Cinnamoyl-memantine hybrids: Synthesis, X-ray crystallography and biological activities

Article abstract of DOI:10.1016/j.molstruc.2021.130147

Herein, framework combinations of antioxidant substituted cinnamic acids and memantine (N-methyl-D-aspartate receptor antagonist) in a new multi-targeted chemical entity were described. The amide bond formation of the memantine hybrids 1–5 was performed by EDC/HOBt coupling reaction. The chemical structures of the synthesized compounds were confirmed by means of melting points, UV, IR, ¹H NMR, ¹³C NMR, and HRMS. Additionally, the crystal structures of memantine hybrids (2–5) were also studied by single-crystal X-ray diffraction. The single-crystal X-ray analysis revealed that the compounds 2, 5 crystallize in a centrosymmetric manner both in monoclinic space group (SG) P2₁/c, (No 14) and in a non-centrosymmetric manner for compounds 3 and 4, SG R3, (No 146) and SG P2₁2₁2₁, (No 19), respectively. Furthermore, preliminary in vitro screenings of their neuroprotective and radical scavenging activities were performed. The radical scavenging activity of synthesized memantine hybrids was measured against 1,1-diphenyl-2-picrylhydrazyl (DPPH^●), hydroxyl (OH^●) and superoxide (O2^●?) radicals and compared with the standard antioxidants (ferulic and sinapic acids). Radical scavenging activity studies show that amongst the tested hybrids, N-sinapoyl amide of memantine (3) emerges as the most potent antioxidant in all tests. Moreover, in vitro evaluation of anti-Alzheimer effects showed that the obtained memantine hybrids displayed neuroprotection in the moderate levels. Generally, they possess a little weaker activity as compared to the positive control memantine. Taken together, our findings reveal that the N-sinapoylamide of memantine (3) can be considered as a promising neuroprotective agent for Alzheimer's disease, acting as well as a potent radical scavenger.

