molecules
Article
Inhibition of Acetylcholinesterase by Novel Lupinine Derivatives
Igor A. Schepetkin 1, Zhangeldy S. Nurmaganbetov 2,3, Serik D. Fazylov 2, Oralgazy A. Nurkenov 2,
Andrei I. Khlebnikov 4 , Tulegen M. Seilkhanov 5 , Anarkul S. Kishkentaeva 2, Elvira E. Shults 6
and Mark T. Quinn 1,*
1 Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59717, USA;
igor@montana.edu
2 Institute of Organic Synthesis and Coal Chemistry, Karaganda 100008, Kazakhstan;
nzhangeldy@yandex.ru (Z.S.N.); iosu8990@mail.ru (S.D.F.); nurkenov_oral@mail.ru (O.A.N.);
anar_kish@mail.ru (A.S.K.)
3 School of Pharmacy, Medical University of Karaganda, Karaganda 100012, Kazakhstan
4 Kizhner Research Center, Tomsk Polytechnic University, 634050 Tomsk, Russia; aikhl@chem.org.ru
5 Laboratory of Engineering Profile NMR Spectroscopy, Sh. Ualikhanov Kokshetau University,
Kokshetau 020000, Kazakhstan; tseilkhanov@mail.ru
6 N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences,
630090 Novosibirsk, Russia
* Correspondence: mquinn@montana.edu
Abstract: Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive
memory loss and cognitive impairment due in part to a severe loss of cholinergic neurons in specific
brain areas. AD is the most common type of dementia in the aging population. Although several
acetylcholinesterase (AChE) inhibitors are currently available, their performance sometimes yields
unexpected results. Thus, research is ongoing to find potentially therapeutic AChE inhibitory agents,
both from natural and synthetic sources. Here, we synthesized 13 new lupinine triazole derivatives
and evaluated them, along with 50 commercial lupinine-based esters of different carboxylic acids, for
AChE inhibitory activity. The triazole derivative 15 [1S,9aR)-1-((4-(4-(benzyloxy)-3-methoxyphenyl)-
1H-1,2,3-triazol-1-yl)methyl)octahydro-2H-quinolizine)] exhibited the most potent AChE inhibitory
Citation: Schepetkin, I.A.; activity among all 63 lupinine derivatives, and kinetic analysis demonstrated that compound 15 was
Nurmaganbetov, Z.S.; Fazylov, S.D.; a mixed-type AChE inhibitor. Molecular docking studies were performed to visualize interaction
Nurkenov, O.A.; Khlebnikov, A.I.; between this triazole derivative and AChE. In addition, a structure-activity relationship (SAR)
Seilkhanov, T.M.; Kishkentaeva, A.S.;
model developed using linear discriminant analysis (LDA) of 11 SwissADME descriptors from the
Shults, E.E.; Quinn, M.T. Inhibition of
50 lupinine esters revealed 5 key physicochemical features that allowed us to distinguish active
Acetylcholinesterase by Novel
Lupinine Derivatives. Molecules 2023, versus non-active compounds. Thus, this SAR model could be applied for design of more potent
28, 3357. https://doi.org/10.3390/ lupinine ester-based AChE inhibitors.
molecules28083357
Keywords: acetylcholinesterase; acetylcholinesterase inhibitor; Alzheimer’s disease; ester; lupinine;
Academic Editor: Carlotta Granchi molecular docking; triazole derivative
Received: 15 March 2023
Revised: 8 April 2023
Accepted: 9 April 2023
Published: 11 April 2023 1. Introduction
Alzheimer’s disease (AD) and other neurodegenerative disorders are predicted to
become the second leading cause of death worldwide because of the increasing elderly
population in most countries. AD produces gradual cognitive dysfunction, including
Copyright: © 2023 by the authors.
difficulty in making decisions, language problems, mood swings, learning, orientation, and
Licensee MDPI, Basel, Switzerland.
This article is an open access article other behavioral issues [1]. The loss of cognitive function due to AD is associated with
distributed under the terms and the rapid hydrolysis of acetylcholine by cholinesterases, including acetylcholinesterase
conditions of the Creative Commons (AChE). Consequently, inhibition of AChE has been proposed to be neuroprotective [2].
Attribution (CC BY) license (https:// Indeed, AChE inhibitors represent the first line of symptomatic drug treatment for mild-to-
creativecommons.org/licenses/by/ moderate AD. AChE inhibitors were initially utilized in the treatment of myasthenia gravis,
4.0/). a neuromuscular condition associated with loss of ACh receptors at the neuromuscular
Molecules 2023, 28, 3357. https://doi.org/10.3390/molecules28083357 https://www.mdpi.com/journal/molecules
Molecules 2023, 28, 3357 2 of 15
junction, followed by skeletal muscle weakening [3,4]. AChE also represents a therapeutic
target for controlling glaucoma, Parkinson’s disease, senile dementia, myasthenia gravis,
and ataxia [3].
Natural products are often used as starting points for drug discovery and have been
considered as the most important resource for the identification of lead compounds due to
their diverse molecular architectures and a wide range of bioactivities [5,6]. Thus, natural
products represent a valuable source from which novel AChE inhibitors may be discov-
ered [4,7], and plant alkaloids, flavonoids, chalcones, xanthones and their derivatives
have been screened for AChE inhibitory activity (e.g., see [8–13]). Considering the paucity
of new AChE inhibitors, we explored the possibility of developing inhibitors based on
transformation of the plant alkaloid lupinine. Lupinine ([(1R,9aR)-octahydro-2H-quinolizin-
1-yl]methane) is found mainly in Lupinus and Anabasis plants [14–16] and is of interest as
a pharmacophore. For example, lupinine has been reported to inhibit the fungal metallo-
protease Mpr1 [17]. Likewise, compounds with octahydro quinolizine nuclei have been
reported as ligands of serotonin receptors 5-HT3 and 5-HT4 [18]. Similar compounds have
also been shown to exhibit antimalarial [19,20], antitubercular [21], and anticholinesterase
activities [22–25]. The quinolizidine nucleus of lupinine is simultaneously bulky and highly
lipophilic and can be used for replacement with heterocyclic groups, or the ring could be
connected with bi- and tricyclic groups to develop AChE inhibitors [25,26]. On the other
hand, it should be noted that simple esters of lupinine have also been reported to exhibit
some anti-AChE activity [23].
We synthesized 13 lupinine-based triazole derivatives and evaluated them, together
with 50 additional commercial lupinine-based esters of different carboxylic acids, for
AChE inhibitory activity. This screening resulted in the identification of some novel
AChE inhibitors, with the most potent being compound 15. Molecular docking allowed
us to characterize 15 for its potential interaction with the AChE binding site. We also
developed a structure-activity relationship (SAR) model to predict AChE inhibitory activity
of lupinine esters.
2. Results and Discussion
2.1. Chemistry
We synthesized three novel compounds by reacting lupinine azide 1 with termi-
nal alkynes 3-(prop-2-yn-1-yl-thio)-1H-1,2,4-triazole-5-amine (2), (2R,2S)-3-methylpent-
4-yne-2,3-diol (3), and 3-ethoxy-4-(prop-2-ynyloxy)benzaldehyde (4) under the condi-
tions of Cu-catalyzed 1,3-dipolar cycloaddition. As a result, (1S,9aR)-1-[(1,2,3-triazole-1-
yl)methyl]octahydro-1H-quinolizines 5–7, which contained various substituents at position
C-4 of the 1,2,3-triazole ring were obtained (Scheme 1). Structures of the synthesized
compounds 5–7 were confirmed by 1H and 13C NMR spectroscopy, mass spectrometry, and
two-dimensional COSY (1H-1H), HMQC (1H-13C) and HMBC (1H-13C) NMR spectroscopy
(see Supplementary Figures S1–S6), which established the homo- and heteronuclear spin-
spin couplings. In describing the spectra, we used the numbering of core atoms shown in
Scheme 1 structure 5.
Reaction of lupinine azide 1 with a diastereomeric mixture of alkyne 3 resulted in
a mixture of unseparated diastereomers 6a,b (ratio 4:1, as in initial alkyne 3). Relative
stereochemistry and the ratio of diastereomers 6a,b was determined by analysis of 1H NMR
data. The proton on the substituent at C-15 of the triazole ring (H-19) appeared at 3.88 and
4.05 ppm (multiplet signals). The other triazole derivatives 8–17 were synthesized as single
compounds, as previously described [27–29]; however, this is the first report of their effects
on AChE activity.
MMoolleeccuulleess 22002233,, 2288,, x3 3F5O7R PEER REVIEW 3 3oof f1155 
 
N NH 24 1
H N 9 10 2
2 N 3
S 8 6 4
N 22 N
2 NH 7 5
2 23
HN 18
21 S H
N 19 16 11
20 17 15 12N
14 13
N N
5
H OH
CuSO  .
4  5H2O, H3C C C
N NaAsc, N N
OH CH3 19a
DMF, 75oC 19a
3 H 18
20
H C 19 OH H
C H + 20 OH
3 19 H
H N 17 H3C
22 C N C 17
N HO N
22 C
1 N H3C N N HO
21 6a H3C N N
21
O 6b
27
O H H3C 26
O O25 N
29
4 CH3 O 21 20
28
22 19 O H
18
H 23 24 17
28a N
7 N N  
SScchheemmee 11.. SSyynntthheessiiss ooff qquuiinnoolliizziinnee--ttrriiaazzoolleess 55––77.. 
22..22.. BBiioollooggiiccaall Reessuullttss 
Wee ssccrreeeeneed oourr lliibbrraarryy ooff llupiiniinee deerriivvaattiivveess ffoorr ttheeiirr eeffffeeccttss oon AChE aaccttiivviittyy iin 
ccomparriisson wwitihth ggalaalnantatmaminine,e a,  kankonwonw AnCAhCEh iEnhiinbhitiobri tuosreuds iend thine ttrheeattmreeantmt oefn mt ioldf  mAlizld-
hAelizmherim’s edri’sedasisee. aTshe.e Tlihberalriyb rwarays wasaseamsbselemdb flreodmfr towmo tsweots soeft scofmcpoomupnodus:n tdhse: tfhirestfi srestt 
csoent tcaoinetadi n1e3d lu1p3inluinpein-binasee-bda tsreidaztorliea zdoelreivdaetriviveast i5v–e1s7 5(–T1a7bl(eT a1b),l ean1d), tahned sethcoensde csoent dcosne-t
tcaointeadin 5e0d l5u0pliunpinine-ibnaes-beads edstersste 1rs8–1687– 6o7f odfidffiefrfernetn ctacrabrobxoyxlyicli cacaicdids scoconntatainininingg aalilipphhaattiicc,, 
aromattiicc,, or heterocyclliic moiiettiies.. SSttrruuccttuurreess ooff tthee llupiiniinee--bbaasseedd eesstteerrss aarree shown iin 
Supplementary TaabblleeS S11. .A ACChhEEi ninhihbiibtoitroyrya caticvtivtyitoyf oafl latlrli atrzioalzeodle rdiveartivivaetsiv, aens,d asntdru sctruurce-s
toufrtehse oaf ctthive ealcutipvien ilnuepidneinriev adteivrievsaatirveesh aorwe nshionwTnab ilne sT1abalneds 12 ,arnedsp 2e, crteivspeleyc.tively. 
Due to the small number of compounds, it is difficult to draw definite conclusions on
Tstarbulcet u1.r Ae-CachtEiv inithyibreitloartyio ancstihviiptys .oHf louwpienvineer,-bitasceadn tbrieanzoltee detrhivaattrievpesl.a cing the methyl group in
compound 12 with a methoxy substituent resulted in AChE inhibitory activity (compound
13), whereas increasing the numNber of methoxy groups in the benzene ring to three resulted
in a loss of activity (compound 14). On the other hand, the presence of a benzyloxy
group in the para position of theHbenzene ring resulted in the maximum activity among all
derivatives (compound 15; IC50 = 7.2 µM). Compound 15 has a 4-benzyloxyphenyl moiety,
which was also preseRnt on seveNral other previously reported AChE inhibitors, including
compounds A–C with IC5N0 vaNlues in th e micromolar/submicromolar range [30–32] (see
Compd. R chemical structuIrCes a(µnMd A) ChE iCnhoimbiptodr. y activity in TableR3 ). AChE inhibitorIyCactivity of
50 50 (µM) 
compound 15 was comparable to that of galantamine (IC50 = 8.2 ± 1.3 µM).
S N A visual inspection of the ester lupinine derivatives showOed that a compound should
5 contaNiHn a suffici1e0n1t.l1y ±b u2l3k.9y R group1(3e .g., compounds 25, 44, and C6H43), or the link51er.8b ±e t9w.2e en
N
the ester oxygen and the terminal hydrophobic moiety should con sist of four chemical
bondNsH(2e .g., compounds 22, 43, and 49) for it to exhibit AChE inhibitory activity. Compound
25 (IC50 = 24.4 µM) bearing a 6,6-dimethyl-6,7-dihydrobenzofuran-4(5H)-one group was
the most active of the lupinine-based esters. Noticeably, the 3,3-dimethylcyclohexanone
substructure in this fragment was also present in previously reported AChE inhibitors [33].
