A aggregation ¼ 57.9% at

10 M; and mGAT2 IC₅₀¼ 19.05 M). Moreover, we also identiﬁed 31 as the

M; mGAT1 IC₅₀¼ 10.96

M, respectively), with high

b-secretase enzyme (BACE1) and amyloid b (Ab)

g

Keywords:

m

Alzheimer’s disease

Multifunctional ligands

Inhibitors

Butyrylcholinesterase

BACE1

m

b

m

most potent mGAT4 and hGAT3 inhibitor (IC₅₀¼ 5.01

m

M and IC₅₀¼ 2.95

m

selectivity over other subtypes. Compounds 36 and 31 represent new anti-Alzheimer agents that can

ameliorate cognitive decline and modify the progress of disease.

GABA transporters

Ab aggregation

̌

1. Introduction

(donepezil, galantamine, rivastigmine and memantine) and one

ﬁxed-dose combination of donepezil and memantine, which was

The discovery of new drug for Alzheimer’s disease (AD) is one of

the biggest challenges in modern medicinal chemistry. Although

we have broadened the knowledge about its etiopathogenesis since

AD was ﬁrst described, effective pharmacotherapy still does not

exist. Current treatments include only four symptomatic drugs

approved in 2014 [1e3]. Marketed drugs are indicated for mild to

severe Alzheimer’s dementia, but they alleviate cognitive and

behavioral symptoms for only a limited period of time (typically a

few months after the start of treatment) [1]. Moreover, when

reading the news about clinical trials for AD, only one word comes

to mind: “failure” [4e6]. However, a series of setbacks have not

discouraged pharmaceutical companies or academic units from

pursuing the goal of a new, innovative therapy for AD. This is not

unusual, considering how great the need is, especially when we

reﬂect on the facts and ﬁgures that describe this disease:

* Corresponding author.

E-mail address: mfmalaws@cyf-kr.edu.pl (B. Malawska).

These authors contributed equally to this work.

1

https://doi.org/10.1016/j.ejmech.2021.113397

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

approximately 50 million cases of dementia currently (including

60e70% with AD), predictions of tripling that number by 2050,

aging societies, extremely unpleasant symptoms that rob patients

of their dignity, and ﬁnally, the awareness that this disease can

affect anyone of us [7,8].

by subsequent cutting of an amyloid precursor protein (APP) by

enzymes

crucial roles in the etiopathogenesis of AD were discovered, BACE1

and -secretase have become targets of numerous research pro-

b-secretase (BACE1) and g-secretase [48e50]. Since their

g

jects aiming to develop inhibitors or modulators [51,52]. Some of

these compounds have entered clinical trials and were tested in

patients at early and mild to moderate stages of AD with fully

AD has a very complex pathogenesis with two main primary

pathological hallmarks: neuroﬁbrillary tangles (NFTs) and amyloid

beta (Ab) plagues that are results of misfolding, aggregation and

developed Ab pathology [53e55]. However, due to the complexity

accumulation of certain proteins, namely tau and amyloid beta. The

accumulation of these plagues and tangles lead to neuronal dam-

age, major synaptic changes, progressive neurodegeneration,

neuronal death, and ﬁnally to dementia [9e13]. Dementia is mainly

related to impaired cholinergic neurotransmission and thus, anti-

AD drugs work as inhibitors of acetylcholinesterase (AChE) and/or

butyrylcholinesterase (BuChE) - enzymes that hydrolyze acetyl-

choline (ACh) in neuron synapses [14,15]. It is worth noting that in

the healthy brain, AChE hydrolyzes the main pool of acetylcholine

with BuChE playing an additional role, but during the progression

of AD, BuChE takes over the role of AChE and is responsible for

almost 80% of the cholinesterase activity. This indicates that BuChE

should be more desirable biological target aiming at cholinergic

neurotransmission in the search for palliative therapies [16e18].

AD is associated not only with dementia and memory impair-

ment related to cholinergic neurotransmission but also with clin-

ical symptoms, including depression, hallucinations, speech

disorders, motor disabilities, and aggressive behavior [19e21]

related to impairment of serotoninergic, excitatory amino acid, or

of AD, it is important to focus not only on disease-modifying targets

and to search for new drugs that alleviate dementia and the

accompanying behavioral and psychological symptoms of demen-

tia as well.

Taking into consideration the multifactorial nature of the dis-

ease, a combination therapy, especially a multitarget strategy, can

be a great perspective [56,57]. The multitarget-directed ligand

(MTDL) approach has been developed as an answer to the in-

efﬁciency of selective compounds in complex diseases such can-

cers, infections, and cardiovascular diseases [58,59]. Currently,

multifunctional agents have also been successful in the ﬁeld of CNS

diseases, e.g., in the treatment of Parkinson’s disease (rasagiline,

saﬁnamide) and schizophrenia (aripiprazole, brexpiprazole), and

more are launched onto the market [60]. These successes give us

hope that the synergic effects resulting from complex action might

be a great solution to the problems of inefﬁcient AD therapy as well.

This strategy indeed allows not only addressing more than one

target but also combining disease-modifying and symptomatic

components in one molecule [61]. This approach has resulted in the

discovery of many multifunctional molecules, the most numerous

of which is the combination of cholinesterase inhibitors with other

biological targets [62e66]. The most valuable are examples those

that combine the pharmacophores responsible for the aggregation

g

-aminobutyric acid (GABA) neurotransmission [22e24]. Although

the role of the GABAergic system in AD was recognized earlier, from

the observation that GABA neurotransmission is progressively

reduced in AD, the results of recent studies have produced large

amounts of new and interesting data [25e28]. GABAergic system

regulate excitatory neurons in brain through GABA release and is

involved in the processes of learning and memory. GABA trans-

porters (GATs) play an important role in the regulation of

GABAergic activity in different brain regions, and changes in their

expression can potentially disrupt the excitatory/inhibitory bal-

ance, contributing to the pathogenesis of AD. It seems particularly

valuable to show GATs as important molecular targets in the search

for new anti-AD agents [29e31]. Until now four GATs have been

identiﬁed and designated hGAT1, hBGT1, hGAT2, and hGAT3 (cor-

responding to mGAT1, mGAT2, mGAT3, and mGAT4 in mice) [32,33].

Individual types of transporters differ in their structure and

occurrence, and their roles in the CNS have not yet been fully un-

derstood [34,35]. Both GAT1 and GAT3 subtypes are highly

expressed in the CNS in different regions of the brain [36e38], in

contrast to GAT2 and BGT1. It has been found hGAT3/mGAT4

transporter, known as the astrocyte-speciﬁc GABA transporter is

signiﬁcantly increased in both human and mouse AD brains [39].

Studies in a transgenic mouse model for AD (5xFAD) and in brain

samples from human AD patients showed the GAT-4 inhibitor

SNAP-5114 signiﬁcantly attenuated tonic GABA currents and

reversed the long-term potentiation impairment and rescued

memory deﬁcits [39]. Moreover, altered expression of GATs in the

human AD brain and the complexity of the changes in their func-

tions over the course of the disease have been shown and thus,

targeting GABA transporters may be a promising solution in AD

treatment [40,41]. GABA transporters inhibitors belong to different

classes of chemical compounds, however, non-amino acid de-

rivatives contain common structural elements (Fig. 1) [31].

of the neurotoxic Ab protein or the tau protein with other targets

[67e70]. It should be emphasized that the design and discovery of a

promising multitarget agent with balanced activity and drug-like

properties pose a challenge [71,72]. However, considering the po-

tential beneﬁts (multifactorial proﬁle of activities, better patient

compliance, lower risk of drug-drug interactions, efﬁcacy, safety),

we should continue our efforts to achieve this goal.

The work presented describes the search for new multifunc-

tional compounds targeting both the causes and symptoms of

neurodegenerative processes in AD (Fig. 2). To address main causes

of the disease, we decided to target protein misfolding by BACE1

enzyme inhibition and Ab and tau aggregation inhibition. Due to

the complexity of the symptoms of AD we focused on the biological

targets that alleviate dementia symptoms (BuChE) and neuropsy-

chiatric symptoms (GATs).

2. Results and discussion

2.1. Design

Recently, we presented a series of broad-acting multifunctional

anti-AD agents targeting the cholinergic system, amyloidopathy,

and tauopathy [73]. Among these hydroxyethylamine derivatives,

we highlighted compound I as the most interesting multifunc-

tional ligand due to its promising and well-balanced proﬁle of ac-

tivities, namely inhibition of eqBuChE (IC₅₀¼ 2.92

(IC₅₀¼ 5.74 M), hBACE1 (IC₅₀¼ 41.60 M), A

(IC₅₀¼ 3.09 M) and tau aggregation (53.8% at 10

m

b

M), hBuChE

aggregation

m

mM). In the study

presented herein we have used compound I as a lead structure in

the development of ﬁrst-in-class multifunctional ligands targeting

Tau tangles, Ab plaques and their production have also become

an attractive biological processes for the development of new anti-

AD drugs [45e47]. The focus is placed on reducing these hallmarks

formation, aggregation or inducing their clearance. Regarding the

BuChE, BACE1 and GABA transporters with A

aggregation properties.

Based on the previous structure-activity relationship (SAR)

analysis and molecular modeling studies we have designed a series

b and tau anti-

Ab, its most aggregable Ab_1-40and Ab_1-42fragments are produced

2

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

Fig. 1. The structures of selected nonamino acid GATs inhibitors [42e44].

diphenylethyl or 3,3-diphenylpropyl which were connected

through an amine (series I) or amide (series II) group with 1-

amino-4-phenylbutan-2-ol-3-yl moiety (fragment B). The third

fragment (fragment C) contained different amino moieties. Due to

the valuable inﬂuence of branched substituents on the anti-

aggregation potency [73], we selected and introduced mainly

benzylamines with branched substituents (tert-butyl, isopropyl,

triﬂuoromethyl) in various positions of the phenyl ring or with

branching on methylene linker with one or two methyl groups. The

results of the docking studies indicated that substituents without

an aromatic ring could be introduced in place of the benzylamine

moiety. Therefore, for numerous derivatives, we replaced the

benzylamine fragment (fragment C) with various nonaromatic

moieties including tert-butyl, 2,2-dimethylpropyl, 2,2,2-

triﬂuoroethyl, and cyclopropylmethylene.

Possessing two chiral centers, the designed compounds may

take one of four possible conﬁgurations: 2S,3R, 2S,3S, 2R,3R, or

2R,3S. Considering the importance of chirality to biological activity,

we decided to assess its impact. We selected and obtained isomers

with the 2S conﬁguration, which is preferable in numerous BACE1

inhibitors based on hydroxyetylenamine core (e.g., GRL-8234 and

GSK188909) [80,81]. We conﬁrmed in molecular modeling studies,

that the binding modes of designed compounds were in line with

that observed in the crystal structure of a BACE1 complex with

active hydroxyetylenamine-based inhibitors [82,83]. The chiral

center with a hydroxyl group (position 3) seemed to be much more

interesting regarding creating crucial interactions (e.g., with the

catalytic dyad), so we decided to focus our exploration here. For

unsubstituted benzylamine derivatives (fragment C) and for the

selected derivatives, we synthesized pairs of diastereoisomers

(2S,3R) and (2S,3S) and evaluated their biological potencies. Due to

the lack of signiﬁcant differences in biological activity for other

designed compounds we obtained just one pure diastereoisomer

(2S, 3R or 2S,3S).

Fig. 2. A strategy for designing multifaceted drugs against AD with possible in-

terventions in dementia symptoms, causes and neuropsychiatric manifestation of

disease.

of modiﬁcations of compound I aiming at improving inhibitory

activity of the compounds against BuChE and BACE1. Analysis of

docking of compound I in the active sites of these enzymes

revealed hydrophobic pockets that stay unoccupied by the com-

pound, that could be excellent space for exploration (Fig. 3).

Therefore, in series I (Fig. 3) an extra benzyl moiety was incorpo-

rated into the hydroxyalkylamine core to interact with these hy-

drophobic pockets. Branching in this fragment can create additional

interactions with Tyr71, Phe108 and Trp115 in the S1 pocket of

BACE1. This should also ensure a better position of the benzhydryl

scaffold in the S2 and S3 pockets and lead to an enhancement in the

inhibitory activity toward BACE1. The introduction of an aromatic

substituent should improve the ﬁt of the ligand to BuChE through

the interaction of the ligand with the free hydrophobic pocket

created by Trp231, Phe329 and Phe398, among others. Moreover,

due to the structural similarity of the designed molecules to the

structures of GABA transporter inhibitors, we decided to examine

their activity toward this biological target [74e78].

2.2. Chemistry

Following the chemical synthesis outlined in Scheme 1, we ob-

tained 41 ﬁnal compounds divided into two groups: amine de-

rivatives (16e27) and amide derivatives (28e56). The ﬁrst step was

the nucleophilic substitution of the starting epoxide (2S, 3S or

2R,3S) with the appropriate amine under basic conditions (catalytic

amount of pyridine). The ring-opening reaction proceeded via the

stereospeciﬁc S_N2 mechanism and created only one isomer. Next,

resulting aminoalcohols 1a-15a were N-BOC deprotected to obtain

the corresponding 1-substituted 1,3-diamino-4-phenylbutan-2-ol

derivatives 1b-15b. The ﬁnal compounds were synthesized using

one of two alternative methods for series I and series II. To obtain

the amine derivatives (16e27), we utilized reductive amination of

the corresponding amine and 2,2-diphenylacetaldehyde or 3,3-

diphenylpropanal using sodium cyanoborohydride in the pres-

ence of acetic acid. The last step to obtain the ﬁnal derivatives of

Further modiﬁcations (series II) included the replacement of

one of the amine moieties with an amide bond placed between the

hydroxyethylamine core and the benzhydrylalkyl fragment (Fig. 3).

This allows the reduction of one protonated group and localization

of a positive charge at only the nitrogen atom of the benzylamine or

a corresponding fragment and could result in stabilization of the

binding mode in the active site of BuChE and BACE1.

The designed compounds from series I and II consist of three

fragments (Fig. 4). For fragment A, we used diphenylmethyl, 2,2-

3

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

Fig. 3. Design strategy of new multifunctional anti-AD agents e BACE1 and BuChE inhibitors. Binding modes of compound I in the active space of BACE1 and BuChE. The aromatic

amino acids included in the unoccupied hydrophobic pockets are shown as sticks. The protein surface is presented according to the Eisenberg hydrophobicity scale using a color

gradient from white (least hydrophobic) to red (most hydrophobic) [79].

dicyclohexylcarbodiimide (DCC) in the presence of base 4-

dimethylaminopyridine (DMAP), which was used as the activator

of the coupling reaction, and the addition of hydroxybenzotriazole

(HOBt) prevented racemization [84]. All ﬁnal products were ob-

tained as pure diastereoisomers, which was conﬁrmed by ¹H NMR

(see Figs. S48 and S49).

2.3. Inhibition of eeAChE, eqBuChE and hBuChE

To evaluate the inhibitory potency of the newly developed

compounds against BuChE from equine serum (eqBuChE) and hu-

man BuChE (hBuChE) as well as to check their selectivity over AChE

from electric eel (eeAChE), we used a method established by Ellman

et al. [85]. In the ﬁrst step, the screening assay was performed, and

we chose 10 mM for eeAChE and eqBuChE and 1 mM for hBuChE as

the concentration of inhibitor. Then, we determined IC₅₀values for

the compounds with the percentage of inhibition against eeAChE

and eqBuChE above 50% and against hBuChE above 80%. As refer-

ence compounds, we used tacrine and donepezil. The results are

presented in Tables 1 and 2.

2.3.1. Series I

Initially, we synthesized and deﬁned the activities of the

unsubstituted benzylamine (fragment C) compounds, (2S,3R)-17

and (2S,3S)-18, which are an exemplary pair of diastereoisomers.

We noticed that both derivatives were active, with similar po-

tencies against hBuChE and slightly different activities in the case of

eqBuChE. Therefore, we assumed that it is unnecessary to develop

all the pairs of diastereomers; instead it allowed us to choose be-

tween either 2S, 3R or 2S, 3S derivatives.

Fig. 4. General structure of the designed compounds.

series II (28e56) was amidation between the previously obtained

primary amines 1b-15b and 2,2-diphenylacetic acid or 3,3-

diphenylpropanoic acid using coupling agents 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide hydrochloride (EDC) or N,N⁰-

All derivatives from series I displayed activity against eqBuChE

4

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

Scheme 1. Reagents and conditions: (i) appropriate amine, pyridine (cat), n-propanol/isopropanol, reﬂux, 16 h; (ii) TFA, DCM, RT, 2 h; (iii) 2,2-diphenylacetaldehyde/3,3-

diphenylpropanal, acetic acid, sodium cyanoborohydride, DCM, RT, 24 h; (iv) 2-diphenylacetic acid/3,3-diphenylpropanoic acid, DMAP, HOBt, EDC/DCC, DCM, RT, 24 h.

Table 1

Inhibition of eeAChE, eqBuChE, hBuChE, and hBACE1 by compounds in series I.

Cmp

R

2,3

n

eeAChE

IC₅₀

M]/% inh.^a

eqBuChE

IC₅₀

M]/% inh.^b

hBuChE

IC₅₀

M]/% inh.^c

hBACE1

IC₅₀

M]/% inh.^d

[m

Series I

16

17

18

19

20

S,R

S,S

0

1

0

1

<10%

0.95 0.05

1.54 0.06

0.65 0.03

0.32 0.01

1.01 0.05

0.32 0.02

71.7% 0.9

0.63 0.02

0.58 0.02

0.80 0.04

42% 11

55.3% 3.8

58.0% 8.5

55.2% 3.8

65.5% 8.5

21

22

S,R

1

<10%

2.04 0.08

1.84 0.08

55.4% 4.7

67.4% 0.9

50.6% 1.7

6.6 0.4

25.9% 6.5

23

24

S,R

0

1

<10%

0.95 0.02

0.98 0.02

0.92 0.04

72.5% 0.5

53.9% 7.1

62% 10

25

S,R

1

<10%

1.60 0.05

53.2% 2.9

71.8% 3.7

26

27

S,S

S,R

1

0

<10%

0.19 0.01

0.59 0.03

68.5% 0.8

0.19 0.01

51.3% 4.3

63.9% 8.0

References

Compound I

Tacrine

21.8% 5.5

0.023 0.0004

0.011 0.0002

Nd

2.92 0.10

0.015 0.0001

1.83 0.04

nd

5.74 0.26

0.034 0.0004

nd

41.6 1.5

nd

Donepezil

Inhibitor IV^e

0.046 0.01

Values are expressed as the means the standard error of the mean (SEM) of at least three experiments (n ¼ 3), each performed in triplicate.

a

IC₅₀inhibitory concentration of electric eel AChE or percent inhibition at an inhibitor concentration of 10

m

M.

IC₅₀inhibitory concentration of BuChE from horse serum or percent inhibition at an inhibitor concentration of 10

IC₅₀inhibitory concentration of human BuChE or percent inhibition at an inhibitor concentration of 1 M.

IC₅₀inhibitory concentration of human recombinant BACE1 and substrate (Rh-EVNLDAEFK-quencher) or percent inhibition at an inhibitor concentration of 50

Calbiochem, Merck, Nottingham, UK. nd; not determined.

b

c

mM.

m

d

e

m

M.

with IC₅₀values ranging from 0.19 to 2.04

the compounds inhibited eeAChE below 10% at 10

they were considered to be highly selective BuChE inhibitors.

m

M. Since almost all of

Among them, we deﬁned 26 as the most potent eqBuChE inhibitor

with an IC₅₀value of 190 nM. Analyzing the SAR, we observed that a

shorter distance between the benzhydryl moiety (fragment A) and

mM (except 22),

5

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

Table 2

Inhibition of eeAChE, eqBuChE, hBuChE, and hBACE1 by compounds in series II.

