Rational design of new multitarget histamine H3 receptor ligands as potential candidates for treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2020.112743

Design and development of multitarget-directed ligands (MTDLs) has become a very important approach in the search of new therapies for Alzheimer's disease (AD). In our present research, a number of xanthone derivatives were first designed using a pharmaco

Full text of DOI:10.1016/j.ejmech.2020.112743

European Journal of Medicinal Chemistry 207 (2020) 112743
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Rational design of new multitarget histamine H3 receptor ligands as
potential candidates for treatment of Alzheimer’s disease
a
,
Dorota Łazewska *, Marek Bajda b, Maria Kaleta a, Paula Zare˛ba b, Agata Doroz-Płonka a,
_
Agata Siwek c, Alaa Alachkar d, Szczepan Mogilski e, Ali Saad d, Kamil Kuder a,
a
b
b
a
ꢀ
Agnieszka Olejarz-Maciej , Justyna Godyn , Dorota Stary , Sylwia Sudoł ,
Małgorzata Wie˛cek a, Gniewomir Latacz a, Maria Walczak f, Jadwiga Handzlik a,
d
b
a
ꢀ
Bassem Sadek , Barbara Malawska , Katarzyna Kiec-Kononowicz
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna Str. 9, 30-688, Krakow, Poland
a
ꢀ
b
c
ꢀ
Department of Physicochemical Drug Analysis, Jagiellonian University Medical College, Medyczna Str. 9, 30-688, Krakow, Poland
ꢀ
Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna Str. 9, 30-688, Krakow, Poland
d Department of Pharmacology & Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 1766, Al Ain, United Arab
Emirates
e
ꢀ
Department of Pharmacodynamic, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna Str. 9, 30-688, Krakow, Poland
f
ꢀ
Department of Toxicology, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna Str 9, 30-688, Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Design and development of multitarget-directed ligands (MTDLs) has become a very important approach
in the search of new therapies for Alzheimer’s disease (AD). In our present research, a number of
xanthone derivatives were first designed using a pharmacophore model for histamine H3 receptor (H3R)
antagonists/inverse agonists, and virtual docking was then performed for the enzyme acetylcholines-
terase. Next, 23 compounds were synthesised and evaluated in vitro for human H3R (hH3R) affinity and
inhibitory activity on cholinesterases. Most of the target compounds showed hH3R affinities in nano-
molar range and exhibited cholinesterase inhibitory activity with IC50 values in submicromolar range.
Furthermore, the inhibitory effects of monoamine oxidases (MAO) A and B were investigated. The results
showed low micromolar and selective human MAO B (hMAO B) inhibition. Two azepane derivatives,
namely 23 (2-(5-(azepan-1-yl)pentyloxy)-9H-xanthen-9-one) and 25 (2-(5-(azepan-1-yl)pentyloxy)-7-
chloro-9H-xanthen-9-one), were especially very promising and showed high affinity for hH3R
Received 23 May 2020
Received in revised form
26 July 2020
Accepted 26 July 2020
Available online 21 August 2020
Keywords:
Xanthone derivatives
Multitarget-directed ligands
Alzheimer’s disease
Histamine H3 receptor
Cholinesterase inhibitors
Monoamine oxidase inhibitors
(Ki
¼
170 nM and 100 nM respectively) and high inhibitory activity for acetylcholinesterase
(IC50 ¼ 180 nM and 136 nM respectively). Moreover, these compounds showed moderate inhibitory
activity for butyrylcholinesterase (IC50 ¼ 880 nM and 394 nM respectively) and hMAO B (IC50 ¼ 775 nM
and 897 nM respectively). Furthermore, molecular docking studies were performed for hH3R, human
cholinesterases and hMAO B to describe the mode of interactions with these biological targets. Next, the
two most promising compounds 23 and 25 were selected for in vivo studies. The results showed sig-
nificant memory-enhancing effect of compound 23 in dizocilpine-induced amnesia in rats in two tests:
step-through inhibitory avoidance paradigm (SIAP) and transfer latency paradigm time (TLPT). In
addition, favourable analgesic effects of compound 23 were observed in neuropathic pain models.
Therefore, compound 23 is a particularly promising structure for further design of new MTDLs for AD.
© 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
In the drug discovery process, the development of novel agents
with potential interactions with multiple therapeutic targets is of
prime importance in the field of multifactorial diseases, e.g. Alz-
* Corresponding author.
E-mail address: dlazewska@cm-uj.krakow.pl (D. Łazewska).
heimer’s disease (AD) [1]. Designing a new drug molecule plays a
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https://doi.org/10.1016/j.ejmech.2020.112743
0223-5234/© 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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D. Łazewska et al. / European Journal of Medicinal Chemistry 207 (2020) 112743
2
very important role in the search for new effective therapeutic
agents. Conventionally, promising lead identification is achieved by
[10]. Recently also promising analgesic effects of histamine H3R
ligands was shown [11]. In the past years, in addition to selective
H3R antagonists/inverse agonists, multitarget (mostly dual acting)
ligands were also obtained. Compounds were designed as struc-
tures exhibiting antagonism/inverse agonism towards H3Rs while
simultaneously affecting other biological targets [12,13]. Some of
the promising structures, namely UW-MD-71 and E244, with dual
activity of H3Rs antagonism and cholinesterase inhibition are
shown in Fig. 1 [14e16]. Recently, another propitious multitarget
compound, namely contilisant (Fig. 1), was reported to show hu-
man H3Rs (hH3Rs) antagonism, cholinesterases and monoamine
oxidases (MAO) A and B inhibition and sigma 1 receptor agonism
[17,18].
experimental high-throughput screening, but it is
a time-
consuming and expensive process [1,2]. Previous experimental
studies have revealed that rational drug design significantly re-
duces the costs associated with synthesis accompanied with an
increased probability that the emerged structures will be recog-
nised as drugs. Thus far, most of the drugs available in the market
were found by chance or coincidence. The current drug discovery
approach includes, among others, strategies based on structure
design, as structure-based drug design is becoming an essential
tool for faster and more cost-efficient lead discovery relative to the
traditional methods, and is especially important for diseases with
complicated aetiologies such as cancer, inflammation, metabolic
syndrome, and neurodegenerative and cardiovascular diseases.
Accordingly, drugs with the capability to simultaneously act on
several biological cells are seen as potential candidates with a high
probability for their successful treatment. However, designing such
compounds is quite challenging, including proper selection of
biological targets, achievement of a satisfactory activity level to-
wards these multiple targets and optimisation of their pharmaco-
kinetic parameters [3].
Xanthones (dibenzo-g-pyrones) are compounds broadly
distributed in nature and exhibit a wide variety of pharmacological
activities, e.g. antibacterial, anti-inflammatory, antitumoral,
glucosidase inhibitory, and cardioprotective [19e22]. One of the
derivatives, namely compound DMXAA (Vadimezan, (5,6-
dimethyl-9-oxo-9H-xanthen-4-yl)-acetic acid), has reached the
stage of clinical trials as a tumour-vascular disrupting agent but
failed in the phase III trial [23]. Recently, some other xanthones as
potential ligands to treat AD were reported to show inhibition of
AD is a neurodegenerative disorder with yet unclear aetiology.
Various factors (genetic, environmental and endogenous) have
been suggested to play a role in its pathogenesis and development
[4].
In AD, cognitive impairments and memory loss are related to
changes in many neurotransmitter systems, especially progressive
damage of cholinergic neural transmission and reduction of
acetylcholine level. Moreover, acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE), which are hydrolysing enzymes of
cholinesterases, MAO and b-amyloid aggregation as well as anti-
oxidant activity [24e26]. Structures of some promising xanthones
with cholinesterase inhibitory activity are shown in Fig. 2 [25,26].
On the basis of these literature data and to continue our previ-
ous studies in this field [16,27,28], we designed new structures as
potential multifunctional ligands for the treatment of AD.
Xanthone-containing compounds were designed on the basis of the
structure of pitolisant (the first H3R ligand that entered the market
as Wakix® [29]) and a xanthone derivative 1 with anticholines-
terase activity (Fig. 3). Compound 1 showed structural elements
that according to the proposed general construction pattern of
histamine H3R antagonists/inverse agonists should be in a mole-
cule: a tertiary basic amine connected via a linker (commonly a
linear chain) to the “eastern” arbitrary fragment [30] (see Fig. 3).
Considering these similarities, we initially designed six xan-
thones (Table 1S, supplementary data) and performed molecular
docking to AChE [16]. The obtained results showed high ChemScore
values (47 < Score < 51) for these compounds (Table 1S, supple-
mentary data). Moreover, we evaluated in silico drug-likeness
(Table 1S, supplementary data) of compounds using the Swis-
sADME web tool and obtained promising molecular properties of
these compounds [31]. Thus, encouraged by these results, we
designed and synthesised (Scheme 1) the entire series of xanthones
(6ꢀ28) and tested these compounds in in vitro assays to evaluate
their hH3R affinity and cholinesterase inhibitory activity. The
binding mode of the obtained compounds with AChE and BuChE
was studied by docking to the enzyme active sites (acetylcholin-
esterase PDB code: 1EVE; butyrylcholinesterase PDB code: 1P0I).
Moreover, as molecular docking into the active site of human MAO
B (hMAO B) showed promising results, all compounds were tested
for hMAO A and hMAO B inhibitory activity. Furthermore, molecular
docking studies were performed for histamine H3R. Next, the two
most promising compounds were selected for in vivo studies to
evaluate their procognitive and analgesic properties. In addition
the membrane permeability of these compounds was tested
in vitro.
acetylcholine, are involved in the formation of a complex with
amyloid and hyperphosphorylated tau protein. The accumulation of
-amyloid deposits and neurofibrillary tangles of phosphorylated
b-
b
tau protein in the brain tissue is also an important factor in the
development of AD [4]. Moreover, literature describes that mono-
amine oxidase (MAO) B activity is increased in the brain of AD
patients [5]. Furthermore, recent studies revealed that MAO B levels
are increased both in astrocytes and neurons, and suggested that
MAO B regulates b-amyloid production via g-secretase [6]. The risk
of AD and other forms of dementia increases with the age the same
as development of pain. AD patients suffer very often not only from
chronic pain, but other types as neuropathic and nociceptive are
also observed [7,8]. Proper pain control in AD is difficult as pain is
usually communicated orally and verbal communication de-
teriorates in elders.
The current cure for AD includes mostly inhibitors of acetyl-
cholinesterase, namely donepezil, rivastigmine, and galantamine
[4]. Their action mainly involves alleviating and slowing down the
course of the disease without the possibility of its effective treat-
ment or prevention. Therefore, it is advisable to search for new
drugs, especially drugs that can influence multiple targets associ-
ated with AD. Consequently, multitarget-directed ligands (MTDLs)
is a promising approach in the search for novel and effective anti-
AD drugs.
Histamine H3 receptors (H3Rs) have received considerable in-
terest in the past several years because of their distribution mainly
in the central nervous system and especially in brain regions
related to memory [9]. Inhibition of the activity of these receptors
causes an increase in the levels of histamine and other neuro-
transmitters, including acetylcholine and dopamine [9].
2. Results and discussion
Pharmacological studies have suggested the use of histamine
H3R antagonists/inverse agonists in the treatment of various hu-
man disorders, e.g. AD, Attention Deficit Hyperactivity Disorder
(ADHD), Parkinson’s disease, schizophrenia, narcolepsy and allergy
2.1. Chemistry
A series of 2-(cycloaminoalkoxy)xanthones was obtained by the
multistep process as shown in Scheme 1. First, 2-hydroxyxanthone

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D. Łazewska et al. / European Journal of Medicinal Chemistry 207 (2020) 112743
3
Fig. 1. Structures of histamine H3 receptor ligands with multitarget activities. eeAChE e AChE from electric eel; eqBuChE- BuChE from equine serum. Data from Ref: UW-DM-71 [14];
E244 [16]; contilisant [17,18].
Fig. 2. Structures and cholinesterase inhibitory activity of xanthone derivatives. eeAChE e AChE from electric eel; eqBuChE- BuChE from equine serum. Data from Ref: 1 [25]; 2 [26].
Fig. 3. Structure of pitolisant, xanthone derivative 1 and designed compounds showing the pharmacophore features for histamine H3 receptor antagonist/inverse agonists.
or 6 (7)-chloro-2-hydroxyxanthone was prepared by coupling of a
proper 2-chlorobenzoic acid with 4-methoxyphenol by Ullmann
condensation followed by cyclisation and demethylation of 2-(4-
methoxyphenoxy) acid in concentrated sulfuric acid [32]. Then,
aqueous solution of sodium hydroxide. The final compounds were
synthesised by amination of 2-(bromoalkoxy)xanthones with an
appropriate amine (piperidines or azepane) in the mixture of
ethanol:water (21:4) as described previously [33]. Oily products
were converted to oxalate salts by reaction with oxalic acid.
O-alkylation with
a,u-dibromoalkanes was performed in an