 
Molecules 2023, 28, x FOR PEER REVIEW 3 of 15 
N NH 24 1
H2N 2
9 10 N 3
S
MoM
22
lecoulelecsu l2e0s2 230, 2238,,  2x8 F, xO FRO PRE EPREE RRE RVEIEVWIE W N N 8 6 4
7 5 3 o3 
2 NH2 23 f o1f5  15 
HN 18
21 S
19 H
N 16 11
20 17 15 12N
N N NH 24
H24 N 10 114 1
NH H N2 9 N13 2
2 3
S 2 9 10 NN N3
S N 22 N 8 6 4
8 6 4 5
2N NHNH 22 N 7 5
2 2 2 23
23 18 7 5
H HNHN21 18
OH 21 S S H
N N 19 11
19 16H
17 16 11
CuSO  .
4  5H2O, 20 15
H C C C 20 17 15 12N
3 12
N N
114 13
NaAsc, 14 N3 N N
N OH CH3 19a N N
DMF, 75oC 19a 5
3 H 18 5
H 20 H
H OHOH H 19 OH 20
3C C H + OH
H C 19 H
H N 17 3 17
CuCSOuS O.  .
 54H 5OH,2O,
N H CH3CC C C 22 C N C
C C N
4 2 22
NN NaNAsaAsc, 3 HO N
c, N N N
1 N OH CH3 H139aC N N HO N N
DMDFM, 7F5, o7C5
oC OH CH3 19a 21 19aH H3C
18 19a
O 3 3 20 H 18 6a H 21
20 6b
H CH3C 19 OHOH H + 20 H
19
C C H + H20 C 19
3 OHOH H
H N 3 17 H C 19 7 H
H 22 17 C 3 C 1
17
N NN 22 C N N C
22
HOHO 22 C C N N
1 N N
1 O HH3CH3C
21 N N N 27
N H C HOHO
6a3 26 H CH3 3C NN N
21 N
6a 21
O 1
O 2
25 N
O O 6b6b
4 CH 29
3 O 21 20
H 28 27
O O H H CH
27
232C3 26 19 O H
26 18
H O2245 17 N N
O O 23
2298a O25 N
4 4 CHCH3 29 O 21 20
3 O 21 20
28
28 N N
22 19 O 7 H
H 22 19 O18 H
18
Scheme 1. Synthesis of quinolizine-triazoles H5–7. 23 24 17
28a 23 24 17 N
28a N
7 N NN N
2.2. Biological Results 7
SchSecmheem 1e.  S1y. nSythnetshiess oisf  oqfu qinuoinliozlizine-triazoles 5–7. 
We screened our librarinye o-tfr ialuzopleins i5n–7e.  derivatives for their effects on AChE activity in 
co2m.22.p .B2a.i orBlioisogolioncga iwlc Railte hRsu eglstuas ltasn tamine, a known AChE inhibitor used in the treatment of mild Alz-
heimeWr’seW  sdec irseceeraneseeend.e  odTu hore ul irlb ilrbiabrrrayar ryoyf   woluf aplsui npaiinsnseien dmee drbielveraidvti avftreiovs emfso  rft otwhr eothi res eierft fsee fcoftesfc  tocsno o mAnp CAohCuEhn aEdc tasi:cv titithvyie ti ynf i irns t set 
coconmctoapminapreiadsro i1nso3 wn l iuwthpi tgihna iglnaanelat-abnmatasimnedein,  ater ,k ian kzonowolenw  AdneC ArhiCvEha iEnti hviniebhsiit bo5ir–t ou1r7s ue (dsTe iadnb  itnlhe et h 1ter)e, t aratenmadtem nteth noetf   osmef icmlodin lAdl A zs-lezt- con-
tahienhiemediem r5’es0r  ’dlsui sdpeiaisnseeian.s eT.-h bTeah sleieb dlria berrysat rewyr asws  1aas8s –sa6esm7se bmolfeb ddle ifdfrf oefmroe mntwt  tocw asore btssoe xtosyf  olcifoc  mcaopcmiodpuson udcnosd:n stth:a etinh feiinr fsgitr  ssatel tis peht atic, 
arcomncotanittniacei,nd eo 1dr3   1hl3ue tpleuirnpoiinnceyin-cbelai-cbs eamdse otdri ietartziioaezlseo.  ldSeet rdruievcraitvtuiavrteivs e 5os– f15 7–th 1(e7T  a(lTubalpebi ln1e)i ,n1 a)e,n- abdna tdshe etdh s ee cssoetencordsn  sdae rts eec tos ncho-onw- n in 
Molecules 2023, 28, 3357 Sutapitnpaeilndee m5d0e  5nl0ut apluriynpi inTneian-bela-ebs eaSds1e .ed sA teeCsrtshe rE1s8  –1in68h7– 6iob7f i otdofi rfdyfei frafeecnrteti vncitat rycba robxfo yaxlilycl litacrc iiaadczsiod clsoe nc dotaneitrnaiivinnagitn iagvl eiapslh,i pa4htnioacdft, i1 c5s,t ruc-
tuareoasmr ooamft iatcht, ieco ,r a ochrte ihtveeert oelcuryopccilyniccil nimce o mdieoetirieeitvsi.ea sSt.it vrSuetcrsut uacrteus rs eohsf o otwhf enth  lieun pl uTinpaiibnnleien-bse a-1bs eaadsne deds  2tee,s rrtsee rsaspr eae rcseth isovhweolnyw .in n  in 
SuSpupplepmleemnetanrtya rTya Tbaleb lSe1 S. 1A. CAhCEh iEn hinibhiitboirtyo rayc taicvtiitvyi toyf  oafl la tlrl itarziaozleo ldee drievraivtiavteivs,e sa,n adn sdt rsutcru- c-
Ttaubtures of the
Trlaeebs 1 loe. fA1 t.hCAehC aEhc  Eitanicvhteivinihb leuit ibipolutorinypi
ryi annceine derivat
act tidiveirtyiv oaft liluvpeipvisni enasir neae rs-ehb soahsweondwine-based tr  itnnr i iaTnza oTblaleb sdl ee1sr a i1vn aand 2, respectively. 
iazole derivativdets i.2v,e rse. spectively. 
TaTblaeb 1le.  A1.C AhCEh iEnh inibhiitboirtyo rayc taivctiitvyi toyf  Noluf pluinpiinnei-nbea-sbeads etdri atrzioalzeo dlee rdievraitvivateisv.e s. 
N N
H
R HNH
R R
N N N
N
N
Compd. R  N NN   IC50 (µM) 
CoCmoCpmodmp.pdd. . R R  IC 50 (µM)   Compd.  R  IICC5I0C(µM)
50 (5µ0 (Mµ)M ) 
S N
S N N O
S NH O O
5 NH CHCH CH3
3
MoMlecoulele5csu  l25e0s 52 230, 2238,,  2x8 F, xO FRO PR PEERN REV NH 101.1 ± 23.9 13 51.8 ± 9.2 
EER REVNIEWNIE W 1100111.01.11 ±.±1 2 ±23 3.29.39 .9 13 1133 3 5511.8.58±1 ±.894 9  .±2o.42 f9   o.12f5   15 
MoMlecoulelecsu l2e0s2 230, 2238,,  2x8 F, xO FRO PRE EPRE ERRE VREIEVWIE W 4 o4f  o1f5  15 
Molecules 2023, 28, x FOR PEER REVIEWNH
N H2N H2 2 4 of 15 
MoMMleocoulleelcecusu ll2ees0s  22300,2 22338,, , 2 2x88 ,F,  xxO  FFROO PRRE  PEPEREEE RRE  RRVEEIEVVWIIEE WW  4 o44f   oo1ff5   1155  O O
O COH3CH3
CHCH3
6 6 6 NN.NA.A.. A. . 3
14 1144 O
O O NN.A.NA. .. A. 
6 6 N.NA.. A. 14 CH
14 O 3
O N.A. 
O O COOHCOH3CH N.A. 
6 N.A. 14 H CH3C 3 3
O O CH
CHC3H
3 O CH 3 N.A. 
3 3
6 N.A. 14 H3CH3C
6 6 N.A. 14 OO O COH N.NA..A . 
O O N.A. 14 H C O
O COH3COH33 N.A. 
3 O CH
7 7 O O CH
O O 3
73.743 ±.4 1 ±6 .176 .7 O
15 15 H3C O O CHC33H
H C 3
3H C O O CH 7.27 ±.2 0 ±.2 0 .2 
7 7 O O O O O 73..7
3
43 ±±.4 1 ±6 ..176 .7 15 155 3
OO O
OO 7.27.±27 0±.2. 20 ±.2 0 .2 
7 O O
O O OCHCH O CH
3 3 73.4 ± 16.7 15 CH 3
O C3H3 7.2 ± 0.2 
87  877   ---C---HCOHO CH
2HOCHH3 3 O O
O 737.7N433 .±.NA44  1 ±..±A6  1.1.766 ..77  151 155  O O 7.277 .±.22 O O 2  0 ±±. 2 00 ..22  
98  98 8 ---C-----(-—CC-O-(H-HC
O
OCH2)HOO2)HCOHO
HH3 N
3 2 H NNN.NA..AA..A...A. . O O
3 2 O O
918  09  ---C--(-CC---10 ---C( H-H)-(
-C)(HH2OHCH3
O(COC3H)C(H2COH3C)3H)2HOC3)H9 NN.NA.A..A. . O O O
98  —3C2(3C2H3)22O2H2 N2CN NN.NA.A..A. . CHCH
8 3
108 1 0 ---C---(C-----(H-C--(H)-COH)O(OC32H)O2(HOCHH) C)NCN NNN.NA.NA.A..A..A. .. 16 16 O O 3 1411.421.2 ± 
---C32H2 OH 2 2 N.A. HO  ± 15.135 .3 
3 2 2 2 2 HO O
9 ---C(C— O CH
H ) OH N.A. 16 16 O HO 3CH3
10 141.421 ±.2 1 ±5 .135 .3 
191  1911  0 ---C--(--C--H(C3)2O(C3 H2 2)2CN N
C(CH (HC3H)23O)2OH NN
3)2O(CH2)2CN N..NA.A.A... A. .. 161 O HOO
6 CH3CH3 CH3
H C 1411.241
10 ±.21 ±5 .315.3 
11011  101  ----C--(C(HC3H)23O)2(OC(HC2H)22C)2NCN NN.NNA.NA...AA.. A... HH3CO 3 CHCH3 CH
161 16 CH3
6  H C CH3 3 CHCH 3 141.2 ± 15.3 
11 CHCH N.A. H C 3
HOHO
H3O CHC3CHH 3 3 1414.21 
3 .±2  1±5 1.35 .3 
3
3
1121 1 121
1  1 
3
CHCH3 N.NN.A.
3 N.A.... AA ..
H3C
 CH
.  CH
17 17 O O CHC3H 3
H C 3 3 68.618 ±.1 2 ±.2 2 .2 
12 12 CH3 N.N
3
A.. A. 17 17 OH3CHO3C CHCH
CH 3
3 3 O O 68.618 ±.1 2 ±.2 2 .2 
12 CHCH
CH 3 N.A. 17 O O O 68.1 ± 2.2 
3 3
121 122  C C N.NN...AA ..  171 177  O O
O O
12 C C N.A. 17 686.816.61±88 .±.121  .2 ±2±.  22 ..22  
O O
C O
CCCCompound 6 was present as a mixture of diastereomers 6a,b (ratio 4:1). N.A.: no activity was observed at the
highest concentration tested (150 µMN).N
The mechanism of AChNENinhibition was determined for the most active compound
15. The Lineweaver–Burk recNHiprocal plot (Figure 1) revealed a series of lines converging
on the same point near theHxNH-aN
NxHis, indicating that 15 caused a mixed type of inhibition, as
expected for dual binding sOiteHOinhibitors of AChE [34,35].