Cmp

R

2,3

n

eeAChE

IC₅₀

M]/% inh.^a

eqBuChE

IC₅₀

M]/% inh.^b

hBuChE

IC₅₀

M]/% inh.^c

hBACE1

IC₅₀

M]/% inh.^d

[

m

[m

Series II

28

29

30

31

32

33

S,R

S,S

S,R

S,S

0

1

0

1

<10%

1.51 0.04

0.23 0.01

1.38 0.04

2.94 0.11

0.11 0.01

0.33 0.01

34.1% 2.2

40.5% 1.1

62.3% 1.6

67.7% 1.8

0.54 0.01

0.35 0.01

56.2% 9.5

66.1% 4.9

35.4 1.6

17.4 0.7

66.2% 2.5

23.4 0.9

34

35

S,R

0

1

<10%

1.55 0.04

1.28 0.04

56.5% 7.0

0.44 0.01

37.0% 9.8

49% 11

36

37

S,R

0

1

2.86 0.14

<10%

0.06 0.01

0.45 0.01

0.02 0.0005

77.3% 4.1

5.9 0.2

6.0 0.2

38

39

40

S,R

S,S

S,R

0

1

5.13 0.22

<10%

24.0% 1.6

0.50 0.02

0.20 0.01

0.65 0.02

0.08 0.002

74.9% 0.9

0.50 0.01

66% 12

3.4 0.2

4.6 0.2

41

42

S,S

0

1

<10%

36.8% 9.7

0.39 0.01

29.5% 6.4

0.67 0.02

<10%

8.6 0.3

8.3 0.2

43

44

S,S

0

1

<10%

3.44 0.12

0.17 0.01

0.20 0.003

0.88 0.03

2.1 0.1

6.2 0.3

45

46

S,R

0

1

<10%

1.82 0.06

8.86 0.74

14.5% 2.5

36.4% 2.7

61.7% 4.9

74.1% 6.2

47

48

S,R

0

1

<10%

45.9% 3.1

3.23 0.08

<10%

13.3% 3.8

76.1% 7.7

8.9 0.5

49

50

S,S

0

1

<10%

0.84 0.02

11.8 0.16

35.5% 8.5

<10%

51

52

S,R

0

1

<10%

0.10 0.01

2.77 0.07

0.45 0.01

46.2% 4.4

40.04% 4.1

33.6 1.6

53

54

S,R

0

1

<10%

26.3% 5.8

19.2% 9.9

<10%

15.4 1.1

24.4% 7.3

55

56

S,R

0

1

<10%

3.21 0.07

3.23 0.07

12.3% 3.6

41.1% 3.2

44.4% 6.5

<10%

References

Compound I

Tacrine

21.8% 5.5

0.023 0.0004

0.011 0.0002

nd

2.92 0.10

0.015 0.0001

1.83 0.04

nd

5.74 0.26

0.034 0.0004

nd

41.6 1.5

nd

Donepezil

Inhibitor IV^e

0.046 0.01

Values are expressed as the means the standard error of the mean (SEM) of at least three experiments (n ¼ 3), each performed in triplicate.

a

IC₅₀inhibitory concentration of electric eel AChE or percent inhibition at an inhibitor concentration of 10

m

M.

IC₅₀inhibitory concentration of BuChE from horse serum or percent inhibition at an inhibitor concentration of 10

IC₅₀inhibitory concentration of human BuChE or percent inhibition at an inhibitor concentration of 1 M.

IC₅₀inhibitory concentration of human recombinant BACE1 and substrate (Rh-EVNLDAEFK-quencher) or percent inhibition at an inhibitor concentration of 50

Calbiochem, Merck, Nottingham, UK. nd; not determined.

b

c

mM.

m

d

e

m

M.

amine bond slightly improved the inhibitory potency (e.g., 19 vs

20). The introduction of branched, aliphatic moieties instead of the

aromatic ring in fragment C was the most beneﬁcial for inhibitory

activity toward eqBuChE (e.g., 26 vs 18).

While testing the inhibitory abilities against hBuChE, for six

derivatives we established IC₅₀values ranging from 190 to 920 nM.

The most active was compound 27 containing a tert-butylmethyl

moiety in fragment A, for which molecular modeling studies were

performed (see Supporting Information).

Compared to starting compound I, all compounds of series I

exhibited higher inhibitory potencies toward eqBuChE and hBuChE.

The most active compound toward eqBuChE (26) is over 15 times

more active than compound I, while the most potent hBuChE in-

hibitor (27) is over 30 times more active than compound I. These

results clearly conﬁrmed that the introduction of a benzyl moiety

into fragment B not only allowed retention of selectivity over AChE

6

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

but also signiﬁcantly improved potency toward BuChE.

decreased at increasing concentrations of the enzyme inhibitor.

2.3.2. Series II

2.5. X-ray crystallography of hBuChE in complex with 51

Similar to series I, the study of compounds of series II started

with investigating the impact of chirality on biological activity. For

compounds with an unsubstituted benzylamine (fragment C), we

synthesized two pairs of diastereoisomers: (S,R)-28 and (S,S)-29

with a diphenylmethyl substituent and (S,R)-30 and (S,S)-31 with a

2,2-diphenylethyl substituent in fragment A. We also obtained a

pair of diastereoisomers for 3-isopropyl substituted compounds in

fragment C ((S,R)-38, (S,S)-39). As the observed activity differences

did not promote any speciﬁc conﬁguration, as previously described,

we decided to synthesize compounds in the 2S, 3R or 2S, 3S con-

ﬁgurations as pure stereoisomers.

To examine possible interactions of the obtained compounds

with the hBuChE, we performed docking studies. Obtained results

predicted two signiﬁcant binding modes for selected compounds:

27 and 51 (see Supporting Information). To answer the bothering

question of what binding mode the molecule 51 adopts in the

hBuChE active site, we decided to conduct crystallographic studies

of this complex. To elucidate the binding mode of 51 with hBuChE,

the crystal structure of this complex was solved at 2.25 Å resolution

(Fig. 5). Crystals were obtained by the hanging drop method and

then soaked with 51 at a ﬁnal ligand concentration of 1 mM (1%

MeOH). As previously observed for other BuChE inhibitors con-

While evaluating inhibitory activity against BuChE, most com-

pounds were found to be moderate to potent eqBuChE inhibitors

taining aromatic parts [62,86e88], p-stacking mainly rules the in-

with established IC₅₀values ranging from 0.06 to 11.8

mM. Com-

teractions of 51 with hBuChE. The di-phenyl-acetamide moiety

(fragment A) engages its benzyl rings in interactions with the ar-

pounds 36 and 51 were the most potent eqBuChE inhibitors (IC₅₀

values 60 nM and 100 nM, respectively), demonstrating IC₅₀values

in the same order of magnitude as tacrine. Almost all active de-

rivatives were revealed as selective inhibitors of eqBuChE. Only 36

and 38 exhibited nonselective inhibitory properties against both

cholinesterases; however still preferable towards eqBuChE.

The SAR analysis for series II brought some interesting conclu-

sions that partially coincide with the results of series I. Regarding

the length of fragment A, we found that the benzhydryl directly

connected to the amide bond of fragment B improved the eqBuChE

inhibitory properties in most cases (e.g., 36 vs 37), similar to that

observed for series I. Second, we noticed that the most active

compounds contained a benzylamine fragment in their structures

(fragment C). Unlike in series I, here, the substitution of benzyl-

amine (fragment C) resulted in an increase in activity in most

cases. It is worth emphasizing that the introduction of a 2-methoxy,

3-tert-butyl or 3-isopropyl substituent signiﬁcantly increased ac-

tivity. In turn, the introduction of one or two methyl groups onto

the methylene of the benzylamine clearly impaired anti-BuChE

potency. In comparison with compounds of series I, the replace-

ment of the benzylamine scaffold with various aliphatic moieties

had a totally different result. Generally, such modiﬁcation did not

have a positive impact. However, surprisingly, 51, which possessed

a benzhydryl as fragment A and a 2,2-dimethylpropyl substituent

as fragment C distinguished itself as a potent inhibitor of eqBuChE.

Comparing the results for series I and II, we observed that our

hypothesis, that focusing all positive charges on just one nitrogen

atom would produce a beneﬁcial effect, was not completely

conﬁrmed. Just in cases of the 2-methoxybenzyl, 3-isopropylbenzyl

and 2,2-dimethylpropyl derivatives, the introduction of an amide

instead of an amine group resulted in an increase in eqBuChE

inhibitory activity.

omatic residues that line the active site gorge through

p-p in-

teractions. A ﬁrst benzyl ring ﬁts in the acyl-binding pocket of

hBuChE and interacts through T-stacking with Trp231 with a

centroid distance of 5.2 Å, and, to a lesser extent, with Phe398, with

a centroid distance of 6.3 Å. The second benzyl ring interacts with

Phe329, with a centroid distance of 4.1 Å, and, to a lesser extent,

with Tyr332, with a centroid distance of 4.9 Å, also through

stacking interactions. The carbonyl of 51 points toward the water

molecule that lies in the oxyanion hole between the O atom of the

p-

g

catalytic serine Ser198 and the backbone nitrogen atoms of Gly116

and Gly117. However, the distance between this water molecule

and the carbonyl oxygen atom is longer than that of a classical H-

bond at 3.5 Å. In the crystallization buffer, the amine functional

group bearing the trimethyl moiety (fragment C) is likely charged,

forming a favorable cation-

tance of 4.3 Å in the hBuChE choline pocket. The ﬁnal benzyl ring

folds back over the diphenyl moiety and engages an additional p-p

p interaction with Trp82, with a dis-

interaction with the benzyl ring already engaged with Trp231 and

Phe398 with a centroid distance of 5.3 Å. Finally, an additional

interaction exists with the hydroxyl group that bridges a water

molecule already engaged with Asn83. In this position, the ligand

occludes the active site gorge, thus preventing substrate access to

the catalytic residues.

Binding mode B of 51 obtained during the in silico studies shows

many similarities to that observed in this crystal. The most

important are undoubtedly the interactions of the 2,2-

diphenylethyl fragment with Trp231, Phe398, Phe329, and Tyr332

and the formation of the cation-p bond between the protonated

amine and Trp82. The differences between the experimental and

predicted in silico modes include the formation of T-shaped aro-

matic intramolecular interactions by the benzyl group and a very

signiﬁcant share of two water molecules that interact with the

polar fragments of 51.

Finally, we examined the biological activities against hBuChE

and for 10 compounds we determined IC₅₀values ranging from 20

to 880 nM. Among them, the most potent was 36, with a 3-tert-

butylbenzylamine in fragment C, which was also the most potent

inhibitor of eqBuChE. Additionally, here, we observed the beneﬁcial

effect of benzylamine substitution in fragment C, especially those

with bulky substituents (3- or 4-tert-butyl, 3-isopropyl).

2.6. Inhibition of hBACE1

The second enzyme biological target for the presented com-

pounds is BACE1. We determined the BACE1 inhibitory properties

using a FRET (ﬂuorescence resonance energy transfer)-based ﬂuo-

rometric assay [73]. As a substrate for human recombinant BACE1

(hBACE1), we used a modiﬁed peptide analog of APP with the

Swedish mutation. First, we performed the initial assay at a

2.4. Kinetic studies of eqBuChE inhibition

To determine the mode of BuChE inhibition, we performed ki-

netic studies of eqBuChE with two of the most potent compounds,

36 and 51. The Lineweaver-Burk and Cornish-Bowden plots (see

Fig. S5 and S6) showed that both derivatives were noncompetitive

eqBuChE inhibitors. The K_mvalues were not affected by changing

the substrate or inhibitor concentrations, whereas the V_maxvalues

screening concentration of 50 mM. Then, we established IC₅₀values

for compounds displaying inhibition percentages above 80%

(Tables 1 and 2). We selected inhibitor IV [89] as a reference

compound with its established IC₅₀value of 46 nM.

Primarily, the proposed modiﬁcations in series I were expected

7

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

Fig. 5. Close-up view of the 2.25 Å X-ray structure of 51 bound to hBuChE. A, top view, B, side view. Critical residues are represented as sticks, with carbon atoms colored green

for BuChE or orange for 51, nitrogen atoms are represented in blue and oxygen atoms appear in red. Water molecules are represented as a red sphere. The gray shape represents the

active site gorge, and the blue mesh represents the 3

cation- interactions are in cyan.

s-contoured omit map of 51. Dashed lines represent key interactions, p-interactions are in magenta, H-bonds are in yellow and

p

to essentially improve the inhibitory potency against BACE1.

Among the tested compounds, the most active was 22, bearing a 3-

isopropylbenzylamine moiety in fragment C, which showed an IC₅₀

value of 6.6

inhibitory potencies toward BACE1 ranging from 41.6% to 71.8% at

50 M. In the preliminary screening test of compounds of series II,

we selected 14 compounds and established IC₅₀values ranging

from 2.1 to 35.4 M. Among them, 43, containing a 4-tert-butyl-

mM. The remaining compounds of series I exhibited

m

benzylamine scaffold as fragment C, was the most potent BACE1

inhibitor.

The SAR analysis clearly showed that the introduction of an

amide bond was crucial for BACE1 binding and improved the

inhibitory potencies in comparison with the compounds of series I

(e.g., 48 vs 25). Regarding fragment A, we observed slightly higher

activities for compounds with 2,2-diphenylethyl moieties than

those with diphenylmethyl moieties (40 vs 38). In regard to frag-

ment C, we noticed that the benzylamine scaffold substituted by

tert-butyl, isopropyl, and triﬂuoromethyl moieties improved the

anti-BACE1 potency compared with the unsubstituted derivatives

(e.g., 39, 43 vs 29). This positive effect was not observed for the 2-

and 3-methoxy substituents or extra methyl groups at the methy-

lene of benzylamine. On the other hand, we noticed that the

replacement of this fragment with an aliphatic moiety decreased

the inhibitory ability with the exception of 52 and 53. Among pairs

of diastereoisomers, we did not observe any meaningful differences

in the inhibitory activities. For this reason, the conﬁguration of

hydroxyl group in position 3 can be considered as not relevant for

BACE-1 inhibition.

Fig. 6. The docking results of 22 (orange) in BACE1 active site visualized along with the

essential amino acids (green). Gray surface represents the boundaries of the active site.

interaction is weakened due to the strong competition of both

protonated amines for access to the catalytic dyad. This leads to the

occurrence of competing conformations, making the modeling re-

sults nonuniform. Among the ten docking repetitions for each

compound, the poses compliant with the assumed binding mode

were the minority of the obtained conformations. It seems that the

key to obtaining the expected results was a good ﬁt of fragment C

into pockets S1’ and S2’. Therefore, with respect to compound I, we

have improved activity for merely one compound (22), while the

others, for which fragment C did not allow for an optimal ﬁt to the

BACE1 active site, exhibited similar or lower potencies.

The binding mode of 43 docked to the BACE1 active site is

shown in Fig. 7. It clearly shows that the introduced amide forms

two hydrogen bonds with Gly223 and the main chain of the ﬂap

(Gln73). Restricting the mobility of this ﬂexible ﬂap promotes the

inhibition of enzyme activity. Moreover, limiting the positive

charge to the amine in the benzylamine fragment signiﬁcantly

increased the reproducibility of the results for the active de-

rivatives. This indicates a preference for compounds to adopt the

expected conformation when binding to BACE1. As seen in the

example of compound 43, adjustment of fragment C to pockets S1’

and S2’ correlates with the strength of the interaction of fragment

B with the catalytic dyad, an essential grip point for hydrox-

yethylamine derivatives. As seen in Fig. 7, molecule 43, with a well-

arranged benzylamine substituent, creates many signiﬁcant

2.7. Docking studies on hBACE1 for compounds 22 and 43

The results for series I indicate that the highest-ranked ligand

poses have the assumed binding mode in which the introduced

benzyl fragment is arranged in hydrophobic pocket S1. In the

example of the most active compound 22 (Fig. 6), we can see that

fragment B forms a series of hydrogen bonds with the amino acids

Asp32, Gly34, Asp221, and Gly223 that are enhanced by the ionic

interaction of both protonated amines with Asp221. The extra ring

participates in the aromatic CH-p interaction with Tyr71. This

contributes to the strong binding of the compound in the vicinity of

the catalytic dyad. However, in the case of many molecules, this

8

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

signiﬁcantly affecting the other mGATs (29 vs 31, 34 vs 35, 45 vs 46,

55 vs 56). Among the tested compounds, we found that unsub-

stituted fragment C derivative 31 was the most active and selective

inhibitor of mGAT4 (mGAT4 IC₅₀¼ 5.01

mM; hGAT3 IC₅₀¼ 2.95

mM),

and the introduction of a methoxy or tert-butyl group slightly

decreased the inhibitory potency (31 vs 35, 36). An extra methyl

group in fragment C and replacement with aliphatic moieties also

impaired this activity (29 vs 45, 51, 53, 55; 31 vs 46, 56). Notably,

there was a positive effect from benzylamine substitution on the

inhibitory potency toward mGATs 1 and 2 in the case of 4-tert-butyl

substituted compound 36.

In summary, the obtained preliminary results seem to be very

promising but require further development. At present, we have

identiﬁed the potent and selective mGAT4 inhibitor 31 and multi-

functional ligands that affect both cholinergic and GABAergic

transmission and BACE1 inhibition exempliﬁed by compound 36. It

is noteworthy that 31 is the most potent GAT3 inhibitor known to

date among its selective inhibitors [31,77].

Fig. 7. The docking results of 43 (orange) in BACE1 active site visualized along with the

essential amino acids (green). Gray surface represents the boundaries of the active site.

interactions (hydrogen bonds with Asp32, Gly34, Asp221, and

Gly223 and an ionic interaction with Asp221) in the vicinity of the

BACE1 catalytic site. In addition, its binding is ampliﬁed by contacts

inside the S1 pocket (Phe108) and with the ﬂap (Tyr71). The

docking results of compound 51 with an aliphatic 2,2-

dimethylpropane substituent indicate that it cannot bind within

the S2’ pocket. Furthermore, the nearby Tyr191 was often a steric

hindrance for aliphatic variants of fragment C. Both effects can

explain the observed decline in activity against BACE1 for this

group of derivatives.

In summary, the biological evaluation conﬁrmed our molecular

modeling studies that branching in hydrophobic space and the

introduction of an amide bond would improve the binding mode in

the active site of BACE1. Both implemented modiﬁcations proved to

be necessary to signiﬁcantly enhance inhibitory activity toward

BACE1.

2.9. Antiaggregation properties

The processes of Ab and tau aggregation leading to the forma-

tion of neuritic plaques and NFTs are considered the main causes of

the development of AD. Since the previously published group of

hydroxyethylamine derivatives, including compound I, displayed

dual antiaggregation properties [73], we evaluated the percent

amyloid aggregation inhibition for all synthetized compounds from

series I and II and the inhibitory activity against tau aggregation for

a few selected derivatives.

2.9.1. Inhibition of Ab_1-42aggregation

2.8. Inhibitory properties toward GAT subtypes

To determine the inhibition of A

ﬂuorometric thioﬂavin-T (ThT) assay in vitro [91] with inhibitors at

a concentration of 10 M. The results are presented in Fig. 8. Except

for ﬁve compounds (16, 28, 29, 41, 52), the remaining compounds

inhibited Ab_1-42aggregation above 20%. The percentages of inhi-

bition were in the range of 34.7e81.4% and 20.5e74.4% for series I

and II, respectively. Among all derivatives, nine (six from series I:

18, 19, 21, 22, 25, 27; three from series II: 35, 46, 51) were found to

b aggregation, we performed a

As mentioned before, cholinergic transmission is impaired

during the development of AD, and dysfunction of the GABAergic

system is also observed. A possible way to improve this disturbance

is through the inhibition of GATs [29,31]. Analyzing the structures

of known GAT inhibitors drew our attention to the group of in-

hibitors possessing general structures and some crucial elements

similar to our newly designed compounds. These are two or three

aromatic rings (hydrophobic fragments) that are connected

through an aliphatic linker to a polar moiety with a protonated

nitrogen atom [78]. Because our compounds consist of these three

fragments, we performed a biological screening of selected de-

rivatives toward four mouse GAT subtypes (mGAT1 to mGAT4). We

tested 14 compounds with variable length of the alkyl linker in

fragment A, the substitution on fragment C, and the presence of

amine or amide bonds. Their inhibitory activities toward the mGATs

m

inhibit Ab aggregation at a level comparable to the well-known

reference resveratrol, with the most potent compound 19 display-

ing an 81.4% inhibitory effect. Analyzing the obtained data, amine

bonds were found to be preferable for the inhibition of amyloid

aggregation. The introduction of alkyl substituents onto the ben-

zylamine fragment did not signiﬁcantly inﬂuence the level of ag-

gregation. Between aliphatic and aromatic derivatives, no clear

structure-activity relationship was observed. However, we

noticed some interesting differences between the pairs of 2S, 3R

and 2S, 3S isomers with 2,2-diphenylethyl moieties in fragment A

and unsubstituted benzylamines in fragment C in both series. In

series I, we found that the 2S, 3S isomer (18) showed approximately

two times stronger inhibition than the 2S, 3R (17), while in series II,

the 2S, 3R isomer (30) was approximately two times more potent

than the 2S, 3S isomer (31). Moreover, analogs with shorter benz-

hydryl moieties (16, 28, 29) in fragment A in both series showed no

were determined at a screening concentration of 100 m

M by [³H]

GABA uptake using human embryonic kidney (HEK-293) cells sta-

bly expressing mouse GABA transporters. For compounds reducing

GABA uptake by at least 50%, IC₅₀values were determined. In

addition, IC₅₀values were determined for the inhibition of (²H₆)

GABA transport by mass spectrometry (MS) transport assays using

COS-7 cells stably expressing hGAT-3. The IC₅₀values and per-

centages of remaining GABA uptake are presented in Table 3.