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Table 1
Biological activity of synthesized compounds (6ꢀ28).
Compound
hH3Ra Ki SEM [nM]
eeAChEb
eqBuChEc
hMAO Bd
hMAO Ad
IC50 SEM [nM]
IC50 SEM [nM]
IC50 SEM [nM]
%Inh. at 1 m
Me
6
7
8
9
172
150 11
1060 21
203
282 21
156
56
148
100
1105 51
3
622
238
358
378
620
342
353
691
387
384
307
9
4
6
8
1
4
8
1
7
9
5
2540 42
983 24
2000 67
1983 50
954 23
978 24
2829 89
685 13
1150 28
2040 51
1470 32
1090 30
1610 32
1100 27
965 18
1510 41
899 23
880 26
1280 34
1498 314
756 154
939 81
6%
5%
4%
8%
8%
1%
11%
2%
4%
42%
28%
1%
22%
5%
6
946 88
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
pitolisant
1821 195
4205 966
1980 299
665 119
1548 23
1830 285
717 81
1864 18
1538 446
673 44
856 205
1642 578
720 119
775 111
2111 467
897 150
8
2
3
8
436
110
448 57
140 11
5
8
697 16
421
386
406
5
5
7
97
8
6%
520 31
465 41
170 10
86
100
496 23
602 11
238 25
561 11
37%
28%
4%
2%
12%
3%
5%
23%
9%
525
180
314
8
3
4
5
1
136 10
394
6
308
235
270
6
5
5
605 10
1240 30
545 10
385
2
975 246
1387 237
(2%)h
30
4
(3.0%)f,g
8400 180
12 3i
tacrine
32 2g
ntj
1.5 0.5g
ntj
ntj
rasagiline
safinamide
clorgiline
ntj
ntj
ntj
ntj
ntj
ntj
25
8
7
ntj
ntj
1
ntj
ntj
ntj
1.8 0.5
a
[3H] Na-Methylhistamine binding assay performed with cell membrane preparation of CHOeK1 cells stably expressing the human H3 receptor; mean value of two in-
dependent experiments SEM.
b
AChE from electric eel; IC50, mean value for three experiments.
BuChE from equine serum; IC50, mean value for three experiments.
Fluorometric Amplex™ Red MAO assay.
c
d
e
%Inhibition of hMAO A at 1
%Inhibition of eeAChE at 10
Data from Ref. [16].
m
m
M; mean values of two independent experiments.
M.
f
g
h
i
%Inhibition of hMAO B at 1
Data from Ref. [35].
mM; mean values of two independent experiments.
j
Not tested.
Compounds were characterised by spectral analysis (1H NMR and/
or 13C NMR and/or IR), mass spectrometry (MS) and elemental
analysis.
elongation from five to six atoms appeared unprofitable for hH3R
affinity. These results are consistent with our previous studies [34].
2.3. Cholinesterase inhibitory activity
2.2. Human histamine H3 receptor affinity
Compounds were tested for acetyl- and butyrylcholinesterase
inhibition using the slightly modified method established by Ell-
man et al. with commercial tacrine as the reference standard [36].
Inhibitory activity was determined against AChE from electric eel
(Electrophorus electricus, eeAChE) and BuChE from equine serum
(eqBuChE). All tested compounds exhibited cholinesterase inhibi-
tion with IC50 values in (sub)micromolar range. Generally, com-
pounds showed higher AChE inhibition (136 nM ꢁ IC50 ꢁ 697 nM)
than BuChE inhibition (394 nM ꢁ IC50 ꢁ 2829 nM). The most potent
eeAChE inhibitors were compounds 23 and 25 with the five-carbon
linker and an azepane moiety, and with IC50 values of 180 nM and
136 nM respectively. Generally, all azepane derivatives (23e28)
were among the most potent AChE inhibitors. The same tendency
was noted in BuChE inhibition. No correlation was observed be-
tween the change of carbon length (five to six) or the position of
chlorine substituent (6- or 7-) in the xanthone ring and inhibition
of cholinesterase activity.
All tested compounds showed affinity for hH3R in sub-
micromolar range. The Ki values varied between compounds from
good (Ki ¼ 56 nM; 12) to weak (Ki ¼ 1105 nM; 15). Most compounds
showed moderate hH3R affinities (Ki: 100e500 nM). The results are
summarised in Table 1. Both the most potent and the least active
hH3R ligands were derivatives of 3-methylpiperidine (12 and 15).
Generally, 3-methylpiperidine (11e16) and azepane derivatives
(23e28) were among the most potent compounds, whereas 4-
methylpiperidine derivatives (except 20) were the weakest li-
gands. The introduction of a chlorine substituent into the xanthone
moiety (6- or 7-position) did not influence hH3R affinity. In the
pentyl derivatives series (7, 12, 13, 18, 19, 24 and 25), the better was
the 6-position, while in the hexyl series (9, 10, 15, 16, 21, 22, 27 and
28), the more potent were derivatives with the 7-chlorine sub-
stituent. Elongation of the carbon chain from five to six atoms
influenced hH3R affinity. Almost all (except 20) the most potent
hH3R ligands (Ki < 100 nM) were pentyl derivatives. Furthermore,
in most cases, the hexyl-linked xanthones were less potent than
their pentyl analogues, e.g. 15 vs 12, 27 vs 24. Thus, carbon chain
Two compounds 23 and 25 with the highest eeAChE inhibitory
potencies (IC50 ꢁ 180 nM) were chosen for the inhibition of human
AChE (hAChE) and human BuChE (hBuChE). Tacrine and donepezil

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D. Łazewska et al. / European Journal of Medicinal Chemistry 207 (2020) 112743
5
Scheme 1. Synthesis way of designed compounds. (i) CH3ONa, Cu2O/Cu, oil, 195e200 ꢂC, 5 h; (ii) concentrated H2SO4, reflux, 48 h; (iii), BrCH2(CH2)nCH2Br, K2CO3, KI, acetone,
reflux, 10e12 h; (iv) amine, K2CO3, KI, ethanol:water (21:4), reflux 12e24 h.
were included as reference compounds. The results (Table 2)
confirmed high inhibitory effects of both compounds for hAChE.
IC50 values were comparable (especially for 25) with the data ob-
tained from eeAChE for 23 and 25 (25: hAChE IC50 ¼ 120 nM vs
2.5. MAO A and B inhibition
MAO A and B inhibitory effects were evaluated using Amplex
Red Monoamine Oxidase kits [37]. Rasagiline (MAO B), safinamide
(MAO B) and clorgiline (MAO A) were used as reference com-
pounds. Initially, all synthesised compounds were tested at the
eeAChE IC50 ¼ 136 nM; 23: hAChE IC50 ¼ 392 nM vs eeAChE IC50
¼
180 nM). Moreover, compound 25 showed hAChE inhibition with
the similar strength as the reference compound tacrine (IC50 ¼ 131
nM). However, the results obtained for hBuChE show weaker
concentration of 1
10 M (first; data not shown) and 1
compounds that showed more than 50% of inhibition activity at the
concentration of 1 M were further evaluated, and their IC50 values
were determined. The results are outlined in Table 1. Most com-
pounds showed moderate to weak inhibition of hMAO
mM for hMAO B and at the concentrations of
m
m
M (next) for hMAO A. All
inhibitory activity than that for eqBuChE (e.g. 25: hBuChE IC50
1595 nM vs eqBuChE IC50 ¼ 394 nM).
¼
m
B
2.4. Kinetic studies of cholinesterases for selected compounds
(IC50 > 380 nM), whereas none of the tested compounds displayed
higher than 50% inhibition potency against hMAO A at the con-
Because of the highest potency towards eeAChE, 23 and 25 were
selected as representatives for kinetic studies for both enzymes
(eeAChE and eqBuChE) to investigate the mechanism of inhibition.
Analysis of the Lineweaver-Burk reciprocal plots (1/V vs 1/S)
showed that compounds 23 and 25 (Figs. 4A and 5A) displayed
linear noncompetitive inhibition, as increased slopes and preserved
intercepts at increasing concentrations of the inhibitor were
observed for both enzymes. Noncompetitive type of inhibition was
further confirmed by CornisheBowden plots (S/V vs I) (Figs. 4B and
5B).
centration of 1
mM. According to these results, the tested com-
pounds showed better inhibition profile against hMAO B than
against hMAO A. The most potent hMAO B inhibitor was compound
26 with an azepane moiety and IC50 of 385 nM. Among (un)
substituted piperidines (6¡22), especially in the pentyl series (6, 7,
11e13 and 17e19), the introduction of 7-chlorine substituent
increased the inhibitory activity towards hMAO B in comparison
with unsubstituted or 6-Cl substituted compounds (7 vs 6; 13 vs 11
vs 12 or 19 vs 17 vs 18). However, such correlation was not seen in
azepane’s series (23e28).
Table 2
2.6. Molecular docking to histamine H3 receptor
Human acetyl- and butyrylcholinesterase inhibitory activity of selected compounds.
Among the four histamine receptors reported to date, crystal
structure for only histamine H1 receptor has been resolved (PDB ID:
3RZE [38]). Therefore, in search for novel histamine H3R ligands,
homology modeling methods are used. In the present study, a
histamine H3R homology model was used to build crystal structure
of M2 muscarinic acetylcholine receptor (PDB ID: 3UON) with the
Compound
human AChE IC50 nM SEM
392 11
human BuChE IC50 nM SEM
23
25
tacrine
donepezil
1278 29
1595 53
34 0.3
nta
120
131
6
2
2
0.1
a
Not tested.

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6
Fig. 4. Lineweaver-Burk (A) and Cornish-Bowden (B) plots illustrating non-competitive eeAChE inhibition by compound 23 and 25. S ¼ substrate (ATC) concentration; V ¼ initial
velocity rate; I ¼ inhibitor concentration.
Fig. 5. Lineweaver-Burk (A) and Cornish-Bowden (B) plots illustrating non-competitive eqBuChE inhibition by compound 23 and 25. S ¼ substrate (BTC) concentration; V ¼ initial
velocity rate; I ¼ inhibitor concentration.
reference ligand pitolisant positioned in the binding pocket, ac-
cording to the data described in literature [39].
and TYR 3947.35. Moreover, the latter one additionally stabilised the
structures through
rivatives, additional
p
-
p
p
stacking. For the “meta” chlorine de-
All the docked compounds were characterised by docking score
values between ꢀ10.019 and ꢀ4.597. For most of them, salt bridge
and/or hydrogen bond formation between protonated amine ni-
trogen and GLU2065.46 was found d a supposedly key histamine
H3R antagonist/inverse agonist interaction (upper case numeration
according to BalesteroseWeinstein [40]). Additionally, previously
described H-bond formation between TYR3746.51 and ether oxygen
was observed (Fig. 6). The position of protonated basic moieties of
the ligands was mostly fixed, independent of the alkyl linker length.
A similar finding was noted for xanthone fragments e for all of the
docked ligands these fragments were placed in a “hydrophobic
cage” formed by PHE193 and TYR189 (ECL2), TYR942.64, TYR912.61
p
-
stacking with TYR942.64 was also present.
In fact, halogen bond formation between chlorine atoms and the
hydroxyl proton of TYR912.61 was observed despite its position in
the xanthone moiety (Fig. S1; Supplementary data). On the other
hand, hydrogen bond is formed between the carbonyl group and
one of the nitrogen atoms of ARG3816.58, which is in agreement of
our previous findings [41].
2.7. Molecular docking to cholinesterases
2.7.1. Molecular docking to acetylcholinesterase
The docking studies for tested compounds allowed us to

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D. Łazewska et al. / European Journal of Medicinal Chemistry 207 (2020) 112743
7
Fig. 6. Compound 25 in histamine H3 homology receptor binding pocket (left) and its ligand-interaction diagram (right). Hydrogen bonds are represented by yellow and halogen
bonds by violet dashed lines. TM5 was partly removed for better viewing purposes. (For interpretation of the references to colour in this figure legend, the reader is referred to the
Web version of this article.)
describe their binding mode in the active site of acetylcholines-
terase (PDB code: 1EVE). Most of them bound to the enzyme in a
similar manner. The basic amines were close to the catalytic triad
and anionic site residues. The alkyl chain created hydrophobic in-
teractions within the anionic site and the acyl pocket. The xanthone
moiety was located between aromatic amino acids from the pe-
ripheral anionic site [42]. The most active compounds 25
(IC50 ¼ 136 nM) and 23 (IC50 ¼ 180 nM) mimicked the binding
mode of donepezil in the acetylcholinesterase active site [43]. Their
best-ranked poses are shown in Fig. 7.
donepezil. On the other hand, compound 23 was observed to form a
hydrogen bond between its oxygen atom from the ether linker and
the hydroxyl moiety of Tyr121 from the peripheral anionic site
(PAS). In the AChE active gorge, the Tyr121 hydroxyl moiety inter-
acted with the abovementioned water molecule 1159. This type of
interaction could be associated with its potent biological activity
and was observed in our previous studies [16].
Tested compounds 23 and 25 contain large dibenzo-g-pyrone
(xanthone) fragments located between PAS residues. These frag-
ments created hydrophobic interactions with aromatic amino acids
such as: Tyr334, Tyr70, Tyr121 and Trp279. Additionally, the rigid
The azepane rings of both the described compounds were
located between His440 from the catalytic triad and anionic site
residues Trp84 and Phe330. The protonated nitrogen atoms inter-
structure of xanthone enabled to create p-p stacking with Trp279.
As water 1254 was important for the binding of donepezil, it was
also found to interact with the carbonyl oxygen atom from
xanthone of compound 23.
acted with aromatic rings through catione
p interactions. More-
over, the nitrogen atoms could form hydrogen bond interactions
with water molecule (1159). Alkyl chains were responsible for hy-
drophobic interactions with Phe330, Phe331, and Phe290 from the
anionic site and the acyl pocket of the enzyme.
For compound 25, the oxygen atom from the ether linker
formed a hydrogen bond with water 1254, and thus, it mimicked
interactions of the oxygen atom from the indanone ring of
2.7.2. Molecular docking to butyrylcholinesterase
The tested compounds were also docked into the active site of
butyrylcholinesterase. The analysis of the binding mode with
BuChE showed the most common and top-ranked poses presented
in Fig. 8. The heterocyclic moiety with tertiary amine was located
Fig. 7. Binding mode of compound 25 (left panel) and compound 23 (right panel) within the acetylcholinesterase active site. Residues coloured in: yellow e catalytic triad, orange e
anionic site, green e peripheral anionic site, purple e acyl pocket. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this
article.)