Full text of DOI:10.1016/j.molstruc.2021.130147

Journal of Molecular Structure 1234 (2021) 130147
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Cinnamoyl-memantine hybrids: Synthesis, X-ray crystallography and
biological activities
a,∗
b
a
a
c
Maya Chochkova , Hailun Jiang , Radoslava Kyoseva , Boyka Stoykova , Elina Tsvetanova ,
d
c
b
b
e
Albena Alexandrova , Rui Liu , Zhuorong Li , Yavor Mitrev , Hristina Dimitrova-Sbirkova ,
f
e
Martin Štícha , Boris Shivachev
a
Faculty of Mathematics and Natural Sciences, South-West University ‘‘Neofit Rilski’’, 66, Ivan Mihailov Str., 2700 Blagoevgrad, Bulgaria
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P. R. China
Laboratory of Free Radical Processes, Institute of Neurobiology, Bulgarian Academyof Sciences, Acad. G. Bonchev Bl.23, 1113 Sofia, Bulgaria
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. Bl. 9, Sofia 1113, Bulgaria
Institute of Mineralogy and Crystallography “Acad. Ivan Kostov”, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bl. 107, 1113 Sofia, Bulgaria
Department of Chemistry, Faculty of Science, Charles University, Prague 2, Czech Republic
b
c
d
e
f
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, framework combinations of antioxidant substituted cinnamic acids and memantine (N-methyl-D-
aspartate receptor antagonist) in a new multi-targeted chemical entity were described. The amide bond
formation of the memantine hybrids 1–5 was performed by EDC/HOBt coupling reaction. The chemical
structures of the synthesized compounds were confirmed by means of melting points, UV, IR, 1H NMR,
13 C NMR, and HRMS. Additionally, the crystal structures of memantine hybrids (2–5) were also studied
by single-crystal X-ray diffraction. The single-crystal X-ray analysis revealed that the compounds 2, 5
crystallize in a centrosymmetric manner both in monoclinic space group (SG) P21/c, (No 14) and in a non-
centrosymmetric manner for compounds 3 and 4, SG R3, (No 146) and SG P2 2 2 , (No 19), respectively.
Received 23 May 2020
Revised 14 February 2021
Accepted 16 February 2021
Available online 25 February 2021
Keywords:
Memantine
sinapic acid
ferulic acid
1
1 1
Furthermore, preliminary in vitro screenings of their neuroprotective and radical scavenging activi-
3
,4-dimethoxycinnamic acid
dihydro-p-coumaric acid
-N,N-dimethylaminocinnamic acid
ties were performed. The radical scavenging activity of synthesized memantine hybrids was measured
●
●
●▬
against 1,1-diphenyl-2-picrylhydrazyl (DPPH ), hydroxyl (OH ) and superoxide (O2 ) radicals and com-
pared with the standard antioxidants (ferulic and sinapic acids). Radical scavenging activity studies show
that amongst the tested hybrids, N-sinapoyl amide of memantine (3) emerges as the most potent antiox-
idant in all tests.
4
X-ray Crystallography
Quantitative nuclear magnetic resonance
Alzheimer’s disease
Moreover, in vitro evaluation of anti-Alzheimer effects showed that the obtained memantine hybrids
displayed neuroprotection in the moderate levels. Generally, they possess a little weaker activity as com-
pared to the positive control memantine.
Taken together, our findings reveal that the N-sinapoylamide of memantine (3) can be considered as
a promising neuroprotective agent for Alzheimer’s disease, acting as well as a potent radical scavenger.
©
2021 Elsevier B.V. All rights reserved.
1. Introduction
Alzheimer’s disease (AD) represents one of the most prevalent
forms of dementia diagnosed by progressive loss of memory, learn-
ing, and other cognitive functions [1]. AD continues to be an insid-
ious, enigmatic, and irreversible degenerative brain disorder [2, 3],
affecting people not just over age 65, but also younger-onset indi-
viduals. According to World Alzheimer Report 2018, around 50 mil-
lion people worldwide have been afflicted with dementia in 2018,
and this number is expected to reach 152 million by 2050. The
global estimated cost of dementia in 2018 is US$ 1 trillion and
may exceed nearly double by 2030 [4]. Thus, due to this serious
health and economic extent of dementia, in particular AD could be
considered as “modern global epidemic”[5].
Abbreviations: Aβ, amyloid-β; AD, Alzheimer’s disease; CA, cinnamic acid; CAPE,
caffeic acid phenethyl ester; ChEIs, cholinesterase inhibitors; CNS, central nerve sys-
tem; DR, deoxyribose; dihydro-p-CoumA, dihydro-p-coumaric acid; 3,4-(OMe)2CA,
3,4-dimethoxycinnamic acid; 4-NMe2CA, 4-N,N-dimethylaminocinnamic acid; DMF,
dimethylformamide; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; EDTA,
ethylene diamine tetraacetic acid; EtOAc, ethyl acetate; FA, ferulic acid; FDA,
Food and Drug Administration; HE, hexane; HOBt, 1-hydroxybenzotriazole; Mem ·
HCl, memantine hydrochloride; NMDAR, N-methyl-D-aspartate receptor antagonist;
NMM, N-methylmorpholine; qNMR, Quantitative nuclear magnetic resonance; SA,
sinapic acid.
∗
Corresponding author.
E-mail address: mayabg2002@yahoo.com (M. Chochkova).
https://doi.org/10.1016/j.molstruc.2021.130147
0
022-2860/© 2021 Elsevier B.V. All rights reserved.