Studying the obtained results, we noticed that amine derivatives

18 and 27 are moderately potent, nonselective GAT inhibitors, and

the replacement of the amine bond by an amide bond resulted in an

impairment of biological activity; however, amide derivatives

improved the selectivity toward mGAT4 (18 vs 31, 27 vs 51).

Considering the inﬂuence of the length of the alkyl linker, a longer

core was found to be better for potency toward mGAT4 without

signiﬁcant activity. Remembering the protein nature of Ab, the

impact of compound chirality on antiaggregation activity should

not come as a surprise; however, the observed reverse de-

pendencies and subtle structural changes that caused considerable

effects on the activity are puzzling and should be explored in future

studies. Overall, we should point out that modiﬁcations imple-

mented in the lead structure allowed us to retain antiaggregation

properties at a level similar to that of compound I.

9

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

Table 3

Inhibition of GABA transporters by selected compounds.

Cmp

IC₅₀SEM [mM]

mGAT1

mGAT2

mGAT3

mGAT4

hGAT3

mGAT1 binding

18

27

29

31

7.59 0.7

26.30

34.67

5.50 0.5

14.13

34.67

5.75 1.2

12.59

52%

5.50 0.8

9.77 0.7

26.91

5.01 1.1

2.95 0.9

19.95

33.11

5.89 0.8

55%

79%

55%

30.90

102%

30.20

85.11

74%

61%

74%

76%

83%

32

34

35

36

45

46

51

53

55

56

87.10

61%

67%

10.96

77%

59%

25.12

92%

51%

25.70

61%

41.69

19.05

60.26

51%

72.44

87%

85%

69%

67%

92%

55%

84%

69%

54%

92%

54%

51%

80%

85%

65%

82%

81%

66%

71%

100%

74%

83%

74.13

35.48

39.81

60%

References

DDPM-859^a

64.57

39.81

75.86

38.02

14.13

1.66 0.1

3.16 0.08

1.35 0.3

1.59 0.04

nd

DDPM-1457^a

3.39 0.2

46.77

Data are given as the mean SEM of three independent experiments that were performed in triplicate. The percent result represents the [³H]GABA uptake or NO-711 binding

in the presence of 100 M inhibitor. Data without SEM implies that only one experiment was performed in triplicate. IC₅₀values are from the GABA uptake assays

(means SEM, n ꢀ 3 for values above 10 M, n ¼ 1 for values between 10 and 100 M), nd; not determined.

The structures are described in Ref. [90].

m

a

Fig. 8. Effects of the synthesized compounds on Ab_1-42and tau aggregation at the screening concentration 10 mM (compounds with percent inhibition below 20% are not shown,

Table S2).

2.9.2. Inhibition of tau aggregation

2.10. Neurotoxicity

We selected 5 derivatives (18, 36, 44, 46, 51) with high inhibi-

tory activities against A

inhibit tau aggregation. Therefore, a ﬂuorometric ThT assay was

performed using the full-length form of the tau protein (0N4R) at

an inhibitor concentration of 10 mM. Two out of ﬁve compounds, 46

and 18, exhibited very high levels of inhibition: 73.1% and 85.8%,

respectively (Fig. 8). Nevertheless, we could not specify which

fragments are important for this biological property since the most

potent derivatives differed greatly in their structure.

b

aggregation to evaluate their ability to

When looking for new anti-AD drugs, both the inhibition of

biological targets in the brain and also a lack of neurotoxicity are

very important features. To evaluate whether our compounds are

safe, we performed an MTT assay with 5 derivatives (18, 36, 44, 46,

51) using murine hippocampal HT22 cells. We determined the cell

viability at two different compound concentrations: 10

30 M. Fig. 9 presents the results expressed as a percentage of cell

viability compared with untreated cells.

The results suggest that the amide derivatives (36, 44, 46, 51)

were safe at a concentration of 10 M but strongly decreased cell

viability at 30 M. The observed variability in neurotoxicity at these

mM and

m

10

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

However, the compounds proved to be safe at a concentration of

1 mM and aliphatic derivative 51 was safe at 10 mM as well (the

concentrations needed for target inhibition). As with the neuro-

toxicity assessment, the observed variability in hepatotoxicity for

compounds 36 and 41 at tested concentrations 1 mM and 10 mM was

signiﬁcant. Clearly, we observed the inﬂuence from the number of

aromatic rings and the physicochemical properties of the com-

pounds on toxicity. A reduction in the number of aromatic rings and

lipophilicity signiﬁcantly improved the safety, which conﬁrmed

that looking for active derivatives with aliphatic moieties in frag-

ment C was a very positive step worth continuing. In conclusion,

the toxicity properties require further optimization and before

undertaking in vivo studies, it seems to be reasonable to verify the

safety of these compounds using additional concentrations (inter-

mediate concentrations, i.e. 3 mM and 5 mM).

Fig. 9. Murine hippocampal HT22 cell viability by MTT assay. Statistical signiﬁcance

(GraphPad Prism 8.0.1) was evaluated by one-way ANOVA followed by Bonferroni’s

comparison test (****p < 0.0001 compared with the negative control).

2.12. Metabolic stability

Very often, MTDLs, because of their worse/suboptimal physi-

cochemical properties, have been found to be not drug-like enough

in comparison to single-target drugs [92]. It is therefore important

to perform ADME-tox studies at an early stage of drug discovery

and development to prove their safety and optimal pharmacoki-

netic parameters. Consequently, we decided to investigate meta-

bolic stability e one of the most critical parameters. In this regard,

we determined the structures of probable metabolites in silico us-

ing MetaSite 6.0.1 software for three selected derivatives with

promising biological proﬁles: 36, 40, 51. The computer predictions

demonstrated that the tert-butyl moiety and the aliphatic carbons

near the nitrogen atom in fragment C and at the para position in

the benzhydryl moiety have the highest probability of metabolism

(Fig. S7). Further, in vitro experiments with human liver micro-

somes (HLMs) were performed. We then obtained UPLC spectra of

the reaction mixtures after 120 min of incubation. The compounds

were mostly metabolically stable (Figs. S8eS13). After incubation,

approximately 70% of the parent compounds remained in the

mixture (36, 79.56%; 40, 67.89%, and 51, 79.17% respectively), while

the unstable reference drug verapamil displayed 30% retention

(Fig. S14). According to the data obtained in vitro, the in silico pre-

dictions and ion fragment analysis we identiﬁed prime amines as

the most probable metabolites in all three cases (see Supporting

Information, Table S3).

two concentrations 10

tested compounds, we determined that 46 was the safest, which

showed 100% cell viability at 10 M and approximately 50% viability

at the higher concentration. Only compound 18 also killed cells at

10 M, which alerted us to the possible toxicity of the amine de-

rivatives and discouraged greater extension of this series of com-

pounds. Generally, amide compounds can be considered nontoxic

at concentrations required for biological activity, and they were the

subject of interest for further ADME-tox studies. However, the

demonstrated toxicity of tested compounds should be taken under

consideration before the further, extended study.

mM and 30 mM was signiﬁcant. Among the

m

2.11. Hepatotoxicity

Due to the chronic nature of AD, it should be assumed that the

therapy will be long-term. It is therefore necessary to determine

the toxicity, especially hepatotoxicity, at a very early stage during

drug discovery. Consequently, we decided to evaluate the cell

viability for three selected amide compounds (36, 40, 51). The

cytotoxic effect was evaluated in hepatoma HepG2 cells after 72 h of

incubation at four different compound concentrations: 1, 10, 50 and

100

mM. The presented results (Fig .1 0) showed a strong hepatotoxic

effect for all derivatives at higher concentrations (50 and 100

mM).

To conclude, the synthesized compounds can be considered

metabolically stable, and indeed, the main metabolic process for

this group of derivatives is N-dealkylation in fragment C observed

in all cases. Additionally, meta-substituted compounds can undergo

carboxylation or hydroxylation reactions.

2.13. Drug-drug interactions einﬂuence on CYP3A4 and CYP2D6

activity

Elderly patients suffering from AD very often have comorbid-

ities, and they need to take many drugs. This leads to the risk of

dangerous drug-drug interactions (DDIs). Most xenobiotics,

including drugs, are metabolized by cytochrome P450s, with

CYP3A4 and CYP2D6 being the most commonly involved. Inﬂuence

on these isoforms (induction or inhibition) may suggest potential

undesired interactions, and thus, it is important to evaluate

whether drug candidates affect cytochromes as early as possible

during development. Therefore, we selected 3 derivatives (36, 40

and 51) to examine their inﬂuence on CYP3A4 and CYP2D6 activity

compared to reference inhibitors (ketoconazole and quinidine). The

results are presented on Figs. 1 1 a nd 12. All compounds, regardless

of whether they contained an aromatic or aliphatic moiety in

fragment C, inhibited both isoforms at a level comparable to the

Fig. 10. The effects of the cytostatic drug doxorubicin (DX), the mitochondrial toxin

carbonyl cyanide 3-chlorophenyl-hydrazone (CCCP) and 36, 40, 51 on hepatoma HepG2

cell viability after 72 h of incubation at 37 ^ꢁC under 5% CO₂. Statistical signiﬁcance

(GraphPad Prism 8.0.1) was evaluated by one-way ANOVA followed by Bonferroni’s

comparison test (****p < 0.0001 compared with the negative control 1% DMSO in

growth media).

11

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

series II, inhibitor 36 was the most potent (hBuChE IC₅₀¼ 20 nM),

showing an IC₅₀that was on the same order of magnitude as the

reference tacrine, with some potency against AChE (eeAChE

IC₅₀¼ 2.86

mM). The obtained crystal structure of the hBuChE

complex with compound 51, which is also a strong enzyme inhib-

itor (hBuChE IC₅₀¼ 450 nM), revealed its binding mode to the

enzyme and allowed comparison with the analysis of the in-

teractions using the molecular docking method. It should also be

noted that for some new derivatives, hBACE1 enzyme inhibitory

activity signiﬁcantly increased compared to the lead structure, with

the most active compound being 43 (hBACE1 IC₅₀¼ 2.1

mM).

Moreover, the antiaggregation properties for A and tau protein

b

were retained and remained at a similar level of activity to that

displayed by compound I.

The crucial achievement of the conducted research is the

demonstration of the inhibitory effects of this group of multifunc-

tional compounds on GABA transporters, which led us to discover

molecules with unique properties. While the role of the GABAergic

system in AD has been shown in various studies, recent data and

the proposed GABAergic hypothesis of AD indicate its possibly

greater contribution with therapeutic potential from molecules

inﬂuencing this system. In our study, GAT inhibitory activity was

shown for the ﬁrst time for multifunctional anti-AD agents. To the

best of our knowledge, among them, we identiﬁed compound 31 as

Fig. 11. The inﬂuence of 36, 40, 51 (10 mM) and the reference inhibitor ketoconazole

(KE) on CYP3A4 activity. Statistical signiﬁcance (****p < 0.0001) was analyzed by

GraphPad Prism 8.0.1 software using one-way ANOVA and Bonferroni’s multiple

comparison posttest. The compounds were examined in triplicate.

the most potent GAT3 inhibitor (mGAT4 IC₅₀¼ 5.01

mM; hGAT3

IC₅₀¼ 2.95

m

M), with high selectivity against other subtypes.

From the point of view of the strategy of searching for a multi-

functional compound against AD, compound 36 is the most inter-

esting and valuable. It exhibits the following activities against the

tested molecular targets: eeAChE IC₅₀

IC₅₀¼ 60 nM; hBuChE IC₅₀¼ 20 nM; hBACE1 IC₅₀¼ 5.9

antiaggregation ¼ 57.9% (at 10 M); mGAT1 IC₅₀¼ 10.96

mGAT2 IC₅₀¼ 19.05 M. It is worth noting that a compound that

¼

2.86

m

M; eqBuChE

M; A

M and

m

b

m

displays such a broad and complex mechanism of action including

the modulation of GATs, potentially affecting the causes and alle-

viating the symptoms of AD was not so far reported among mul-

titarget agents. The inﬂuence on cholinergic and GABAergic

transmission and the pathological amyloid pathway make this

molecule unique and original. Furthermore, the preliminary results

from the in vitro preclinical studies of the selected active com-

pounds that determined their potential toxicity, hepatotoxicity,

metabolic stability, and drug-drug interactions, are a good indica-

tion and a promising starting point for further development of this

group of compounds and extended studies in vivo.

Fig. 12. The inﬂuence of 36, 40, 51 (10 mM) and the reference inhibitor quinidine (QD)

on CYP2D6 activity. Statistical signiﬁcance (****p < 0.0001) was analyzed by GraphPad

Prism 8.0.1 software using one-way ANOVA and Bonferroni’s multiple comparison

posttest. The compounds were examined in triplicate.

reference compounds at a concentration of 10 mM, and therefore,

they showed a high risk of DDIs. Further development of this group

of compounds requires the improvement of this property.

3. Conclusions

4. Experimental section

Multiple factors that inﬂuence the development of AD as well as

the initiation of changes in the brain many years before the onset of

disease symptoms are potential causes of a lack of effective therapy

and difﬁculties in planning and conducting appropriate preclinical

and clinical trials. The MTDL strategy applied in this research could

be a chance for the discovery of a new medicine and a proper

approach to treat this complex disease. With the support of mo-

lecular modeling methods, we have implemented rational modiﬁ-

cations of the lead structure compound I that resulted in a

signiﬁcant improvement in the biological activity of new multidi-

rectional molecules. It should be highlighted that as a result of this

work, we obtained two series of hydroxyethylamine derivatives,

including interesting compounds with a balanced activity proﬁle

affecting both symptoms and causes of AD and molecules with a

potent inhibitory effect toward just one target.

4.1. Chemistry

4.1.1. General methods

All of the reagents were purchased from commercial suppliers

and were used without further puriﬁcation. Tetrahydrofuran (THF)

and dichloromethane (DCM) were distilled under nitrogen imme-

diately before use. The drying agent used for THF was sodium/

benzophenone ketyl, and for DCM, calcium hydride. Reactions were

monitored by thin-layer chromatography carried out on aluminium

sheets precoated with silica gel 60 F₂₅₄(Merck). For the TLC and

ﬂash chromatography following solvents were used: dichloro-

methane (DCM), methanol (MeOH), diethyl ether (Et₂O), chloro-

form (CHCl₃), ethyl acetate (EtOAc), petroleum ether (PET), 25%

ammoniaꢂwater solution, acetonitrile (CH₃CN). Flash chromatog-

raphy was performed on Isolera™ Spectra (Biotage) using Merck

Regarding the enzyme BuChE, structural changes in both series

greatly ameliorated the level of inhibition. Among amine de-

rivatives, the most potent hBuChE inhibitor 27 was over 30 times

more active (hBuChE IC₅₀¼ 190 nM) than compound I, whereas in

silica gel 60 (63e200

HPLC puriﬁcation was performed on LC-4000 Jasco using a Phe-

nomenex Luna C8 (5

m, 15 ꢃ 21.2 mm) column and CH₃CN/H₂O

mm) as a stationary phase. The reverse-phase

m

12

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

gradient with 0.1% HCOOH as a mobile phase. The purity of the ﬁnal

(0.093 g, 1.48 mmol) in DCM (5 mL) was performed according to the

compounds was determined using an analytical RPLC-MS on Wa-

procedure C. Puriﬁcation: washing reaction mixture with water

ters Acquity TQD using an Aquity UPLC BEH C18 column (1.7

m

m,

(3 ꢃ 30 mL), ﬂash column chromatography (DCM/MeOH/NH_3(aq)

,

2.1 ꢃ 100 mm) at 214 and 254 nm CH₃CN/H₂O gradient with 0.1%

HCOOH was used as the mobile phase at a ﬂow rate of 0.3 mL/min.

All the compounds showed purity above 95%. ¹H NMR and ¹³C NMR

spectra were recorded on Varian Mercury 300 MHz or Jeol

500 MHz. The chemical shifts are reported in ppm and were

referenced to residual solvent signals (¹H, CDCl₃at 7.26 ppm,

DMSO‑d₆at 2.50 ppm; ¹³C, CDCl₃at 77.2 ppm). Signal multiplicities

are represented by the following abbreviations: s (singlet), br. s

(broad singlet), br. d (broad doublet), d (doublet), dd (doublet of

doublets), ddd (doublet of doublet of doublets), dt (doublet of

triplets), t (triplet), td (triplet of doublets), tdd (triplet of doublet of

doublets), q (quartet), dq (doublet of quartets), qd (quartet of

doublets), and m (multiplet). Mass spectra (MS) were recorded on

UPLC-MS/MS system consisting of a Waters ACQUITY UPLC (Waters

Corporation, Milford, MA, USA) coupled to a Waters TQD mass

spectrometer (electrospray ionization mode ESI-tandem

quadrupole).

9.5/0.5/0.05 v/v/v). Yield: 0.095 g (28.5%), TLC (DCM/MeOH/NH_3(aq)

9.5/0.5/0.05 v/v/v) R_f¼ 0.33, MW 450.63, formula: C₃₁H₃₄N₂O, MS

m/z 451.3 (M þ H^þ), ¹H NMR (300 MHz, CDCl₃)

d ppm 6.97e7.49 (m,

20H), 3.97 (t, J ¼ 7.62 Hz, 1H), 3.69e3.85 (m, 3H), 3.19 (dd, J ¼ 11.72,

6.45 Hz, 1H), 3.08 (dd, J ¼ 11.72, 8.21 Hz, 1H), 2.91 (dt, J ¼ 9.23,

4.47 Hz, 1H), 2.65e2.82 (m, 3H), 2.53 (dd, J ¼ 14.07, 9.38 Hz, 1H),

2.16 (br. s, 3H); ¹³C NMR (75 MHz, CDCl₃)

d ppm 142.9, 142.2, 139.6,

138.4, 128.6, 128.5, 128.5, 128.3, 128.3, 127.9, 127.8, 127.1, 126.5,

126.4, 126.3, 69.6, 62.3, 53.8, 52.7, 51.5, 50.9, 35.6.