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8
Fig. 9. Binding mode of compound 26 within human monoamine oxidase B active site.
Residues coloured in yellow, green - cofactor FAD. (For interpretation of the references
to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 8. Binding mode of compound 25 within butyrylcholinesterase active site. Resi-
dues coloured in: yellow e catalytic triad, orange e anionic site, green e peripheral
anionic site, purple e acyl pocket. (For interpretation of the references to colour in this
figure legend, the reader is referred to the Web version of this article.)
We observed significant changes in the inhibitory activity in
comparison with compounds from our previous work [44]. This
could be related to the differences in the binding mode. For the
between amino acids from PAS and the anionic site. The alkyl linker
interacted with hydrophobic amino acids from the anionic site. The
xanthone fragment interacted with amino acids of the catalytic
triad and with the acyl pocket residues [43].
most active inhibitor of hMAO
B
described previously
(IC50 ¼ 4.5 nM), the pyrrolidine ring was close to the cofactor (FAD).
This binding mode in the active site of enzyme seems to be crucial
to obtain strong inhibitory activity. The differences in the potency
of this series of compounds could be explained by the lack of a
hydrogen bond with Tyr435. This type of interaction was not
observed for structures with high IC50 values, such as 6, 24 or 28.
Compound 25 (IC50
¼
394 nM) bound to the butyr-
ylcholinesterase active site as follows: the azepane ring of the
compound showed hydrophobic interactions with Tyr332 from the
peripheral anionic site. A flexible five-carbon alkyl chain enabled to
bend the molecule. Consequently, the protonated nitrogen atom
could form catione
p interaction with the aromatic ring of
xanthone. Moreover, the carbonyl oxygen atom in position 9 of the
xanthone ring interacted by hydrogen bonds with amino acids from
the catalytic triad: His438 and Ser198. These interactions were
observed in our previous study for azepane derivatives with 6-
methylchromone moiety. This H-bond network could explain the
high inhibitory activity. Additionally, the aromatic ring with the
chlorine substituent interacted with hydrophobic amino acids such
as Phe329 and Trp231 and with branched-chain amino acids from
the acyl pocket: Val288 and Leu286. This binding mode was similar
in comparison with the 6-methylchromone group [16].
2.9. In vivo studies of compounds 23 and 25
2.9.1. Analgesic effect of selected compounds
2.9.1.1. Formalin test in mice. Subcutaneous injection of formalin
results in a stimulation and then damage of sensory endings. The
nociceptive response of animals consists of two phases: phase I is
associated with immediate activation of nociceptors and phase II
leads to sensitisation of spinal reflex circuits. Moreover, it has been
suggested that formalin injection results in pathological changes
that resemble those observed in nerve injury and neuropathic pain
[45]. In our previous studies, we showed that opioids attenuate the
nociceptive response in both phases, whereas NSAIDs preferen-
tially diminish the nociceptive response in phase II of the test [46].
Furthermore, we showed that histamine H1 receptor, H3R and H4
receptor antagonists also attenuated nociceptive response, thus
showing promising efficacy especially in the late phase [46e48].
The same observation was made by Hamzeh Gooshchi et al. [49]
and Dou et al., [50]. In formalin-induced pain in rats, H3R ligands
like thioperamide [49] and 2-methyl-7-(3-morpholinopropoxy)-
3,4-dihydroisoquinolin-1(2H)-one [50] significantly suppressed
pain in the late phase. Now, as continuation of our previous studies,
we investigated analgesic properties of the two most promising
xanthone derivatives: 23 and 25. As shown in Fig. 10, the intra-
peritoneal (i.p.) administration of compound 23 prior to the sub-
cutaneous injection of formalin significantly attenuated the
nociceptive response in mice in both phases of the test. Its ED50
value in phase I was 24.7 mg/kg, whereas the ED50 value in phase II
was 11.3 mg/kg. Compound 23 was more effective in both phases
than the second investigated compound 25 (phase I:
ED50 ¼ 27.3 mg/kg and phase II: ED50 ¼ 22.5 mg/kg). These results
suggest promising analgesic properties of compound 23, which
were subsequently confirmed in other models of pain.
2.8. Molecular docking to MAO B
All the tested compounds showed a similar arrangement within
the human MAO B active site (PDB code: 4A79). Heterocyclic
moieties with a tertiary basic amine were located on the edge of the
lipophilic pocket. The alkyl linker interacted with the enzyme
through hydrophobic interactions. The dibenzo-g-pyrone frag-
ments were located near the cofactor (FAD). For the top-ranked
poses, the calculated ChemScore values were between 49.75 and
56.37.
Fig. 9 shows the binding of 26, the most active inhibitor of hMAO
B (IC50 ¼ 385 nM), with the enzyme active site. The azepane ring of
the inhibitor interacted with hydrophobic amino acids such as
Phe103, Trp119 and Ile199. Moreover, the protonated nitrogen atom
could form cation-p interactions with Trp119. The alkyl chain of
compound 26 created hydrophobic interactions with Ile316,
Leu167, Leu171, Ile199 and Ile198. The xanthone ring was close to
the FAD and formed aromatic ring interactions with the residues of
Tyr326, Tyr435, Phe343, Tyr60, and Tyr398. Binding within the
hMAO B active site was stabilised by hydrogen bonds between the
oxygen atom of xanthone and the hydroxyl group of Tyr435 for
tested compound 26.

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Fig. 11. Effect of compound 23 on carrageenan-induced oedema (A), mechanical
hyperalgesia (B) and on thermal hyperalgesia (C) developed after subplantar injection
of 1% carrageenan in rats. Data are expressed as means S.E.M. for 6 animals. 0 - the
initial reaction considered as the nociceptive reaction before carrageenan adminis-
tration. Statistical significance compared to vehicle-treated animals (control):
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ^ p < 0.05, ^^ p < 0.01 (for the dose
of 22.5 mg/kg). Statistical analysis: two-way ANOVA post hoc Bonferroni test. Statis-
tical significance in different time points of control group: #p < 0.05, ##p < 0.01,
###p < 0.001. Statistical analysis: one-way ANOVA followed by Dunnett’s Multiple
Comparison Test.
Fig. 10. Antinociceptive activity of compounds 23 and 25 in the formalin test. Results
are shown as time of licking in phase I (0e5 min after intraplantar injection of
formalin) and in phase II (15e30 min after formalin injection). Each value represents
the mean S.E.M. for 8e10 animals. Statistical analysis: one-way ANOVA followed by
post hoc Dunnett’s test. Statistical significance compared to vehicle-treated animals
(Tween): *p < 0.05, **p < 0.01, ***p < 0.001. C-control group.
2.9.1.2. Carageenan-induced paw oedema and mechanical and ther-
mal hypernociception in rats. Subplantar injection of carrageenan
induced oedema and mechanical and thermal hyperalgesia. Fig. 11
(panel A) shows the time course of rat paw oedema development in
the control group and groups pretreated with compound 23. The
tested compound significantly reduced paw oedema at the doses of
22.5 mg/kg and 30 mg/kg. Pretreatment with compound 23 at the
dose of 22.5 mg/kg reduced paw oedema by 36.1%, 32.8%, 26.5%,
19.37% and 22.4% at 1, 2, 3, 6 and 24 h following carrageenan in-
jection respectively. The effect of the higher dose of 30 mg/kg was
more significant (77.9%, 53.3%, 52.1%, 44.9% and 57.8% of oedema
reduction respectively). Thus, as oedema is one of the most sig-
nificant symptoms of inflammation, we concluded that compound
23 has some anti-inflammatory activity. Fig.11 (panel B) shows that
the subplantar injection of carrageenan decreased the withdrawal
threshold (mechanical hyperalgesia). The reduction was 88.4%,
83.0% and 90.9% of the initial reaction at 3, 6 and 24 h respectively.
Pretreatment with compound 23 inhibited mechanical inflamma-
tory hyperalgesia as observed by an increased withdrawal
threshold. The most pronounced effect (118.0% 9.0% of the initial
reaction) was observed at the dose of 30 mg/kg 3 h after inflam-
mation induction and was maintained for 24 h. A statistically sig-
nificant effect was also observed at the dose of 22.5 mg/kg;
however, it was weaker (94.6% 4.5%). Fig. 11 (panel C) shows that
the subplantar injection of carrageenan-induced thermal hyper-
algesia as observed by decreased latency of nociceptive response
for radiant heat stimulation. The initial latency of the control group
(before carrageenan injection) was 12.61 0.6 s and was signifi-
cantly reduced to 71.4%, 63.6%, 50.3%, 43.2% and 83.3% of the initial
value at 1, 2, 3, 6 and 24 h following carrageenan injection
respectively. Compound 23 was significantly active at the dose of
30 mg/kg. The initial latency in that group was 9.43 0.5 s. The
administration of 23 changed the values to 130.0%, 102.8%, 96.0%,
92.0% and 116.0% of the initial value at 1, 2, 3, 6 and 24 h following
carrageenan injection respectively. This shows that compound 23
was able to not only attenuate inflammatory hyperalgesia but also
show analgesic effect for heat stimulus.
2.9.1.3. Oxaliplatin-induced neuropathic pain in mice. To investigate
the analgesic activity of compound 23 in a neuropathic pain model,
we tested it in oxaliplatin (OXPT)-induced peripheral neuropathy,
in which mechanical and cold allodynia results from impaired
regulation of the activity of ion channels among others [51]. The

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10
neuropathy develops within a few hours after OXPT administration,
which is characterised as the early phase of neuropathic pain. The
second late phase develops in few days after OXPT administration.
Thus, we tested compound 23 at 3 h and 7 days post neuropathy
induction. We investigated the influence of compound 23 on tactile
allodynia by using the von Frey method. Three hours after the
administration of OXPT, we observed a significant decrease in the
value of mean force that caused paw withdrawal reaction
(1.82 0.05 g) (61.2% of the baseline) compared to that in non-
treated animals (2.97
0.04 g) (the baseline). Seven days after
OXPT administration, the pain threshold was 1.94 0.04 (65.3% of
the baseline). The observed tactile allodynia was attenuated by
compound 23, which elevated pain sensitivity threshold in the
early phase in a dose-dependent manner (Fig. 12) (2.63 0.08 g,
2.69 0.07 g and 2.84 0.07 g at the dose of 15 mg/kg, 22.5 mg/kg
and 30 mg/kg, which correspond to 88.5%, 90.6% and 95.6% of the
baseline value respectively). In the late phase, the values of pain
threshold were as follows: 2.55
0.06 g, 2.63
0.08 g and
2.98 0.06 g, which correspond to 85.9%, 88.5% and 100.0% of the
baseline value respectively. These results indicate that compound
23 has the potential for relieving particularly neuropathic pain (all
the tested doses were significantly active) and should be further
tested in that direction.
Fig. 13. Effects of compounds 23 and 25 treatment on DIZ-induced memory deficits in
inhibitory avoidance paradigm. Average STL time measured on the first training day
before the delivery of foot shock (black columns), and average STL time measured on
the test day (grey columns). ##P < 0.001 for average STL time compared with that of
the SAL-treated group. *P < 0.05 for average STLs compared with the DIZ-treated
group. **P < 0.001 for average STLs compared with the DIZ-treated group. &P < 0.05
for average STLs compared with the 23 (2.5 mg)-treated group. $P < 0.05 for average
STLs compared with the 23 (5 mg)-treated group. The data are expressed as the
mean SEM (n ¼ 7).
2.9.2. Memory-enhancing effects of compounds 23 and 25
Compounds 23 and 25 were assessed in rats with dizocilpine-
induced amnesia by i.p. administration of the tested compounds
at the dose range of 1.25e5 mg/kg. To assess the memory-
enhancing effect of compounds 23 and 25, step-through inhibi-
tory avoidance paradigm (SIAP) in step-through passive avoidance
test and transfer latency paradigm time (TLPT) in elevated plus
maze (EPM) test were used.
variance showed that acute systemic pretreatment with 23 (1.25,
2.5 and 5 mg/kg), 25 (1.25, 2.5 and 5 mg/kg) or DOZ (1 mg/kg)
exhibited a significant effect on STLs [F(8,54) ¼ 290.963; P < 0.001].
As compared to the saline-treated group, subsequent post hoc an-
alyses showed that DIZ (0.1 mg/kg) decreased STL time
[F(1,12) ¼ 1858.03; P < 0.001]. In addition, tested compound 23 (1.25,
2.5 and 5 mg/kg) and DOZ (1 mg/kg) exerted significant memory-
enhancing effect on STLs as compared to DIZ-treated group
([F(1,12) ¼ 262.72; P < 0.001], [F(1,12) ¼ 118.8498597; P < 0.05],
[F(1,12) ¼ 25.40; P < 0.05], and [F(1,12) ¼ 1073.05; P < 0.001])
respectively (Fig. 13). The procognitive effects observed for com-
pound 23 was dose-dependent, as the dose of 1.25 mg/kg was
significantly higher than that of 2.5 mg in providing memory-
enhancing effect [F(1,12) ¼ 37.66; P < 0.05] (Fig. 13). Furthermore,
the memory-enhancing effect provided by the dose of 2.5 mg of 23
was significantly higher than that observed with 5 mg of the same
compound [F(1,12) ¼ 16.07; P < 0.05]. Contrary to this result, com-
pound 25 failed to provide procognitive effects when administered
in the same dose range (all P > 0.05).
2.9.2.1. Memory-enhancing effects of compounds 23 and 25 in SIAP.
To determine the procognitive effect of tested compounds 23 and
25, 9 groups were acutely pretreated with either Saline þ Saline,
dizocilpine (DIZ, 0.1 mg) þ Saline, DIZ (0.1 mg) þ 23 (1.25 mg), DIZ
(0.1 mg) þ 23 (2.5 mg), DIZ (0.1 mg) þ 23 (5 mg), DIZ (0.1 mg) þ 25
(1.25 mg), DIZ (0.1 mg) þ 25 (2.5 mg), DIZ (0.1 mg) þ 25 (5 mg) and
DIZ þ Donepezil (DOZ, 1 mg) 30e45 min prior to the test session,
and their effects on DIZ-induced memory deficits were assessed by
determining the STLs to enter the dark box (Fig. 13) [15,52e58].
When injected before the retention test, statistical analysis of
2.9.2.2. Memory-enhancing effects of compounds 23 and 25 on TLPT
in the EPM test. The procognitive effects of tested compounds 23
and 25 on memory deficits of DIZ-treated rats in EPM paradigm
were assessed in the nine treatment groups as described for SIAP.
When injected before, on the test day, statistical analysis of vari-
ance showed that acute systemic pretreatment with 23 (1.25, 2.5
and 5 mg/kg), 25 (1.25, 2.5 and 5 mg/kg) or DOZ (1 mg/kg) exhibited
a significant effect on TLT [F(8,54) ¼ 5.35; P < 0.001]. Further sub-
sequent post hoc analyses indicated that DIZ-treated rats exhibited
significantly increased transfer latency time (TLT) time when
compared with control SAL-treated rats [F(1,12) ¼ 10.28; P < 0.01]
(Fig. 14). Moreover, the results showed that acute systemic pre-
treatment with compound 23 (1.25, 2.5 and 5 mg/kg, i.p.) and DOZ
(1 mg/kg) counteracted memory impairment of DIZ-treated ani-
mals by significantly decreasing TLT ([F(1,10) ¼ 14.20; P < 0.05],
[F(1,10) ¼ 14.20; P < 0.05], [F(1,10) ¼ 14.20; P < 0.05], and
[F(1,10) ¼ 14.20; P < 0.05]) respectively as compared to that in DIZ-
Fig. 12. Antiallodynic effects of compound 23 in the tactile allodynia evaluated in the
von Frey test in oxaliplatin-induced model of peripheral neuropathy. Results compared
to vehicle-treated group (Pre 23). Statistical analysis: repeated measures analysis of
variance (ANOVA), followed by Dunnett’s post hoc comparison: ***p
****p < 0.0001.
< 0.001,