Lupinine and all synthHeOsiHzOed derivatives (5–17) were evaluated for their cytotoxicity
in vitro using human THP H
O O-1 mORonRocytic cells. These compounds had no cytotoxicity when
Compd. R tested at conc entrations uOpOtOo O5
O0RµRM. Thus, the lupininRe derivatives reportedICher (eµcMou) ld be
Compd. R 50
CoCmopmdp. d. R R used for fu r the r biologicaOl evaluation in cell cultureRa nd in vivo models. IC50 (µM) 
22 22 39.359 ±.5 7 ±.2 7 .2   R R R ICI50C (5µ0 M(µ)M ) 
Co2m2 2p2d . R  
CHCH 39.359 ±.5 7 ±.2 7 .2 OOO  R RR
O O O
O 3 3 O OR IC50 (µM) 
CoCCmoo2mpm2d pp.dd.. R RR CHCH  3   O O OR IC50 (µM) 
O O CHC3
3 H 3 9.5 ± 7.2  
3  OR R ICI5C0
22 39 44 68 (5.90µ  (M±µ 5M). 8)  
2252 22 52  O CCHHC33H3 2349.23.4549  ±..±.455  3  7 ±±±..4  2 37 ..42  
44 O O 68.9 ± 5.8 
7 .2 44 44 68.698 ±.9 5 ±.8 5 .8 
25 25 O CHCCCHHH3 24.244 ±.4 3 ±.4 3 .4 O
O O 3 33 44 O O O OO 68.9 ± 5.8 
25 O O CH3 24.4 ± 3.4 
O O CHC3H 444 444  686.6988 .±.99  5 ±±. 8 5.8 
252 255  
3
Cl 242.244 .±.44
 
Cl   3 ±.4 3 .4 H C O O 5.8 
O ± 3.4 H3C3 O O CHC3H3
Cl Cl H3CH3CO O O OCHC3H3
41 41 O OO Cl 99.949 ±.4 1 ±2 .132 .3 49 49 H C O O CH 1111.131 ±.3 1 ±2 .152 .5 
41 41 NHNH Cl Cl 99.949 ±.4 1 ±2 .132 .3 49 49 3 3
H C O O CH 111.131 ±.3 1 ±2 .152 .5 
41 NHNH CCl lC
Cl 3 3
Cl l 99.4 ± 12.3 49 H3HC3C O O O O CHC3H3 111.3 ± 12.5 
414 4
H
11  3CNHHH
3CO O Cl 99.4 ± 12.3 49 CHCH 111.3 ± 12.5 
H3C 3CO O 999.49 .±4  1±2 1.32 .3 494 9 3 3
CH 11
CH 11.31 .±3  1±2 1.52 .5 
43 43 N NNHH
Cl
H3C O Cl Cl 89.869 ±.6 7 ±.7 7 .7 64 64 3 3
CH 74.714 ±.1 6 ±.8 6 .8 
43 43 H C O 89.869 ±.6 7 ±.7 7 .7 64 64 3 74.1 ±
H CH C CHCH 74.1 ± 6.8 6 .8 
3
43 H3HC3C O O 89.6 ± 7.7 64 3 3 3 33
H3CH3C CH
CCH3 H  
CH3 3 74.1 ± 6.8 
434 433  898.8699 .±.66  7 ±±.  77 ..77  64 3  74.1 ± 6.8 
DuDeu toe  ttoh eth sem samlla nllu nmubmebr eorf6  oc4of6  4
H
mco pmopuonudnsd, ist, H 
3iCt is difficul CH
is Cdifficult tot  tdor ad3wr CH a wde dfienfiitnei ct7eo4 nc7.1oc4 nl.±u1c  s6l±iu. o86sn .i8os  nosn o n 
strsutcrutuDcrteuDe-rau etcoe-ta  ittcvohti iettvyh si etmry es almraleltal inaolltun inomsuhnbmsehbri peorsf  .oc Hof mcoowpmoepvuoenrud,n siHtd,  3icHsCta,  i3ips. However, it can 3bniCste   db insei ofd fntiiecofdfutie clttduh t laCottht H t
3
 dCaro3eHrt   adp3rw erl aap cwdliaen cdfgiien tfgihitn eetih  cmteeo  enmctochelnyutchlsl yiguolrs noigosur nopsun  op n  
ins ticrnsuo tmccruotDupmcrouteupue-ro anteuocd-tan  ti1cvhdt2ie it 1v yws2im tr iywetah lrlai eltta hlni ao mutanim oesmhtnbheisepohtrhsx io.poy fHxs  sc.y ouH wbsmousewptbviosteutuvrie,tne unidrtet, s  cnir,at te in ctsr a uiebsnlse t dub enildeftof e ntiidcenou d tiAlen ttd h CtA oathh tCdE arhr etaiE pnrw elhiap nidcblhiaeinitfcbgoiiinr ttiyoght er etay h ccm oetai necvmttcihitlevuyyti hslt( iyygco olrn( omgcsour -ompnu - p 
pionsput irocnnuouDd cmcn tuo1DuDdp3emru uo) 1et,ep3ueo -w a) ot ntt,ocuo hdwt  nteiet vh1hr dsie2eem ta  1yr sswse2ma mr a ilienwlsata lh nclailil nrlut  anehincmo aur umnasembmi saenmhsbtbrgihie enp otrtrghsfh x o . oceot yfHohf  xn  cmcseyououo n wpmmbsuouespbpmutvbeoioenstbruuutr deio,nent rsfiund dt, om  etsics ftn,a,r e m iiteinst tsh  r  eidbiuseostie lsh xdftd ufoeyniiilfdxc fotfgfuey itirciedcl nogutdu  u rtil Alntotp h t u CtsdoAao phri t dnCasd Er r whriet anhaipE wn wdetl ah ie b dncidefebiihe enbnififtgiiebziontnne ieritinz hytotcee ero an  y crncmcoei octna iernlcvguitcctnhi lsitltuvguoiyos i ls tttn i(iohgycos rr not n(oeohmscseunr o o oe-p mnen   - 
rseptisrsnosuetptu rlscrcotnuutoeuucdlcmdtnrt eu 1udpid3r-nro ae) 1e ,iuc-3a- nawtan) i c,lcvda hotwtii  iestvlv1yshroi2i t estro yaysrewf s e rl ro aaaieietnftsclhl ia ctaaoi itrntacvniieoct oisaimrtnvhnysesiisea tnhph(tyschgisii popn. o( tsmHcgshx.o.  ye otHpHmh  wnosoeopuue wwnobmnvuuesdeebvtmnvr iee,1tde bruri4r t ,eo,)1   ci.rnif4 tat   Oot)m cnc .fa rna  eObemnn tset hn buehb no telethtxo h  nenoyotedoexot  gdh toytieree nt todgdhr u rAe tahtophrhtCau  saarhn hpteita dn Esrprn e, e ilt dpinahtpnhc, lelt haiaeh tnbcih cebigpien nibn r ttggepoehz s nretrtehyenhz mnse eaec n  mercmenetit cnhei oervetgytfi hinh  lotta yygogf l  l rb tta( gogohce ruo rbnrtoomhpe-uuenr p- ep-e   
zinrypezi linosconryuoxe  lcmolscyntouox edmpgmlydt ro1 e popgu3idonuor)n u,o pui dawnun n  i pdl1hdnao2  e   si1t l1rnsowh2e2  s eao tiwswh tsf ph  eioiaatn tfhracph ca t aa r miaracvpe a taimeom itsvptsyhieieion ttot(siyhghcoxi o otoynt(ixhmcox  oysoeynpum  f  ns sbotuupuhsfbm tobnetisuhst dbtubtnei ieteet 1durubn4  oezet)1 fn.ne4r  tmnzOte)  .eresr eeun Oetr sselhtiuntun heor llgedttixtneh  ey ordiged entg    hsi orrinuAenot hslr uCAt AueehplrCdCtas Ee hh nii dnnEaidEn  n ,it t ihnhidhntni he,ehbth  eh tbiiimht bebeopei niatrrmt oyzxoepre siarmyanyxec n esiautmae cicrmcvenitutn iici tvomvegayfi ict  toyt(aoyicf  cv  o(tb(taichmciet voroybnemi-m et-eyn- - -
apzmroyapepuozmlosoonyuuuxodglloynnt  e1dxa dggd3ly   1lr)1 a  ,3oigd3 lnw)lu)re , ,o dpr ahwwui  evlihprhonaie esv tiratirsnaevhse t ao eeatinvshf s p  eiceia(nanrs ccrpec octa(raairmc versepoaaiiponmt ssyposiginp ionu t(tgosicgnhoi ou tdettnhminh  on e1deonpu 5 n fn om;1 ou tu5IhfmbCm; ne tIdhb brCb  e eo=1er rf bn4  o 7om=)ezf..f n2e   m7eOm50 n zt.µ2eheneMn  to tµrhthexih)Mon oy.re xgx iC) gny y.or ro g tgCeogmh sruroeuoeopmrulsuo stuh50 pep uildasostn ne  ui ditindnhn    ,t1e dti hthn5 t hbeh 1e eh te5 bhb na  meepzhsnn e ramazenzsx ese eia4naemn x-rn ebeii4u ncm er-remignbinu n ezot gmgnoayf   ct zlt taotoayhi  xc vltrbtoyhtehieitx-revrnyey ei- et-ey   
praehzmprseryeuaehnlosmsloeyutnuxnelolgl dytytn eme  lagd idlno mr l a  oiindealnoult  e  iypladearo,  it  eslviylonwrsoa,is  svtohtswsiha fvi o cteoheaihf fvsic p c  aeta(hwicscrcv t aotaii(wi tvmcsvpyo iaiot ptamsy(ysolc  is poua(t(oicomcln sooudnopmmn   roo1deppu5fs or on;e1etu uhn5dIsnCn;ete   dn1 dI5bo0C4 t  1 en)1=5.4no0 4   )7Onzs)=...e e   2nOv7Ons  .eeµent2nvrh M   raetµtihlrhn ) Mao.eego l tC )  toho.rho teetCehtmhrshre oue heprmrlp rat oh erhnpuedpaadonvnr nu,idei dndontv ,h u,1 di ttoe5htsh h ul 1eypehe5s  rm al pepyrhsersa  raepaexrssn esoi e4mepcarn-net obue4c creoed-metnb fe  o e zodAaafnyf  c CzlbaAatoyi eh vxbCblnEoyieeh-t xn-ynEy- - -
zpyazhzlmpoyeyxlhnolooenyxx nlgyg y  rm algog lroulrm ooipdeuuo etpiipyrne i, ti v iyntnwah,   ttethihwhv ipeceha h sprip  ca(ahw crrpao aa owm psps oaipotasssoil ioisuttanioinolo  sndonopf    ro 1otepf5hfs r ;te tehIh nsCbeet e 5 nb0bno tee=z nnn eo 7znzns.eee2 nnv  srµeei vMn rrraeiginl)nr  .agr goeCl   trsrhoeuemestslhrutuep elldptortee  rudidepnn v ri dienitnoh v  1uteith5oh s meluehy  smaa mlxsyra eia amxpxr ieio4mumpr-mbtuoueerm dmante c z daAtayic cvlCtoAtiiihtvxvCyiEyit th-y yE   
ampaahmomenonognyn glag l  mlaa ldllol e iddereitevyrrai,iv vtiwaavttehiivvsiec e(hssc   o((wcmcooampmso ppuaoolnsuudonn  d1dp5  r1;1e 55Is;C;e  InI5C0Ct  =50o
50   7=n=.   277s ..µ2e2v M µµeMr)M.a l)C) ..  ooCCmtohompemrop pupoonruudennv d1dio5  1u 1h55s a lhyhsa  aasrs  e 4aap-  ob44r-e-btbneeezdnny zzlAoyyxlClooyhxx-yEy- -
phppehhneeynnl yymll  momieootiiyeet,t yyw,,  hwwihchhiicc hhw  awwsa asas l saaolls soop  rpeprsreesnseetn nott  noo nns e svseeevvreaerlr aaoll  tohotethhre erpr  rpeprvreeiovvuiioosuulyss llyyr e rpreeopprotoerrdttee ddA  CAAhCCEhh EE  
Molecules 2023, 28, x FOR PEER REVIEW 4 of 15 
MMMoloeocule
Molellececuculul
sle es2s 0 22200322,3 32,, 8 22,8 8x,,   xFx O FFOROR RP  PEPEERER RR  REREVEVIVEIIEWEWW   4 44o  foo f1es 2023, 28, x FOR PEER REVIEW 4 of f 1 
511 55  
Moolleeccuulleess  22002233,,  2288,,  xx  FFOR  PEER  REVIIEW  44
5 
  ooff  1155  
O
CH3
OOO
6 N.A. 14 O CH
O O CH
CCH3H3
3 3 N.A. 
666 6   NNNN..A. CH3
A..AA.  ..  1114144 4  
3
O CHO3O NN.
6  N..A..  14  
 H N
3C OO NN.AA..AA..  ..  
OO COCH
. . 
OO CH3 .A. 
H 3
H
H3C3C CHH3
O H3C3C CH 3
O 3 CH33
H33C OOO
7 OO O
OO 73.4 ± 16.7 15 O CCHO CH
O CH3H3
3 3 7.2 ± 0.2 
7 OO OO
77 7   OO 7773
CH3
733.3.4.4 ± 
3
 O CH .4.4  ± 
± ± 11  16166.6.77..7 7  1155  OO 77.2.2 ± ± 0
7 O O 73.4 ±  16..7  
 1155   OO 77..2.215 7.2   ±
 ±
±   0
  0.0.2.2 .2.2
  
O 3
O O 0.2   
8 ---CHO2OHCCHCH3H3 N.A.
CH O
3
9 888 8   ---C-(--
3
C------C
CH O
 ---
-H
-C-
CCH3H)H2O2OOHHH33 NN.ANN.A...AA.2 .. OO
10 989 9   ---C(C-H-----C-3----
H2OH N.A.
-)CC2(OC(
CCH(CH322H)O32)O2H)O2HCHN NN.AN(CH ) OH NN.A..
..A.AA. .
.. OO OO
9 3 2 . 16 OO O CH3 141.2 ± 15.3 
110910  
 ---C(CH3)2OH N.A.
 -----C-C(C--(--CH--CH3(()C2)OHO(C33())C2H2OH N..A O
100  -----C-C(C(CHH3) )2 O(CHH2)22)CN
3 2 2)22CCNN NNN.A..A. .
.. HO
A. 16 CCHH3 3 141.2 ± 15.3 
11  --- 16 141.2 ± 15.3 
10 ---C((CH3
33))2O22O(((CCHH2)
22))2C22CNN N.NAN....A A... 1166  HHOO CH CCHH3
 3 3 1141.2 ±
16 HO CH 14411...22   ±±   1
 155.3.3  
HHCO 33 15..3  
11111 1   NNN.A..AA. 
3
.. 
HO CH3C CHCH
CH3H3
H C 3 3
11  CH N.A. H C
3 N..A..  H3H3
3C3C CCHCH3H333
12 N.A. 17 O H33C CCHH
CH3 3
CCHH 3
CCH3H3 3 68.1 ± 2.2 
3 3
1112122 2   
CH33 NNNN..AA..AA..  ..  1117177 7   
OO O 6688.1.1 ± ± 2 2.2.2  
Molecules 202 3, 28, 3357 O
. .  O 6688..1.1 ±
12 N.A. 17 O O   5± o 2 f2..12.52   O
C OO 68.1 ± 2.2 
O
C
CCC
TabCle 2. AChE inhibitory activity of lupinine-based esters of different carboxylic acids.