4.1.4.2. (2R,3S)-1-(benzylamino)-3-((3,3-diphenylpropyl)amino)-4-

phenylbutan-2-ol (17). The reaction of (2R,3S)-3-amino-1-(benzy-

lamino)-4-phenylbutan-2-ol (1b) (0.200 g, 0.74 mmol) with 3,3-

diphenylpropanal (0.156 g, 0.74 mmol) in the presence of acetic

acid (0.067 g, 1.11 mmol) and sodium cyanoborohydride (0.093 g,

1.48 mmol) in DCM (5 mL) was performed according to the pro-

cedure C. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 30 mL), ﬂash column chromatography (DCM/MeOH/NH_3(aq)

,

4.1.2. Procedure for the synthesis of compounds 1a-15a (procedure

A)

9.5/0.5/0.05 v/v/v). Yield: 0.061 g (17.7%), TLC (DCM/MeOH/NH_3(aq)

9.5/0.5/0.05 v/v/v) R_f¼ 0.26, MW 464.65, formula: C₃₂H₃₆N₂O, MS

m/z 465.4 (M þ H^þ), ¹H NMR (300 MHz, CDCl₃)

d ppm 7.05e7.36 (m,

Tert-butyl ((S)-1-((S)-oxiran-2-yl)-2-phenylethyl)carbamate or

tert-butyl ((S)-1-((R)-oxiran-2-yl)-2-phenylethyl)carbamate (1.0

equiv.), corresponding amine (1, 1.1 or 2 equiv.), and a catalytic

amount of pyridine in n-propanol, isopropanol or ethanol were

reﬂuxed for 16 h. Then, the solvent was evaporated and the

resulting residue was puriﬁed by ﬂash column chromatography

20H), 3.70e3.96 (m, 3H), 3.65 (dt, J ¼ 7.03, 4.10 Hz, 1H), 2.37e2.84

(m, 8H), 1.75e2.33 (m, 4H), ¹³C NMR (75 MHz, CDCl₃)

d ppm 144.2,

137.9, 129.2, 129.1, 128.8, 128.8, 128.7, 128.7, 128.5, 128.0, 127.7,

126.6, 126.5, 126.3, 68.5, 62.2, 53.0, 50.3, 48.5, 45.8, 35.3, 35.2.

using

puriﬁcation).

a mixture of DCM and MeOH (gradient or isocratic

4.1.4.3. (2S,3S)-1-(benzylamino)-3-((3,3-diphenylpropyl)amino)-4-

phenylbutan-2-ol (18). The reaction of (2S,3S)-3-amino-1-(benzy-

lamino)-4-phenylbutan-2-ol (2b) (0.150 g, 0.55 mmol) with 3,3-

diphenylpropanal (0.117 g, 0.55 mmol) in the presence of acetic

acid (0.050 g, 0.83 mmol) and sodium cyanoborohydride (0.070 g,

1.11 mmol) in DCM (4 mL) was performed according to the pro-

cedure C. Puriﬁcation: washing reaction mixture with water

4.1.3. Procedure for the synthesis of compounds 1be15b (procedure

B)

To the solution of appropriate BOC-protected compound (1.0

equiv.) in DCM, TFA (5 mL/1 mmol substrate) was added dropwise.

The reaction mixture was stirred at room temperature for 2 h.

When the reaction was ﬁnished, the solvent and TFA were evapo-

rated under reduced pressure, producing a residue that was then

dissolved in ammonia water solution and extracted with DCM. The

organic extracts were combined, dried over anhydrous Na₂SO₄,

ﬁltered and concentrated under vacuum.

(3 ꢃ 30 mL), ﬂash column chromatography (DCM/MeOH/NH_3(aq)

,

9.5/0.5/0.05 v/v/v). Yield: 0.053 g (20.4%), TLC (DCM/MeOH/NH_3(aq)

9.5/0.5/0.05 v/v/v) R_f¼ 0.24, MW 464.65, formula: C₃₂H₃₆N₂O, MS

m/z 465.4 (M þ H^þ), ¹H NMR (300 MHz, CDCl₃)

d ppm 7.14e7.35 (m,

18H), 7.06e7.13 (m, 2H), 3.93 (t, J ¼ 7.91 Hz, 1H), 3.86 (d, J ¼

12.89 Hz, 1H), 3.75 (d, J ¼ 13.50 Hz, 1H), 3.48e3.55 (m, 1H),

2.56e2.97 (m, 9H), 2.43 (dt, J ¼ 11.72, 7.33 Hz, 1H), 2.12 (q, J ¼

Detailed procedures for the synthesis of intermediates 1a-15a

and 1be15b are described in Supporting Information

7.42 Hz, 2H); ¹³C NMR (75 MHz, CDCl₃)

d ppm 144.5, 144.4, 138.8,

138.4, 129.3, 128.6, 128.5, 128.5, 128.4, 127.7, 127.3, 126.4, 126.3, 69.1,

62.4, 53.5, 52.4, 48.7, 45.7, 36.9, 35.8.

4.1.4. Procedure for the synthesis of compounds 16e27 (procedure

C)

To the solution of 1-substituted 1,3-diamino-4-phenylbutan-2-

ol derivative (1.0 equiv.) in anhydrous DCM (10 mL/1 mmol sub-

strate) 2,2-diphenylacetaldehyde or 3,3-diphenylpropanal (0.8e1.0

equiv.) and acetic acid (1e1.5 equiv.) were added. The reaction

mixture was stirred at room temperature for 0.5 h and then sodium

cyanoborohydride (1.8e2.5 equiv.) was added portionwise. When

the reaction was ﬁnished, the reaction mixture was washed with

water. The organic phase was then dried over anhydrous Na₂SO₄,

ﬁltered and concentrated under vacuum. The crude product was

puriﬁed by ﬂash column chromatography using a mixture of DCM

and MeOH or by HPLC preparative system using an acetonitrile and

water mixture.

4.1.4.4. (2S,3S)-3-((2,2-diphenylethyl)amino)-1-((2-methoxybenzyl)

amino)-4-phenylbutan-2-ol (19). The reaction of (2S,3S)-3-amino-

1-((2-methoxybenzyl)amino)-4-phenylbutan-2-ol (3b) (0.150 g,

0.50 mmol) with 2,2-diphenylacetaldehyde (0.098 g, 0.50 mmol) in

the presence of acetic acid (0.045 g, 0.75 mmol) and sodium cya-

noborohydride (0.057 g, 0.90 mmol) in DCM (4 mL) was performed

according to the procedure C. Puriﬁcation: washing reaction

mixture with water (3 ꢃ 30 mL), ﬂash column chromatography

(DCM/MeOH/NH_3(aq), 9.5/0.5/0.05 v/v/v). Yield: 0.030 g (12.5%), TLC

(DCM/MeOH/NH_3(aq), 9.5/0.5/0.05 v/v/v) R_f¼ 0.22, MW 480.65,

formula: C₃₂H₃₆N₂O₂, MS m/z 481.4 (M þ H^þ), ¹H NMR (300 MHz,

CDCl₃)

7.62 Hz, 1H), 3.77e3.85 (m, 4H), 3.56e3.64 (m, 1H), 3.15e3.48 (m,

5H), 3.04e3.14 (m, 1H), 2.72e2.90 (m, 3H), 2.55e2.71 (m, 2H), ¹³

NMR (75 MHz, CDCl₃) ppm 157.6, 142.8, 138.5, 130.3, 129.2, 129.1,

d

ppm 7.06e7.39 (m, 17H), 6.83e6.97 (m, 2H), 3.98 (t, J ¼

4.1.4.1. (2R,3S)-1-(benzylamino)-3-((2,2-diphenylethyl)amino)-4-

phenylbutan-2-ol (16). The reaction of (2R,3S)-3-amino-1-(benzy-

lamino)-4-phenylbutan-2-ol (1b) (0.200 g, 0.74 mmol) with 2,2-

diphenylacetaldehyde (0.145 g, 0.74 mmol) in the presence of

acetic acid (0.067 g, 1.11 mmol) and sodium cyanoborohydride

C

d

128.7, 128.6, 128.5, 128.0, 127.9, 126.8, 126.6, 126.4, 126.3, 126.1,

125.9, 120.6, 110.4, 68.7, 62.5, 55.3, 52.5, 51.9, 51.7, 48.3, 36.9.

13

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European Journal of Medicinal Chemistry 218 (2021) 113397

4.1.4.5. (2S,3S)-3-((3,3-diphenylpropyl)amino)-1-((2-

methoxybenzyl)amino)-4-phenylbutan-2-ol (20). The reaction of

(2S,3S)-3-amino-1-((2-methoxybenzyl)amino)-4-phenylbutan-2-

ol (3b) (0.200 g, 0.67 mmol) with 3,3-diphenylpropanal (0.141 g,

0.67 mmol) in the presence of acetic acid (0.060 g, 1.00 mmol) and

sodium cyanoborohydride (0.105 g, 1.67 mmol) in DCM (4 mL) was

performed according to the procedure C. Puriﬁcation: washing

reaction mixture with water (3 ꢃ 30 mL), ﬂash column chroma-

tography (DCM/MeOH/NH_3(aq), 9.5/0.5/0.05 v/v/v). Yield: 0.072 g

(21.9%), TLC (DCM/MeOH/NH_3(aq), 9.5/0.5/0.05 v/v/v) R_f¼ 0.38, MW

494.68, formula: C₃₃H₃₈N₂O₂, MS m/z 495.2 (M þ H^þ), ¹H NMR

chromatography (3e10% MeOH in DCM), HPLC preparative system

(20e90% water in acetonitrile). Yield: 0.038 g (14.6%), TLC (DCM/

MeOH/NH_3(aq), 9/1/0.1 v/v/v) R_f¼ 0.36, MW 464.65, formula:

C

₃₂H₃₆N₂O, MS m/z 465.2 (M þ H^þ), ¹H NMR (300 MHz, CDCl₃)

ppm 7.00e7.43 (m, 18H), 6.82e6.93 (m, 2H), 4.75 (br. s, 2H), 3.96

d

(t, J ¼ 7.62 Hz, 1H), 3.63e3.85 (m, 2H), 3.10e3.39 (m, 2H), 2.92e3.10

(m, 3H), 2.61 (dd, J ¼ 12.60, 4.98 Hz,1H), 2.43 (dd, J ¼ 13.48, 4.69 Hz,

1H), 2.13e2.28 (m, 1H), 1.43 (d, J ¼ 6.45 Hz, 2H), 1.24e1.35 (m, 1H),

¹³C NMR (75 MHz, CDCl₃)

d ppm 170.2, 142.6, 142.1, 137.7, 128.9,

128.8, 128.6, 128.5, 128.3, 127.9, 127.7, 127.3, 126.6, 126.5, 68.5, 62.9,

58.1, 53.4, 51.7, 48.0, 36.2, 22.0.

(300 MHz, CDCl₃)

d ppm 7.06e7.33 (m, 17H), 6.81e6.94 (m, 2H),

4.03 (d, J ¼ 12.89 Hz, 1H), 3.81e3.95 (m, 4H), 3.80 (s, 3H), 3.66 (br. s,

1H), 3.48 (s, 2H), 3.13 (dd, J ¼ 12.60, 3.81 Hz,1H), 2.56e2.85 (m, 4H),

2.40e2.51 (m, 1H), 1.98e2.10 (m, 2H); ¹³C NMR (75 MHz, CDCl₃)

4.1.4.9. (2R,3S)-3-((3,3-diphenylpropyl)amino)-4-phenyl-1-(((S)-1-

phenylethyl)amino)butan-2-ol (24). The reaction of (2R,3S)-3-

amino-4-phenyl-1-(((S)-1-phenylethyl)amino)butan-2-ol

(10b)

d

ppm 157.6, 144.1, 137.8, 130.9, 130.2, 129.3, 128.7, 128.6, 127.7,

(0.160 g, 0.56 mmol) with 3,3-diphenylpropanal (0.095 g,

0.45 mmol) in the presence of acetic acid (0.051 g, 0.84 mmol) and

sodium cyanoborohydride (0.088 g, 1.66 mmol) in DCM (2.5 mL)

was performed according to the procedure C. Puriﬁcation: washing

reaction mixture with water (3 ꢃ 30 mL), ﬂash column chroma-

tography (3e10% MeOH in DCM), HPLC preparative system

(20e90% water in acetonitrile). Yield: 0.040 g (14.7%), TLC (DCM/

MeOH/NH_3(aq), 9/1/0.1 v/v/v) R_f¼ 0.38, MW 478.68, formula:

126.6, 126.4, 122.8, 120.9, 110.7, 66.0, 63.6, 55.6, 52.5, 48.8, 48.4,

45.5, 36.3, 35.2.

4.1.4.6. (2R,3S)-3-((3,3-diphenylpropyl)amino)-1-((3-

methoxybenzyl)amino)-4-phenylbutan-2-ol (21). The reaction of

(2R,3S)-3-amino-1-((3-methoxybenzyl)amino)-4-phenylbutan-2-

ol (4b) (0.200 g, 0.67 mmol) with 3,3-diphenylpropanal (0.140 g,

0.67 mmol) in the presence of acetic acid (0.060 g, 1.00 mmol) and

sodium cyanoborohydride (0.084 g, 1.33 mmol) in DCM (4 mL) was

performed according to the procedure C. Puriﬁcation: washing

reaction mixture with water (3 ꢃ 30 mL), ﬂash column chroma-

tography (DCM/MeOH/NH_3(aq), 9.5/0.5/0.05 v/v/v). Yield: 0.070 g

(21.2%), TLC (DCM/MeOH/NH_3(aq), 9.5/0.5/0.05 v/v/v) R_f¼ 0.27, MW

494.68, formula: C₃₃H₃₈N₂O₂, MS m/z 495.2 (M þ H^þ), ¹H NMR

C

₃₃H₃₈N₂O, MS m/z 479.3 (M þ H^þ), ¹H NMR (300 MHz, CDCl₃)

ppm 7.43e7.51 (m, 2H), 7.30e7.39 (m, 3H), 7.00e7.26 (m, 13H),

d

6.83 (d, J ¼ 6.45 Hz, 2H), 4.74 (d, J ¼ 9.38 Hz,1H), 4.16 (q, J ¼ 6.84 Hz,

1H), 3.85e3.95 (m, 1H), 3.40e3.69 (m, 2H), 2.46e3.14 (m, 9H), 2.37

(dd, J ¼ 12.02, 10.26 Hz, 1H), 1.74 (d, J ¼ 7.03 Hz, 3H); ¹³C NMR

(75 MHz, CDCl₃) d ppm 143.3, 143.2, 135.9, 135.9, 129.3, 128.9, 128.7,

127.9, 127.7, 127.0, 126.5, 126.5, 66.9, 61.9, 59.2, 48.7, 48.6, 45.6, 31.5,

31.4, 20.6.

(500 MHz, CDCl₃)

d ppm 7.77 (br. s, 3H), 7.08e7.26 (m, 14H),

6.97e7.01 (m, 2H), 6.90 (s, 1H), 6.85 (dd, J ¼ 8.31, 2.00 Hz, 2H), 4.09

(d, J ¼ 7.45 Hz, 1H), 3.95 (d, J ¼ 13.17 Hz, 1H), 3.75e3.83 (m, 2H),

3.73 (s, 3H), 3.11e3.17 (m, 1H), 2.76e2.84 (m, 2H), 2.53e2.70 (m,

4.1.4.10. (2R,3S)-3-((3,3-diphenylpropyl)amino)-4-phenyl-1-((2-

phenylpropan-2-yl)amino)butan-2-ol (25). The reaction of (2R,3S)-

3-amino-4-phenyl-1-((2-phenylpropan-2-yl)amino)butan-2-ol

(11b) (0.200 g, 0.67 mmol) with 3,3-diphenylpropanal (0.140 g,

0.67 mmol) in the presence of acetic acid (0.060 g, 1.01 mmol) and

sodium cyanoborohydride (0.076 g, 1.21 mmol) in DCM (4 mL) was

performed according to the procedure C. Puriﬁcation: washing

reaction mixture with water (3 ꢃ 30 mL), ﬂash column chroma-

tography (DCM/MeOH/NH_3(aq), 9.5/0.5/0.05 v/v/v). Yield: 0.074 g

(22.4%), TLC (DCM/MeOH/NH_3(aq), 9.5/0.5/0.05 v/v/v) R_f¼ 0.31, MW

492.71, formula: C₃₄H₄₀N₂O, MS m/z 493.3 (M þ H^þ), ¹H NMR

4H), 2.24 (q, J ¼ 7.45 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃)

d ppm

169.4, 160.1, 143.8, 137.2, 130.1, 129.1, 128.8, 128.7, 127.7, 127.7, 126.9,

126.6, 121.7, 114.9, 114.6, 67.8, 62.5, 55.3, 51.4, 49.5, 48.8, 45.7, 33.4.

4.1.4.7. (2R,3S)-3-((3,3-diphenylpropyl)amino)-1-((3-

isopropylbenzyl)amino)-4-phenylbutan-2-ol (22). The reaction of

(2R,3S)-3-amino-1-((3-isopropylbenzyl)amino)-4-phenylbutan-2-

ol (6b) (0.114 g, 0.37 mmol) with 3,3-diphenylpropanal (0.077 g,

0.37 mmol) in the presence of acetic acid (0.033 g, 0.55 mmol) and

sodium cyanoborohydride (0.042 g, 0.66 mmol) in DCM (2.5 mL)

was performed according to the procedure C. Puriﬁcation: washing

reaction mixture with water (3 ꢃ 30 mL), ﬂash column chroma-

tography (3e10% MeOH in DCM). Yield: 0.035 g (18.9%), TLC (DCM/

MeOH/NH_3(aq), 9/1/0.1 v/v/v) R_f¼ 0.37, MW 506.73, formula:

(500 MHz, CDCl₃)

d

ppm 7.46 (d, J ¼ 7.45 Hz, 2H), 7.35 (t, J ¼ 7.73 Hz,

2H), 7.06e7.28 (m,14H), 7.00 (d, J ¼ 6.87 Hz, 2H), 3.78 (t, J ¼ 7.73 Hz,

1H), 3.64 (dt, J ¼ 7.30, 3.51 Hz, 1H), 3.48 (br. s, 3H), 2.82 (td, J ¼ 6.87,

4.58 Hz, 1H), 2.58e2.66 (m, 1H), 2.41e2.53 (m, 5H), 1.99e2.13 (m,

2H), 1.57 (d, J ¼ 10.31 Hz, 6H); ¹³C NMR (126 MHz, CDCl₃)

d ppm

C

d

₃₅H₄₂N₂O, MS m/z 507.3 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

ppm 7.03e7.34 (m, 19H), 3.70e3.93 (m, 3H), 3.66 (dt, J ¼ 7.88,

144.7, 144.6, 138.3, 129.3, 128.7, 128.6, 128.6, 127.9, 127.10 126.5,

126.3, 126.3, 126.1, 69.6, 62.1, 57.1, 48.7, 45.7, 45.5, 35.8, 35.6, 29.1,

28.5.

3.79 Hz, 1H), 2.89 (dq, J ¼ 13.82, 7.04 Hz, 1H), 2.54e2.84 (m, 5H),

2.15e2.53 (m, 3H), 2.00e2.13 (m, 2H), 1.25 (d, J ¼ 6.30 Hz, 6H), two

NH not detected; ¹³C NMR (126 MHz, CDCl₃)

d

ppm 149.3, 144.6,

4.1.4.11. (2S,3S)-1-(tert-butylamino)-3-((3,3-diphenylpropyl)amino)-

4-phenylbutan-2-ol (26). The reaction of (2S,3S)-3-amino-1-(tert-

butylamino)-4-phenylbutan-2-ol (12b) (0.100 g, 0.42 mmol) with

3,3-diphenylpropanal (0.089 g, 0.42 mmol) in the presence of acetic

acid (0.038 g, 0.63 mmol) and sodium cyanoborohydride (0.053 g,

0.85 mmol) in DCM (3 mL) was performed according to the pro-

cedure C. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 30 mL), ﬂash column chromatography (3e10% MeOH in DCM).

Yield: 0.017 g (9.2%), TLC (DCM/MeOH/NH_3(aq), 9/1/0.1 v/v/v)

R_f¼ 0.31, MW 430.64, formula: C₂₉H₃₈N₂O, MS m/z 431.5 (M þ H^þ),

138.7, 129.3, 128.7, 128.6, 128.6, 128.5, 127.8, 127.8, 126.6, 126.6,

126.3, 126.3, 125.9, 125.4, 69.1, 61.9, 54.0, 51.1, 48.6, 45.7, 35.8, 35.6,

34.2, 24.1.