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11
(compounds 9,10,15,16, 21, 22, 27 and 28) and Egan’s (15,16, 21, 22,
27 and 28) rule. None of the compounds complied with the rule of
Muegge (one violation). Violation of only one rule does not exclude
the chance of a molecule to become a drug. Moreover, according to
the results, all compounds should cross the blood-brain barrier, be
absorbed through the intestine and be transported by P-glycopro-
teins. Toxicity of the designed compounds was evaluated using
ProToxII tool [60]. The programme predicted (Table S4; supple-
mentary data) high doses of oral toxicity (LD50 > 244 mg/kg) and
potential toxic behaviour to immune system (due to immunotox-
icity). Further, the SwissADME programme [31,59] determined that
compounds 6e28 should have no pan-assay interference liability
(PAINS). It is worth to underline that apart from generally similar
and beneficial ADMET properties for both compounds 23 and 25,
the active in vivo compound 23 displayed log P lower than the
inactive 25, and thus closer to the advised values for drug-likeness
compounds.
Fig. 14. Effects of compounds 23 and 25 treatment on DIZ-induced memory deficits in
elevated plus maze test. Average of TLT measured on the first training day (black
columns), and average of TLT measured on the test day (grey columns), namely 24 h
after the training day, and 30e45 min after DIZ or tested compounds injection.
#P < 0.01 vs saline-treated group. *P < 0.05 vs DIZ-treated rats. Values are expressed as
the mean þSEM (n ¼ 7).
2.10.2. Permeability of BBB of compounds 23 and 25
In order to explain the differences in in vivo activity of com-
pounds 23 and 25 the ability of these compounds to cross blood
brain barrier (BBB) was checked using PAMPA. PAMPA is widely
known experimental method for an estimation of compound’s
biological membrane permeability, including BBB penetration via
passive transport. The artificial membrane imitates barriers for
ligand absorption with a good correlation to in vivo conditions. The
study was performed in accordance to the previously described
method [61]. According to the manufacturer’s guidelines, com-
pounds can be classified as permeable if Pe ꢃ 1.5 ꢄ 10ꢀ6 cm/s [62].
Caffeine was used as the highly permeable reference drug. Results
indicated that both tested compounds were permeable, but dis-
played a lower (23; Pe ¼ 5.6 ꢄ 10ꢀ6 cm/s) or much lower (25;
Pe ¼ 2.2 ꢄ 10ꢀ6 cm/s) permeability coefficient values than that of
caffeine (Pe ¼ 15.1 ꢄ 10ꢀ6 cm/s; Table 3). More than twice weaker
permeability of compound 25 in comparison with 23 could explain
the observed difference in in vivo activity.
treated rats, and no significant difference was observed in the
procognitive effect provided by compound 23 (1.25, 2.5 and 5 mg/
kg) (all P > 0.05) (Fig. 14). However, compound 25 failed to atten-
uate the impaired memory of DIZ-treated rats (all P > 0.05).
On the basis of the results obtained for the in vivo memory-
enhancing assessments, it can be concluded that acute systemic
pretreatment with compound 23 significantly mitigated the
memory deficits associated with systemic administration of DIZ in
two different in vivo test models (Figs. 13 and 14). The memory-
enhancing effects observed for the novel H3R ligand 23 were
consistent with earlier studies in which nonimidazole-based H3R
antagonists showed procognitive effects in rats [52e54,57]. How-
ever, compound 25 failed to attenuate the memory deficits in the
same sets of behavioural experiments.
3. Conclusions
On the basis of preliminary in silico studies, a series of xanthone
derivatives was designed, synthesised and biologically evaluated
in vitro and in vivo. For the first time, xanthones are described here
as MTDL with antagonistic affinities for hH3R and inhibitory activity
for cholinesterases and hMAO B. Most of the twenty three com-
pounds showed affinities to both hH3R and AChE in the nanomolar
concentration range. Additionally, the compounds could also
inhibit BuChE and hMAO B but with weaker strength than that for
hH3R and AChE. Furthermore, none of the compounds displayed
any inhibitory activity towards hMAO A. Thus, as described here,
xanthones are promising novel multifunctional agents that can
improve not only cognitive symptoms of AD by hampering H3R
activity and inhibiting cholinesterases (AChE and BuChE) but also
slow down the progression of AD by inhibiting hMAO B. In addition,
in vivo studies showed analgesic and procognitive activity of tested
2.10. Studies on drug-likeness of compounds 23 and 25
Taking into consideration a very similar pharmacological profile
in vitro together with very little structural dissimilarity between 23
and 25, the apparent discrepancy for both compounds in the
pharmacological action in vivo is an intriguing question. The only
structural difference for the compounds is seen in the presence (25)
or absence (23) of chlorine substituent at the aromatic ring. This
suggests the important role for hydrophobicity that is responsible
for drug-like ADME properties of compounds, in particular, those
associated with membrane penetration. Thus, drug-likeness
studies, with special accent on membrane permeability for com-
pounds 23 and 25, were performed in search for a reason of the
activity difference.
2.10.1. In silico ADMET and drug-likeness properties
ADMET properties of were calculated for all synthesised com-
pounds using a free accessible web tool: SwissADME [31,59]. The
calculated data are shown in Tables S2 and S3 (supplementary
data). All compounds exhibited good physicochemical properties,
except slightly high predicted lipophilicity (log P in the range of
4.57e5.53). SwissADME estimates drug-likeness based on five
Table 3
Permeability coefficient of compounds 23 and 25.
Compound
Pea,b [10ꢀ6 cm/s] SD
23
25
Caffeine
5.6 1.1
2.2 0.3
15.1 0.4
ꢀ
commonly used rules (Lipinski, Ghose, Veber, Egan and Muegge) to
a
Tested in triplicate.
For permeable compounds Pe is equal or higher than 1.5 ꢄ
ꢀ
b
filter molecules. All compounds satisfied the rules of Lipinski and
Veber.
A
few compounds showed one violation in Ghose’s
10ꢀ6cm/s.

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12
compounds 23 and 25. However, in all tests, compound 23 showed
the higher effect probably due to better drug-likeness properties
than compound 25, especially, membranes penetration, including
BBB. Procognitive properties of 23 were comprehended in two
behavioural paradigms, namely SIAP and TLPT tests, where 23
significantly ameliorated memory impairments induced by DIZ
(especially at the dose of 1.25 mg/kg). In pain models (formalin test,
carrageenan-induced hypernociception and oxaliplatin-induced
neuropathic pain), compound 23 showed significant analgesic ef-
fect. Thus, compound 23 can be a promising lead structure in the
search for MTDL for future therapeutic management of AD.
of CH2Cl2 and purified by extraction with 1% solution of NaHCO3
(twice). The inorganic solution was acidified with 10% HCl and
white solid precipitate: 5.5 g, yield 39%, Mp: 115e118 ꢂC.
2-Hydroxyxanthone (4a). 2-(4-methoxyphenoxy)benzoic acid
(22 mmol, 5.37 g) was refluxed on the water bath with concen-
trated sulfuric acid (30 mL) for 2 h. Then the mixture was poured
into the icy water and the formed solid was precipitated washed
with water, 0.5% NaOH, water and crystallized from ethanol. Yellow
solid, Mp: 235e240 ꢂC, yield 45% (2.26 g) (Mp: 240.2e240.9 ꢂC
(ethanol) [63]).
6-Chloro-2-hydroxyxanthone
(4b).
Synthesis
as
2-
hydroxyxanthone from 4-chloro-2-(4-methoxyphenoxy)benzoic
acid (11 mmol, 3.1 g) in 14 mL of concentrated sulfuric acid. Yellow
solid, Mp: 210e218 ꢂC, yield 32% (0.87 g).
4. Experimental
4.1. Chemistry
7-Chloro-2-hydroxyxanthone
(4c).
Synthesis
as
2-
hydroxyxanthone from 5-chloro-2-(4-methoxyphenoxy)benzoic
acid (18 mmol, 5.04 g) in 25 mL of concentrated sulfuric acid. Yel-
low solid, Mp: 254e250 ꢂC, yield 85% (3.8 g).
2-(5-Bromopentyloxy)-9H-xanthen-9-one (5a). To the solution of
0.3 g (7.5 mmol) in 12 mL of water 2-hydroxyxanthone (4a) was
added (7.5 mmol; 1.69 g). The mixture was stirred for a few minutes
and then 1,5-dibromopentane (7.5 mmol; 1.73 g) was added and
refluxed for 7 h. After cooling down the precipitate was filtered off,
washed with water and purified by flash chromatography (CH2Cl2).
Obtained white solid, Mp: 91-93 ꢂC, yield 49% (1.34 g).
2-(5-Bromopentyloxy)-6-chloro-9H-xanthen-9-one (5b). Synthe-
sis as 2-(5-bromopentyloxy)-9H-xanthen-9-one from 6-chloro-2-
hydroxyxanthone (4b) (5 mmol; 1.23 g) in NaOH solution
(5 mmol; 0.2 g in 10 mL of water) with 1,5- dibromopentane
(5 mmol; 1.15 g). Refluxed for 5 h. After cooling down the precip-
itate was filtered off, washed with water and the crude product was
used for the synthesis without further purification. Light yellow
solid, (0.8 g). The reaction was repeated. Obtained light yellow solid
(0.5 g).
2-(5-Bromopentyloxy)-7-chloro-9H-xanthen-9-one (5c). Synthe-
sis as 2-(5-bromopentyloxy)-9H-xanthen-9-one from 7-chloro-2-
hydroxyxanthone (4c) (6 mmol; 1.48 g) in NaOH solution
(6 mmol; 0.24 g in 10 mL of water) with 1,5- dibromopentane
(6 mmol; 1.38 g). Refluxed for 12 h. After cooling down the pre-
cipitate was filtered off, washed with water and the crude product
was used for the synthesis without further purification. White solid
(1.75 g).
All starting materials, reagents and solvents were obtained from
commercial suppliers (Sigma Aldrich, Alfa Aesar or Chempur) and
were used without further purification. Reaction progress was
monitored by thin layer chromatography (TLC) on silica gel 60 F254
plates and the spots were visualized by UV light and treatment with
Dragendorff reagent (solvent system: methylene chloride: meth-
anol 9:1). Melting points (Mp) were determined using MEL-TEMP II
apparatus (LD Inc. Long Beach, CA, USA) and are uncorrected. NMR
(1H and 13C) spectra were recorded in DMSO‑d6 on Varian-Mercury-
VX 300 MHz PFG (Varian, Palo Alto, CA, USA) or Avance III HD
400 MHz (Bruker; Germany) spectrometers or FT-NMR 500 MHz
spectrometer (Jeol Ltd, Akishima, Tokyo, Japan). Chemical shifts (d)
are expressed in parts per million (ppm). Data are reported
chemical shift, multiplicity (s, singlet; d, dublet; t, triplet; q, quar-
tet; qu, quintet; m, multiplet; br, broad), approximate coupling
constants J expressed in Hertz (Hz), number of protons. Recorded
NMR data are available in Supplementary Materials. IR spectra were
measured as KBr pellets on FT/IR Nicolet iS5 spectrometer (Ther-
moScientific). Mass spectra (LC/MS) were performed on Waters TQ
Detector Mass Spectrometer (Water Corporation, Milford, CT, USA).
Retention times (tR) are given in minutes. UPLC/MS analysis
confirmed purity (>95%) of all compounds (except 9e93.01%,
21e93.91% and 27e94.98%). Elemental analyses (C, H, N) were
performed on an Elemental Analyser Vario El III (Hanau, Germany)
and agreed with theoretical values within 0.4%.
4.1.1. Synthesis of starting materials
2-(6-Bromohexyloxy)-9H-xanthen-9-one (5d). Synthesis as 2-(5-
bromopentyloxy)-9H-xanthen-9-one from 2-hydroxyxanthone
(4a) (10 mmol; 2.26 g) in NaOH solution (10 mmol; 0.4 g in
15 mL of water) with 1,6- dibromohexane (10 mmol; 2.44 g).
Refluxed for 9 h. After cooling down the precipitate was filtered off,
washed with water and ethanol. The crude product was used for
the synthesis without further purification. White solid (2.6 g).
2-(6-Bromohexyloxy)-6-chloro-9H-xanthen-9-one (5e). Synthesis
as 2-(5-bromopentyloxy)-9H-xanthen-9-one from 6-chloro-2-
hydroxyxanthone (4b) (6 mmol; 1.48 g) in NaOH solution
(6 mmol; 0.24 g in 10 mL of water) with 1,6- dibromohexane
(6 mmol; 1.46 g). Refluxed for 5.5 h. After cooling down the pre-
cipitate was filtered off, washed with water and ethanol. The crude
product was used for the synthesis without further purification.
White solid (1 g).
2-(6-Bromohexyloxy)-7-chloro-9H-xanthen-9-one (5f). Synthesis
as 2-(5-bromopentyloxy)-9H-xanthen-9-one from 6-chloro-2-
hydroxyxanthone (4c) (6 mmol; 1.48 g) in NaOH solution
(6 mmol; 0.24 g in 10 mL of water) with 1,6- dibromohexane
(6 mmol; 1.46 g). Refluxed for 5.5 h. After cooling down the pre-
cipitate was filtered off, washed with water and ethanol. The crude
product was used for the synthesis without further purification.
White solid (1.4 g).
2-(4-Methoxyphenoxy)benzoic acid (3a). To freshly prepared so-
dium methoxide (100 mmol Na in 100 mL of absolute methanol)
was added 2-chlorobenzoic acid (50 mmol, 7.83 g), 4-
methoxyphenol (60 mmol, 7.45 g) copper (0.39 mmol, 0.025 g)
and copper (I) oxide (0.17 mmol, 0.025 g) and warming on the
water bath until dissolution. Then methanol was evaporated and to
the rest paraffin oil (50 mL) was added and heated to 210 ꢂC for 2 h.
After cooling down paraffin oil was decanted and 50 mL od xylene
was added and the precipitate was filtered, dissolved in hot water
and refluxed for a few minutes with activated carbon. The filtrate
was acidified with concentrated hydrochloric acid (HCl). The pre-
cipitate was crystallized with ethanol. White solid, Mp: 145e147 οC,
yield 44% (5.4 g).
4-Chloro-2-(4-methoxyphenoxy)benzoic acid (3b). Synthesised as
described for 2-(4-Methoxyphenoxy)benzoic acid from 2,4-
dichlorobenzoic acid (50 mmol, 9.55 g). White solid, Mp:
200e204 οC, yield 45% (6.2 g).
5-Chloro-2-(4-methoxyphenoxy)benzoic acid (3c). Synthesised as
described for 2-(4-methoxyphenoxy)benzoic acid from 2,5-
dichlorobenzoic acid (50 mmol, 9.55 g). After acidification with
concentrated hydrochloric acid as the solid did not precipitate
water was decanted from oily residue and then dissolved in 50 mL