Molecules 2023, 28, x FOR PEER REVIEW N 5 of 15 
 NNNN
N
H
inhibitors, including compOo
HuHHHHnds A–C with IC50 values in the micromolar/submicromolar 
range [30–32] (see chemicaOlO OOstructures and AChE inhibitory activity in Table 3). AChE 
inhibitory activity of coOmpouORnd 15 was comparable to that of galantamine (IC50 = 8.2 ± 1.3 
Compd. R µM).  OOO O RRRR R IC50 (µM) 
CC2Co2omompd. R IC50 (µM) O ComRpd. R IC50 (µM)
CCoom mmpppdpdd.d... RRRR A visual3 i9 n . 5 s ±p 7e.c2t ion of t  h  e ester lupinine derRRiRR v   atives showed tIhICICICaC50t5 0 (a( µ50 (µ( µµcMMMoM)m) ) ) 22 50  pound should 
Co2mp
2222 2  
d.. —(CH
 O R2)3C
coCnHl 39.35  39± 7.2  O  I 50 ( ) 
3
 tain a suffic3i39.95..5 5±  ± ±7  7.72..2 2  
 O R IC50 (µM) 
22 9e.n5 t±l y7 .b2 ulky R group (e.g., compounds 25, 44, and 64), or the linker between 
OO CHC3CHH 39..5  ±  7..2  OO OO
25 OO the eCC
CCHsH
3Ht3
3e3H3 r oxy2g4e.4n an 44 OO 68.9 ± 5.8 
CH  ± 3.4d  the terminal hydroOphobOiOcO moiety should consist of four chemical 
O 333 444 686.89.9± ±5 .58.8 
2252255  bondCCHH3
255 3
  CsH 33(e.g., 2c4o.242m242±44.4.4p4..4 34 ±o.±  ± 4± 3u3  3.3.n44..4 4d   s 22, 4434, 4   a 44 nd 49) for it to exhibit AChE in6h66688i
88.b9...
99
.9i 
 ± 5.8 
O   ±t ±o  5 r5. .8 
± 5..8y8    activity. Com-
25  
Opound 25 (IC50 =24 2..44  ±.4  3 µ..4M  ) bearing a 6,6-dimethyl-6,7-dihydrobenzofuran-4(5H)-one group 
OOO H C O O CH
OwasC lthe most active of the lupinine-base3 3
Hd CestOers. NOoticCeHably, the 3,3-dimethylcyclohexa-
41 Cl
none CsCClull bstru9c9.t4u ±r e1 2i.n3 H3 C O O CH3
Cll  this fra4g9m ent was aHlsH3
3oC3C pOrOesent OiOn CpCHrH3
H3C O O CH3e3
3viou1s1ly1. 3r e±p 1o2.r5t ed AChE inhibi-
4441411 1  NH torsC l[33]. 99.949±9.94..4 14±
3
 2 ± ±1.3  12
3
12.23..3 3  4449499 9  11111.131.13±..3 3± 1 ± ±21  .12512.25..5 5  
411   99..4  ± 12
NNHH Cl  
H C NHO TCChlle mech9a9n.4i s±m 12...33   4499   111..3  ±  12..5  
3 NH Cl
NH Cll  of AChE inhibition was deterCmHi3ned for the1 m11o.3s ±t  1a2c.5ti ve compound 
H
43 HH3H
C3CCC
O
O1O3 3 O5. The Linew89e.6a ±v e7.r7– Burk re6c4i procal plot (Figure 1) rCCeCHCHvH3He33aled a s7e4r.1ie ±s  6o.8f  lines converging 
H33C O 3
4443433 3   on the same po8889i89n9.9.66.t.6 6 ± ± n ± ± 77 e 7.7.7a7..7 7r    the x-a666x4644 i4 s  , indicHa3tCing that 15 caCH
CH 33
3 used a mixe7774d744.4.1 1.t.1 1 y±±  ± p6 6.e .   .   .  ±  6 6443 .88..8 8 o  f inhibition, as 
expected for8 d9.u68a9±.l6 7b .±7i n7.d7 ing site 6i4n hibitors HoHH3HfC3C CAC ChE [34C,3CHH5]. 747.14±.1 6.
3 ± 86..8  
3
3 3 CCHH3 3 
Due to the small number of compounds, it His33 Cdifficult to dCraHw3 3   
 definite conclusions on 
structuDrDeDu-ueau ecet to tito vot hi ttthehy 
Due to the ees
r
 s m s
e
msm
lmat
aalal
i
ll
o
l lln ln 
n unsuumu
h
mmm
ibps
bbeberee
. 
rror
H
 o  ofof 
ofc fc 
wo cocomo
emmvper,
mppopoouo
 uiunutnn
 dncdds
ad,sns i,,  t bi e noted that repl
s, it iti ti s isisd sd  didififfiffifficficiucuculultlt ltt tt o toto od d  drdrararaw
aawcw di nw d ded
gfeei fthe
efinfinininititeit
 
et
m eec c o c
ethy
ocononncnclclcul
llu sugissroioionounsnp sos   onon n  
ins ctssrotturmrucutcpcDuttouruuerre-enea  -ttd-acoa tc  c1itttvth2iivie vtwi y itssty miyrt e hrrale ealllalllat  a intmotiuionoemnsnthsbshiohepixirrpsp y o.ss  H.ff.s    HcucHoobowomswwtepiveteouvevureee,rn ri,,dt  i  itscrst ,ea ,c c isianattun  niibls st ed
structure-activit  be bd eeni i nffo nffiotinioecct tudeAedl ldttC h   tttthoahatE  ad tr t rrie rnrapeewhplpail  bldacaicectniofifnginirng ygtii h  ttttahehc   ccmtoivneitccthyll
uuysions on 
e meth
str ct r - cti i e meth (yslsyc iilglo  grgnmorrssou-o  oupunp p    
poinsuiitn nnrc u doccco mo1tmmu3p)rpo,pe wherea
in compoou-ouaununcndntddi dv 1 1 i1212t
ty2 ysw
    irrre2  wwnietc
llhlaartionsh
iitth hetta iia oa asm n imnmssehg
i ipthsse..   Hnuomwbevere 
C woithm ap moeteuethithtpnohosoxodx.xyxyHy  y s1 os ususw5ububbse,bstsv tsitcitetitu
r
iotrr
,
utou,e
,f u  
i 
ein
t
eiment
t  t cnncct 
ea
ct ra
n
t tr ern
hrs
 
ee  o
bube not
ssxueluty l elnt tdegoedr tt
eioendud  pA  td  iinn tt
h
 AhsAC 
aaiCChn
ttt h  E
r hrrt
e
Eeh 
p
Eip en
l
 i lil
a
nhba
c
nheicchb
iinii
n
inbibzt
g 
igoiett  
t
orntot
hee methyl group 
esnulttread tin AnC (hμE Minh)ibitoryhryry e
 yar    imacantccigetvtit titvihvotiyit ttyl lyh rreoe  
repsiiuonl tced in a  activity  ( g(c (c(rococomomumm-p-- -
pppoou oucunonmu2unn0d
d d1  1
p  1313o3)3),u)),
 ) w,,n los
 w wwdhhh e
 hs1re e2eorr ae few asaa sisicttiereas i tn inhincnc 
v rcaceirr t reaem
yeaasa
 
sise
(snicttiningh
ongg o
mgt t hx tt
pheyhohe e
 enss u n nun
numbudmssmb tt1it4umbibebtereue
)
r ro er
.   onfoO fmttfn rof m   mrme
 eteseshetthuth
eohll oxttooexyxtdy hyg   e girignrorr  o uhoAupuaCpnsp hsdis nE i,in n  t iih tnthehhe ebiip  bbebrineieettnzosnzerrzyenen  ecnae eercc  i rttorniiiivfngn igia gtt  yo tbto  ot(e( hcc tnthorh-ermre e--e  
zyrlrper
o
erseoe
xussyulunt legsultltdetde
re odd1 di3
u n i)pin ,,n  a w  ia nalh o  lletosohrrse ssoa   posfso a fiiafr n acaacct ccriprtvteioivaivtsissyiititit in a loss of activityy ny
i
 (
o (g c(
n
c(oc 
 ctt oomho
of  the
mmemppnpopoououmunu
 nbdnbde de1n rr 14z  1o4)e4.ff)n )  nd 14). .mO. 
e OO nernt tiOnn ht
nhoogexx  ryyoe   gtsghurrreooltruue phdpsas s i n iniinnd   t t,tth hhteehe   e bmb eanxziemenuee m rriin agc  tttiov  th  tthhee  ootthheerr  hhaanndd,,  tthhee ittyrr ee  
amzrryoelnssougxl lyttae lgdl r  doiineur  paiv  ilalnot sistvssh e oes f fp ( caocrctatmii vpiiottoysui  t(n(iccodon m1 o5pf; oItuCh1ned 0 b=  0e1 n74.)z)2..e   OnµeMn   rtt)tihh.n eeCg    oortmtethhsepuerrorl  t uhhenadandn id dn1,, , 5  tttth hheeea  
ep p nprprzererseesenssneenc nreciceneo g ofo tfafo  a  abt  hbebrneeen-ne-- 
zzyyllooxxyy  ggrroouupp  iinn  tthhee  ppaarraa  ppoossiittiioonn  ooff  tth5h0ee  bbeennzzeennee  rriinngg  rreessuulltteed  iinn  tthee  ms p  mmarre axa4ssi
e-embnneucccneme z  oo yafffl c  oaatx ben-
xxiimmuumm  aacci  tvtybiivi-vetinyitty --y  
phazeynllyoolx ym  gorrioeutyp,   iwin h tthiceh   pwaarraaas  p aolssiiotti iopnnr 
azamamymmoloononxngngyg g  a ag alalrlll oll dl du dedepreeri rirviivnivava atattthivtiviveve seesp s sa( (c r(c(oacoc omompmmopppopoiutouiunuonndndd d
 eo1o s
f
 15ff1e
   ;tnt thte  o bne n55 szeevneer a rrli inog
 15; ;hI; IC IeCIC C5b05 0 e= = n= = 7z 7 7.e7.22.n.2 2 µe µ µ MµMrMiM)n).).g 
tC  h rrroeeemrsss uuplllttorteeuddvn   iiidoinnu    1tstth5hly eeh    mamresaa paxxo iiimr4mt-ebuudemm nA z  activity 
15 . C
n  ll eri ti s (c  ; I 5050  .  )).  .C Co
oommmpppooouuunnnddd  1 11555  h hhaa  asss s
 a
 a a
  44--bbeennazyCzccylythotliiloxvEoxyii xtty-yy 
pahmenoylg  malol idetyri, vwathivicehs (wcoams paolsuon dp r1e5s;e InCt o =n 7s.e2v µerMal) . oCthoemr pporu   4--benzylloxy--- 
ppphhheeennnyyyll l  mmmool oiieiei etttyt
yy,, 
,
 ,
  wwwhhhiicchh  wwaass  aallssoo  pprreesseenn5t5t00  oonn  sseevveerraall  ootthheerr  pp -
phenyl moiety, whiiiccchh   wwaasss   aalllsssoo   pprrreessseennttt   oonn   ssseevveerrraalll   oottthheerrr   pprerernveevdvivio ioio1uou5usus lslshylyly ay  rs re rerapeep pop4oor-ortbrtreteetdeednd dz  A yAAAClCoChxhEyE-  
rrevii
ChE
ousslly  rreporrtted  AChhEE   
 
50
10
25
5
0
0
0.00 0.05 0.10
1/[S]
 
Figure 1. Representative double-reciprocal Lineweaver–Burk plot illustrating the mixed-type mecha-
Fignuisrme o1f. ARCephEreinsehnibtiatitoinveb ydcooumbploeu-rnedc1ip5.rocal Lineweaver–Burk plot illustrating the mixed-type mech-
anism of AChE inhibition by compound 15. 
Table 3. Chemical structures and AChE inhibitory activities of lupinine derivative 15 and previ-
ously reported AChE inhibitors with a 4-benzyloxyphenyl moiety [36–38]. 
Name Chemical Structure IC50 (μM) 
N N
N
H
Compound 15 7.2 
O
N
O
H3C  
H3C
NH
N N
Compound A 11.8 
N O
 
 
1/V
MMoolleeccuulleess  22002233,,  2288,,  xx  FFOORR  PPEEEERR  RREEVVIIEEWW  55  ooff  1155  
iinnhhiibbiittoorrss,,  iinncclluuddiinngg  ccoommppoouunnddss  AA––CC  wwiitthh  IICC50  vvaalluueess  iinn  tthhee  mmiiccrroo50 mmoollaarr//ssuubbmmiiccrroommoollaarr  
rraannggee  [[3300––3322]]  ((sseeee  cchheemmiiccaall  ssttrruuccttuurreess  aanndd  AACChhEE  iinnhhiibbiittoorryy  aaccttiivviittyy  iinn  TTaabbllee  33))..  AACChhEE  
iinnhhiibbiittoorryy  aaccttiivviittyy  ooff  ccoommppoouunndd  1155  wwaass  ccoommppaarraabbllee  ttoo  tthhaatt  ooff  ggaallaannttaammiinnee  ((IICC50 = 8.2 ± 1.3 
50 = 8.2 ± 1.3 
µµMM))..  