4.1.4.8. (2R,3S)-3-((2,2-diphenylethyl)amino)-4-phenyl-1-(((S)-1-

phenylethyl)amino)butan-2-ol (23). The reaction of (2R,3S)-3-

amino-4-phenyl-1-(((S)-1-phenylethyl)amino)butan-2-ol

(10b)

(0.160 g, 0.56 mmol) with 2,2-diphenylacetaldehyde (0.088 g,

0.45 mmol) in the presence of acetic acid (0.051 g, 0.84 mmol) and

sodium cyanoborohydride (0.088 g, 1.66 mmol) in DCM (2.5 mL)

was performed according to the procedure C. Puriﬁcation: washing

¹H NMR (300 MHz, CDCl₃)

d

ppm 7.09e7.32 (m, 15H), 3.94 (t, J ¼

7.91 Hz,1H), 3.42 (dt, J ¼ 7.33, 3.96 Hz,1H), 2.40e2.85 (m, 10H), 2.12

reaction mixture with water (3

ꢃ

30 mL), ﬂash column

(q, J ¼ 7.62 Hz, 2H), 1.09 (s, 9H); ¹³C NMR (75 MHz, CDCl₃)

d ppm

14

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

144.6, 139.0, 129.3, 128.5, 127.7, 126.2, 69.9, 62.3, 50.6, 48.7, 45.8,

45.7, 37.3, 36.2, 28.7.

4.15e4.23 (m, 1H), 3.70 (d, J ¼ 13.17 Hz, 1H), 3.64 (d, J ¼ 13.75 Hz,

1H), 3.58e3.62 (m, 1H), 2.88 (d, J ¼ 8.02 Hz, 2H), 2.64 (dd, J ¼ 12.32,

3.72 Hz, 1H), 2.39 (dd, J ¼ 12.60, 9.74 Hz, 1H), 1.98 (br. s, 2H); ¹³C

4.1.4.12. (2R,3S)-3-((2,2-diphenylethyl)amino)-1-(neopentylamino)-

4-phenylbutan-2-ol (27). The reaction of (2R,3S)-3-amino-1-(neo-

pentylamino)-4-phenylbutan-2-ol (13b) (0.150 g, 0.60 mmol) with

2,2-diphenylacetaldehyde (0.118 g, 0.60 mmol) in the presence of

acetic acid (0.054 g, 0.90 mmol) and sodium cyanoborohydride

(0.068 g, 1.08 mmol) in DCM (4 mL) was performed according to the

procedure C. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 30 mL), ﬂash column chromatography (5% MeOH in DCM).

Yield: 0.044 g (16.9%), TLC (DCM/MeOH, 9/1 v/v) R_f¼ 0.39, MW

430.64, formula: C₂₉H₃₈N₂O₂, MS m/z 431.3 (M þ H^þ), ¹H NMR

NMR (126 MHz, CDCl₃) d ppm 172.0, 139.3,139.3,138.0,129.5,128.9,

128.9,128.8,128.7, 128.6,128.6,128.2,127.4, 127.3, 127.2, 126.6, 68.9,

59.4, 53.6, 52.5, 51.8, 38.6.

4.1.5.3. N-((2S,3R)-4-(benzylamino)-3-hydroxy-1-phenylbutan-2-

yl)-3,3-diphenylpropanamide (30). The reaction of (2R,3S)-3-

amino-1-(benzylamino)-4-phenylbutan-2-ol (1b) (0.200 g,

0.74 mmol) with 3,3-diphenylpropanoic acid (0.167 g, 0.74 mmol)

in the presence of DMAP (0.045 mg, 0.37 mmol) and DCC (0.305 g,

1.48 mmol) in DCM (5 mL) was performed according to the pro-

cedure D. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 30 mL), ﬂash column chromatography (5% MeOH in DCM).

Yield: 0.130 g (36.7%), TLC (DCM/MeOH, 9/1 v/v) R_f¼ 0.30, MW

478.64, formula: C₃₂H₃₄N₂O₂, MS m/z 479.5 (M þ H^þ),¹H NMR

(500 MHz, CDCl₃) d ppm 7.12e7.36 (m, 10H), 7.00e7.10 (m, 5H), 3.97

(d, J ¼ 19.48 Hz, 2H), 3.34e3.43 (m, 2H), 3.13e3.27 (m, 3H),

3.00e3.09 (m, 1H), 2.75e2.88 (m, 2H), 2.50e2.64 (m, 2H), 1.05 (s,

9H), 0.98 (s, 2H); ¹³C NMR (126 MHz, CDCl₃)

d ppm 129.1, 129.0,

128.9, 128.7, 128.6, 128.0, 127.9, 126.8, 68.1, 63.2, 60.8, 53.3, 52.7,

51.4, 36.7, 31.1, 27.6.

(500 MHz, CDCl₃) d ppm 7.33e7.42 (m, 5H), 7.04e7.33 (m, 15H),

6.32 (d, J ¼ 8.59 Hz, 1H), 4.48 (dd, J ¼ 9.45, 6.59 Hz, 1H), 4.13 (br. s,

2H), 3.95 (qd, J ¼ 8.50, 4.30 Hz, 1H), 3.74 (d, J ¼ 13.17 Hz, 1H), 3.56

(dt, J ¼ 8.02, 4.01 Hz, 1H), 3.49 (d, J ¼ 12.60 Hz, 1H), 2.85e2.98 (m,

2H), 2.75e2.84 (m, 3H), 2.28 (dd, J ¼ 13.17, 4.58 Hz, 1H); ¹³C NMR

4.1.5. Procedure for the synthesis of compounds 28e56 (procedure

D)

To the solution of 2,2-diphenylacetic acid or 3,3-

diphenylpropanoic acid (1.0 equiv.) in anhydrous DCM (10 mL/

1 mmol substrate) the following reagents in sequence were added:

DMAP (0.5 equiv.), HOBt (1.3 equiv.) and EDC or DCC (1.3e2 equiv.).

The reaction mixture was stirred at room temperature for 0.5 h and

then appropriate 1-substituted 1,3-diamino-4-phenylbutan-2-ol

derivative (1.0 equiv.) was added and stirring was overnight

continued. When the reaction was ﬁnished, the reaction mixture

was washed with water. The organic phase was than dried over

anhydrous Na₂SO₄, ﬁltered and concentrated under vacuum. The

crude product was puriﬁed by ﬂash column chromatography using

a mixture of DCM and MeOH or by HPLC preparative system using

an acetonitrile and water mixture.

(126 MHz, CDCl₃) d ppm 173.1,143.4,143.2,137.0,129.8,129.4,129.3,

129.2, 128.9, 128.8, 127.9, 127.6, 126.9, 126.8, 126.8, 69.2, 52.5, 52.2,

50.2, 47.3, 42.4, 36.1.

4.1.5.4. N-((2S,3S)-4-(benzylamino)-3-hydroxy-1-phenylbutan-2-

yl)-3,3-diphenylpropanamide (31). The reaction of (2S,3S)-3-amino-

1-(benzylamino)-4-phenylbutan-2-ol (2b) (0.150 g, 0.55 mmol)

with 3,3-diphenylpropanoic acid (0.126 g, 0.55 mmol) in the pres-

ence of DMAP (0.034 mg, 0.28 mmol) and DCC (0.231 g, 1.11 mmol)

in DCM (4 mL) was performed according to the procedure D. Pu-

riﬁcation: washing reaction mixture with water (3 ꢃ 30 mL), ﬂash

column chromatography (5e6% MeOH in DCM). Yield: 0.089 g

(32.2%), TLC (DCM/MeOH, 9/1 v/v) R_f¼ 0.35, MW 478.64, formula:

C

d

₃₂H₃₄N₂O₂, MS m/z 479.3 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

ppm 7.13e7.34 (m, 20H), 5.99 (d, J ¼ 8.59 Hz, 1H), 4.55 (t, J ¼

4.1.5.1. N-((2S,3R)-4-(benzylamino)-3-hydroxy-1-phenylbutan-2-

yl)-2,2-diphenylacetamide (28). The reaction of (2R,3S)-3-amino-1-

(benzylamino)-4-phenylbutan-2-ol (1b) (0.200 g, 0.74 mmol) with

2,2-diphenylacetic acid (0.157 g, 0.74 mmol) in the presence of

DMAP (0.045 g, 0.37 mmol) and DCC (0.305 g, 1.48 mmol) in DCM

(5 mL) was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 30 mL), ﬂash column

chromatography (5% MeOH in DCM). Yield: 0.103 g (30.0%), TLC

(DCM/MeOH, 9/1 v/v) R_f¼ 0.30, MW 464.61, formula: C₃₁H₃₂N₂O₂,

8.02 Hz, 1H), 3.89e3.96 (m, 1H), 3.51e3.59 (m, 3H), 2.84e2.94 (m,

2H), 2.83 (br. s, 2H), 2.73 (d, J ¼ 7.45 Hz, 2H), 2.31 (dd, J ¼ 12.60,

4.01 Hz, 1H), 2.02 (dd, J ¼ 12.32, 10.02 Hz, 1H); ¹³C NMR (126 MHz,

CDCl₃)

d ppm 171.0, 143.7, 139.2, 138.2, 129.4, 128.7, 128.7, 128.5,

128.2, 128.1, 127.8, 127.5, 126.6, 126.6, 126.5, 67.7, 53.3, 52.1, 51.5,

47.6, 43.3, 38.5.

4.1.5.5. N-((2S,3S)-3-hydroxy-4-((2-methoxybenzyl)amino)-1-

phenylbutan-2-yl)-2,2-diphenylacetamide (32). The reaction of

(2S,3S)-3-amino-1-((2-methoxybenzyl)amino)-4-phenylbutan-2-

ol (3b) (0.120 g, 0.40 mmol) with 2,2-diphenylacetic acid (0.085 g,

0.40 mmol) in the presence of DMAP (0.024 g, 0.20 mmol) and DCC

(0.165 g, 0.80 mmol) in DCM (4 mL) was performed according to the

procedure D. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 30 mL), ﬂash column chromatography (5% MeOH in DCM).

Yield: 0.092 g (46.3%), TLC (DCM/MeOH, 9/1 v/v) R_f¼ 0.26, MW

494.64, formula: C₃₂H₃₄N₂O₃, MS m/z 495.3 (M þ H^þ), ¹H NMR

MS m/z 465.4 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

d ppm 7.21e7.37

(m, 14H), 7.01e7.06 (m, 4H), 6.93e6.98 (m, 2H), 5.99 (d, J ¼ 9.17 Hz,

1H), 4.82 (s, 1H), 4.22e4.30 (m, 1H), 3.74e3.80 (m, 1H), 3.65e3.73

(m, 1H), 3.55 (td, J ¼ 6.01, 3.44 Hz, 1H), 3.25 (br. s, 2H), 2.95 (dd, J ¼

14.32, 4.58 Hz, 1H), 2.69e2.74 (m, 1H), 2.61e2.69 (m, 2H); ¹³C NMR

(126 MHz, CDCl₃) d ppm 172.7, 139.3, 139.1, 139.0,129.4,129.1, 129.0,

128.9, 128.8, 128.7, 128.6, 128.6, 127.5, 127.4, 127.2, 126.6, 70.8, 59.3,

54.0, 53.7, 50.6, 36.6.

4.1.5.2. N-((2S,3S)-4-(benzylamino)-3-hydroxy-1-phenylbutan-2-

yl)-2,2-diphenylacetamide (29). The reaction of (2S,3S)-3-amino-1-

(benzylamino)-4-phenylbutan-2-ol (2b) (0.200 g, 0.74 mmol) with

2,2-diphenylacetic acid (0.157 g, 0.74 mmol) in the presence of

DMAP (0.045 g, 0.37 mmol) and DCC (0.305 g, 1.48 mmol) in DCM

(5 mL) was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 30 mL), ﬂash column

chromatography (5% MeOH in DCM). Yield: 0.047 g (13.8%), TLC

(DCM/MeOH, 9/1 v/v) R_f¼ 0.35, MW 464.61, formula: C₃₁H₃₂N₂O₂,

(500 MHz, CDCl₃) d ppm 7.19e7.32 (m, 12H), 7.09e7.16 (m, 4H), 7.07

(dd, J ¼ 7.45, 1.72 Hz, 1H), 6.84e6.92 (m, 2H), 6.13 (d, J ¼ 9.17 Hz,

1H), 4.86 (s, 1H), 4.11e4.19 (m, 1H), 3.79 (s, 3H), 3.67 (s, 2H),

3.60e3.66 (m, 1H), 2.90 (d, J ¼ 8.02 Hz, 2H), 2.58 (dd, J ¼ 12.60,

3.44 Hz, 1 H), 2.34 (dd, J ¼ 12.60, 10.31 Hz, 1H), 2.03 (br. s, 2H); ¹³

C

NMR (126 MHz, CDCl₃) d ppm 157.8, 155.9, 137.0, 135.5, 129.4, 128.9,

128.8, 128.7, 128.6, 128.1, 128.1, 126.9, 80.1, 72.8, 55.4, 53.6, 48.4,

46.1, 38.7, 28.3.

MS m/z 465.4 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

d

ppm 7.17e7.35

4.1.5.6. N-((2S,3S)-3-hydroxy-4-((2-methoxybenzyl)amino)-1-

phenylbutan-2-yl)-3,3-diphenylpropanamide (33). The reaction of

(m, 16H), 7.06e7.13 (m, 4H), 6.01 (d, J ¼ 9.17 Hz, 1H), 4.84 (s, 1H),

15

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

(2S,3S)-3-amino-1-((2-methoxybenzyl)amino)-4-phenylbutan-2-

ol (3b) (0.120 g, 0.40 mmol) with 3,3-diphenylpropanoic acid

(0.091 g, 0.40 mmol) in the presence of DMAP (0.024 g, 0.20 mmol)

and DCC (0.165 g, 0.80 mmol) in DCM (4 mL) was performed ac-

cording to the procedure D. Puriﬁcation: washing reaction mixture

with water (3 ꢃ 30 mL), ﬂash column chromatography (5% MeOH in

DCM). Yield: 0.048 g (23.7%), TLC (DCM/MeOH, 9/1 v/v) R_f¼ 0.27,

MW 508.66, formula: C₃₃H₃₆N₂O₃, MS m/z 509.4 (M þ H^þ), ¹H NMR

chromatography (5% MeOH in DCM), HPLC preparative system

(20e90% water in acetonitrile). Yield: 0.198 g (62.2%), TLC (DCM/

MeOH/NH_3(aq),9/1/0.1 v/v/v) R_f¼ 0.39, MW 520.72, formula:

C

₃₅H₄₀N₂O₂, MS m/z 521.3 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

ppm 7.66 (br. s, 2H), 7.37e7.42 (m, 2H), 7.19e7.30 (m, 11H),

d

7.13e7.17 (m, 1H), 6.99e7.03 (m, 2H), 6.96 (td, J ¼ 4.73, 1.43 Hz, 3H),

6.17 (d, J ¼ 8.02 Hz, 1H), 4.78 (s, 1H), 4.19 (qd, J ¼ 8.59, 4.01 Hz, 1H),

3.93 (d, J ¼ 13.17 Hz, 1H), 3.81 (d, J ¼ 12.60 Hz, 2H), 3.10 (dd, J ¼

14.32, 4.01 Hz, 1H), 3.00 (dd, J ¼ 12.89, 2.58 Hz, 1H), 2.64e2.75 (m,

(500 MHz, CDCl₃)

d

ppm 7.11e7.30 (m, 16H), 6.95 (dd, J ¼ 7.16,

1.43 Hz, 1H), 6.77e6.84 (m, 2H), 6.32 (d, J ¼ 9.17 Hz, 1H), 4.55 (t, J ¼

8.02 Hz, 1H), 3.88e3.95 (m, 1H), 3.73 (s, 3H), 3.50e3.57 (m, 2H),

3.42e3.48 (m, 1H), 2.85e2.97 (m, 2H), 2.77 (dd, J ¼ 13.75, 9.16 Hz,

1H), 2.70 (dd, J ¼ 13.17, 6.30 Hz, 1H), 2.48 (br. s, 2H), 2.24 (dd, J ¼

12.60, 3.44 Hz, 1H), 2.00 (dd, J ¼ 12.32, 10.60 Hz, 1H); ¹³C NMR

2H), 1.30 (s, 9H); ¹³C NMR (126 MHz, CDCl₃)

d ppm 173.3, 169.4,

152.2, 138.9, 138.8, 137.3, 131.3, 129.4, 129.0, 128.9, 128.8, 128.8,

128.7, 127.4, 127.3, 127.2, 126.9, 126.7, 126.2, 69.3, 58.8, 53.3, 52.2,

50.2, 36.0, 34.8, 31.4.

(126 MHz, CDCl₃)

d

ppm 171.1, 157.6, 143.7, 143.7, 138.3, 130.3, 129.5,

4.1.5.10. N-((2S,3R)-4-((3-(tert-butyl)benzyl)amino)-3-hydroxy-1-

phenylbutan-2-yl)-3,3-diphenylpropanamide (37). The reaction of

(2R,3S)-3-amino-1-((3-(tert-butyl)benzyl)amino)-4-phenylbutan-

2-ol (5b) (0.200 g, 0.61 mmol) with 3,3-diphenylpropanoic acid

(0.138 g, 0.61 mmol) in the presence of DMAP (0.037 g, 0.31 mmol),

HOBt (0.107 g, 0.80 mmol) and EDC (0.152 g, 0.80 mmol) in DCM

(8 mL) was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 10 mL), ﬂash column

chromatography (5% MeOH in DCM). Yield: 0.073 g (22.4%), TLC

(DCM/MeOH/NH_3(aq),9/1/0.1 v/v/v) R_f¼ 0.33, MW 534.74, formula:

128.6, 128.5, 128.1, 127.8, 126.6, 126.5, 126.4, 120.7, 110.5, 67.3, 55.3,

52.3, 51.2, 48.6, 47.6, 43.2, 38.4.

4.1.5.7. N-((2S,3R)-3-hydroxy-4-((3-methoxybenzyl)amino)-1-

phenylbutan-2-yl)-2,2-diphenylacetamide (34). The reaction of

(2R,3S)-3-amino-1-((3-methoxybenzyl)amino)-4-phenylbutan-2-

ol (4b) (0.400 g, 1.33 mmol) with 2,2-diphenylacetic acid (0.282 g,

1.33 mmol) in the presence of DMAP (0.082 g, 0.67 mmol), HOBt

(0.234 g, 1.73 mmol) and EDC (0.332 g, 1.73 mmol) in DCM (20 mL)

was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 30 mL), ﬂash column

chromatography (5% MeOH in DCM). Yield: 0.322 g (50.4%), TLC

C

d

₃₆H₄₂N₂O₂, MS m/z 535.3 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

ppm 7.46 (s, 1H), 7.40 (d, J ¼ 8.02 Hz, 1H), 7.32 (t, J ¼ 7.73 Hz, 1H),

7.10e7.29 (m, 16H), 6.63 (d, J ¼ 7.45 Hz, 1H), 4.50 (dd, J ¼ 9.45,

6.59 Hz, 1H), 4.01 (qd, J ¼ 8.31, 4.87 Hz, 1H), 3.77 (d, J ¼ 13.17 Hz,

1H), 3.56e3.61 (m, 1H), 3.51 (d, J ¼ 12.60 Hz, 1H), 2.94e3.02 (m,

2H), 2.87 (dd, J ¼ 14.89, 8.02 Hz, 1H), 2.74e2.83 (m, 2H), 2.31 (dd,

J ¼ 12.60, 4.58 Hz, 1H), 1.35 (s, 9H), NH, OH not detected; ¹³C NMR

(DCM/MeOH, 9.5/0.5 v/v) R_f

C

¼

0.24, MW 494.64, formula:

₃₂H₃₄N₂O₃, MS m/z 495.2 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

d

ppm 7.18e7.30 (m, 10H), 7.00e7.07 (m, 4H), 6.94 (dd, J ¼ 6.87,

2.86 Hz, 2H), 6.78e6.85 (m, 3H), 6.18 (d, J ¼ 8.59 Hz, 1H), 4.81 (s,

1H), 4.24 (dd, J ¼ 6.30, 4.01 Hz, 1H), 3.64e3.86 (m, 6H), 3.53e3.63

(m, 2H), 2.93 (dd, J ¼ 14.32, 4.58 Hz, 1H), 2.55e2.72 (m, 3H), ¹³C

(126 MHz, CDCl₃) d ppm 172.9,152.1,143.5,143.4,137.6,129.4,128.9,

128.8, 128.7, 128.6, 128.1, 127.7, 126.8, 126.8, 126.6, 126.0, 69.7, 53.1,

52.7, 50.5, 47.4, 42.5, 36.2, 34.9, 31.4.