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D. Łazewska et al. / European Journal of Medicinal Chemistry 207 (2020) 112743
13
4.1.2. General procedure for the synthesis of final compounds 6e28
A proper bromoalkoxy-9H-xanthen-9-one (1 mmol) was added
to the solution of a proper amine (2 mmol) in the mixture of
ethanol (21 mL) and water (4 mL) in the presence of potassium
carbonate (3 mmol) and KI (catalytic amount) and refluxed for
10e20 h. After cooling to RT the solid was filtered off. The filtrate
was concentrated in vacuo and purified as described previously
[33].
61.96; H, 6.00; N, 2.78%. Found: C, 62.01; H, 5.78; N, 2.71%.
2-chloro-7-(6-(piperidin-1-yl)hexyloxy)-9H-xanthen-9-one oxa-
late (10). Synthesis from 2-(6-bromohexyloxy)-7-chloro-9H-
xanthen-9-one (5f) (0.41 g, 0.001 mol) and piperidine (0.17 g,
0.002 mol). After purification was obtained 0.22 g of oil. Raw
product was transformed into oxalic acid salt yielding 0.20 g (40%)
of final compound. Mp: 190e193 ꢂC. C24H28NO3Cl x C2H2O4 (MW ¼
503.97). 1H NMR (400 MHz, DMSO‑d6)
d
: 8.12 (d, J ¼ 2.20 Hz, 1H),
2-(5-(piperidin-1-yl)pentyloxy)-9H-xanthen-9-one oxalate (6).
Synthesis from 2-(5-bromopentyloxy)-9H-xanthen-9-one (5a)
(0.36 g, 1 mmol) and piperidine (0.17 g, 2 mmol). After purification
was obtained 0.3 g of oil. Raw product was transformed into oxalic
acid salt yielding 0.32 g (70%) of final compound. Mp: 167e169 ꢂC.
7.92 (dd, J ¼ 2.42, 9.02 Hz, 1H), 7.75 (d, J ¼ 8.80 Hz, 1H), 7.62e7.71
(m, 1H), 7.45e7.60 (m, 2H), 4.10 (t, J ¼ 6.16 Hz, 2H), 3.89 (br s, 4H),
2.90e3.05 (m, 2H), 1.61e1.87 (m, 8H), 1.43e1.59 (m, 4H), 1.38 (d, J ¼
7.04 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d: 175.33, 164.74,155.77,
154.57, 150.65, 135.60, 128.95, 125.86, 125.22, 122.01, 121.66, 121.23,
120.40, 106.81, 68.55, 56.36, 52.53, 28.73, 26.29, 25.49, 23.73, 23.12,
21.97. LC-MS: purity 99.28% tR ¼ 6.35, (ESI) m/z [MþH]þ 414.24.
Anal. calcd. for C26H30NO7Cl: C, 61.96; H, 6.00; N, 2.78%. Found: C,
61.98; H, 5.77; N, 2.66%.
C
23H27NO3 x C2H2O4 (MW ¼ 455.49). 1H NMR (400 MHz, DMSO‑d6)
d
: 8.13e8.26 (m, 1H), 7.82e7.92 (m,1H), 7.66 (t, J ¼ 8.10 Hz, 2H), 7.56
(d, J ¼ 2.7 Hz, 1H), 7.44e7.52 (m, 2H), 4.10 (t, J ¼ 6.5 Hz, 2H),
2.84e3.27 (m, 6H), 1.64e1.92 (m, 8H), 1.40e1.63 (m, 4H). 13C NMR
(101 MHz, DMSO‑d6)
d
: 176.24, 165.02, 155.98, 155.52, 150.68,
2-(5-(3-methylpiperidin-1-yl)pentyloxy)-9H-xanthen-9-one oxa-
late (11). Synthesis from 2-(5-bromopentyloxy)-9H-xanthen-9-one
(5a) (0.36 g, 0.001 mol) and 3-methylpiperidine (0.20 g, 0.002 mol).
After purification was obtained 0.31 g of oil. Raw product was
transformed into oxalic acid salt yielding 0.34 g (72%) of final
compound. Mp: 156e158 ꢂC. C24H29NO3 x C2H2O4 (MW ¼ 469.52).
135.85, 126.40, 125.43, 124.68,121.96,120.95,120.26,118.65,106.92,
68.30, 56.28, 52.48, 28.51, 23.45, 23.24, 23.06, 22.01. IR (cmꢀ1):
1652.10 (-C¼O), 1210.62 (¼CeOeC-), 1025.30 (¼CeOeC-). LC-MS:
purity 100% tR ¼ 5.17, (ESI) m/z [MþH]þ 366.25. Anal. calcd. for
C
25H29NO7: C, 65.92; H, 6.42; N, 3.07%. Found: C, 65.67; H, 6.56; N,
3.00%.
7-chloro-2-(5-(piperidin-1-yl)pentyloxy)-9H-xanthen-9-one oxa-
1H NMR (400 MHz, DMSO‑d6)
d
: 8.20 (dd, J ¼ 1.17, 8.22 Hz, 1H),
7.83e7.92 (m, 1H), 7.65 (t, J ¼ 8.41 Hz, 2H), 7.56 (d, J ¼ 2.74 Hz, 1H),
7.42e7.52 (m, 2H), 4.10 (t, J ¼ 6.26 Hz, 2H), 3.26e3.44 (m, 2H),
2.90e3.09 (m, 2H), 2.63e2.78 (m, 1H), 2.45 (t, J ¼ 11.74 Hz, 1H),
1.61e1.98 (m, 8H), 1.39e1.56 (m, 2H), 0.98e1.15 (m, 1H), 0.89 (d,
late (7). Synthesis from 2-(5-bromopentyloxy)-7-chloro-9H-
xanthen-9-one (5c) (0.39 g, 0.001 mol) and piperidine (0.17 g,
0.002 mol). After purification was obtained 0.19 g of oil. Raw
product was transformed into oxalic acid salt yielding 0.17 g (35%)
of final compound. Mp: 198e200 ꢂC. C23H26NO3Cl x C2H2O4
J ¼ 6.65 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6)
d: 176.23, 165.05,
155.97, 155.51, 150.68, 135.84, 126.39, 125.43, 124.67, 120.26, 118.64,
106.92, 68.30, 57.91, 51.96, 30.58, 28.52, 23.45, 23.25, 19.06. IR
(cmꢀ1): 1653.43 (-C¼O), 1213.02 (¼CeOeC-), 1025.45 (¼CeOeC-).
LC-MS: purity 100% tR ¼ 5.52, (ESI) m/z [MþH]þ 380.21. Anal. calcd.
for C26H31NO7: C, 66.51; H, 6.65; N, 2.98%. Found: C, 66.03; H, 6.90;
N, 2.91%.
(MW ¼ 489.94). 1H NMR (400 MHz, DMSO‑d6)
: 8.11 (d, J ¼ 2.2 Hz,
d
1H), 7.91 (dd, J ¼ 2.2, 8.8 Hz, 1H), 7.74 (d, J ¼ 8.8 Hz, 1H), 7.66 (d,
J ¼ 8.8 Hz,1H), 7.43e7.59 (m, 2H), 4.00e4.21 (m, 2H), 2.85e3.23 (m,
5H), 1.60e1.93 (m, 9H), 1.49 (d, J ¼ 6.6 Hz, 4H). 13C NMR (101 MHz,
DMSO‑d6) d: 175.31, 164.89, 155.73, 154.56, 150.66, 135.60, 128.95,
125.85, 125.21, 121.99, 121.65, 121.22, 120.40, 106.83, 68.34, 56.32,
52.55, 28.49, 23.51, 23.22, 23.14, 22.03. LC-MS: purity 100%
tR ¼ 5.95, (ESI) m/z [MþH]þ 400.21. Anal. calcd. for C25H28NO7Cl: C,
61.28; H, 5.76; N, 2.86%. Found: C, 61.53; H, 5.48; N, 2.83%.
6-chloro-2-(5-(3-methylpiperidin-1-yl)pentyloxy)-9H-xanthen-9-
one oxalate (12). Synthesis from 2-(5-bromopentyloxy)-6-chloro-
9H-xanthen-9-one (5b) (0.40 g, 0.001 mol) and 3-methylpiperidine
(0.20 g, 0.002 mol). After purification was obtained 0.09 g of oil.
Raw product was transformed into oxalic acid salt yielding 0.07 g
(14%) of final compound. Mp: 138e141 ꢂC. C24H28NO3Cl x C2H2O4
2-(6-(piperidin-1-yl)hexyloxy)-9H-xanthen-9-one oxalate (8).
Synthesis from 2-(6-bromohexyloxy)-9H-xanthen-9-one (5d)
(0.38 g, 0.001 mol) and piperidine (0.17 g, 0.002 mol). After puri-
fication was obtained 0.15 g of oil. Raw product was transformed
into oxalic acid salt yielding 0.18 g (38%) of final compound. Mp:
212e215 ꢂC. C24H29NO3 x C2H2O4 (MW ¼ 469.52). 1H NMR
(MW ¼ 503.97). 1H NMR (400 MHz, DMSO‑d6)
: 8.17 (d, J ¼ 8.6 Hz,
d
1H), 7.82 (d, J ¼ 1.6 Hz, 1H), 7.61 (d, J ¼ 9.0 Hz, 1H), 7.43e7.54 (m,
3H), 4.09 (t, J ¼ 6.3 Hz, 2H), 3.25e3.45 (m, 2H), 2.92e3.07 (m, 2H),
2.72 (t, J ¼ 11.3 Hz, 1H), 2.39e2.48 (m, 1H), 1.64e1.94 (m, 8H),
(400 MHz, DMSO‑d6)
d
: 8.21 (d, J ¼ 7.0, 1.17 Hz, 1H), 7.79e7.96 (m,
1.40e1.55 (m, 2H), 0.98e1.14 (m, 1H), 0.89 (d, J ¼ 6.7 Hz, 3H). 13
C
1H), 7.39e7.74 (m, 5H), 4.11 (t, J ¼ 6.10 Hz, 2H), 3.59e3.73 (m, 4H),
NMR (101 MHz, DMSO‑d6) d: 175.54, 165.05, 156.23, 155.74, 150.58,
2.65e2.90 (m, 2H), 1.26e1.85 (m, 14H). 13C NMR (101 MHz,
140.04, 128.28, 125.55, 125.22, 121.98, 120.24, 119.84, 118.49, 106.94,
68.33, 57.89, 51.95, 30.56, 28.51, 23.43, 23.24, 19.06. LC-MS: purity
DMSO‑d6) d: 164.75, 159.85, 155.99, 155.57, 150.68, 135.88, 126.40,
125.47, 124.70, 121.98, 120.96, 120.29, 118.66, 106.87, 79.65, 72.71,
68.54, 58.54, 56.49, 53.20, 28.83, 26.58, 25.62, 19.03, 13.53. LC-MS:
purity 99.06% tR ¼ 5.47, (ESI) m/z [MþH]þ 380.27. Anal. calcd. for
100% tR
¼
6.31, (ESI) m/z [MþH]þ 414.17. Anal. calcd. for
C26H30NO3Cl: C, 61.96; H, 6.00; N, 2.78%. Found: C, 62.26; H, 5.83; N,
2.81%.
C
26H31NO7: C, 66.51; H, 6.65; N, 2.98%. Found: C, 66.70; H, 6.64; N,
7-chloro-2-(5-(3-methylpiperidin-1-yl)pentyloxy)-9H-xanthen-9-
one oxalate (13). Synthesis from 2-(5-bromopentyloxy)-7-chloro-
9H-xanthen-9-one (5c) (0.39 g, 0.001 mol) and 3-methylpiperidine
(0.20 g, 0.002 mol). After purification was obtained 0.24 g of oil.
Raw product was transformed into oxalic acid salt yielding 0.22 g
(44%) of final compound. Mp: 194e197 ꢂC. C24H28NO3Cl x C2H2O4
2.94%.
6-Chloro-2-(6-(piperidin-1-yl)hexyloxy)-9H-xanthen-9-one oxa-
late (9). Synthesis from 2-(6-bromohexyloxy)-6-chloro-9H-
xanthen-9-one (5e) (0.41 g, 0.001 mol) and piperidine (0.17 g,
0.002 mol). After purification was obtained 0.05 g of oil. Raw
product was transformed into oxalic acid salt yielding 0.05 g (9%) of
(MW ¼ 503.97). 1H NMR (400 MHz, DMSO‑d6)
: 8.12 (d, J ¼ 2.20 Hz,
d
final compound. Mp: 213e216 ꢂC. C24H28NO3Cl
x
C2H2O4
1H), 7.91 (dd, J ¼ 8.8, 2.6 Hz, 1H), 7.74 (d, J ¼ 9.2 Hz, 1H), 7.67 (d,
J ¼ 8.8 Hz, 1H), 7.55 (d, J ¼ 2.6 Hz, 1H), 7.51 (dd, J ¼ 9.0, 2.9 Hz, 1H),
4.11 (t, J ¼ 6.2 Hz, 2H), 3.24e3.43 (m, 2H), 2.89e3.07 (m, 2H), 2.70
(br s, 1H), 2.44 (br s, 1H), 1.58e1.94 (m, 8H), 1.39e1.56 (m, 2H),
0.97e1.13 (q, J ¼ 11.89 Hz,1H), 0.89 (d, J ¼ 6.6 Hz, 3H). LC-MS: purity
99.07% tR ¼ 6.22, (ESI) m/z [MþH]þ 414.24. Anal. calcd. for
(MW ¼ 503.97). 1H NMR (500 MHz, DMSO‑d6)
d
: 8.15 (d, J ¼ 8.59 Hz,
1H), 7.82 (d, J ¼ 1.72 Hz, 1H), 7.60 (d, J ¼ 9.16 Hz, 1H), 7.40e7.55 (m,
3H), 4.06 (t, J ¼ 6.44 Hz, 2H), 3.04e3.26 (m, 3H), 2.73e3.02 (m, 3H),
1.54e1.82 (m, 8H), 1.16e1.50 (m, 6H). LC-MS: purity 93.01%
tR ¼ 6.39, (ESI) m/z [MþH]þ 414.17. Anal. calcd. for C26H30NO7Cl: C,