AA  vviissuuaall  iinnssppeeccttiioonn  ooff  tthhee  eesstteerr  lluuppiinniinnee  ddeerriivvaattiivveess  sshhoowweedd  tthhaatt  aa  ccoommppoouunndd  sshhoouulldd  
ccoonnttaaiinn  aa  ssuuffffiicciieennttllyy  bbuullkkyy  RR  ggrroouupp  ((ee..gg..,,  ccoommppoouunnddss  2255,,  4444,,  aanndd  6644)),,  oorr  tthhee  lliinnkkeerr  bbeettwweeeenn  
tthhee  eesstteerr  ooxxyyggeenn  aanndd  tthhee  tteerrmmiinnaall  hhyyddrroopphhoobbiicc  mmooiieettyy  sshhoouulldd  ccoonnssiisstt  ooff  ffoouurr  cchheemmiiccaall  
bboonnddss  ((ee..gg..,,  ccoommppoouunnddss  2222,,  4433,,  aanndd  4499))  ffoorr  iitt  ttoo  eexxhhiibbiitt  AACChhEE  iinnhhiibbiittoorryy  aaccttiivviittyy..  CCoomm--
ppoouunndd  2255  ((IICC50  ==  2244..44  µµ50 MM))  bbeeaarriinngg  aa  66,,66--ddiimmeetthhyyll--66,,77--ddiihhyyddrroobbeennzzooffuurraann--44((55HH))--oonnee  ggrroouupp  
wwaass  tthhee  mmoosstt  aaccttiivvee  ooff  tthhee  lluuppiinniinnee--bbaasseedd  eesstteerrss..  NNoottiicceeaabbllyy,,  tthhee  33,,33--ddiimmeetthhyyllccyycclloohheexxaa--
nnoonnee  ssuubbssttrruuccttuurree  iinn  tthhiiss  ffrraaggmmeenntt  wwaass  aallssoo  pprreesseenntt  iinn  pprreevviioouussllyy  rreeppoorrtteedd  AACChhEE  iinnhhiibbii--
ttoorrss  [[3333]]..  
TThhee  mmeecchhaanniissmm  ooff  AACChhEE  iinnhhiibbiittiioonn  wwaass  ddeetteerrmmiinneedd  ffoorr  tthhee  mmoosstt  aaccttiivvee  ccoommppoouunndd  
1155.. TThhee  LLiinneewweeaavveerr––BBuurrkk  rreecciipprrooccaall  pplloott  ((FFiigguurree  11))  rreevveeaalleedd  aa  sseerriieess  ooff  lliinneess  ccoonnvveerrggiinngg
oonn  tthhee  ssaammee  ppooiinntt  nneeaarr  tthhee  xx--aaxxiiss,,  iinnddiiccaattiinngg  tthhaatt  1155  ccaauusseedd  aa  mmiixxeedd  ttyyppee  ooff  iinnhhiibbiittiioonn,,  aass
eexxppeecctteedd  ffoorr  dduuaall  bbiinnddiinngg  ssiittee  iinnhhiibbiittoorrss  ooff  AACChhEE  [[3344,,3355]]..
Compound 15, concentration (μM)
2200
Compound 15, concentration (μM)
110000
1155
10 5500
10
5 2255
5
00
00
00..0000 00..0055 00..1100
11//[[SS]]
Molecules 2023, 28, 3357 6 of 15
FFiigguurree  11..  RReepprreesseennttaattiivvee  ddoouubbllee--rreecciipprrooccaall  LLiinneewweeaavveerr––BBuurrkk  pplloott  iilllluussttrraattiinngg  tthhee  mmiixxeedd--ttyyppee  mmeecchh--
aanniissmm  ooff  AACChhEE  iinnhhiibbiittiioonn  bbyy  ccoommppoouunndd  1155..  
Table T3.aCblhee m3.ical structur
Table 3.  CChheemmiiccaall  ss
etrsuacntudrAe ChE inhibitory activities of lupinine derivative 15 an
tructuress  aanndd  AACChhEE  iinnhhiibbiittoorryy  aaccttiivviittiieess  ooff  lluuppiinniinnee  ddeerriivvaa
dtivper e1v5i oaunsdly previ-
reportoeudsAlyC rhE inhibitors with a 4-benzyloxyphenyl tive 15 and previ-
ously reeppoorrtteedd  AACChhEE  iinnhhiibbiittoorrss  wwiitthh  aa  44--bbeennzzyy
mlooxiyeptyhe[3n6y–l 3m8].
loxyphenyl mooiieettyy  [[3366––3388]]..  
NaNmaeme  Chemical Structure IC5 (µM)
Name  I0ICC50 (μM) 
50 (μM) 
N
N N
N
N
N
H
CCComoommpoppuoonuudnn1
H
dd5  1155  7.2 77O ..22  
O
N
N O
H C O
H 3
3C
H3C
H3C NH
N NH
N N
Compound A N 11.8 
Molecules 2023, 28, x FOR PEER REVCICEoWmom popuonudnAd A N O 11.811.8 6 of 15 
Molecules 2023, 28, x FOR PEER REVIEW N O 6 of 15 
S
N S
N
CComompoun
Compopu
d
on
Bd S N 1.2
un Bd  B 1.2 
H S
3C H N O O
O 1.2 
H3C N H O
N N
N
O
O
NH NH
NH NH O
Compound C O 0.6 
ComCpoomunpdoCund C 0.6 0.6 
N O
N H O
3C
H3C
CH
C3H3
Chemical names: Compound 15, (1S,9aR)-1-((4-(4-(benzyloxy)-3-methoxyphenyl)-1H-1,2,3-triazol-1-
yl)methyl)octahydro-2H-quinolizine; Compound A, (E)-2-((2-(4-(benzyloxy)phenyl)hydrazineylidene)methyl)-
1-methylbenzimidazole; Compound B, (E)-5-((E)-4-(benzyloxy)benzylidene)-2-((5-ethyl-1,3,4-thiadiazol-2-
yl)imino)thiazolidLinu-4p-oinein; Ce omanpdou nadllC ,s(yEn)-t3h-(e4s-(ibzeendzy ldoxeyr)i-v3-amtievtheosx y(p5h–e1n7y)l )-Nw-e(2r-e(( 2-emveatlhuyalqtueidn olfino-r4 - their 
yl)amino)ethcyyl)taoctroylxaLimcuiipdtyei.n iinn ev itarnod u sainllg  shynthe
cytotoxicity in vitro usingu hmuamns iaTzeHdP -d1 emriovnatoicvyetsi c (c5e–l1ls7.)  Twheesree  coemvapluoautnedds  hfoard their 
cytotoxicity when tested a n THP-1 monocytic cells. These compounds ha dno n o 
2.3. MolecudleacrryivDtoaottcoivkxieincsgi trye pwhen tested t atc ocnocnecnetnratrtiaotniosn up to 50 μM. Thus, the lupinine 
derivatives reoproterdte dh ehree rec ocuoludl db eb eu suesde dfo rfo 
sf uurtph erto b i5o0lo gμiMca.l  eTvhaulsu,a ttihoen  ilnu pcienliln e 
We pceurfloturmree adnmd iolecular do
culture andn  ivni vvoiv mo omdceklisn. g of compound 15 inr tofutrhteheAr CbhiEolobginicdailn gevsaitleu(aPtiDonB in cell 
code 4EY7) using the Rosetta ligaonddedlso. cking protocol implemented in the ROSIE server,
which acc2o.u3n. tMs for full flexibilit
2.3. Moleoclueclaurl aDr oDckoicnkgin 
y of the main chain and side-chain residues in the vicinity
of the dockingWaere pae[3
g 
We prf6o]r.mAedcc morodliencgultaor oduorckminogd oelfi ncogmrepsouulntsd,  the best docking pose of
compound(P1D5Bh caoddae c4eaErlfYcou7rlm)a tueedsdi nmigno tlteehrcefua lcRaeor sedenotetcarkg ilyniggoa fnofd−  c2do4mo.0cp5koiknucgna d
15 1 into the AChE binding site 
lp/rmo5t ooilnc.toolI  ntihmtehp iAlseCmphoeEsne tb,eitdnh deinin gth sei te 
ligand form(sPHD-Bb ocnoddsew 4iEthYr7e)s iudsuinegs  Ttyhre3 3R7o(swetitah tlhigeapnadr tdicoipckaitniogn porfobtotcholb eimnzpylleomxyenatned in the 
methoxy oRxORySgOIeESnI Esae tsrovemrevrs,e) ,rw,T yhwrich1h2ic 4ha(c waccoictuhonumtns etfstoh rfo xfuyllo xflyegxibni)l,itayn odfP thhee2 m95a(iwn icthhatiwn oannidtr osigdeen-chain 
atoms of trheesreitdrsiuadezusoe lisen  ihtnhe teeh rveoi cvciyincciiltneyi) toy(Ff  oitgfh uterh ede o2d)c.okTcin
r 
khgfu alrl efal e[x3i6b]i.l iAtyc coofr dthineg m toa ionu crh maiond aend side-chain 
iensge agreenae [r3a6l ]f.e Aatures of the l
the b ccording to ioguarn md-obdilniendlgiinn rgge sreuslutsl,t s, 
site interactitohnes ebsceta sdnt obdceokcrinkesginp gpo onpssoeibs oelef fcoormth-
kcal/mol. In this pose, t hoef  cloigm
pe-opAuoCnudhn Ed1 i5n1 hh5 aihbdia tdao  rcaya clacacultcliauvtiletaydte odinf ticneortmfearpcfaeoc ueenn edenr1ge5ryg. oyFfo r−24.05 
reference, thkec2aDl/mdioalg. rIanm tohfisli gpaonsde-, rethcep ltaiognradinn dftoe rrfoamcrtsmi oHn-sboontdaisn ewditohn dreosidues Tyr337  of −24.05 
cking of compo (uwnidth the 
15 into ACphapEratiirsctiispchiapotiwaotnnio inno fS oufb popbthloet benzy s H-bonds with residues Tyr337 (with the 
mh enbteanrzyloyFxloiygx uyar neadSn 7d.m emthet-hox-oyx yo xoyxgyegne na toamtos
me m),s ),T yTry1r2142 4( w(iwthit h 
For co
(Fmtpheatohrxaoytx ioyvx eoyxpgyuegnrep)n,o a)s,ne asdn, dPw hPeeh2me925o9 d(5we l(iewtdhit tthhw rtowe enoio tntrhiotergoregnpe ranet voaimtoousm soslyf  otrfh eteph toerr ittarezidaozlAeo lchehe hEteeritnoerc-oyccylec)l e) 
hibitors (A–
res(
iCgF)uigwruei rteh2 )m.ponsible2
 )To. hlTeechsueels aegr etgno for the ACeepnroaelrlo agfhE inle yafeatuantrauelosre gsoofu st
hibitory a cotif
h ttoe c
vithyeloi o 
gm
fl
aignpado comn
u-dbn-idnbdi1ni5dng[in3 6gs– it3se8i t]ein( Ttinaerbtaelecrat3ico)t.nioTsn hsce acna nb eb e 
docking compu
diraegsrp
toantisoinbslef oforrt htheese AliCghanEd isnhleidbittoordyocking poses ppoosuitniod
am of ligand-receptor interaction asc toibvtitay of compounn d1e5 d1. 5wF.o iFtrho riren fr-teehfr-eeenArecCnech, eEth, teh 2eD 2 D 
binding sitedsimilarly to 15 (Figure 3), and with interface
AChiaEg risa msh oowf lnig iannd-receptor interactions obietnaneiendreg doie nos ndin oddciokccianktiginn ggo fs otcrfo ocmnogpmbopiunondudinn d1g5 :1 i5n tino to 
−23.34 (comApCouhnEd isA s)h,o−w2n4 . i7Sn0u S(pcupoplmpemlpeoemunentnadrtayBr F)y,i agFnuigdrue− rSe27 S3. 7.6. 5 kcal/mol (compound C).
11/V/V
Molecules 2023, 28, 3357Molecules 2023, 28, x FOR PEER REVIEW 7 of 15 7 of 15
 
Molecules 2023, 28, x FOR PEER REVIEW  8 of 15 
 Figure 2. DocFkiginurge p2.o Dsoecskoinfg cpoomsesp ofu cnomdp1o5unind 1A5 Cinh AEC(hPED (PBDcBo cdoede4 4EEY77)).. HH-b-obnodns darse ashreowsnh oinw bnluein blue
dashed lines. dRaeshseidd uliensesw. Rietshidinue2s. w5 iÅthion f2.e5a Åc hofp eoacshe paorsee avries ivbislieb.le. 
For comparative purposes, we modeled three other previously reported AchE inhib-
itors (A–C) with molecular topology analogous to compound 15 [36–38] (Table 3). The 
docking computations for these ligands led to docking poses positioned within the AChE 
binding site similarly to 15 (Figure 3), and with interface energies indicating strong bind-
ing: −23.34 (compound A), −24.70 (compound B), and −23.65 kcal/mol (compound C). 