NMR (126 MHz, CDCl₃)

d 172.8, 159.9, 140.2, 139.2, 139.1, 137.7,

129.6, 129.4, 129.1, 129.0, 128.9, 128.8, 128.7, 127.4, 127.2, 126.6,

120.9, 114.1, 113.1, 70.8, 59.2, 55.3, 53.9, 53.5, 50.5, 36.6.

4.1.5.11. N-((2S,3R)-3-hydroxy-4-((3-isopropylbenzyl)amino)-1-

phenylbutan-2-yl)-2,2-diphenylacetamide (38). The reaction of

(2R,3S)-3-amino-1-((3-isopropylbenzyl)amino)-4-phenylbutan-2-

ol (6b) (0.153 g, 0.49 mmol) with 2,2-diphenylacetic acid (0.104 g,

0.49 mmol) in the presence of DMAP (0.030 g, 0.25 mmol), HOBt

(0.086 g, 0.64 mmol) and EDC (0.122 g, 0.64 mmol) in DCM (8 mL)

was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 10 mL), ﬂash column

chromatography (5e8% MeOH in DCM), Yield: 0.108 g (43.5%), TLC

(DCM/MeOH/NH_3(aq),9/1/0.1 v/v/v) R_f¼ 0.51, MW 506.69, formula:

4.1.5.8. N-((2S,3R)-3-hydroxy-4-((3-methoxybenzyl)amino)-1-

phenylbutan-2-yl)-3,3-diphenylpropanamide (35). The reaction of

(2R,3S)-3-amino-1-((3-methoxybenzyl)amino)-4-phenylbutan-2-

ol (4b) (0.400 g, 1.33 mmol) with 3,3-diphenylpropanoic acid

(0.301 g, 1.33 mmol) in the presence of DMAP (0.082 g, 0.67 mmol),

HOBt (0.234 g, 1.73 mmol) and EDC (0.332 g, 1.73 mmol) in DCM

(20 mL) was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 30 mL), ﬂash column

chromatography (5% MeOH in DCM). Yield: 0.360 g (53.1%), TLC

C

₃₄H₃₈N₂O₂, MS m/z 507.7 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

ppm 7.21e7.31 (m, 10H), 7.15e7.20 (m, 2H), 7.11 (d, J ¼ 7.45 Hz,

(DCM/MeOH, 9.5/0.5 v/v) R_f

C

¼

0.27, MW 508.66, formula:

d

₃₃H₃₆N₂O₃, MS m/z 509.2 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

1H), 7.03 (d, J ¼ 7.45 Hz, 4H), 6.93e6.99 (m, 2H), 6.05 (d, J ¼ 8.02 Hz,

1H), 4.83 (s, 1H), 4.22e4.30 (m, 1H), 4.22 (br. s, 2H), 3.79 (d, J ¼

13.00 Hz,1H), 3.70 (d, J ¼ 13.00 Hz,1H), 3.59e3.65 (m,1H), 2.97 (dd,

J ¼ 14.03, 4.30 Hz, 1H), 2.90 (dt, J ¼ 13.75, 6.87 Hz, 1H), 2.79 (dd, J ¼

d

ppm 7.12e7.28 (m, 14H), 7.06 (d, J ¼ 6.87 Hz, 2H), 6.79e6.90 (m,

3H), 6.17 (d, J ¼ 8.59 Hz, 1H), 4.47 (t, J ¼ 8.02 Hz, 1H), 3.99e4.07 (m,

1H), 3.74e3.85 (m, 5H), 3.60e3.65 (m, 1H), 3.50e3.55 (m, 1H),

3.35e3.41 (m, 1H), 2.72e2.88 (m, 4H), 2.53 (dd, J ¼ 12.60, 3.44 Hz,

1H), 2.37 (dd, J ¼ 12.32, 5.44 Hz, 1H), ¹³C NMR (126 MHz, CDCl₃)

12.32, 2.58 Hz, 1H), 2.62e2.71 (m, 2H), 1.25 (d, J ¼ 6.87 Hz, 6H); ¹³

C

NMR (126 MHz, CDCl₃) d ppm 172.8, 149.4, 139.1, 139.0, 137.6, 129.4,

d

171.7, 159.9, 143.6, 143.5, 139.7, 137.6, 129.7, 129.4, 128.7, 128.7,

129.1, 129.0, 128.9, 128.8, 128.7, 127.4, 127.2, 127.0, 126.6, 126.2,

125.8, 70.6, 59.2, 53.8, 53.5, 50.6, 36.4, 34.1, 24.1, 24.1.

128.6, 128.0, 127.7, 126.7, 126.6, 126.6, 120.8, 114.3, 113.1, 70.1, 55.3,

53.4, 53.1, 50.5, 47.4, 43.2, 36.4.

4.1.5.12. N-((2S,3S)-3-hydroxy-4-((3-isopropylbenzyl)amino)-1-

phenylbutan-2-yl)-2,2-diphenylacetamide (39). The reaction of

(2S,3S)-3-amino-1-((3-isopropylbenzyl)amino)-4-phenylbutan-2-

ol (7b) (0.090 g, 0.29 mmol) with 2,2-diphenylacetic acid (0.061 g,

0.29 mmol) in the presence of DMAP (0.018 g, 0.14 mmol), HOBt

(0.051 g, 0.37 mmol) and EDC (0.072 g, 0.37 mmol) in DCM (5 mL)

was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 10 mL), ﬂash column

chromatography (5% MeOH in DCM), Yield: 0.051 g (35.0%), TLC

4.1.5.9. N-((2S,3R)-4-((3-(tert-butyl)benzyl)amino)-3-hydroxy-1-

phenylbutan-2-yl)-2,2-diphenylacetamide (36). The reaction of

(2R,3S)-3-amino-1-((3-(tert-butyl)benzyl)amino)-4-phenylbutan-

2-ol (5b) (0.200 g, 0.61 mmol) with 2,2-diphenylacetic acid (0.130 g,

0.61 mmol) in the presence of DMAP (0.037 g, 0.31 mmol), HOBt

(0.107 g, 0.80 mmol) and EDC (0.152 g, 0.80 mmol) in DCM (8 mL)

was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 10 mL), ﬂash column

16

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

(DCM/MeOH, 9/1 v/v) R_f¼ 0.42, MW 506.69, formula: C₃₄H₃₈N₂O₂,

NMR (500 MHz, CDCl₃) d ppm 7.52e7.55 (m, 2H), 7.47e7.49 (m, 1H),

MS m/z 507.4 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

d

ppm 7.18e7.31

7.45 (d, J ¼ 8.02 Hz, 1H), 7.14e7.26 (m, 13H), 7.04 (d, J ¼ 7.45 Hz, 2H),

5.72 (br. d, J ¼ 8.59 Hz, 1H), 4.48 (t, J ¼ 8.02 Hz, 1H), 4.01e4.10 (m,

1H). 3.61e3.72 (m, 2H), 3.20e3.37 (m, 1H), 2.90e3.13 (m, 1H),

2.76e2.81 (m, 4H), 2.41e2.46 (m, 1H), 2.34e2.40 (m, 1H), NH not

(m, 12H), 7.13 (t, J ¼ 7.50 Hz, 3H), 7.07e7.11 (m, 3H), 7.04 (d, J ¼

7.45 Hz,1H), 6.00 (d, J ¼ 8.59 Hz,1H), 4.85 (s,1H), 4.16e4.23 (m,1H),

3.68 (d, J ¼ 13.00 Hz, 1H), 3.57e3.64 (m, 2H), 2.86e2.92 (m, 3H),

2.66 (dd, J ¼ 12.03, 4.01 Hz, 1H), 2.39 (dd, J ¼ 12.32, 10.02 Hz, 1H),

1.81 (br. s, 2H), 1.25 (d, J ¼ 6.87 Hz, 6H); ¹³C NMR (126 MHz, CDCl₃)

detected; ¹³C NMR (126 MHz, CDCl₃)

d

ppm 171.5, 143.5, 143.5,

140.4, 137.4, 131.7, 130.9 (q, J ¼ 31.99 Hz), 129.4, 129.1, 128.7, 128.7,

127.9, 127.7, 126.8, 126.7, 125.1 (q, 4.02 Hz), 123.9 (q,

d

ppm 172.0, 149.3, 139.6, 139.4, 139.3, 138.1, 129.5, 128.9, 128.9,

J

¼

128.8, 128.7, 128.6, 127.3, 127.2, 126.5, 126.3, 125.6, 125.4, 68.8, 59.4,

53.8, 52.5, 51.9, 38.6, 34.1, 24.1, 24.1.

J ¼ 272.20 Hz),124.2 (q, J ¼ 3.62 Hz), 70.3, 53.3, 53.0, 50.5, 47.4, 43.3,

36.3.

4.1.5.13. N-((2S,3R)-3-hydroxy-4-((3-isopropylbenzyl)amino)-1-

phenylbutan-2-yl)-3,3-diphenylpropanamide (40). The reaction of

(2R,3S)-3-amino-1-((3-isopropylbenzyl)amino)-4-phenylbutan-2-

ol (6b) (0.150 g, 0.48 mmol) with 3,3-diphenylpropanoic acid

(0.109 g, 0.48 mmol) in the presence of DMAP (0.029 g, 0.24 mmol),

HOBt (0.084 g, 0.62 mmol) and EDC (0.120 g, 0.62 mmol) in DCM

(8 mL) was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 10 mL), ﬂash column

chromatography (5% MeOH in DCM), Yield: 0.096 g (38.5%), TLC

(DCM/MeOH/NH_3(aq),9/1/0.1 v/v/v) R_f¼ 0.41, MW 520.72, formula:

4.1.5.16. N-((2S,3S)-4-((4-(tert-butyl)benzyl)amino)-3-hydroxy-1-

phenylbutan-2-yl)-2,2-diphenylacetamide (43). The reaction of

(2S,3S)-3-amino-1-((4-(tert-butyl)benzyl)amino)-4-phenylbutan-

2-ol (9b) (0.150 g, 0.46 mmol) with 2,2-diphenylacetic acid (0.098 g,

0.46 mmol) in the presence of DMAP (0.028 g, 0.23 mmol), HOBt

(0.081 g, 0.60 mmol) and EDC (0.115 g, 0.60 mmol) in DCM (8 mL)

was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 10 mL), ﬂash column

chromatography (5e10% MeOH in DCM). Yield: 0.129 g (53.9%), TLC

(DCM/MeOH, 9.5/0.5 v/v) R_f

C

¼

0.24, MW 520.72, formula:

C

₃₅H₄₀N₂O₂, MS m/z 521.4 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

ppm 7.14e7.34 (m, 17H), 7.10 (d, J ¼ 7.45 Hz, 2H), 6.21 (d, J ¼

₃₅H₄₀N₂O₂, MS m/z 521.5 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

d

ppm 7.34 (d, J ¼ 8.02 Hz, 2H), 7.21e7.30 (m, 9H), 7.16e7.20 (m, 4H),

8.02 Hz, 1H), 4.49 (dd, J ¼ 9.16, 7.45 Hz, 1H), 4.01e4.09 (m, 1H), 3.75

(d, J ¼ 13.17 Hz, 1H), 3.58 (d, J ¼ 12.60 Hz, 1H), 3.43e3.48 (m, 1H),

2.78e2.96 (m, 5H), 2.70 (dd, J ¼ 12.60, 2.86 Hz, 1H), 2.34 (dd, J ¼

12.89, 4.87 Hz, 1H), 1.27 (dd, J ¼ 6.87, 1.72 Hz, 6H), NH, OH not

7.05e7.13 (m, 4H), 6.42 (d, J ¼ 9.17 Hz, 1H), 4.90 (s, 1H), 4.59 (br. s,

2H), 4.21 (q, J ¼ 7.45 Hz, 1H), 3.75e3.82 (m, 1H), 3.67 (d, J ¼

13.00 Hz, 1H), 3.62 (d, J ¼ 13.00 Hz, 1H), 2.88 (dd, J ¼ 7.45, 4.58 Hz,

2H), 2.64 (dd, J ¼ 12.20, 5.00 Hz, 1H), 2.55 (dd, J ¼ 12.03, 9.74 Hz,

detected; ¹³C NMR (126 MHz, CDCl₃)

d

ppm 172.5, 149.7, 143.6,

1H), 1.31 (s, 9H); ¹³C NMR (126 MHz, CDCl₃)

d ppm 172.5, 151.1,

143.4, 137.5, 129.4, 129.0, 128.8, 128.7, 128.6, 128.0, 127.7, 127.6,

126.8, 126.8, 126.7, 126.7, 69.8, 53.1, 52.8, 50.5, 47.4, 42.7, 36.2, 34.1,

24.1, 24.1.

139.4, 139.3, 138.0, 129.5, 129.1, 128.9, 128.8, 128.7, 128.6, 127.3,

127.2, 126.6, 125.8, 68.7, 59.0, 52.8, 52.6, 51.3, 38.2, 34.6, 31.4.

4.1.5.17. N-((2S,3S)-4-((4-(tert-butyl)benzyl)amino)-3-hydroxy-1-

phenylbutan-2-yl)-3,3-diphenylpropanamide (44). The reaction of

(2S,3S)-3-amino-1-((4-(tert-butyl)benzyl)amino)-4-phenylbutan-

2-ol (9b) (0.200 g, 0.61 mmol) with 3,3-diphenylpropanoic acid

(0.138 g, 0.61 mmol) in the presence of DMAP (0.037 g, 0.31 mmol),

HOBt (0.107 g, 0.80 mmol) and EDC (0.152 g, 0.80 mmol) in DCM

(8 mL) was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 10 mL), ﬂash column

chromatography (5% MeOH in DCM). Yield: 0.130 g (39.8%), TLC

(DCM/MeOH/NH_3(aq),9/1/0.1 v/v/v) R_f¼ 0.53, MW 534.74, formula:

4.1.5.14. N-((2S,3R)-3-hydroxy-1-phenyl-4-((3-(triﬂuoromethyl)

benzyl)amino)butan-2-yl)-2,2-diphenylacetamide (41). The reaction

of

(2R,3S)-3-amino-4-phenyl-1-((3-(triﬂuoromethyl)benzyl)

amino)butan-2-ol (8b) (0.338 g, 1.00 mmol) with 2,2-

diphenylacetic acid (0.212 g, 1.00 mmol) in the presence of DMAP

(0.061 g, 0.50 mmol), HOBt (0.175 g, 1.30 mmol) and EDC (0.249 g,

1.30 mmol) in DCM (10 mL) was performed according to the pro-

cedure D. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 10 mL), ﬂash column chromatography (5e10% MeOH in DCM).

Yield: 0.117 g (22.0%), TLC (DCM/MeOH, 9/1 v/v) R_f¼ 0.5, MW

532.61, formula: C₃₂H₃₁F₃N₂O₂, MS m/z 533.3 (M þ H^þ), ¹H NMR

C

₃₆H₄₂N₂O₂, MS m/z 535.4 (M þ H^þ), ¹H NMR (300 MHz, CDCl₃)

ppm 7.07e7.41 (m, 19H), 6.76 (d, J ¼ 8.79 Hz, 1H), 6.39 (br. s, 2H),

d

(500 MHz, CDCl₃)

d

ppm 7.50e7.57 (m, 2H), 7.41e7.46 (m, 2H),

4.59 (dd, J ¼ 9.38, 6.45 Hz, 1H), 3.93e4.04 (m, 1H), 3.67e3.81 (m,

2H), 3.48 (d, J ¼ 12.89 Hz, 1H), 2.99 (dd, J ¼ 14.65, 9.38 Hz, 1H), 2.85

(dd, J ¼ 14.65, 6.45 Hz, 1H), 2.72 (d, J ¼ 7.62 Hz, 2H), 2.38 (d, J ¼

7.28e7.31 (m, 4H), 7.21e7.26 (m, 5H), 7.19 (br. d, J ¼ 6.87 Hz, 2H),

7.07 (br. d, J ¼ 6.30 Hz, 2H), 7.03 (br. d, J ¼ 6.87 Hz, 2H), 6.87 (br. d,

J ¼ 4.01 Hz, 1H), 6.13 (br. d, J ¼ 9.16 Hz, 1H), 4.86 (s, 1H), 4.17e4.35

(m, 1H), 3.56e3.79 (m, 4H), 2.85e2.96 (m, 2H), 2.60e2.67 (m, 1H),

7.03 Hz, 2H), 1.27 (s, 9H); ¹³C NMR (126 MHz, CDCl₃)

d ppm 171.9,

151.6,143.7,143.5,137.9,129.4,128.9,128.8,128.7,128.6,128.1,127.8,

126.7, 126.6, 126.6, 125.9, 67.7, 52.3, 52.1, 50.6, 47.5, 42.8, 38.0, 34.7,

31.3.

2.33e2.55 (m, 1H); ¹³C NMR (126 MHz, CDCl₃)

d ppm 172.2, 140.6,

139.3, 139.2, 137.9, 131.5, 130.9 (q, J ¼ 32.00 Hz), 129.5, 129.3, 129.0,

129.0, 128.9, 128.9, 128.8, 128.8, 128.6, 127.4, 127.3, 126.6, 124.8 (q,

J ¼ 3.82 Hz),124.2 (q, J ¼ 272.82 Hz),124.2 (q, J ¼ 3.60 Hz), 69.3, 59.3,

53.2, 52.6, 52.0, 38.4.

4.1.5.18. N-((2S,3R)-3-hydroxy-1-phenyl-4-(((S)-1-phenylpropan-2-

yl)amino)butan-2-yl)-2,2-diphenylacetamide (45). The reaction of

(2R,3S)-3-amino-4-phenyl-1-(((S)-1-phenylethyl)amino)butan-2-

ol (10b) (0.160 g, 0.56 mmol) with 2,2-diphenylacetic acid (0.119 g,

0.56 mmol) in the presence of DMAP (0.034 g, 0.28 mmol) and DCC

(0.232 g, 1.12 mmol) in DCM (5 mL) was performed according to the

procedure D. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 10 mL), ﬂash column chromatography (5% MeOH in DCM).

Yield: 0.140 g (50.6%), TLC (DCM/MeOH, 9.5/0.5 v/v) R_f¼ 0.15, MW

478.64, formula: C₃₂H₃₄N₂O₂, MS m/z 479.4 (M þ H^þ), ¹H NMR

4.1.5.15. N-((2S,3R)-3-hydroxy-1-phenyl-4-((3-(triﬂuoromethyl)

benzyl)amino)butan-2-yl)-3,3-diphenylpropanamide (42). The reac-

tion of (2R,3S)-3-amino-4-phenyl-1-((3-(triﬂuoromethyl)benzyl)

amino)butan-2-ol (8b) (0.338 g, 1.00 mmol) with 3,3-

diphenylpropanoic acid (0.226 g, 1.00 mmol) in the presence of

DMAP (0.061 g, 0.50 mmol), HOBt (0.175 g, 1.30 mmol) and EDC

(0.249 g, 1.30 mmol) in DCM (10 mL) was performed according to

the procedure D. Puriﬁcation: washing reaction mixture with

water (3 ꢃ 10 mL), ﬂash column chromatography (5e10% MeOH in

DCM). Yield: 0.184 g (33.7%), TLC (DCM/MeOH, 9/1 v/v) R_f¼ 0.46,

MW 546.63, formula: C₃₃H₃₃F₃N₂O₂, MS m/z 547.2 (M þ H^þ), ¹H

(500 MHz, CDCl₃)

d ppm 7.13e7.43 (m, 15H), 6.86e7.02 (m, 5H),

5.95 (d, J ¼ 8.59 Hz, 1H), 4.74e4.82 (m, 1H), 4.26 (tdd, J ¼ 9.02, 9.02,

6.59, 5.16 Hz, 1H), 3.65e3.72 (m, 1H), 3.41e3.51 (m, 1H), 2.83e2.90

(m, 1H), 2.52e2.58 (m, 1H), 2.42e2.49 (m, 1H), 1.88e1.96 (m, 1H),

17

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

1.68 (dt, J ¼ 14.03, 3.58 Hz, 1H), 1.28e1.41 (m, 3H), 1.04e1.17 (m,

with 2,2-diphenylacetic acid (0.108 g, 0.51 mmol) in the presence of

DMAP (0.039 g, 0.32 mmol), HOBt (0.112 g, 0.83 mmol) and EDC

(0.159 g, 0.83 mmol) in DCM (8 mL) was performed according to the

procedure D. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 10 mL), ﬂash column chromatography (5e10% MeOH in DCM),

HPLC preparative system (20e90% water in acetonitrile). Yield:

0.061 g (22.4%), TLC (DCM/MeOH/NH_3(aq),9/1/0.1 v/v/v) R_f¼ 0.21,

MW 430.59, formula: C₂₈H₃₄N₂O₂, MS m/z 431.3 (M þ H^þ), ¹H NMR

1H), ¹³C NMR (126 MHz, CDCl₃)

d 172.7, 139.1, 137.6, 129.3, 129.1,

129.0, 128.9, 128.8, 128.7, 127.4, 127.3, 127.2, 127.0, 126.8, 126.6, 70.7,

58.8, 53.8, 34.0, 25.7, 25.0, 24.1.