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D. Łazewska et al. / European Journal of Medicinal Chemistry 207 (2020) 112743
14
C26H30NO7Cl: C, 61.96; H, 6.00; N, 2.78%. Found: C, 62.03; H, 5.76; N,
2.73%.
(101 MHz, DMSO‑d6) d: 176.2, 165.1, 156.0, 155.5, 150.7, 135.8, 126.4,
125.4, 124.7, 121.9, 120.9, 120.3, 118.6, 106.9, 68.3, 56.0, 51.9, 30.4,
28.5, 23.6, 23.3, 21.3. IR (cmꢀ1): 1658.68, (-C¼O), 1214.77
(¼CeOeC-), 1018.57 (¼CeOeC-). LC-MS: purity 99.34% tR ¼ 5.50,
(ESI) m/z [MþH]þ 380.21. Anal. calcd. for C26H31NO7: C, 66.51; H,
6.65; N, 2.98%. Found: C, 66.17; H, 6.92; N, 2.85%.
2-(6-(3-Methylpiperidin-1-yl)hexyloxy)-9H-xanthen-9-one oxa-
late (14). Synthesis from 2-(6-bromohexyloxy)-9H-xanthen-9-one
(5d) (0.38 g, 0.001 mol) and 3-methylpiperidine (0.20 g,
0.002 mol). After purification was obtained 0.28 g of oil. Raw
product was transformed into oxalic acid salt yielding 0.28 g (58%)
6-Chloro-2-(5-(4-methylpiperidin-1-yl)pentyloxy)-9H-xanthen-
9-one oxalate (18). Synthesis from 2-(5-bromopentyloxy)-6-chloro-
9H-xanthen-9-one (5b) (0.40 g, 0.001 mol) and 4-methylpiperidine
(0.20 g, 0.002 mol). After purification was obtained 0.05 g of oil.
Raw product was transformed into oxalic acid salt yielding 0.03 g
(6%) of final compound. Mp: 168e171 ꢂC. C24H28NO3Cl x C2H2O4
of final compound. Mp: 194e197 ꢂC. C25H31NO3
x
C2H2O4
(MW ¼ 483.56). 1H NMR (500 MHz, DMSO‑d6)
d: 8.16 (dd, J ¼ 1.43,
8.02 Hz, 1H), 7.79e7.89 (m, 1H), 7.62 (t, J ¼ 8.88 Hz, 2H), 7.52 (d,
J ¼ 3.15 Hz, 1H), 7.39e7.47 (m, 2H), 4.05 (t, J ¼ 6.44 Hz, 2H),
3.21e3.36 (m, 2H), 2.92 (t, J ¼ 7.59 Hz, 2H), 2.66 (t, J ¼ 10.46 Hz, 1H),
2.41 (t, J ¼ 11.46 Hz, 1H), 1.70e1.88 (m, 4H), 1.55e1.69 (m, 4H), 1.44
(qu, J ¼ 7.52 Hz, 2H), 1.32 (qu, J ¼ 7.30 Hz, 2H), 1.00 (q, J ¼ 11.93 Hz,
(MW ¼ 503.97). 1H NMR (400 MHz, DMSO‑d6)
: 8.19 (d, J ¼ 8.61 Hz,
d
1H), 7.86 (d, J ¼ 1.57 Hz, 1H), 7.64 (d, J ¼ 9.00 Hz, 1H), 7.45e7.59 (m,
3H), 4.11 (t, J ¼ 6.26 Hz, 2H), 3.37 (d, J ¼ 10.96 Hz, 2H), 2.93e3.05
(m, 2H), 2.82 (br s, 2H), 1.66e1.87 (m, 6H), 1.60 (br s, 1H), 1.42e1.53
(m, 2H), 1.26e1.41 (m, 2H), 0.92 (d, J ¼ 6.65 Hz, 3H). IR (cmꢀ1):
1656.54 (-C¼O), 1214.03(¼CeOeC-), 1074.77 (¼CeOeC-). LC-MS:
purity 100% tR ¼ 6.29, (ESI) m/z [MþH]þ 414.17. Anal. calcd. for
1H), 0.84 (d, J ¼ 6.59 Hz, 3H). 13C NMR (126 MHz, DMSO‑d6)
d: 176.3,
165.1, 156.1, 155.6, 150.7, 136.0, 126.5, 125.5, 124.8, 122.0, 121.0,
120.4, 118.7, 106.9, 68.6, 58.0, 58.0, 57.9, 52.1, 30.6, 28.8, 26.4, 25.6,
23.7, 19.1. LC-MS: purity 100% tR ¼ 5.82, (ESI) m/z [MþH]þ 394.30.
Anal. calcd. for C27H33NO3: C, 67.06; H, 6.88; N, 2.90%. Found: C,
67.40; H, 6.83; N, 2.89%.
C26H30NO7Cl: C, 61.96; H, 6.00; N, 2.78%. Found: C, 61.54; H, 5.86; N,
6-chloro-2-(6-(3-methylpiperidin-1-yl)hexyloxy)-9H-xanthen-9-
one oxalate (15). Synthesis from 2-(6-bromohexyloxy)-6-chloro-
9H-xanthen-9-one (5e) (0.4 g, 0.001 mol) and 3-methylpiperidine
(0.20 g, 0.002 mol). After purification was obtained 0.17 g of oil.
Raw product was transformed into oxalic acid salt yielding 0.16 g
(31%) of final compound. Mp: 193e196 ꢂC. C25H30NO3Cl x C2H2O4
2.67%.
7-Chloro-2-(5-(4-methylpiperidin-1-yl)pentyloxy)-9H-xanthen-
9-one oxalate (19). Synthesis from 2-(5-bromopentyloxy)-7-chloro-
9H-xanthen-9-one (5c) (0.40 g, 0.001 mol) and 4-methylpiperidine
(0.20 g, 0.002 mol). After purification was obtained 0.22 g of oil.
Raw product was transformed into oxalic acid salt yielding 0.21 g
(42%) of final compound. Mp: 205e208 ꢂC. C24H28NO3Cl x C2H2O4
(MW ¼ 518.00). 1H NMR (400 MHz, DMSO‑d6)
d: 8.08e8.25 (m, 1H),
7.86 (d, J ¼ 1.76 Hz, 1H), 7.59e7.70 (m, 1H), 7.39e7.58 (m, 3H), 4.10
(t, J ¼ 6.39 Hz, 2H), 3.22e3.42 (m, 2H), 2.97 (br s, 2H), 2.69 (d,
J ¼ 12.32 Hz, 1H), 2.35e2.48 (m, 1H), 1.58e1.88 (m, 7H), 1.43e1.55
(m, 2H), 1.31e1.41 (m, 2H), 1.17e1.28 (m, 1H), 0.99e1.08 (m, 1H),
0.89 (d, J ¼ 6.17 Hz, 3H). IR (cmꢀ1): 1616.51 (-C¼O), 1213.87
(¼CeOeC-), 1073.96 (¼CeOeC-). LC-MS: purity 95.19% tR ¼ 6.65,
(ESI) m/z [MþH]þ 428.19. Anal. calcd. for C27H32NO7Cl: C, 62.60; H,
6.23; N, 2.70%. Found: C, 62.29; H, 5.96; N, 2.52%.
(MW ¼ 503.97). 1H NMR (400 MHz, DMSO‑d6)
d: 8.13 (br s, 1H), 7.92
(d, J ¼ 7.9 Hz, 1H), 7.63e7.81 (m, 2H), 7.41e7.62 (m, 2H), 4.12 (br s,
2H), 3.29e3.44 (m, 3H), 3.01 (br s, 2H), 2.84 (br s, 1H), 1.17e2.03 (m,
11H þ H of solvent EtOH), 0.93 (d, J ¼ 5.3 Hz, 3H). LC-MS: purity
98.24% tR ¼ 6.27, (ESI) m/z [MþH]þ 414.24. Anal. calcd. for
C26H30NO7Cl: C, 61.96; H, 6.00; N, 2.78%. Found: C, 61.90; H, 5.75; N,
2.64%.
2-(6-(4-Methylpiperidin-1-yl)hexyloxy)-9H-xanthen-9-one oxa-
2-Chloro-7-(6-(3-methylpiperidin-1-yl)hexyloxy)-9H-xanthen-9-
one oxalate (16). Synthesis from 2-(6-bromohexyloxy)-7-chloro-
9H-xanthen-9-one (5f) (0.41 g, 0.001 mol) and 3-methylpiperidine
(0.20 g, 0.002 mol). After purification was obtained 0.20 g of oil.
Raw product was transformed into oxalic acid salt yielding 0.18 g
(35%) of final compound. Mp: 164e166 ꢂC. C25H30NO3Cl x C2H2O4
late (20). Synthesis from 2-(6-bromohexyloxy)-9H-xanthen-9-one
(5d) (0.38 g, 0.001 mol) and 4-methylpiperidine (0.20 g,
0.002 mol). After purification was obtained 0.25 g of oil. Raw
product was transformed into oxalic acid salt yielding 0.23 g (48%)
of final compound. Mp: 186e189 ꢂC. C25H31NO3
x
C2H2O4
(MW ¼ 483,54). 1H NMR (500 MHz, DMSO‑d6)
d
: 8.16 (dd, J ¼ 1.1,
(MW ¼ 518.00). 1H NMR (400 MHz, DMSO‑d6)
d: 8.09 (d, J ¼ 2.3 Hz,
8.0 Hz, 1H), 7.90e7.78 (m, 1H), 7.62 (t, J ¼ 8.6 Hz, 2H), 7.52 (d,
J ¼ 2.9 Hz, 1H), 7.41e7.47 (m, 2H), 4.05 (t, J ¼ 6.3 Hz, 2H), 3.32 (d,
J ¼ 10.3 Hz, 2H), 2.86e2.98 (m, 2H), 2.78 (br s, 2H), 1.67e1.78 (m,
4H), 1.49e1.67 (m, 3H), 1.37e1.49 (m, 2H), 1.23e1.37 (m, 4H), 0.87
(d, J ¼ 6.3 Hz, 3H). LC-MS: purity 98.95% tR ¼ 5.90, (ESI) m/z [MþH]þ
394.23. Anal. calcd. for C27H33NO7: C, 67.06; H, 6.88; N, 2.90%.
Found: C, 67.35; H, 6.91; N, 2.86%.
1H), 7.89 (dd, J ¼ 2.7, 9.0 Hz, 1H), 7.72 (d, J ¼ 9.0 Hz, 1H), 7.63 (d,
J ¼ 9.0 Hz, 1H), 7.42e7.55 (m, 2H), 4.08 (t, J ¼ 6.5 Hz, 2H), 3.25e3.44
(m, 2H), 2.88e3.04 (t, 2H), 2.70 (t, J ¼ 10.96 Hz, 1H), 2.44 (t,
J ¼ 11.7 Hz, 1H), 1.60e1.94 (m, 8H), 1.48 (qu, J ¼ 7.3 Hz, 2H),
1.30e1.41 (qu, J ¼ 7.04, Hz 2H), 1.05 (q, J ¼ 12.27 Hz, 1H), 0.89 (d,
J ¼ 6.7 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6)
d: 175.3, 165.0, 155.7,
154.5, 150.6, 135.5, 128.9, 125.8, 125.2, 122.0, 121.6, 121.2, 120.3,
106.8, 68.5, 57.9, 52.0, 30.6, 28.7, 26.3, 25.5, 23.7, 19.1. IR (cmꢀ1):
1655.97 (-C¼O), 1295.19 (¼CeOeC-), 1022.74 (¼CeOeC-). LC-MS:
purity 99.33% tR ¼ 6.61, (ESI) m/z [MþH]þ 428.38. Anal. calcd. for
6-Chloro-2-(6-(4-methylpiperidin-1-yl)hexyloxy)-9H-xanthen-9-
one oxalate (21). Synthesis from 2-(6-bromohexyloxy)-6-chloro-
9H-xanthen-9-one (5e) (0.41 g, 0.001 mol) and 4-methylpiperidine
(0.20 g, 0.002 mol). After purification was obtained 0.16 g of oil. Raw
product was transformed into oxalic acid salt yielding 0.16 g (31%)
of final compound. Mp: 187e190 ꢂC. C25H30NO3Cl x C2H2O4
C
27H32NO7Cl: C, 62.60; H, 6.23; N, 2.70%. Found: C, 62.29; H, 6.13; N,
2.60%.
2-(5-(4-methylpiperidin-1-yl)pentyloxy)-9H-xanthen-9-one oxa-
(MW ¼ 518.00). 1H NMR (400 MHz, DMSO‑d6)
: 8.20 (d, J ¼ 8.36 Hz,
d
late (17). Synthesis from 2-(5-bromopentyloxy)-9H-xanthen-9-one
(5a) (0.36 g, 0.001 mol) and 4-methylpiperidine (0.20 g, 0.002 mol).
After purification was obtained 0.28 g of oil. Raw product was
transformed into oxalic acid salt yielding 0.27 g (58%) of final
compound. Mp: 175e177 ꢂC. C24H29NO3 x C2H2O4 (MW ¼ 469.52).
1H), 7.86 (s, 1H), 7.60e7.73 (m, 1H), 7.40e7.58 (m, 3H), 4.03e4.17
(m, 2H), 3.26e3.47 (m, 3H), 2.99 (br s, 2H), 2.84 (br s, 1H), 1.72e1.85
(m, 4H), 1.67 (br s, 2H), 1.31e1.53 (m, 6H), 1.01e1.10 (m, 1H), 0.92 (d,
J ¼ 6.16 Hz, 3H). IR (cmꢀ1): 1609.59 (-C¼O), 1215.36 (¼CeOeC-),
1074.05 (¼CeOeC-). LC-MS: purity 93.91% tR ¼ 6.65, (ESI) m/z
[MþH]þ 428.13. Anal. calcd. for C27H32NO3Cl: C, 62.60; H, 6.23; N,
2.70%. Found: C, 62.37; H, 6.48; N, 2.74%.
1H NMR (400 MHz, DMSO‑d6)
d
: 8.20 (dd, J ¼ 1.17, 7.83 Hz, 1H),
7.80e7.93 (m, 1H), 7.65 (t, J ¼ 8.80 Hz, 2H), 7.55 (d, J ¼ 3.13 Hz, 1H),
7.41e7.51 (m, 2H), 4.09 (t, J ¼ 6.26 Hz, 2H), 3.37 (d, J ¼ 11.35 Hz, 2H),
2.93e3.07 (m, 2H), 2.83 (t, J ¼ 11.54 Hz, 2H), 1.66e1.87 (m, 6H), 1.60
(br s, 1H), 1.31e1.53 (m, 4H), 0.92 (d, J ¼ 6.26 Hz, 3H). 13C NMR
2-Chloro-7-(6-(4-methylpiperidin-1-yl)hexyloxy)-9H-xanthen-9-
one oxalate (22). Synthesis from 2-(6-bromohexyloxy)-7-chloro-
9H-xanthen-9-one (5f) (0.41 g, 0.001 mol) and 4-methylpiperidine