 
 
FigFuirgeu 3re. S3u. pSuerpiemripmopsoesde dodcokcikningg ppoosseess ooff ccompoundss 1155( g(grerenen),)A, A(y (eylleolwlo)w, B), (Bli g(hlitgbhltu bel)u, aen),d and C 
(maCge(mntag) einnt aA) iCnhAEC (hPED(BP DcoBdceo d4eEY4E7)Y. 7T).heT hceo-ccor-ycsrytasltlailzliezde dlilgigaanndd ddoonneepezil is sshhoowwnni nint htihnin red 
sticrkesd. Tsthicek rse. siTdhueerse esimdubersaceimngb rtahcein hgytdhreohpyhdorboipch poobcickepto ocfk eAtCohf EA CarheE vaisreibvleis. iTblhee.  pTohseitpionsist iofn sthe co-
crysotfatlhliezecdo- cdroynsteaplleizeild adnodn eApCezhiEl  arnedsiAduCehsE croersriedsupeosncdor troes tphoenird “tonatthievier”“ nloactiavteio”nlo icna tihoen 4inEYth7e struc-
ture4.E Y7 structure.
It is noteworthy that the 4-benzyloxyphenyl moieties of all docked inhibitors and the 
N-benzylpiperidine fragment of the co-crystallized ligand donepezil occupy the same 
area of space in the hydrophobic pocket surrounded by residues Trp86, Gly120, Gly121, 
Tyr124, Tyr133, Tyr337, Phe338, His447, and Gly448 (Figures 2 and 3), although the bond-
ing patterns of A–C differed from those of compound 15. These molecules formed H-
bonds with Tyr124 (compounds A and C), Tyr337 (compound A), His447, Ser293, and 
Arg296 (compound B). In addition, the terminal cyclic moieties of these inhibitors, includ-
ing the quinolizidine heterocycle of compound 15 and the indanone fragment of 
donepezil, match well with each other in the AChE binding site. It should be noted that 
for the investigated compounds, most of the above-mentioned residues surrounding the 
pocket are among the top ten residues tightly interacting with the ligands, according to 
the partial MolDock scores as evaluated by the “Energy Inspector” tool of Molegro 6.0 
software, which is due to significant van der Waals interactions of the molecules with 
these residues. In terms of the reported AChE functional domains [37], the subpocket res-
idues identified belong to important functional domains, including the catalytic triad 
(His447), the anionic domain (Trp86, Tyr337, Phe338), and the oxyanion domain (Gly121). 
Additionally, we obtained high partial MolDock scores for Tyr124 and Trp286, which are 
located in the peripheral anionic site at the entrance of the binding gorge [37]. 
2.4. Classification SAR Model 
Lupinine derivatives containing an ester moiety are, in general, more synthetically 
accessible. Thus, we used the lupinine ester derivatives to build an SAR model using lin-
ear discriminant analysis (LDA) to determine if it would be helpful for further drug design 
within this subgroup of substituted lupinines. LDA is a statistical technique used to cate-
gorize data points into two or more classes using a linear formalism [38]. The compounds 
containing an ester or carbamate moiety were separated into two classes (“Active” and 
“NA”) according to the data shown in Table 2, which includes only active AChE inhibitors 
found within the entire set of the lupinine esters (see Supplementary Table S1). Selected 
physicochemical and ADME parameters calculated using the SwissADME online tool 
 
Molecules 2023, 28, 3357 8 of 15
It is noteworthy that the 4-benzyloxyphenyl moieties of all docked inhibitors and the
N-benzylpiperidine fragment of the co-crystallized ligand donepezil occupy the same area
of space in the hydrophobic pocket surrounded by residues Trp86, Gly120, Gly121, Tyr124,
Tyr133, Tyr337, Phe338, His447, and Gly448 (Figures 2 and 3), although the H-bonding
patterns of A–C differed from those of compound 15. These molecules formed H-bonds
with Tyr124 (compounds A and C), Tyr337 (compound A), His447, Ser293, and Arg296
(compound B). In addition, the terminal cyclic moieties of these inhibitors, including the
quinolizidine heterocycle of compound 15 and the indanone fragment of donepezil, match
well with each other in the AChE binding site. It should be noted that for the investigated
compounds, most of the above-mentioned residues surrounding the pocket are among
the top ten residues tightly interacting with the ligands, according to the partial MolDock
scores as evaluated by the “Energy Inspector” tool of Molegro 6.0 software, which is due
to significant van der Waals interactions of the molecules with these residues. In terms of
the reported AChE functional domains [37], the subpocket residues identified belong to
important functional domains, including the catalytic triad (His447), the anionic domain
(Trp86, Tyr337, Phe338), and the oxyanion domain (Gly121). Additionally, we obtained
high partial MolDock scores for Tyr124 and Trp286, which are located in the peripheral
anionic site at the entrance of the binding gorge [37].
2.4. Classification SAR Model
Lupinine derivatives containing an ester moiety are, in general, more synthetically
accessible. Thus, we used the lupinine ester derivatives to build an SAR model using linear
discriminant analysis (LDA) to determine if it would be helpful for further drug design
within this subgroup of substituted lupinines. LDA is a statistical technique used to catego-
rize data points into two or more classes using a linear formalism [38]. The compounds
containing an ester or carbamate moiety were separated into two classes (“Active” and
“NA”) according to the data shown in Table 2, which includes only active AChE inhibitors
found within the entire set of the lupinine esters (see Supplementary Table S1). Selected
physicochemical and ADME parameters calculated using the SwissADME online tool
were considered as independent variables (predictors) for LDA analysis along with two
manually defined structural descriptors Nam and Q. Based on the 11 selected predictors, the
LDA models in the form of classification functions (1) and (2) were built by STATISTICA
6.0 software with the “Best subset” option switched on.
We found that the best subset included 5 of the 11 descriptors (D1–D5, Table 2), which
were sufficient for good LDA classification of the compounds, with 41 of the 50 lupi-
nine derivatives classified correctly as AChE inhibitors (the class “Active”) or inactive
compounds (the class “NA”). The values of SwissADME descriptors appearing in the
classification functions are shown in Table S2 (see Supplementary Materials).
The best subset of predictors included molecular weight (MW), number of rotat-
able bonds (Nrot), molar refractivity (MR), water solubility measure SILICOS-IT Log Sw
(sLogS) [39], and the indicator Q of the quaternary carbon atom. This relatively simple
LDA model can be expressed by the following two classification functions:
F(Active) = a0 + a1·D1 + a2·D2 + . . . + a5·D5 (1)
F(NA) = b0 + b1·D1 + b2·D2 + . . . + b5·D5 (2)
where D1–D5 are the values of descriptors from the best subset; a0, b0 are the intercepts
from Table 4; a1–a5, b1–b5 are coefficients of the linear classification functions from the
corresponding columns of Table 4.
Molecules 2023, 28, 3357 9 of 15
Table 4. Physicochemical descriptors from the best subset and the corresponding coefficients of
classification functions.
Coefficient of Classification Functions
Descriptor
Active NA
Intercept −61.838 −51.489
D1 MW 0.064 0.046
D2 Nrot −0.848 −0.133
D3 MR 1.450 1.354
D4 sLogS 9.175 8.580
D5 Q −7.863 −5.265
Abbreviations: molecular weight (MW), number of rotatable bonds (Nrot), molar refraction (MR), water solubility
characteristic (sLogS), and indicator variable for the quaternary sp3 carbon atom (Q).
According to the LDA model, a compound is classified as active if F (Active) > F
(NA), and vice versa. Hence, the influence of each predictor can be evaluated based on the
corresponding pair coefficients in the two classification functions. For example, a higher
molecular weight favors activity because the coefficient for MW is larger in F (Active).
The same refers to molar refractivity and water solubility. Conversely, higher molecular
flexibility and the presence of a quaternary carbon atom disfavor activity in view of lower
(more negative) coefficients for Nrot and Q predictors in F (Active).
The classification matrix for the investigated compounds is shown in Table 5. Ac-
cording to this matrix, the LDA model correctly classifies 6 of 7 (85.7%) active AChE
inhibitors and 35 of 43 (81.4%) inactive compounds. In spite of the noticeable number of
false positives among the “NA” class, a total of 82.0% of the compounds were recognized
properly by the model. The per-compound classifications are presented in Table S3 (see
Supplementary Materials).
Table 5. Classification matrices for the LDA model built based on 50 lupinine ester derivatives.
Percent Non-Active Active
(Calculated) (Calculated)
Non-active
(observed) 81.4 35 8
Active (observed) 85.7 1 6
Total 82.0 36 14
The number of compounds correctly classified by the model is indicated in bold.
The single compound which was erroneously classified as inactive (19) contains a
chorine atom at the terminal position of the ester tail. This is a significant structural
difference from other active lupinine derivatives, which contain cyclic substructures at the
terminal position of each molecule.
Leave-one-out (LOO) validation of the model (i.e., predicting the activity of a discarded
compound by a model built on the basis of the remaining 49 molecules) showed that 32 out
of 43 inactive compounds (74.1%) and 5 out of 7 active compounds (71.4%) were correctly
predicted (74 % in the total set).
The SAR model based on physicochemical descriptors of the lupinine-based esters
revealed key features distinguishing AChE inhibitors versus non-active compounds. One
of the weak points of the model is the imbalanced character of the data set, which contained
many more inactive compounds than active ones. However, the reasonable quality and
predictive ability of the model, as well as the simplicity and rapidity of the calculations
associated with the LDA algorithm, suggest promise in using this model for large database
mining and virtual screening of lupinine-based AChE inhibitors.
Molecules 2023, 28, 3357 10 of 15
3. Experimental Section
3.1. Chemistry
1H and 13C NMR spectra were recorded on a JNN-ECA Jeol 400 spectrometer (fre-
quency 399.78 and 100.53 MHz, respectively) with deuterated dimethyl sulfoxide (DMSO-
d6) as the solvent. The chemical shifts were measured with reference to signals of the
residual protons or carbon atoms of DMSO-d6. The multiplicity of signals in the 13C NMR
spectra was determined from spectra recorded in the J-modulation mode (JMOD). The
assignment of signals in the 1H and 13C NMR spectra were confirmed by two-dimensional
homonuclear (1H-1H COSY) and heteronuclear 1H-13C (HMBC, HSQC) spectroscopy and
literature data for quinolizine. High-resolution mass spectra were recorded on a Thermo-
Scientific DFS spectrometer (evaporator temperature of 200–250 ◦C, electron ionization
70 eV). Melting points were determined on a Mettler Toledo FP900 system. The process of
chemical reactions was monitored by thin-layer chromatography (TLC) on Sorbfil UV-254
plates using CH3Cl and CH3Cl–EtOH (10:1) as eluents. The plates were visualized with
iodine vapor and ultraviolet (UV) light (254 nm). The reaction products were isolated by
recrystallization or column chromatography using Acros silicagel (0.035–0.240 mm) and
CHCl3 and CHCl3–EtOH (100:1→10:1) as eluents.
Alkynes of 3-(prop-2-yn-1-yl-thio)-1H-1,2,4-triazole-5-amine (2), (2R,2S)-3-methylpent-4-
yne-2,3-diol (4:1, diastereomeric mixture) (3) and 3-ethoxy-4-(prop-2-ynyloxy)benzaldehyde
(4) were purchased from Alfa Aesar.
Compounds 1 and 8–17 were synthesized as described previously [27–29]. The synthe-
sized structures were confirmed by analytical and spectral data. Sample purity was >99%.
(–)-Lupinine (m.p. 69–71 ◦C (EtOH), [α] 25
D –30.5◦ (c 0.41, MeOH); (literature data: m.p.
68–69 ◦C (EtOH), [α] 25
D –23.5◦) [40] was isolated from the Anabasis aphyla L., as described
previously [41].
Lupinine azide 1 was obtained from lupinine in two stages, as described previ-
ously [28]. Briefly, the reaction of (–)-lupinine with methanesulfonyl chloride in the presence
of Et3N in CH2Cl2 resulted in (1R,9aR)-(octahydro-2H-quinolizine-1-yl)methyl methane-
sulfonate, which was treated with NaN3 in dimethylformamide (DMF), resulting in the
organic quinolizine azide (1) [29].
3.1.1. General Procedure for Compounds (5–7)
A mixture of lupinine azide (1) (0.29 g, 1.5 mmol), substituted acetylene [3-(prop-2-yn-
1-yl-thio)-1H-1,2,4-triazole-5-amine (2), (2S)-3-methylpent-4-yne-2,3-diol (3), and 3-ethoxy-
4-(prop-2-ynyloxy)benzaldehyde (4) (1.35 mmol), CuSO4 × 5H2O (0.017 g, 0.0675 mmol)
and sodium ascorbate (0.013 g, 0.0675 mmol) in DMF (6 mL) was stirred at 75 ◦C for 6–8 h
using TLC monitoring. After cooling, the residue was filtered, washed with hexane, and
dried. Triazoles 5–7 were isolated from the residue by chromatography on silicagel (eluent:
CH3Cl, CH3Cl–EtOH, 100:1→ 10:1).