4.1.5.19. N-((2S,3R)-3-hydroxy-1-phenyl-4-(((S)-1-phenylethyl)

amino)butan-2-yl)-3,3-diphenylpropanamide (46). The reaction of

(2R,3S)-3-amino-4-phenyl-1-(((S)-1-phenylethyl)amino)butan-2-

ol (10b) (0.160 g, 0.56 mmol) with 3,3-diphenylpropanoic acid

(0.127 g, 0.56 mmol) in the presence of DMAP (0.034 g, 0.28 mmol)

and ECC (0.216 g, 1.12 mmol) in DCM (5 mL) was performed ac-

cording to the procedure D. Puriﬁcation: washing reaction mixture

with water (3 ꢃ 10 mL), ﬂash column chromatography (5% MeOH in

DCM). Yield: 0.130 g (47.0%), TLC (DCM/MeOH, 9.5/0.5 v/v) R_f¼ 0.14,

MW 492.66, formula: C₃₃H₃₆N₂O₂, MS m/z 493.7 (M þ H^þ), ¹H NMR

(500 MHz, CDCl₃)

d

ppm 7.18e7.25 (m, 9H), 7.13 (t, J ¼ 6.87 Hz, 4H),

6.96 (br. s, 2H), 6.76 (br. s, 1H), 4.87 (s, 1H), 4.36e4.45 (m, 1H), 4.27

(d, J ¼ 7.45 Hz, 1H), 2.79e3.00 (m, 3H), 2.73 (m, J ¼ 10.31 Hz, 1H),

1.22 (s, 9H), NH, OH not detected; ¹³C NMR (126 MHz, CDCl₃)

d ppm

173.1,169.5,139.3,139.1,137.6,129.4,129.0,128.6,128.6,127.2,127.0,

126.6, 69.0, 58.6, 56.9, 52.8, 45.1, 38.2, 25.7.

(300 MHz, CDCl₃)

d

7.10e7.40 (m, 18H), 6.97 (dd, J ¼ 1.76, 7.62 Hz,

4.1.5.23. N-((2S,3S)-4-(tert-butylamino)-3-hydroxy-1-phenylbutan-

2-yl)-3,3-diphenylpropanamide (50). The reaction of (2S,3S)-3-

amino-1-(tert-butylamino)-4-phenylbutan-2-ol (12b) (0.150 g,

0.64 mmol) with 3,3-diphenylpropanoic acid (0.115 g, 0.51 mmol)

in the presence of DMAP (0.039 g, 0.32 mmol), HOBt (0.112 g,

0.83 mmol) and EDC (0.159 g, 0.83 mmol) in DCM (8 mL) was

performed according to the procedure D. Puriﬁcation: washing

reaction mixture with water (3 ꢃ 10 mL), ﬂash column chroma-

tography (5e10% MeOH in DCM), HPLC preparative system

(20e90% water in acetonitrile). Yield: 0.041 g (14.6%), TLC (DCM/

MeOH/NH_3(aq),9/1/0.1 v/v/v) R_f¼ 0.2, MW 444.62, formula:

2H), 5.87 (d, J ¼ 8.79 Hz, 1H), 4.44 (t, J ¼ 7.91 Hz, 1H), 3.99e4.11 (m,

1H), 3.66 (q, J ¼ 6.84 Hz, 1H), 3.19e3.27 (m, 1H), 2.78 (d, J ¼ 8.21 Hz,

2H), 2.72 (dd, J ¼ 7.03,14.65 Hz,1H), 2.59 (dd, J ¼ 5.27, 14.07 Hz,1H),

2.62 (br. s, 2H), 2.43 (dd, J ¼ 3.52, 12.89 Hz, 1H), 2.26 (dd, J ¼ 5.27,

12.89 Hz, 1H), 1.40 (d, J ¼ 6.45 Hz, 3H); ¹³C NMR (126 MHz, CDCl₃)

d

171.3, 143.5, 143.5, 137.3, 129.3, 128.6, 128.6, 128.5, 127.8, 127.6,

127.4, 126.7, 126.6, 126.6, 126.4, 69.8, 58.5, 53.0, 48.8, 47.4, 43.4,

36.3, 23.7.

4.1.5.20. N-((2S,3R)-3-hydroxy-1-phenyl-4-((2-phenylpropan-2-yl)

amino)butan-2-yl)-2,2-diphenylacetamide (47). The reaction of

(2R,3S)-3-amino-4-phenyl-1-((2-phenylpropan-2-yl)amino)butan-

2-ol (11b) (0.160 g, 0.54 mmol) with 2,2-diphenylacetic acid

(0.114 g, 0.54 mmol) in the presence of DMAP (0.033 g, 0.27 mmol)

and DCC (0.221 g, 1.12 mmol) in DCM (5 mL) was performed ac-

cording to the procedure D. Puriﬁcation: washing reaction mixture

with water (3 ꢃ 10 mL), ﬂash column chromatography (5% MeOH in

DCM). Yield: 0.092 g (34.8%), TLC (DCM/MeOH, 9.5/0.5 v/v)

R_f¼ 0.21, MW 492.66, formula: C₃₃H₃₆N₂O₂, MS m/z 493.5

C

d

₂₉H₃₆N₂O₂, MS m/z 445.7 (M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

ppm 8.43 (br. s, 1H), 7.12e7.30 (m, 14H), 7.04 (d, J ¼ 9.17 Hz, 1H),

6.06 (br. s, 2H), 4.58 (dd, J ¼ 8.88, 6.59 Hz, 1H), 4.17 (q, J ¼ 8.02 Hz,

1H), 3.98 (d, J ¼ 8.02 Hz, 1H), 3.00 (dd, J ¼ 14.89, 9.16 Hz, 1H), 2.83

(dd, J ¼ 14.89, 6.87 Hz, 1H), 2.73e2.80 (m, 2H), 2.58e2.69 (m, 2H),

1.18 (s, 9H); ¹³C NMR (126 MHz, CDCl₃)

d ppm 171.8, 144.0, 143.6,

137.6, 129.2, 128.7, 128.6, 128.6, 127.9, 127.8, 126.7, 126.6, 126.5, 68.2,

56.5, 52.6, 46.9, 44.8, 42.4, 38.3, 25.8.

(M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

d

ppm 7.16e7.43 (m, 15H),

4.1.5.24. N-((2S,3R)-3-hydroxy-4-(neopentylamino)-1-phenylbutan-

2-yl)-2,2-diphenylacetamide (51). The reaction of (2R,3S)-3-amino-

6.88e7.02 (m, 5H), 4.72e4.83 (m, 3H), 4.17e4.26 (m, 1H), 3.29e3.37

(m, 1H), 2.98 (dd, J ¼ 14.32, 4.01 Hz, 1H), 2.57e2.67 (m, 1H), 2.36 (d,

J ¼ 4.01 Hz, 2H), 1.39e1.52 (m, 6H), 1.16e1.38 (br. s, 1H), ¹³C NMR

1-(neopentylamino)-4-phenylbutan-2-ol

(13b)

(0.140

g,

0.56 mmol) with 2,2-diphenylacetic acid (0.119 g, 0.56 mmol) in the

presence of DMAP (0.034 g, 0.28 mmol) and DCC (0.231 g,

1.12 mmol) in DCM (5 mL) was performed according to the pro-

cedure D. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 30 mL), ﬂash column chromatography (5% MeOH in DCM).

Yield: 0.075 g (30.3%), TLC (DCM/MeOH 9.5/0.5 v/v) R_f¼ 0.18, MW

444.62, formula: C₂₉H₃₆N₂O₂, MS m/z 445.4 (M þ H^þ), ¹H NMR

(126 MHz, CDCl₃)

d 172.6, 139.0, 137.5, 129.4, 129.0, 128.9, 128.9,

128.8, 128.7, 128.5, 127.4, 127.3, 126.8, 126.6, 126.1, 100.0, 70.8, 59.3,

53.1, 44.5, 36.5, 29.4, 28.5.

4.1.5.21. N-((2S,3R)-3-hydroxy-1-phenyl-4-((2-phenylpropan-2-yl)

amino)butan-2-yl)-3,3-diphenylpropanamide (48). The reaction of

(2R,3S)-3-amino-4-phenyl-1-((2-phenylpropan-2-yl)amino)butan-

2-ol (11b) (0.160 g, 0.54 mmol) with 3,3-diphenylpropanoic acid

(0.121 g, 0.54 mmol) in the presence of DMAP (0.033 g, 0.27 mmol)

and DCC (0.221 g, 1.12 mmol) in DCM (5 mL) was performed ac-

cording to the procedure D. Puriﬁcation: washing reaction mixture

with water (3 ꢃ 10 mL), ﬂash column chromatography (5% MeOH in

(500 MHz, CDCl₃)

d

ppm 7.20e7.29 (m, 9H), 7.06 (dd, J ¼ 7.45,

2.29 Hz, 2H), 7.03 (dd, J ¼ 7.73, 1.43 Hz, 2H), 6.96e7.00 (m, 2H), 5.94

(d, J ¼ 8.59 Hz,1H), 4.81 (s,1H), 4.24e4.31 (m,1H), 3.54 (td, J ¼ 6.59,

3.44 Hz, 1H), 3.67 (br. s, 2H), 3.06 (dd, J ¼ 14.03, 4.30 Hz, 1H),

2.69e2.79 (m, 3H), 2.40 (d, J ¼ 11.46 Hz, 1H), 2.34 (d, J ¼ 11.46 Hz,

1H), 0.93 (s, 9H); ¹³C NMR (126 MHz, CDCl₃)

d ppm 172.7, 139.1,

DCM). Yield: 0.105

g

(38.7%), TLC (DCM/MeOH, 9.5/0.5 v/v)

139.0, 137.6, 129.5, 129.0, 128.9, 128.8, 128.8, 128.7, 127.4, 127.3,

126.6, 70.4, 62.1, 59.2, 53.5, 52.6, 36.4, 31.5, 27.7.

R_f¼ 0.24, MW 506.69, formula: C₃₄H₃₈N₂O₂, MS m/z 507.6

(M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

d ppm 6.91e7.50 (m, 20H),

5.70 (d, J ¼ 8.59 Hz, 1H), 4.44 (t, J ¼ 8.02 Hz, 1H), 4.02e4.15 (m, 1H),

3.17 (br. s, 1H), 2.73e2.87 (m, 3H), 2.65 (dd, J ¼ 14.32, 5.16 Hz, 1H),

2.26 (dd, J ¼ 12.60, 2.86 Hz,1H), 2.14 (dd, J ¼ 12.32, 4.30 Hz,1H),1.51

(s, 3H), 1.45 (s, 3H), 1.15e1.37 (m, 2H), ¹³C NMR (126 MHz, CDCl₃)

4.1.5.25. N-((2S,3R)-3-hydroxy-4-(neopentylamino)-1-phenylbutan-

2-yl)-3,3-diphenylpropanamide (52). The reaction of (2R,3S)-3-

amino-1-(neopentylamino)-4-phenylbutan-2-ol (13b) (0.140 g,

0.56 mmol) with 3,3-diphenylpropanoic acid (0.127 g, 0.56 mmol)

in the presence of DMAP (0.034 g, 0.28 mmol) and DCC (0.231 g,

1.12 mmol) in DCM (5 mL) was performed according to the pro-

cedure D. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 30 mL), ﬂash column chromatography (5e8% MeOH in DCM).

Yield: 0.128 g (49.8%), TLC (DCM/MeOH, 9/1 v/v) R_f¼ 0.28, MW

458.65, formula: C₃₀H₃₈N₂O₂, MS m/z 459.6 (M þ H^þ), ¹H NMR

d

171.5, 143.7, 143.5, 137.4, 129.5, 128.7, 128.6, 128.5, 127.8, 127.7,

127.0, 126.7, 126.7, 126.6, 126.0, 70.0, 56.6, 52.8, 47.5, 44.5, 43.5, 36.1,

29.0, 28.7.

4.1.5.22. N-((2S,3S)-4-(tert-butylamino)-3-hydroxy-1-phenylbutan-

2-yl)-2,2-diphenylacetamide (49). The reaction of (2S,3S)-3-amino-

1-(tert-butylamino)-4-phenylbutan-2-ol (12b) (0.150 g, 0.64 mmol)

(500 MHz, CDCl₃) d ppm 7.16e7.30 (m, 13H), 7.11e7.14 (m, 2H), 6.02

18

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

(d, J ¼ 8.02 Hz, 1H), 4.51 (dd, J ¼ 9.16, 6.87 Hz, 1H), 4.07e4.16 (m,

1H), 3.33 (ddd, J ¼ 7.73, 4.87, 3.44 Hz, 1H), 2.77e2.93 (m, 4H), 2.53

(dd, J ¼ 12.60, 3.44 Hz, 1H), 2.28e2.35 (m, 2H), 2.14 (d, J ¼ 12.03 Hz,

1H), 0.98 (s, 9H), NH, OH not detected; ¹³C NMR (126 MHz, CDCl₃)

4.1.5.29. N-((2S,3R)-4-((cyclopropylmethyl)amino)-3-hydroxy-1-

phenylbutan-2-yl)-3,3-diphenylpropanamide (56). The reaction of

(2R,3S)-3-amino-1-((cyclopropylmethyl)amino)-4-phenylbutan-2-

ol (15b) (0.350 g, 1.50 mmol) with 3,3-diphenylpropanoic acid

(0.338 g, 1.50 mmol) in the presence of DMAP (0.091 g, 0.75 mmol),

HOBt (0.262 g, 1.94 mmol) and EDC (0.372 g, 1.94 mmol) in DCM

(25 mL) was performed according to the procedure D. Puriﬁcation:

washing reaction mixture with water (3 ꢃ 30 mL), ﬂash column

chromatography (5e10% MeOH in DCM). Yield: 0.380 g (48.4%), TLC

(DCM/MeOH/NH_3(aq),9.5/0.5/0.05 v/v/v) R_f¼ 0.28, MW 442.60,

formula: C₂₉H₃₄N₂O₂, MS m/z 443.8 (M þ H^þ), ¹H NMR (500 MHz,

d

ppm 171.7, 143.6, 143.5, 137.6, 129.5, 128.7, 128.7, 128.6, 128.0,

127.7, 126.7, 126.6, 126.6, 69.8, 61.9, 53.0, 52.1, 47.4, 43.0, 36.5, 31.4,

27.8.

4.1.5.26. N-((2S,3R)-3-hydroxy-1-phenyl-4-((2,2,2-triﬂuoroethyl)

amino)butan-2-yl)-2,2-diphenylacetamide (53). The reaction of

(2R,3S)-3-amino-4-phenyl-1-((2,2,2-triﬂuoroethyl)amino)butan-

2-ol (14b) (0.200 g, 0.76 mmol) with 2,2-diphenylacetic acid

(0.129 g, 0.61 mmol) in the presence of DMAP (0.047 mg,

0.38 mmol), HOBt (0.134 g, 1.00 mmol) and EDC (0.190 g,

1.00 mmol) in DCM (8 mL) was performed according to the pro-

cedure D. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 10 mL), ﬂash column chromatography (5% MeOH in DCM).

Yield: 0.246 g (70.7%), TLC (DCM/MeOH, 9/1 v/v) R_f¼ 0.6, MW

456.51, formula: C₂₆H₂₇F₃N₂O₂, MS m/z 457.3 (M þ H^þ), ¹H NMR

CDCl₃)

d

ppm 7.06e7.28 (m, 15H), 6.22 (d, J ¼ 8.59 Hz, 1H), 4.71 (br.

s, 1H), 4.48 (dd, J ¼ 9.16, 7.45 Hz, 1H), 4.06 (qd, J ¼ 8.02, 5.16 Hz, 1H),

3.41e3.46 (m, 1H), 2.99 (s, 1H), 2.74e2.92 (m, 4H), 2.61 (dd,

J ¼ 12.60, 3.44 Hz, 1H), 2.52 (dd, J ¼ 12.32, 6.59 Hz, 1H), 2.33 (dd,

J ¼ 12.60, 5.73 Hz, 1H), 2.09 (dd, J ¼ 12.60, 7.45 Hz, 1H), 0.85e0.99

(m, 1H), 0.41e0.56 (m, 2H), 0.04e0.22 (m, 2H), ¹³C NMR (126 MHz,

CDCl₃)

d ppm 173.5, 143.5, 143.2, 137.6, 129.4, 128.8, 128.7, 128.1,

127.6, 126.8, 126.7, 126.6, 69.5, 53.2, 53.0, 50.7, 47.1, 41.9, 36.3, 7.4,

4.5, 4.2.

(500 MHz, CDCl₃)

d ppm 7.22e7.33 (m, 9H), 7.02e7.08 (m, 4H),

6.93e6.99 (m, 2H), 5.67 (d, J ¼ 8.59 Hz, 1H), 4.84 (s, 1H), 4.18e4.27

(m, 1H), 3.46 (s, 2H), 3.08e3.19 (m, 2H), 3.04 (dd, J ¼ 14.03, 4.30 Hz,

1H), 2.86 (dd, J ¼ 12.32, 3.15 Hz, 1H), 2.65e2.75 (m, 2H), 1.62 (br. s,

4.2. Molecular modeling

1H); ¹³C NMR (126 MHz, CDCl₃)

d

ppm 172.9, 139.0, 138.8, 137.4,

All 3D structures of compounds were prepared with the

appropriate stereoisomerism in CORINA Classic online tool [Mo-

lecular Networks GmbH, Germany and Altamira, LLC, USA]. Sub-

sequently, atom types and protonation states were checked and

Gasteiger-Marsili charges were assigned using Sybyl-X1.1 [Tripos,

St. Louis, MO, USA]. Before docking, hBACE1 complex (PDB: 4D8C

chain C) and hBuChE (PDB: 1P0I chain A) were prepared using

PlayMolecule ProteinPrepare web service at pH 7.4 [93]. Molecular

docking was performed with GOLD 5.3 [The Cambridge Crystallo-

graphic Data Center, Cambridge, UK] based on our previously

developed docking algorithms [94,95]. Binding sites were deﬁned

as all amino acid residues within 10 Å from NVP-BXD552 (BXD)

present in 4D8C complex or 20 Å from glycerol (GOL) present in

1P0I complex. We applied the automatic genetic algorithm settings

for very ﬂexible ligands. For each ligand, we received 10 confor-

mations sorted by GoldScore. In the case of docking to 1P0I, chosen

water molecules were kept, allowing the program to decide on

their participation in ligand binding (toggle option) [95]. Further

rescoring of selected ligand poses was carried out using the KDEEP

and PRODIGY-LIGAND services [96,97]. The stability of the ﬁnal

complexes was tested by MD simulation using tools available on the

PlayMolecule website [98]. Final results were visualized by PyMOL

0.99rc2 [DeLano Scientiﬁc LLC, Palo Alto, CA, USA].