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D. Łazewska et al. / European Journal of Medicinal Chemistry 207 (2020) 112743
15
(0.20 g, 0.002 mol). After purification was obtained 0.20 g of oil.
Raw product was transformed into oxalic acid salt yielding 0.18 g
(35%) of final compound. Mp: 182e185 ꢂC. C25H30NO3Cl x C2H2O4
purification was obtained 0.26 g of oil. Raw product was trans-
formed into oxalic acid salt yielding 0.20 g (42%) of final compound.
Mp: 180e182 ꢂC. C25H31NO3 x C2H2O4 (MW ¼ 483.54). 1H NMR
(MW ¼ 518.00). 1H NMR (400 MHz, DMSO‑d6)
d
: 8.11 (d, J ¼ 2.7 Hz,
(400 MHz, DMSO‑d6)
d
: 8.20 (dd, J ¼ 1.17, 8.22 Hz,1H), 7.81e7.93 (m,
1H), 7.91 (dd, J ¼ 2.7, 9.0 Hz, 1H), 7.74 (d, J ¼ 9.0 Hz, 1H), 7.66 (d,
J ¼ 9.4 Hz, 1H), 7.45e7.58 (m, 2H), 4.10 (t, J ¼ 6.25 Hz, 2H),
3.30e3.40 (m, 2H), 2.97 (t, J ¼ 7.8 Hz, 2H), 2.65e2.85 (br s, 2H),
1.71e1.86 (m, 4H), 1.55e1.72 (m, 3H), 1.43e1.54 (m, 2H), 1.27e1.42
(m, 4H), 0.92 (d, J ¼ 6.3 Hz, 3H). IR (cmꢀ1): 1666.59 (-C¼O), 1295.97
(¼CeOeC-), 1043.16 (¼CeOeC-). LC-MS: purity 97.92% tR ¼ 6.64,
(ESI) m/z [MþH]þ 428.38. Anal. calcd. for C27H32NO7Cl: C, 62.60; H,
6.23; N, 2.70%. Found: C, 62.21; H, 6.11; N, 2.51%.
1H), 7.65 (t, J ¼ 8.41 Hz, 2H), 7.55 (d, J ¼ 2.74 Hz, 1H), 7.42e7.51 (m,
2H), 4.09 (t, J ¼ 6.26 Hz, 2H), 3.20 (br s, 4H), 2.95e3.08 (m, 2H),
1.54e1.89 (m, 12H), 1.43e1.53 (m, 2H), 1.31e1.41 (m, 2H). 13C NMR
(101 MHz, DMSO‑d6)
d: 176.24, 165.10, 155.98, 155.55, 150.66,
135.84, 126.39, 125.44, 124.67, 121.96, 120.95, 120.26, 118.64, 106.87,
68.49, 56.70, 53.98, 28.76, 26.31, 23.52. IR (cmꢀ1): 1654.09 (-C¼O),
1626.78, 1216.75 (¼CeOeC-), 1025.42 (¼CeOeC-). LC-MS: purity
97.44% tR ¼ 5.77, (ESI) m/z [MþH]þ 394.23. Anal. calcd. for
2-(5-(Azepan-1-yl)pentyloxy)-9H-xanthen-9-one oxalate (23).
Synthesis from 2-(5-bromopentyloxy)-9H-xanthen-9-one (5a)
(0.36 g, 0.001 mol) and azepane (0.20 g, 0.002 mol). After purifi-
cation was obtained 0.30 g of oil. Raw product was transformed into
oxalic acid salt yielding 0.32 g (68%) of final compound. Mp:
140e142 ꢂC. C24H29NO3 x C2H2O4 (MW ¼ 469.52). 1H NMR
C27H33NO7: C, 67.06; H, 6.88; N, 2.90%. Found: C, 66.91; H, 6.70; N,
2.75%.
2-(6-(azepan-1-yl)hexyloxy)-6-chloro-9H-xanthen-9-one oxalate
(27). Synthesis from 2-(6-bromohexyloxy)-6-chloro-9H-xanthen-
9-one (5e) (0.41 g, 0.001 mol) and azepane (0.20 g, 0.002 mol). After
purification was obtained 0.09 g of oil. Raw product was trans-
formed into oxalic acid salt yielding 0.07 g (14%) of final compound.
Mp: 190e193 ꢂC. C25H30NO3Cl x C2H2O4 (MW ¼ 518.00). 1H NMR
(400 MHz, DMSO‑d6)
d
: 8.20 (dd, J ¼ 7.83, 1.17 Hz,1H), 7.84e7.91 (m,
1H), 7.65 (t, J ¼ 8.80 Hz, 2H), 7.55 (d, J ¼ 3.13 Hz, 1H), 7.44e7.51 (m,
2H), 4.09 (t, J ¼ 6.26 Hz, 2H), 3.37 (d, J ¼ 11.35 Hz, 2H), 2.95e3.03
(m, 2H), 2.83 (m, J ¼ 11.54 Hz, 2H), 1.67e1.84 (m, 6H), 1.60 (br s, 1H),
1.32e1.53 (m, 4H), 0.92 (d, J ¼ 6.26 Hz, 3H). 13C NMR (101 MHz,
(400 MHz, DMSO‑d6)
d
: 8.19 (d, J ¼ 8.61 Hz, 1H), 7.85 (d, J ¼ 1.56 Hz,
1H), 7.64 (d, J ¼ 9.00 Hz, 1H), 7.46e7.58 (m, 3H), 4.08e4.13 (m, 2H),
3.19e3.35 (m, 3H), 2.95e3.18 (m, 3H), 1.79 (br s, 6H), 1.56e1.70 (m,
6H), 1.44e1.52 (m, 2H), 1.33e1.42 (m, 2H). 13C NMR (101 MHz,
DMSO‑d6) d: 176.24, 165.13, 155.98, 155.52, 150.68, 135.84, 126.39,
125.43, 124.67, 121.96, 120.95, 120.26, 118.64, 106.92, 68.33, 56.65,
54.00, 28.53, 26.53, 23.88, 23.54, 23.24. IR (cmꢀ1): 1652.51 (-C¼O),
1212,53 (¼CeOeC-), 1027.28 (¼CeOeC-). LC-MS: purity 100%
tR ¼ 5.42, (ESI) m/z [MþH]þ 380.21. Anal. calcd. for C26H31NO7: C,
66.51; H, 6.65; N, 2.98%. Found: C, 66.42; H, 6.88; N, 2.87%.
DMSO‑d6) d 175.6, 164.8, 156.3, 155.8, 150.6, 140.1, 128.3, 125.6,
125.3, 122.0, 120.3, 119.9, 118.5, 107.0, 68.6, 56.7, 54.1, 28.7, 26.4,
26.3, 25.5, 24.0, 23.5. IR (cmꢀ1): 1614.41 (-C¼O), 1214.76 (¼CeOeC-
), 1074.28 (¼CeOeC-). LC-MS: purity 94.98% tR ¼ 6.57, (ESI) m/z
[MþH]þ 428.19. Anal. calcd. for C27H32NO7Cl: C, 62.60; H, 6.23; N,
2.70%. Found: C, 62.23; H, 6.25; N, 2.53%.
2-(5-(Azepan-1-yl)pentyloxy)-6-chloro-9H-xanthen-9-one oxa-
late (24). Synthesis from 2-(5-bromopentyloxy)-6-chloro-9H-
xanthen-9-one (5b) (0.36 g, 0.001 mol) and azepane (0.20 g,
0.002 mol). After purification was obtained 0.16 g of oil. Raw
product was transformed into oxalic acid salt yielding 0.16 g (32%)
of final compound. Mp: 139e141 ꢂC. C24H28NO3Cl x C2H2O4
2-(6-(Azepan-1-yl)hexyloxy)-7-chloro-9H-xanthen-9-one oxalate
(28). Synthesis from 2-(6-bromohexyloxy)-7-chloro-9H-xanthen-
9-one (5f) (0.41 g, 0.001 mol) and azepane (0.20 g, 0.002 mol). After
purification was obtained 0.20 g of oil. Raw product was trans-
formed into oxalic acid salt yielding 0.16 g (31%) of final compound.
Mp: 172e175 ꢂC. C25H30NO3Cl x C2H2O4 (MW ¼ 518.00). 1H NMR
(MW ¼ 503.97). 1H NMR (400 MHz, DMSO‑d6)
8.15 (d, J ¼ 8.61 Hz,
d
1H), 7.80 (d, J ¼ 1.96 Hz, 1H), 7.59 (d, J ¼ 9.00 Hz, 1H), 7.54e7.42 (m,
3H), 4.08 (t, J ¼ 6.26 Hz, 2H), 3.31e3.14 (m, 4H), 3.12e2.98 (m, 2H),
1.89e1.68 (m, 8H), 1.60 (br s, 4H), 1.54e1.39 (m, 2H). 13C NMR
(400 MHz, DMSO‑d6)
d
: 8.08 (d, J ¼ 2.3 Hz, 1H), 7.89 (dd, J ¼ 2.3,
9.0 Hz, 1H), 7.71 (d, J ¼ 9.0 Hz, 1H), 7.62 (d, J ¼ 9.0 Hz,1H), 7.39e7.55
(m, 2H), 4.07 (t, J ¼ 6.3 Hz, 2H), 3.20 (br. s. 4H), 2.93e3.09 (m, 2H),
1.78 (m, 6H), 1.64e1.73 (m, 2H), 1.60 (m, 4H), 1.42e1.53 (qu, 2H),
(101 MHz, DMSO‑d6)
d: 176.24, 165.13, 155.98, 155.52, 150.68,
135.84, 126.39, 125.43, 124.67, 121.96, 120.95, 120.26, 118.64, 106.92,
68.33, 56.65, 54.00, 28.53, 26.53, 23.88, 23.54, 23.24. IR (cmꢀ1):
1656.96 (-C¼O), 1616.48, 1213.44 (¼CeOeC-), 1074.49 (¼CeOeC-).
LC-MS: purity 100% tR ¼ 6.21, (ESI) m/z [MþH]þ 414.17. Anal. calcd.
for C26H30NO7Cl: C, 61.96; H, 6.00; N, 2.78%. Found: C, 61.62; H,
5.85; N, 2.65%.
2-(5-(Azepan-1-yl)pentyloxy)-7-chloro-9H-xanthen-9-one oxa-
late (25). Synthesis from 2-(5-bromopentyloxy)-7-chloro-9H-
xanthen-9-one (5c) (0.40 g, 0.001 mol) and azepane (0.20 g,
0.002 mol). After purification was obtained 0.22 g of oil. Raw
product was transformed into oxalic acid salt yielding 0.21 g (42%)
of final compound. Mp: 183e186 ꢂC. C24H28NO3Cl x C2H2O4
1.29e1.42 (def qu, 2H). 13C NMR (101 MHz, DMSO‑d6)
d: 175.24,
165.13, 155.73, 154.51, 150.58, 135.53, 128.90, 125.79, 125.18, 121.60,
121.16, 120.33, 106.75, 68.53, 56.69, 53.96, 28.75, 26.52, 23.48. IR
(cmꢀ1): 1665.94 (-C¼O), 1295.11(¼CeOeC-), 1022.80 (¼CeOeC-).
LC-MS: purity 99.68% tR ¼ 6.59, (ESI) m/z [MþH]þ 428.38. Anal.
calcd. for C25H30NO3Cl x C2H2O4: C, 62.60; H, 6.23; N, 2.70%. Found:
C, 62.32; H, 6.20; N, 2.60%.
4.2. In vitro biological studies
4.2.1. Radioligand binding assay to histamine H3 receptor
(MW ¼ 503.97). 1H NMR (400 MHz, DMSO‑d6)
d
: 8.05 (d,
Affinity to human histamine H3 receptor was evaluated in a
J ¼ 2.35 Hz, 1H), 7.87 (dd, J ¼ 9.00, 2.74 Hz, 1H), 7.69 (d, J ¼ 9.00 Hz,
1H), 7.60 (d, J ¼ 8.61 Hz, 1H), 7.43e7.54 (m, 2H), 4.07 (t, J ¼ 6.26 Hz,
2H), 3.15e3.31 (m, 4H), 2.97e3.12 (m, 2H), 1.89 (m, 8H), 1.60 (br s,
radioligand binding assay with Na-methylhistamine as a radio-
_
ligand in CHO K1 cells as described by Łazewska et al. [44]. Ten mM
stock solutions of tested compounds were prepared in DMSO. Serial
dilutions of compounds were prepared in 96-well microplate in
assay buffers using automated pipetting system epMotion 5070
(Eppendorf). Each compound was tested in 6 concentrations from
10ꢀ5 to 10ꢀ10 M (final concentration). All assays were carried out in
duplicate. Radioactivity was counted in MicroBeta2 scintillation
counter (PerkinElmer). Data were fitted to a one-site curve-fitting
equation with GraphPad Prism 6 (GraphPad Software, San Diego,
CA, USA) and Ki values were estimated from the ChengꢀPrusoff
equation [64].
4H), 1.47 (qu, J ¼ 7.34 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d:
175.19, 165.23, 155.67, 154.47, 150.56, 135.49, 128.89, 125.75, 125.15,
121.90, 121.55, 121.13, 120.29, 106.76, 68.36, 56.62, 53.94, 28.52,
26.55, 23.83, 23.46, 23.25LC-MS: purity 100% tR ¼ 6.16, (ESI) m/z
[MþH]þ 414.25. Anal. calcd. for C26H30NO7Cl: C, 61.96; H, 6.00; N,
2.78%. Found: C, 62.08; H, 5.79; N, 2.74%.
2-(6-(Azepan-1-yl)hexyloxy)-9H-xanthen-9-one oxalate (26).
Synthesis from 2-(6-bromohexyloxy)-9H-xanthen-9-one (5d)
(0.38 g, 0.001 mol) and azepane (0.20 g, 0.002 mol). After