3.1.2. 3-((1-(((1S,9aR)-Octahydro-1H-quinolizine-1-yl)methyl)-1H-1,2,3-triazole-4-
yl)methylthio)-1H-1,2,4-triazole-5-amine (5)
Yield 0.22 g (75.86%). Dark-brown powder, m.p. 177–179 ◦C (decomp.). 1H NMR spec-
trum (DMSO-d6), δ, ppm: 1.15–1.66 m (10H, H-3ax,3eq, 4ax,4eq,7ax,7eq,8ax,8eq,9ax,9eq),
1.89 s (2H, H-2ax,10ax), 2.05 s (2H, H-5,6), 2.75 s (2H, H-2eq,10eq), 4.19 s (2H, H-17,17),
4.41 s (2H, H-11,11), 5.97 s (2H, H-24,24), 7.90 s (1H, H-16), 11.95 br. s (1H, H-21). 13C
NMR spectrum (DMSO-d6), δ, ppm: 20.67 (C-3), 24.70 (C-8,9), 26.73 (C-17), 28.85 (C-4,7),
39.27 (C-5), 48.76 (C-11), 57.38 (C-2,10), 64.17 (C-6), 124.09 (C-16), 144.44 (C-15) and 156.20
(C-19,22). Mass spectrum, m/z (Irel., %) (2): 348.2 (7.23), 232.2 (17.40), 151.1 (100.0), 96.0
(21.73), 55.0 (16.38). Found m/z: 348.1839 [M]+. C15H24N8S. Calculated m/z: 348.1838.
Molecules 2023, 28, 3357 11 of 15
3.1.3. (2R,S)-2-(1-(((1S,9aR)-Octahydro-1H-quinolizine-1-yl)methyl)-1H-1,2,3-triazole-4-
yl)butane-2,3-diol (6)
Yield 0.25 g (86.20%). Cream-colored, m.p. 158–161 ◦C. 1H NMR (DMSO-d6), δ, ppm:
1.03 d (3H, H-22,22,22), 1.04–1.95 m (10H, H-3ax,3eq, 4ax,4eq,7ax,7eq,8ax,8eq,9ax,9eq),
1.52 s (3H, H-21,21,21), 1.93–1.98 m (2H, H-2ax,10ax), 1.97–2.06 m (1H, H-6), 2.15–2.21 m
(1H, H-5), 2.81 s (2H, H-2eq,10eq), 3.41 br. s (2H, H-18,22), 4.46–4.57 m (2H, H-11,11), 7.47 s
(1H, H-16). 13C NMR (DMSO-d6), δ, ppm: 17.86 (C-22), 22.98 (C-21), 24.32 (C-3), 24.88 (C-8),
25.38 (C-9), 26.20 (C-4), 29.73 (C-7), 39.26 (C-5), 57.23 (C-2,10), 64.27 (C-6), 73.07 (C-17), 74.54
(C-19), 121.89 (C-16), 151.81 (C-15). Mass spectrum, m/z (Irel., %): 308.3 (12.45), 219.2 (1.66),
151.1 (100.0), 98.0 (9.77), 43.2 (6.68). Found m/z: 308.2207 [M]+. C16H28N4O2. Calculated
m/z: 308.2211.
3.1.4. 3-Ethoxy-4-((1-(((1S,9aR)-octahydro-1H-quinolizine-1-yl)methyl)-1H-1,2,3-triazole-
4-yl)methoxy)benzaldehyde (7)
Yield 0.23 g (76.66%). White powder, m.p. 166–168 ◦C. 1H NMR (DMSO-d6), δ, ppm:
1.12–1.23 m (3H, H-4ax, H-7ax, H-3ax), 1.27 t (3H, H-27,27,27, 3J 7.6 Hz), 1.34–1.41 m (2H,
H-4eq, H-7eq), 1.46–1.49 m (2H, H-8ax, H-8eq), 1.47–1.51 m (2H, H-11,11), 1.63–1.74 m (3H,
H-2ax, H-10ax, H-3eq), 1.92–1.95 m (1H, H-6), 2.70–2.72 m (2H, H-2eq, H-10eq), 2.09 br. s
(1H, H-5), 4.03 q (2H, H-26,26, 3J 7.6 Hz), 5.23 s (2H, H-17,17), 7.32 d (1H, H-24, 3J 9.2 Hz),
7.48 d (1H, H-23, 3J 9.2 Hz), 8.24 s (1H, H-16), 9.79 s (1H, H-28). 13C NMR (DMSO-d6), δ,
ppm: 15.13 (C-27), 21.82 (C-3), 25.03 (C-8), 25.77 (C-9), 25.98 (C-4), 26.36 (C-7), 39.07 (C-5),
57.09 (C-2,10), 48.34 (C-11), 62.60 (C-17), 64.67 (C-6), 64.86 (C-26), 111.57 (C-21), 113.4 (C-24),
126.26 (C-23,16), 130.49 (C-22), 149.09 (C-19), 153.50 (C-20), 191.96 (C-28). Mass spectrum,
m/z (Irel., %): 398.3 (28.87), 256.2 (4.49), 151.1 (100.0), 84.9 (24.34), 55.0 (23.62). Found m/z:
398.2312 [M]+. C22H30N4O3. Calculated m/z: 398.2314.
3.2. Commercial Compounds
Fifty lupinine-based esters of different carboxylic acids (compounds 18–67) were pur-
chased from the Vitas-M laboratory (Champaign, IL, USA). All compounds were dissolved
in DMSO at a stock concentration of 10 mM and stored at −20 ◦C.
3.3. AChE Inhibition Assay
The inhibitory effect of test compounds and galantamine (Tocris Bioscience, San Francisco,
CA, USA) on AChE activity was determined using an AChE inhibitor screening kit from
the Sigma-Aldrich Chemical Co., (St. Louis, MO, USA). The kit is based on an improved
Ellman method, whereby thiocholine produced from AChE activity forms a yellow color
with 5,5′-dithiobis(2-nitrobenzoic acid), and the intensity the color (412 nm) is proportional
to the enzyme activity. The concentration of compound required to cause 50% inhibition
(IC50) was determined by graphing the % inhibition of enzyme activity versus the logarithm
of concentration of the test compound using 5–7 tested concentrations.
3.4. Cytotoxicity Assay
The cytotoxicity of the synthesized compounds was analyzed using a CellTiter-Glo
Luminescent Cell Viability Assay Kit (Promega, Madison, WI, USA) according to the
manufacturer’s protocol. Human THP-1 monocytic cells obtained from ATCC (Manassas,
VA, USA) were cultured in RPMI 1640 medium (Mediatech Inc., Herndon, VA, USA)
supplemented with 10% (v/v) FBS, 100 µg/mL streptomycin, and 100 U/mL penicillin. For
the cytotoxicity assay, the cells were cultured at a density of 104 cells/well with different
concentrations of the test compounds added (3, 6.125, 12.5, 25, 50 µM; final concentration
of DMSO was 1%) for 24 h at 37 ◦C and 5% CO2. Following treatment, substrate was added
to the cells, and the samples were analyzed with a Fluoroscan Ascent FL microplate reader.
Molecules 2023, 28, 3357 12 of 15
3.5. Molecular Docking
Docking of compounds into the acetyl cholinesterase binding site (structure 4EY7
from the Protein Data Bank) was performed using the ROSIE server [42]. The docking
area was chosen around the geometric center of co-crystallized donepezil (1-benzyl-4-[(5,6-
dimethoxy-1-indanon-2-yl)methyl]piperidine) occupying the binding site of the receptor in
the 4EY7 structure. For each of the investigated compounds, up to 2000 ligand conformers
were generated with the BCL algorithm [43] switched on. The number of intermediately
generated docking poses was set to 2000. Other options were used as defaults within the
ROSIE ligand docking protocol, which accounted for full flexibility of the main chain and
side-chains for residues in the vicinity of the docking area [36]. After completion of the
computations, PDB files containing the best poses of compounds docked into AChE were
downloaded from the server and imported into Molegro Virtual Docker 6.0 (MVD) for
visualization and analysis using the built-in “Pose Organizer” tool of MVD.
3.6. Linear Discriminant Analysis (LDA)
Structures of the 50 lupinine-based esters were built using ChemOffice 2016, rep-
resented as SMILES strings, and imported into the SwissADME online tool [39]. The
calculated physicochemical and ADME parameters were subjected to correlation analysis
to select descriptors with low mutual pairwise correlations. The following descriptors
were selected: molecular weight (MW), fraction of sp3 carbon atoms (Csp3), number of
rotatable bonds (Nrot), number of hydrogen bond donors and acceptors (NHBD and NHBA,
respectively), molar refractivity (MR), topological polar surface area (tPSA), consensus
LogP (cLogP), and water solubility SILICOS-IT Log Sw (sLogS). Two structural indica-
tors were added, which indicated absence or presence (value of 0 or 1, respectively) of
an amide unit -C(O)NH- or a quaternary sp3 carbon atom (descriptors Nam and Q, re-
spectively). Although useful computational methods have been developed for finding
molecular subunits (e.g., [44]), the Nam and Q indicators were assigned manually because
of their simplicity. The data sheet containing columns with the values of the independent
predictors enumerated above was supplemented with a column indicating compound
activity (values “Active” or “NA”) as a categorical dependent variable. The resulting data
sheet was imported in STATISTICA 6.0 program (StatSoft, Inc., Tulsa, OK, USA), and the
LDA procedure was performed with the “Best subset” option switched on using equal
prior probabilities for the compound classes. All 50 lupinine-based esters were used as
a training set. To validate the model, the leave-one-out (LOO) procedure was performed
by sequentially discarding one of the compounds and predicting its activity class (i.e., the
dependent categorical variable) by an LDA model obtained on the basis of the remaining
49 compounds.
4. Conclusions
We identified compound 15 as a novel AChE inhibitor and showed that it exhibited
mixed-type inhibitory activity. Molecular docking modeling indicated that compound 15
meets structural requirements necessary to reproduce important intermolecular interactions
described in the literature as fundamental for AChE inhibition. Thus, this compound could
be a promising candidate for evaluation in AD models. Our results also indicate that the
4-benzyloxyphenyl moiety attached to different molecular scaffolds can play an important
role in ligand binding to AChE due to the interaction with the receptor subpocket. This
finding, as well as the derived classification SAR model, may be useful in the design of
other novel AChE inhibitors.
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/molecules28083357/s1, Supplementary Figures S1–S3. Schemes
of correlations in the COSY and HMQC spectra of 5–7. Supplementary Figure S4.1. 1H NMR spectrum of 5.
Supplementary Figure S4.2. 13C NMR spectrum of 5. Supplementary Figure S4.3. The mass spectrum of 5.
Supplementary Figure S5.1. 1H NMR spectrum of 6a,b. Supplementary Figure S5.2. 13C NMR spectrum
Molecules 2023, 28, 3357 13 of 15
of 6a,b. Supplementary Figure S5.3. The mass spectrum of 6a,b. Supplementary Figure S6.1. 1H
NMR spectrum of 7. Supplementary Figure S6.2. 13C NMR spectrum of 7. Supplementary Figure S6.3.
The mass spectrum of 7. Supplementary Table S1. Chemical structures of lupinine-based esters of dif-
ferent carboxylic acids under investigation. Supplementary Figure S7. 2D diagram of ligand-receptor
interactions obtained on docking of compound 15 in AChE. Blue dashed lines—hydrogen bonding
interactions. Red dashed line—steric interaction. Supplementary Table S2. Chemical formulas,
selected ADME parameters of the lupinine-based esters calculated with SwissADME web tool, and
manually added indicator variable for the quaternary sp3 carbon atom (Q). Supplementary Table S3.
Experimentally determined, calculated, and LOO-predicted classes for AChE inhibitory activity of
the lupinine-based esters of different carboxylic acids.
Author Contributions: I.A.S., Z.S.N. and M.T.Q. conceived and designed the project; Z.S.N., S.D.F.,
O.A.N., T.M.S., A.S.K. and E.E.S. synthesized and characterized compounds; I.A.S. performed the
enzymatic assay; A.I.K. conducted the molecular modeling study; I.A.S., Z.S.N., S.D.F., O.A.N.,
A.I.K., T.M.S., A.S.K. and E.E.S. analyzed and interpreted the data; I.A.S., Z.S.N., A.I.K. and M.T.Q.
drafted and revised the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was supported in part by National Institutes of Health IDeA Program grant
GM103474; USDA National Institute of Food and Agriculture Hatch project 1009546; the Montana State
University Agricultural Experiment Station; project No. AP08855567 under the grant funding from the
Committee of Science of the Ministry of Education and Science of the Republic of Kazakhstan, and the
Tomsk Polytechnic University Development Program (Project Priority-2030-NIP/IZ-009-0000-2023).
Data Availability Statement: Data are contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of synthesized compounds are available from the authors.
References
1. Robichaud, A.J. Approaches to palliative therapies for Alzheimer’s disease. Curr. Top. Med. Chem. 2006, 6, 553–568. [CrossRef]
[PubMed]
2. Umar, T.; Meena, R.; Mustehasan; Kumar, P.; Khan, A.A. Recent updates in the development of small molecules as potential
clinical candidates for Alzheimer’s disease: A review. Chem. Biol. Drug Des. 2022, 100, 674–681. [CrossRef] [PubMed]
3. Sepehri, S.; Saeedi, M.; Larijani, B.; Mahdavi, M. Recent developments in the design and synthesis of benzylpyridinium salts:
Mimicking donepezil hydrochloride in the treatment of Alzheimer’s disease. Front. Chem. 2022, 10, 936240. [CrossRef] [PubMed]
4. Mukherjee, P.K.; Satheeshkumar, N.; Venkatesh, P.; Venkatesh, M. Lead finding for acetyl cholinesterase inhibitors from natural
origin: Structure activity relationship and scope. Mini Rev. Med. Chem. 2011, 11, 247–262. [CrossRef]
5. Ahn, K. The worldwide trend of using botanical drugs and strategies for developing global drugs. BMB Rep. 2017, 50, 111–116.