129.3, 129.0, 128.9, 128.9, 128.8, 127.5, 127.3, 126.8, 125.7 (q,

J ¼ 280.80 Hz), 71.3, 59.3, 53.6, 51.2, 50.8 (q, J ¼ 31.39 Hz), 36.6.

4.1.5.27. N-((2S,3R)-3-hydroxy-1-phenyl-4-((2,2,2-triﬂuoroethyl)

amino)butan-2-yl)-3,3-diphenylpropanamide (54). The reaction of

(2R,3S)-3-amino-4-phenyl-1-((2,2,2-triﬂuoroethyl)amino)butan-

2-ol (14b) (0.200 g, 0.76 mmol) with 3,3-diphenylpropanoic acid

(0.138 g, 0.61 mmol) in the presence of DMAP (0.047 mg,

0.38 mmol), HOBt (0.134 g, 1.00 mmol) and EDC (0.190 g,

1.00 mmol) in DCM (8 mL) was performed according to the pro-

cedure D. Puriﬁcation: washing reaction mixture with water

(3 ꢃ 10 mL), ﬂash column chromatography (5% MeOH in DCM).

Yield: 0.213 g (59.4%), TLC (DCM/MeOH, 9/1 v/v) R_f¼ 0.59, MW

470.54, formula: C₂₇H₂₉F₃N₂O₂, MS m/z 471.4 (M þ H^þ), ¹H NMR

(500 MHz, CDCl₃) d ppm 7.17e7.32 (m, 13H), 7.05e7.10 (m, 2H), 5.51

(d, J ¼ 8.59 Hz, 1H), 4.50 (t, J ¼ 7.73 Hz, 1H), 4.05 (dq, J ¼ 8.59,

6.49 Hz, 1H), 3.22e3.28 (m, 1H), 3.12e3.16 (m, 1H), 2.99e3.12 (m,

2H), 2.77e2.83 (m, 3H), 2.47 (d, J ¼ 4.58 Hz, 2H), 1.68 (br. s, 2H); ¹³

C

NMR (126 MHz, CDCl₃) d ppm 171.6, 143.5, 143.5, 137.2, 129.4, 128.7,

128.7, 128.7, 128.0, 127.6, 126.8, 126.7, 125.5 (q, J ¼ 278.86 Hz), 70.6,

52.8, 50.9, 50.8 (q, J ¼ 31.40 Hz), 47.5, 43.3, 36.3.

4.1.5.28. N-((2S,3R)-4-((cyclopropylmethyl)amino)-3-hydroxy-1-

phenylbutan-2-yl)-2,2-diphenylacetamide (55). The reaction of

(2R,3S)-3-amino-1-((cyclopropylmethyl)amino)-4-phenylbutan-2-

ol (15b) (0.130 g, 0.56 mmol) with 2,2-diphenylacetic acid (0.119 g,

0.56 mmol) in the presence of DMAP (0.034 g, 0.28 mmol) and DCC

(0.231 g, 1.12 mmol) in DCM (10 mL) was performed according to

the procedure D. Puriﬁcation: washing reaction mixture with

water (3 ꢃ 10 mL), ﬂash column chromatography (5% MeOH in

DCM). Yield: 0.120 g (48.4%), TLC (DCM/MeOH/NH_3(aq),9.5/0.5/0.05

v/v/v) R_f¼ 0.24, MW 428.58, formula: C₂₈H₃₂N₂O₂, MS m/z 429.6

4.3. Biological evaluation

All ﬁnal compounds were tested for known classes of pan assay

interference compounds (PAINS). According to SwissADME, none of

them contain any structural fragments recognized as PAINS [99].

4.3.1. The in vitro inhibitory activity toward cholinesterases

(eeAChE, eqBuChE, hBuChE)

The reagents used to perform the experiments and eeAChE and

eqBuChE were purchased from SigmaeAldrich (Steinheim, Ger-

many); hBuChE isolated from human plasma was from Vivonics

(Bedford, MA, USA). Ellman’s spectrophotometric assay [85], with

small modiﬁcations, was utilized for testing inhibitory activities of

the synthesized compounds against the cholinesterases, according

to the protocol described elsewhere [73]. Brieﬂy, 5 U/mL aqueous

stock solutions of the enzymes (eeAChE, eqBuChE, and hBuChE)

were diluted before use to the ﬁnal concentrations of 0.384 U/mL.

Stock solutions of the tested compounds were prepared in DMSO

(M þ H^þ), ¹H NMR (500 MHz, CDCl₃)

d ppm 7.16e7.32 (m, 10H),

7.01e7.09 (m, 4H), 6.97 (dd, J ¼ 6.87, 2.86 Hz, 1H), 6.02 (d,

J ¼ 8.59 Hz, 1H), 4.81 (s, 1H), 4.24 (td, J ¼ 4.58, 2.29 Hz, 1H), 3.55 (td,

J ¼ 6.30, 3.44 Hz, 1H), 3.33 (br. s, 2H), 3.00e3.07 (m, 1H), 2.64e2.78

(m, 3H), 2.49 (dd, J ¼ 12.32, 6.59 Hz,1H), 2.33 (dd, J ¼ 12.60, 7.45 Hz,

1H), 0.83e0.92 (m, 1H), 0.40e0.51 (m, 2H), 0.05e0.14 (m, 2H), ¹³

C

NMR (126 MHz, CDCl₃) d ppm 172.6, 149.5, 139.3, 137.9, 129.4, 129.1,

128.9, 128.8, 128.7, 128.6, 128.5, 127.3, 127.2, 126.5, 106.7, 70.9, 59.2,

54.7, 54.0, 51.3, 39.1, 36.5, 10.7, 3.7, 3.5.

19

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A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

and diluted (in demineralized water) prior to use to the desired

concentration. At ﬁrst, the target compound or water or mixture of

4.3.3. Kinetics of eqBuChE inhibition for selected compounds

The most potent eqBuChe inhibitors were selected for the ki-

netic studies. The protocol described above was followed, based on

Ellman’s method [85]. An aqueous substrate BTC stock solution

(0.02125 M) was diluted before use to give six ﬁnal concentrations

in the wells: 0.3, 0.24, 0.18, 0.12, 0.06, and 0.04 mM. Six different

concentrations of each inhibitor were prepared, resulting in the

percent of enzyme inhibition between 30% and 80%. For each

concentration of the target compound, all six BTC solutions were

used to perform the assays. V_maxand K_mvalues of the

MichaelisꢂMenten kinetics were calculated by nonlinear regres-

sion from substrateꢂvelocity curves. Also, Lineweaver-Burk and

Cornish-Bowden plots were obtained, both using linear regression

in GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). Each

experiment was performed in triplicate.

DMSO/water in appropriate ratio; i.e. blank samples (25

mL) was

incubated in 0.1 M phosphate buffer (0.1 M, pH ¼ 8.0, 200

mL) with

DTNB (0.0025 M, 20 mL) and the enzyme (20 mL; eeAChE, eqBuChe

or hBuChE). The experiments were performed at room temperature

(25 ^ꢁC) for eeAChE or eqBuChE and 36 ^ꢁC for the hBuChE. Following

the 5 min of pre-incubation step, the ﬁnal reactions were started by

adding ATC or BTC solutions (depending on the enzyme used). After

the next 5 min, the changes in absorbance were measured at

412 nm, using a microplate reader (EnSpire Multimode; Perki-

nElmer, Waltham, MA, USA). All the target compounds were tested

at the screening concentrations of 10 mM or 1 mM for inhibitory

potencies toward animal cholinesterases or human enzyme,

respectively. Based on equation 100-(S/B) ꢃ 100 (where S and B

were the respective enzyme activities with and without the test

sample, respectively) the percent of inhibition of each enzyme for

each compound was calculated. Compounds with the enzyme

4.3.4. X-ray crystallography of hBuChE in complex with 51

4.3.4.1. Crystallization. Crystals of hBuChE were obtained using

recombinant enzyme produced in Chinese ovary cells as described

earlier [100] and puriﬁed with a BuChE speciﬁc afﬁnity chroma-

tography (Hupresin; CHEMFORASE, Rouen, France) followed by size

exclusion chromatography (Superdex 200, GE Healthcare) [101].

Crystals were obtained using the hanging drop method at 293 K

using a 12 mg/mL protein solution with crystallization buffer (0.1 M

MES, pH ¼ 6.5, 2.15 M (NH₄)₂SO₄)as crystallization buffer. Com-

pound 51 was initially solubilized in methanol at 0.1 M concen-

tration. Ligand/protein complex was obtained by soaking crystals

with a 1 mM 51 solution in crystallization buffer (1% MeOH ﬁnal

concentration). Crystals were cryo-protected in a solution (1 mM

51, 1% MeOH,0.1 M MES, pH ¼ 6.5, 2.15 M (NH₄)₂SO₄, 20% Glycerol)

before ﬂash cooling into liquid nitrogen.

inhibitory activities at 10

mM higher than 50% (eeAChE or eqBuChe),

and at 1 M higher than 80% (hBuChE) were further evaluated to

m

obtain IC₅₀values. The IC₅₀values were determined based on the

enzymes’ inhibitory activities in the seven different concentrations

of each compound, resulting in inhibition between 5% and 95%.

Calculations were made using nonlinear regression (GraphPad

Prism 5; GraphPad Software, San Diego, CA, USA) by plotting the

residual enzyme activities against the applied inhibitor concen-

tration. Tacrine and donepezil were used as the reference com-

pounds. All the experiments were performed in triplicate.

4.3.2. The in vitro inhibitory activity toward human recombinant

BACE1

BACE1 ﬂuorescence resonance energy transfer (FRET) Assay Kit

(Panvera, Madison, WI, USA) was purchased from Life Technologies

(Warsaw, Poland) and used according to the protocol described in

our previous paper [73]. The 384-well black microplates and a

microplate reader (EnSpire Multimode; PerkinElmer, Waltham,

MA, USA) were utilized to perform the experiments. The wave-

length was optimized for the 553 nm excitation and 576 nm

emission. The stock solutions of the target compounds were pre-

pared in DMSO and diluted with assay buffer (50 mM sodium ac-

etate; pH ¼ 4.5) prior to use. The ﬁnal reaction mixture was

composed of BACE1 substrate (Rh-EVNLDAEFK-quencher, based on

4.3.4.2. Structure determination. X-ray diffraction data were

collected on the PROXIMA-2A beamline of the SOLEIL Synchrotron

(Saint Aubin, France) at 100 K. Images recorded on an EIGER X 9 M

(Dectris) detector were processed with XDS [102] using I/sigma>1

and CC1/2 as selection criteria. Data analysis was realized using the

Phenix software suite [103]. An initial model was obtained by

molecular replacement using Phaser-MR and the hBuChE structure

(PDB entry 1P0I) devoid of any ligand, glycans or water molecules.

Electron density was observed in the active site gorge and allowed

unambiguous ﬁtting of 51. Ligand geometry restraints were pro-

cessed with Phenix eLBOW [104] using the semi-empirical quan-

tum mechanical method (AM1) and assuming the calculated

charged position at pH 6.5 [105]. The model was reﬁned by iterative

cycles of model building using Coot [106] and reﬁnement using

Phenix. reﬁne. hBuChE structure in complex with 51 was deposited

into the Protein Data Bank under accession number 7AMZ.

the Swedish mutant of APP, 10

buffer; i.e. blank sample (10

baculovirus-expressed BACE1, 1 U/mL, 10

was incubated at 25 ^ꢁC for 60 min, then the stop solution (2.5 M

sodium acetate, 10 L) was added to stop the reaction. The ﬂuo-

m

L), the tested compound or assay

L) and the enzyme (puriﬁed

L). The reaction mixture

m

rescence signal was read at 576 nm. Based on the [1 e (S60 e S0)/

(C60 e C0)] ꢃ 100 equation, the percent of BACE1 inhibition was

calculated. S0 and S60 were ﬂuorescence intensities of the tested

sample (enzyme, substrate, target compound) at the beginning of

the reaction and after 60 min respectively; C0 and C60 were

analogical ﬂuorescence intensities of the blank sample (enzyme,

substrate, buffer). The compounds were screened at a concentra-

4.3.5. Inhibitory properties toward GAT subtypes

The inhibitory activities of obtained compounds toward four

subtypes of mGAT were determined at a screening concentration of

100 mM and in the case of sufﬁcient potency characterized in full

scale [³H] GABA uptake assays according to a procedure described

before [107]. The inhibition of hGAT3 for the most potent com-

pounds was studied in full scale competition experiments as

described [108]. MS binding assays for mGAT1 were performed as

reported in Ref. [109]. The screening concentration for tested

tion of 50

mM. For the most potent structures (at least 80% of

enzyme inhibition) IC₅₀values were determined. The IC₅₀values of

the reference and target compounds were determined, based on

the BACE1 inhibition results for six diverse concentrations of each

compound (resulting in 10%e95% of enzyme inhibition). Nonlinear

regression (GraphPad Prism 5, GraphPad Software, San Diego, CA,

USA) was used in calculations. The residual enzyme activities were

plotted against the applied inhibitor concentration. BACE1 Inhibitor

IV (Calbiochem, Merck, Nottingham, UK) was used as the reference

compound. All the reactions were performed in triplicate.

compounds was 100 mM.

4.3.6. Inhibition of A

b aggregation

To evaluate antiaggregating properties against A

b

amyloid

thioﬂavin-T (ThT) ﬂuorometric assay was performed [91]. The in-

hibition of Ab_1e42aggregation was measured ﬂuorimetrically as

described previously [1 10]. Brieﬂy, HFIP-pretreated Ab_1e42(Merck

20

ꢀ

A. Pasieka, D. Panek, J. Jonczyk et al.

European Journal of Medicinal Chemistry 218 (2021) 113397

Millipore, Darmstadt, Germany) at 1.5

mM, the test compound

toxin CCCP (10

mM) were used as a positive controls.

(10 mM ﬁnal concentration) and Thioﬂavin-T (10 mM ﬁnal concen-

tration) were incubated at room temperature in 96-well microplate

covered with aluminum foil with continuous shaking for 36e48 h.

The ﬂuorescence intensity (l_ex¼ 440 nm; l_em¼ 490 nm) was

measured every 3 min (Synergy™ H4 plate reader, BioTek In-

struments, Inc. VT, USA). The assay was run in quadruplicates. To

exclude possible interference with the assay readout, absorbance

4.3.9.2. Metabolic stability. The in silico prediction of most probable

sites of metabolism was done by MetaSite 6.0.1 software (Molecular

Discovery Ltd, Hertfordshire, UK). The metabolic pathways deter-

mination in vitro was performed by 120 min compounds incubation

with human liver microsomes (HLMs) purchased from Sigma-

Aldrich, St. Louis, MO, USA. The reactions were conducted in

10 mM TriseHCl buffer (pH 7.4) at 37 ^ꢁC in the presence of NADPH

Regeneration System (Promega, Madison, WI, USA). UPLC/MS an-

alyses were done by Waters ACQUITY TQD system with the TQ

Detector (Waters, Milford, USA).

spectra of the compounds were recorded at 10

mM compound

concentration, and none of the compounds showed any signiﬁcant

absorbance (>0.05 AU) in the range 400e500 nm.

4.3.7. Inhibition of tau aggregation

Tau aggregation was monitored using ThT ﬂuorometric assay.

4.3.9.3. Inﬂuence on CYP activity. The potential drug-drug in-

teractions were predicted using CYP3A4 and CYP2D6 assays pur-

chased from Promega (Madison, WI, USA). The compounds were

Tau protein (0N4R) (10

tested compounds (10

m

M) and heparin (5

m

M) with or without the

mM) were dissolved in 30

mL 25 mM PBS (pH

6.8), which were incubated at 37 ^ꢁC for 72 h. And then the samples

tested in triplicate in concentration 10

mM. The study was per-

were diluted to a ﬁnal volume of 200

mL PBS (pH 6.8) containing

formed with use of the 1 M concentration of respective reference

m

5

mM ThT. After binding to ThT for 5 min at rt in the dark, the

CYP inhibitors: KE and QD.

ﬂuorescence signal was measured (excitation wavelength at

440 nm and emission wavelength at 480 nm with a 5 nm slit width)

on a monochromator based multimode microplate reader (Tecan

Infnite M1000). The ﬂuorescence intensities were recorded, and the

percentage of inhibition on aggregation was calculated by the

following expression: (1ꢂIFi/IFc) ꢃ 100%, in which IFi and IFc were

the ﬂuorescence intensities obtained for absorbance in the pres-

ence and absence of compounds, respectively, after subtracting the

Author contributions

The manuscript was written through contributions of all au-

thors. All authors have given approval to the ﬁnal version of the

manuscript.

Declaration of competing interest

background ﬂuorescence of the ThT solution (5

mM). Methylene

blue was used as a reference compound.

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

4.3.8. Toxicity e MTT assay

For the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetra-

zolium bromide) colorimetric assay [1 1 1], HT22 cells, which was an

immortalized cell line from hippocampus of mice, were cultured in

96-well plates at the concentration of 1 ꢃ 10⁵/mL. Cells were

cultured in DMEMs with 10% FBS and incubated at 37 ^ꢁC, 5% CO₂

Acknowledgments

This work was supported by the National Science Center of

Poland (grant UMO-2016/21/B/NZ7/01744) and the European

Cooperation in Science and Technology COST Action CA15135 and

Slovenian Research Agency e ARRS (grants P1-0208 and L1-8157).

Authors would like to thank the SOLEIL synchrotron for long term

beamtime access 20181022 (IBS BAG). FC, JD and XB were funded by

and then, were incubated with compounds (10 and 30 mM) for 24 h.

Then, MTT (5 mg/mL, 10 mL) was added and cells were incubated for

more 2 h at 37 ^ꢁC. The water soluble MTT reagent was converted to

an insoluble formazan product by viable cells. Next, the precipi-

tated formazan was dissolved with DMSO¼ (100

mL). After shaking

ꢀ ꢀ

the French Ministry of Armed Forces (Direction Generale de

at room temperature for 10 min, the absorbance was measured

through a multi-mode reader (Bio-Tek) at 570 nm. Results were

expressed as the percentage of cell viability in comparison with

nontreated cells which were considered controls (CT). The statis-

tical signiﬁcance was analyzed by GraphPad Prism 8 software using

one-way ANOVA, followed by Bonferroni’s Comparison Test.

ꢀ

l’Armement and Service de Sante des Armees, grant NBC-5-C-

4210). J.K. acknowledges the support of the Czech Science Foun-

dation (20e29633J).

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113397.

4.3.9. In vitro ADME-tox properties

All performed assays and protocols used for determination of

ADME-Tox parameters were described previously [1 12e1 14]. Sta-

tistical signiﬁcances were analyzed by GraphPad Prism™ 8 soft-

ware using One-way ANOVA and Bonferroni’s Multiple Comparison

Post Test. All references used during this study: carbonyl cyanide 3-

chlorophenyl-hydrazone (CCCP), doxorubicin (DX), ketoconazole

(KE), quinidine (QD) and verapamil (VER) were obtained from

Sigma-Aldrich (St. Louis, MO, USA).

Abbreviations

AD

Alzheimer’s disease

ATC

acetylthiocholine

BTC

butyrylthiocholine

DTNB

GATs

MTDL

NFTs

PAINS

5,5⁰-Dithiobis-(2-nitrobenzoic acid)

GABA transporters

multi-target-directed ligands

neuroﬁbrillary tangles

pan-assay interference compounds

4.3.9.1. Hepatotoxicity assays. Hepatotoxicity was estimated using

hepatoma HepG2 (ATCC® HB-8065™) cell line. The CellTiter 96®

AQueous Non-Radioactive Cell Proliferation Assay (MTS) used for

estimation of cells viability was purchased from Promega (Madison,

WI, USA). The compounds were tested in quadruplicate at four

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Article Doi

Discovery of multifunctional anti-Alzheimer's agents with a unique mechanism of action including inhibition of the enzyme butyrylcholinesterase and γ-aminobutyric acid transporters

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Full text of DOI:10.1016/j.ejmech.2021.113397

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