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D. Łazewska et al. / European Journal of Medicinal Chemistry 207 (2020) 112743
16
4.2.2. Inhibition of cholinesterases
4.3.2. Molecular docking to acetylcholinesterase,
Inhibitory activities of the tested compounds against cholines-
terases were measured using a spectrophotometric method as
described by Ellman et al. [36] with small modifications [16].
Firstly, compounds were tested at a screening concentration of
butyrylcholinesterase and monoamine oxidase B
Docking studies were performed according to the previously
validated procedures [42,44]. The tested compounds were pre-
pared in Corina online. Next, bond and atom types were checked.
Charges and hydrogen atoms were added in Sybyl 8.0 (Tripos). Li-
gands were docked into the active site of enzymes: acetylcholin-
esterase (PDB code: 1EVE), butyrylcholinesterase (PDB code: 1P0I)
and human monoamine oxidase B (PDB code: 4A79). Before dock-
ing, crystal structures of enzymes were downloaded from Protein
Data Bank and prepared as follow: the hydrogen atoms were added,
all histidine were protonated at Nε atoms. Ligand and water mol-
ecules were removed, except water molecules 1159, 1249 and 1254
in AChE. Binding sites were defined within the radius of 10 Å, 20 Å
and 12 Å from the reference ligands e donepezil, butyrate and
pioglitazone for AChE, BuChE and hMAO B, respectively. Docking
studies were performed twice for each enzyme in GOLD 5.1 soft-
ware. Standard parameters of genetic algorithm with population
size 100 and operation number 100,000 were applied. For each
ligand 2 ꢄ 10 poses were generated and sorted according to the
ChemScore value. Binding modes were analyzed in PyMOL
software.
10 mM. To determine IC50 value, the absorbance measured at six
different concentrations of inhibitor was converted to % inhibition
of enzyme and plotted against the applied inhibitor concentration,
using nonlinear regression with GraphPad Prism 5 (GraphPad
Software, San Diego, CA, USA). Donepezil and tacrine were used as
the reference compounds. All assays were performed in triplicate.
4.2.3. Kinetic studies of eeAChE and eqBuChE inhibition
The kinetic studies were performed with compounds 23 and 25,
using Ellman’s method [36], modified for 96-well microplates [16].
Inhibitors were tested in the concentrations giving enzyme activ-
ities between 30% and 80%. For each concentration of the inhibitor,
substrate (ATC/BTC) was added at concentrations of 0.3, 0.24, 0.18,
0.12, 0.06, and 0.04 mM in the wells. Each experiment was per-
formed in triplicate. Vmax and Km values of the MichaelisꢀMenten
kinetics were calculated by nonlinear regression from
substrateꢀvelocity curves. Lineweaver-Burk and Cornish-Bowden
plots were calculated using linear regression in GraphPad Prism 5
(GraphPad Software, San Diego, CA, USA).
4.4. In vivo pharmacological studies
4.4.1. Analgesic effect of selected compounds
4.4.1.1. Animals. The experiments were carried out on adult male
Albino Swiss mice (CD-1, 18e25 g) and male Wistar rats (Krf:(WI)
WU), 180e250 g) obtained from the Animal House of the Faculty of
Pharmacy, Jagiellonian University Medical College. Animals were
housed in plastic cages in room at a constant temperature of 20
2 ꢂC, under light/dark (12:12) cycle and had free access to standard
pellet diet and water. Experimental protocols were proved by the
4.2.4. Inhibition of monoamine oxidase A and B
Inhibition of MAO isoenzymes was evaluated by a fluorometric
method using Amplex Red Monoamine Oxidase kits (ThermoFisher
_
Scientific A12214, Waltham, MA, USA) as described by Łazewska
et al. [44]. For all tests recombinant human MAO B and MAO A
(from Sigma AldrichM7441 and M7316, Darmstadt, Germany) were
used. Inhibitory activity of compounds was measured in the pres-
ꢀ
Local Ethics Committee on Animal Experimentation in Krakow,
ence of p-tyramine (200
m
M). As references were used for MAO-B:
M, rasagiline 1 M, safinamide 1 M, and for MAO-A
M. For hMAO B inhibitory activity compounds were
M. Then, IC50 were obtained from
Poland (Resolution No: 139/2017 and 179/2017). All animal pro-
cedures were in accordance with the European Union Directive
(2010/63/EU) and the Polish regulations.
pargyline 10
clorgyline 1
m
m
m
m
tested in a concentration of 1
m
concentrationeresponse curves. All calculations were made in
Microsoft Excel and GraphPad Prism 5 (GraphPad Software, San
Diego, CA, USA). All experiments were performed in duplicate, and
4.4.1.2. Formalin test in mice. The procedure used was essentially
the same as that described previously [46,47]. Briefly, the pain was
induced by the intraplantar injection of 20 mL of 2.5% formalin so-
data are expressed as mean
ments. For hMAO A inhibition compounds were tested first at a
concentration of 10 M, and then at a concentration of 1 M. None
of compounds showed in these screenings inhibition higher that
50%. Thus, no further studies were performed.
SEM of 2e5 independent experi-
lution into the mice right hind paw. The total time (in s) spent on
licking the injected paw during periods of 0e5 min (early phase,
neurogenic) and 15e30 min (late phase, inflammatory) was
measured and was considered as an indicator of nociceptive
behaviour. Before formalin injection mice were treated i.p. with:
vehicle (10 mL/kg, negative control) or investigated compounds at
the doses of 15, 22.5 and 30 mg/kg.
m
m
4.3. Molecular modeling
4.4.1.3. Carageenan-induced paw oedema, mechanical and thermal
hypernociception in rats. The acute, local inflammation and paw
oedema was induced by subplantar injection of 0.1 mL of 1%
carrageenan (made in PBS) into the rat right hind paw. The paw
volume was measured by the dislocation of the water column of the
plethysmometer (Plethysmometr 7140, Ugo Basile). The pain
pressure threshold was used to measure the hyperalgesic response
to mechanical stimuli. Increasing pressure was applied to the dorsal
surface of the right hind paw by an automated gauge (Analgesy
Meter 37215, Ugo Basile) according to the method of Randall and
Selitto. The intensity of the applied force, in grams, was recorded
when the paw was withdrawn (withdrawal threshold). The
hyperalgesic response to thermal stimuli was determined by using
a plantar test apparatus (Commat Ltd. Turkey). Rats were placed
individually in plexiglas chambers and allowed to acclimatize for
20e30 min before testing. The radiant heat was positioned under
4.3.1. Molecular docking to histamine H3 receptor
€
For docking purposes, Schrodinger Maestro Suite (v. 11.5.011,
Release 2018e01) was used [65]. Ligands were built in their ionized
forms (protonated N1 piperidine nitrogen atom, structure
charge þ1) and their bioactive conformations were generated using
ConfGen module [66,67] (water environment, target number of
conformers e 20). Binding site was centered on ligand placed in
homology model (pitolisant). Docking to rigid form of receptor was
performed using Glide module [68e70] (extra precision, flexible
ligand sampling, max 5 poses per conformer). Ligands were rated
according their position in binding pocket, interactions with
binding pocket amino acids, as well as the docking score value.
€
Ligand interaction diagrams were generated using Schrodinger
Maestro, ligand-receptor visualizations were generated using UCSF
Chimera [71].

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D. Łazewska et al. / European Journal of Medicinal Chemistry 207 (2020) 112743
17
the chamber floor directly beneath the hind paw and the latency to
paw withdrawal was automatically recorded by a photocell and an
electronic timer [48].
rat was placed at the distal ending of one of the open arms facing
away from central platform. Transfer latency time (TLT), the time
required for the rat to enter any of the closed arms with its four
paws, was measured with a cut off-time at 80s. After the animal
entered the closed arm by 10 s the rat was returned to its home
cage. Memory retention was examined 24 h later (test day),
applying the same protocol of the first day, however, evaluating
different test groups (as explained for SIAP) administered with DIZ
(0.1 mg/kg, i.p.), different doses of 23 (1.25, 2.5, and 5 mg/kg, i.p.),
different doses of 25 (1.25, 2.5, and 5 mg/kg, i.p.), or reference drug
donepezil (DOZ, 1 mg/kg, i.p.). Prolonged retention of transfer la-
tency time (TLT) was considered as an indicator of impaired
memory as previously described [54,57,73,74].
The data were analyzed for normality through assessment of the
sample distribution or skewness where ꢀ1.3 to þ1.3 was consid-
ered to be normally distributed. After the results had passed the
tests for normality, they were analyzed where applicable using
one- or two-way ANOVA followed by Tukey’s post hoc test. For
statistical comparisons, the software package SPSS 25.0 (IBM
Middle East, Dubai, UAE) was used. The results are expressed as the
mean and standard error of the mean (SEM). P-values, 0.05 were
considered to indicate statistical significance.
4.4.1.4. Oxaliplatin-induced
neuropathic
pain
in
mice.
Neuropathy was induced by the administration of a single dose
(10 mg/kg) of Oxaliplatin (OXPT) dissolved in a 5% glucose solution
(Polfa Kutno, Poland). To assess the sensitivity to mechanical
stimuli the von Frey test was carried out using the electronic von
Frey unit (Bioseb, France). The apparatus was supplied with a single
flexible filament, which was used to apply increasing force (from
0 to 10 g) against the plantar surface of the hind paw of the mouse.
The crossing of pain threshold resulted in the paw withdrawal and
subsequent recording of the mechanical pressure that evoked the
nocifensive response. The measurement was done before the OXPT
administration, 3 h and 7 days afterwards. The compounds were
administrated to the animals with tactile allodynia observed as a
statistically significant decrease in pain threshold. On the day of the
experiment, each mouse was placed in an observation chamber
with a wire mesh bottom and was allowed to habituate for 1 h.
After the habituation period, the mouse pain threshold was tested 3
times alternately with at least 30 s gap between each measure-
ment. The mean of these three consecutive measurements was
taken as a baseline value. The mice with tactile allodynia were i.p.
pretreated with the tested compound and 30 min later the animals
were tested again according to the same procedure [72].
4.5. Permeability
The PAMPA Plate System Gentest™ was used for estimation of
compounds passive penetration through cell membranes and was
provided by Corning (Tewksbury, MA, USA). The assay was per-
formed in accordance to the manufacturer recommendations and
was previously described by our research group [61]. Briefly, 10 mM
4.4.2. Memory-enhancing effects of test compounds 23 and 25
4.4.2.1. Animals. Inbred male Wistar rats were used in the current
study to assess memory-enhancing effects of test compounds 23
and 25. The rats were obtained from the central animal facility in
UAE University. The animals were kept in a specific room with
controlled temperature and humidity (24 ꢂC 2 ꢂC and 55% 15%,
respectively), 12/12-h light/dark sequence, and free access to food
and water. All the rats were 6e8 weeks of age and weighed
189e230 g. The behavioural experiments assessing memory were
performed each day between 09:00 a.m. and 01:00 p.m. by the
same investigator in a blinded manner; all procedures were per-
formed in accordance with the guidelines of the European Com-
munities Council Directive of November 24, 1986 (86/609/EEC) and
approval was obtained from the Institutional Animal Ethics Com-
mittee in College of Medicine and Health Sciences/United Arab
Emirates University (A30-13) before the experiments were started.
All efforts were made to minimize the total number of the animals
used and their pain.
stocks (DMSO) of tested compounds were diluted to 200 mM in PBS
(phosphate-buffered saline, pH 7.4) and added to the donor wells.
Wells on the receiver plate were filled only with PBS. After 5 h of
incubation at the room temperature the concentrations of tested
compounds in wells were estimated using the LC/MS method with
an internal standard. Mass spectra (LC/MS) were performed on API
3200 mass spectrometer (Sciex, Framingham, MA, USA) coupled to
HPLC Shimadzu LC-20AT (Shimadzu, Duisburg, Germany). The
assay was performed in triplicate. The permeability coefficient Pe
was calculated according to the formulas described in the literature
[62,75] and compared to the high permeable reference caffeine.
Declaration of competing interest
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.
4.4.2.2. Step-through inhibitory avoidance paradigm (SIAP).
Memory deficit was assessed by step through inhibitory avoidance
apparatus (Step-through Cage, 7550; Ugo Basile, Comerio, Italy), as
described previously [15,52e58]. The experiment consists of two
trials (training and testing), in the training day each rat was placed
in the white compartment and after 30 s habituation time the door
was raised automatically and once the rat with all his four paws
entered the dark compartment the door was closed and a foot
shock of 0.4 mA (20 Hz, 8.3 ms) was delivered to the grid floor for
3 s, and step-through latency (STL) time was measured. Immedi-
ately after the foot shock rats were returned to its home cage. Rats
that had step through latency more than 60 s were excluded from
the experiment. 24 h later, in the test day STL time was measured in
the same way with no foot shock, however the cut off-time was
increased to 300 s. The reduction of STL time was used as an in-
dicator for memory impairment.
Acknowledgments
This research was funded by National Science Centre, Poland
grant: No UMO-2016/23/B/NZ7/02327 (DŁ) (synthesis and in vitro
studies). In vivo analgesic studies were financially supported by
Jagiellonian University Medical College grant no. N42/DBS/000047
(SM). Support for in vivo procognitive studies was provided to BS by
College of Medicine and Health Sciences and the Office of Graduate
Studies and Research, United Arab Emirates University (UAEU
Program for Advanced Research, grant number#31M310). Further,
the authors (DŁ, KK-K) acknowledge the contribution of EU-COST
action CA18133 (“European Research Network on Signal
Transduction”).
Appendix A. Supplementary data
4.4.2.3. Transfer latency paradigm time (TLPT) in elevated plus maze
test. During the acquisition trial in the first day (training day) each
Supplementary data to this article can be found online at

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18
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