[CrossRef]
6. Butler, M.S. Natural products to drugs: Natural product-derived compounds in clinical trials. Nat. Prod. Rep. 2008, 25, 475–516.
[CrossRef]
7. Ayaz, M.; Nawaz, A.; Naz, F.; Ullah, F.; Sadiq, A.; Islam, Z.U. Phytochemicals-based Therapeutics against Alzheimer’s Disease:
An Update. Curr. Top. Med. Chem. 2022, 22, 1811–1820. [CrossRef]
8. Suciati; Poerwantoro, D.; Widyawaruyanti, A.; Ingkaninan, K. Acetylcholinesterase inhibitory activity of extract and fractions
from the root of Rauvolfia serpentine (L.) Bth.ex Kurz. J. Basic Clin. Physiol. Pharmacol. 2021, 32, 313–317. [CrossRef]
9. Tomassini, L.; Ventrone, A.; Frezza, C.; Fabbri, A.M.; Fortuna, S.; Volpe, M.T.; Cometa, M.F. Phytochemical analysis of
Viburnum davidii Franch. and cholinesterase inhibitory activity of its dihydrochalcones. Nat. Prod. Res. 2021, 35, 5794–5800.
[CrossRef]
10. Saenkham, A.; Jaratrungtawee, A.; Siriwattanasathien, Y.; Boonsri, P.; Chainok, K.; Suksamrarn, A.; Namsa-Aid, M.; Pattanapra-
teeb, P.; Suksamrarn, S. Highly potent cholinesterase inhibition of geranylated xanthones from Garcinia fusca and molecular
docking studies. Fitoterapia 2020, 146, 104637. [CrossRef]
11. Hou, Y.; Wang, M.; Sun, C.; Peng, C.; Zhang, Y.; Li, X. Tunicyclin L, a cyclic peptide from Psammosilene tunicoides: Isolation,
characterization, conformational studies and biological activity. Fitoterapia 2020, 145, 104628. [CrossRef]
12. Lou, H.; Yi, P.; Hu, Z.; Li, Y.; Zeng, Y.; Gu, W.; Huang, L.; Yuan, C.; Hao, X. Polycyclic polyprenylated acylphloroglucinols with
acetylcholinesterase inhibitory activities from Hypericum perforatum. Fitoterapia 2020, 143, 104550. [CrossRef]
13. Tuzimski, T.; Petruczynik, A. Determination of Anti-Alzheimer’s Disease Activity of Selected Plant Ingredients. Molecules 2022,
27, 3222. [CrossRef]
Molecules 2023, 28, 3357 14 of 15
14. Omodeo-Salè, F.; Cortelezzi, L.; Basilico, N.; Casagrande, M.; Sparatore, A.; Taramelli, D. Novel antimalarial aminoquinolines:
Heme binding and effects on normal or Plasmodium falciparum-parasitized human erythrocytes. Antimicrob. Agents Chemother.
2009, 53, 4339–4344. [CrossRef]
15. Van Eijk, J.L.; Radema, M.H. Virgiboidine, A New Alkaloid from Virgilia oroboides and Virgilia divaricata. Planta Med. 1982,
44, 224–226. [CrossRef]
16. Suzuki, H.; Murakoshi, I.; Saito, K. A novel O-tigloyltransferase for alkaloid biosynthesis in plants. Purification, characterization,
and distribution in Lupinus plants. J. Biol. Chem. 1994, 269, 15853–15860. [CrossRef]
17. Aaron, P.A.; Vu, K.; Gelli, A. An Antivirulence Approach for Preventing Cryptococcus neoformans from Crossing the Blood-Brain
Barrier via Novel Natural Product Inhibitors of a Fungal Metalloprotease. mBio 2020, 11, e01249-20. [CrossRef]
18. Sparatore, A.; Cagnotto, A.; Sparatore, F. Quinolizidinyl derivatives of 2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxylic acid
and 1-homolupinanoyl benzimidazolones as ligands for 5-HT3 and 5-HT4 receptors. Farmaco 1999, 54, 248–254. [CrossRef]
19. Basilico, N.; Parapini, S.; Sparatore, A.; Romeo, S.; Misiano, P.; Vivas, L.; Yardley, V.; Croft, S.L.; Habluetzel, A.; Lucantoni, L.; et al.
In vivo and in vitro activities and ADME-tox profile of a quinolizidine-modified 4-aminoquinoline: A potent anti-P. falciparum
and anti-P. vivax blood-stage antimalarial. Molecules 2017, 22, 2102. [CrossRef]
20. Rusconi, C.; Vaiana, N.; Casagrande, M.; Basilico, N.; Parapini, S.; Taramelli, D.; Romeo, S.; Sparatore, A. Synthesis and
comparison of antiplasmodial activity of (+), (-) and racemic 7-chloro-4-(N-lupinyl)aminoquinoline. Bioorg. Med. Chem. 2012,
20, 5980–5985. [CrossRef]
21. Vazzana, I.; Novelli, F.; Sparatore, F.; Sparatore, A.; Fadda, G.; Manca, C. Quinolizidine derivatives with antitubercular activity.
Farmaco 1994, 49, 105–110. [PubMed]
22. Basova, N.E.; Kormilitsyn, B.N.; Perchenok, A.; Rozengart, E.V.; Saakov, V.S.; Suvorov, A.A. Reversible lupininin inhibitors of
cholinesterases of mammalian blood and of optical ganglia of individuals of the commander squid Berryteuthis magister from
different zones of species areal. Zh. Evol. Biokhim. Fiziol. 2012, 48, 213–218. [PubMed]
23. Rozengart, E.V. Bisalkaloid derivatives of dicarboxylic acids on the basis of lupinine, anabasine, and cytisine as reversible
cholinesterase inhibitors. Dokl. Biochem. Biophys. 2003, 388, 39–42. [CrossRef] [PubMed]
24. Tasso, B.; Catto, M.; Nicolotti, O.; Novelli, F.; Tonelli, M.; Giangreco, I.; Pisani, L.; Sparatore, A.; Boido, V.; Carotti, A.; et al.
Quinolizidinyl derivatives of bi- and tricyclic systems as potent inhibitors of acetyl- and butyrylcholinesterase with potential in
Alzheimer’s disease. Eur. J. Med. Chem. 2011, 46, 2170–2184. [CrossRef] [PubMed]
25. Tonelli, M.; Catto, M.; Tasso, B.; Novelli, F.; Canu, C.; Iusco, G.; Pisani, L.; Stradis, A.D.; Denora, N.; Sparatore, A.; et al.
Multitarget Therapeutic Leads for Alzheimer’s Disease: Quinolizidinyl Derivatives of Bi- and Tricyclic Systems as Dual Inhibitors
of Cholinesterases and β-Amyloid (Aβ) Aggregation. ChemMedChem 2015, 10, 1040–1053. [CrossRef]
26. Obaid, R.J.; Naeem, N.; Mughal, E.U.; Al-Rooqi, M.M.; Sadiq, A.; Jassas, R.S.; Moussa, Z.; Ahmed, S.A. Inhibitory potential of
nitrogen, oxygen and sulfur containing heterocyclic scaffolds against acetylcholinesterase and butyrylcholinesterase. RSC Adv.
2022, 12, 19764–19855. [CrossRef]
27. Nurmaganbetov, Z.S.; Nurkenov, O.A.; Fazylov, S.D.; Mukusheva, G.K.; Gazaliev, A.M.; Muldakhmetov, Z.M. Synthesis of
1,2,3-triazolo-quinolizidines based on the quinolizidine alkaloid lupinine. Chem. J. Kazakhstan 2021, 3, 108–118. [CrossRef]
28. Nurmaganbetov, Z.S.; Savelyev, V.A.; Gatilov, Y.V.; Nurkenov, O.A.; Seidakhmetova, R.B.; Shulgau, Z.T.; Mukusheva, G.K.;
Fazylov, S.D.; Shults, E.E. Synthesis and analgesic activity of 1-[(1,2,3-triazol-1-yl)methyl]quinolizines based on the alkaloid
lupinine. Chem. Heterocycl. Compd. 2021, 57, 911–919. [CrossRef]
29. Nurmaganbetov, Z.S.; Fazylov, S.D.; Turdybekov, K.M.; Nurkenov, O.A.; Turdybekov, D.M.; Mukusheva, G.K.; Minayeva, Y.V.;
Khabdolda, G. Synthesis and structure of 4-substituted (1S,9aR)-1-[(1,2,3-triazol-1-yl)-methyl]octahydro-1H-quinolysines of
lupinine. Bull. Univ. Karaganda Chem. 2022, 2, 12–22. [CrossRef]
30. Khodja, I.A.; Boulebd, H.; Bensouici, C.; Belfaitah, A. Design, synthesis, biological evaluation, molecular docking, DFT calcu-
lations and in silico ADME analysis of (benz)imidazole-hydrazone derivatives as promising antioxidant, antifungal, and anti-
acetylcholinesterase agents. J. Mol. Struct. 2020, 1218, 128527. [CrossRef]
31. Aggarwal, N.; Jain, S.; Chopra, N. Hybrids of Thiazolidin-4-Ones and 1,3,4-Thiadiazole: Synthesis and Biological Screening of A
Potential New Class of Acetylcholinesterae Inhibitors. Biointerface Res. Appl. Chem. 2022, 12, 2800–2812.
32. Mo, J.; Yang, H.; Chen, T.; Li, Q.; Lin, H.; Feng, F.; Liu, W.; Qu, W.; Guo, Q.; Chi, H.; et al. Design, synthesis, biological evaluation,
and molecular modeling studies of quinoline-ferulic acid hybrids as cholinesterase inhibitors. Bioorg. Chem. 2019, 93, 103310.
[CrossRef]
33. Lotfi, S.; Rahmani, T.; Hatami, M.; Pouramiri, B.; Kermani, E.T.; Rezvannejad, E.; Mortazavi, M.; Fathi Hafshejani, S.; Askari, N.;
Pourjamali, N.; et al. Design, synthesis and biological assessment of acridine derivatives containing 1,3,4-thiadiazole moiety as
novel selective acetylcholinesterase inhibitors. Bioorg. Chem. 2020, 105, 104457. [CrossRef]
34. Catto, M.; Pisani, L.; Leonetti, F.; Nicolotti, O.; Pesce, P.; Stefanachi, A.; Cellamare, S.; Carotti, A. Design, synthesis and biological
evaluation of coumarin alkylamines as potent and selective dual binding site inhibitors of acetylcholinesterase. Bioorg. Med.
Chem. 2013, 21, 146–152. [CrossRef] [PubMed]
35. Luz, R.; Almeida, R.B.M.; Albuquerque, M.M.S.; Cerqueira, A.P.M.; Tavares, J.F.; Silva, M.S.D.; Filho, R.B.; Dos Santos Junior,
M.C.; Branco, A.; Botura, M.B. Two new dilactonized glycerol glycosides of the dual anticholinesterase active extract from Ocotea
daphnifolia using bioguided fractionation and molecular docking studies. Chem. Biol. Drug Des. 2022, 101, 855–864. [CrossRef]
Molecules 2023, 28, 3357 15 of 15
36. DeLuca, S.; Khar, K.; Meiler, J. Fully Flexible Docking of Medium Sized Ligand Libraries with RosettaLigand. PLoS ONE 2015,
10, e0132508. [CrossRef] [PubMed]
37. Atanasova, M.; Stavrakov, G.; Philipova, I.; Zheleva, D.; Yordanov, N.; Doytchinova, I. Galantamine derivatives with indole
moiety: Docking, design, synthesis and acetylcholinesterase inhibitory activity. Bioorg. Med. Chem. 2015, 23, 5382–5389. [CrossRef]
38. Mitteroecker, P.; Bookstein, F. Linear Discrimination, Ordination, and the Visualization of Selection Gradients in Modern
Morphometrics. Evol. Biol. 2011, 38, 100–114. [CrossRef]
39. Daina, A.; Michielin, O.; Zoete, V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal
chemistry friendliness of small molecules. Sci. Rep. 2017, 7, 42717. [CrossRef]
40. Winterfeld, K.; Holschneider, F.W. Über die Konstitution des Lupinins (I. Mitteil). Chem. Ber. Recl. 1931, 64, 137–150. [CrossRef]
41. Nurkenov, O.A.; Nurmaganbetov, Z.S.; Fazylov, S.D.; Satpaeva, Z.B.; Turdybekov, K.M.; Seilkhanov, T.M.; Talipov, S. Synthesis,
Structure, and Properties of New Lupinine O-Acyl Derivatives. Chem. Nat. Compd. 2019, 55, 506–508. [CrossRef]
42. Lyskov, S.; Chou, F.C.; Conchúir, S.; Der, B.S.; Drew, K.; Kuroda, D.; Xu, J.; Weitzner, B.D.; Renfrew, P.D.; Sripakdeevong, P.; et al.
Serverification of molecular modeling applications: The Rosetta Online Server that Includes Everyone (ROSIE). PLoS ONE 2013,
8, e63906. [CrossRef]
43. Kothiwale, S.; Mendenhall, J.L.; Meiler, J. BCL::Conf: Small molecule conformational sampling using a knowledge based rotamer
library. J. Cheminform. 2015, 7, 47. [CrossRef]
44. Mukherjee, G.; Braka, A.; Wu, S. Quantifying functional-group-like structural fragments in molecules and its applications in drug
design. J. Chem. Inf. Model. 2023, 63, 2073–2083. [CrossRef]
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