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
Beyond the genetically and age-related risk factors, to the com-
plex etiopathogenesis of AD is implicated either the other hall-
marks, such as the accumulation of amyloid β (Aβ) plaques,
the formation of intraneuronal neurofibrillary tangles (NFTs) of
a hyperphosphorylated form of the tau protein, dyshomeostasis
of biometals, decreased of acetylcholine (ACh) level and oxidative
stress [1, 6]. Interestingly, the infectious etiology of AD has been
continuously debated over the past three decades [7-9].
Recently, by following a linking strategy were merged me-
mantine and antioxidant ferulic acid. Interestingly, lengthening
of the spacer between two pharmacophoric functions up to six
methylenes led to the most potent NMDAR antagonist, being only
three times less active than the parent memantine [28].
Based on the multifactorial etiology of AD and enforces from
multi-target strategy for achievement of anti-AD lead candidates
[16], herein, we combined into one hybrid molecule a memantine
core with the antioxidant cinnamic acids.
However, to date, no effective cure for AD is available since the
known drugs modestly alleviate the symptoms but none of them
are able to halt the progression and to overcome the fatal character
of the disease [10].
Furthermore, the synthesized compounds were subjected to a
preliminary in vitro evaluation of their neuroprotective and radical
scavenging activities.
Despite the first Food and Drug Administration (FDA) ap-
proval of tacrine (tetrahydroaminoacridine) as cholinesterase in-
hibitor (ChEI), its use was withdrawn from the pharmaceutical
market soon after its hepatotoxicity was apparent [11]. There-
fore, the current therapeutic arsenal against AD has been lim-
ited only to three clinically approved ChEIs: galantamine [12], ri-
vastigmine [13], donepezil [14], as well as the first and the only
N-methyl- D-aspartate receptor antagonist (NMDAR) – memantine
2
. Experimental section
2
.1. Materials and equipment
Reagents and solvents were purchased mostly from Thermo
Fisher Scientific
- Bulgaria or Sigma Aldrich (FOT, Bulgaria)
and were used without further purification. Analytical thin-layer
chromatography (TLC) was carried out using Merck silica gel
(
3,5-dimethyl-1-adamantylamine; Mem). The latter has a distinct
6
0F254 plates. Visualization was accomplished with UV light
mode of action compared with that of ChEIs. Its protective role
in the brain is thought to be associated with the regulation of
the activity of glutamate. Alzheimer’s brain cells release more glu-
tamate than normal leading to excitoneurotoxicity and neuronal
cell death [15]. Regardless of the tremendous efforts of medicinal
chemists and growing investments to treat this dreadful disorder,
the development of an efficient drug and therapeutic strategy is
needed. Indeed, over the last two decades, the hybrid-based ap-
proach has played a cardinal role to combat different diseases with
complex etiologies, including AD [6]. Therefore, various encourag-
ing results for hybrids directed to diverse AD targets have been
identified and developed at a pre-clinical level [5, 16]. Undoubt-
edly, these ligands comprise properties as metal chelators, antioxi-
dant or anti-inflammatory activity, or prevention of Aβ or tau ag-
gregation. Due to the huge impact of AD affected elderly popula-
tion, the oxidative stress theory of aging has also been a decisive
moment for progression of disease [17]. Indeed, the beneficial ef-
fects of antioxidants against pathological states and diseases with
oxidative stress etiology such as AD, cancer and etc. have been
proven [18].
(
λ=254 nm) and then with Ce-PMo reagent: 10 g Ce (SO ) , 25 g
4
2
H [P(Mo O ) ] x H O, 940 mL H O, 60 mL conc. H SO ) solution
3
3
10
4
2
2
2
4
followed by heating. The synthesized compounds were separated
by column chromatography using silica gel (Acros Organics, mesh
3
5–70).
Melting points were measured in glass capillary tubes on a
„
Stuart SMP 10“apparatus and are uncorrected. UV spectra of the
ꢀ
compounds were measured with an ‘‘Agilent 8453 ’ UV–vis spec-
trophotometer. Attenuated total reflectance infrared spectroscopy
(
ATR-IR) measurements were performed using Thermo Scientific
Nicolet iS10 FT-IR device with ID5 ATR accessory (diamond crys-
tal). All NMR spectra were recorded on Bruker Avance II+ spec-
trometer (14.09 T magnet), operating at 600.01 MHz 1H frequen-
cies, equipped with 5 mm BBO probe with z-gradient coil. The tem-
perature is maintained at 293 K, using Bruker B-VT 3000 tempera-
ture unit with airflow of 535 L/h. All chemical shifts are reported
in parts per million (ppm), referenced against tetramethylsilane
(
TMS, 0.00 ppm) or using the residual solvent signal of 2.5 ppm for
DMSO).
Two dimensional (2D) 1H/ 1H COZY and (2D) 1H/ 13C HSQC
Hydroxycinnamic acids (e. g. ferulic, sinapic, caffeic acids and
etc.) and their derivatives (esters, amides) are compounds of pro-
found interest due to their plethora of activities, most of them as-
sociated to their antioxidant properties [19]. Moreover, remarkable
anti-aggregating activities have been reported in the literature for
caffeic acid and its phenethyl ester (CAPE) [20]. Recently, it has
been demonstrated that divalent caffeic amides inhibit amyloid-
β fibrillization [21, 22]. In this sense, such compounds can also
serve as beneficial fragments for the combination therapy of vari-
ous neurological disorders [23].
spectra were recorded using standard Bruker pulse sequences
cosygpmfqf and hsqcedetgpsp.3.
The purity of the compounds was assessed by using quantita-
tive 1H measurements, using trimethoxybenzene as internal stan-
dard and was found to be >95% in all cases. Details on sample
preparation, experimental conditions and results can be found in
the Supplementary materials.
Electrospray mass spectrometry (ESI-MS) experiments were ac-
quired on Bruker Compact QTOF-MS (Bruker Daltonics, Bremen,
Germany) and controlled by the Compass 1.9 Control software. The
data analysis was performed and the monoisotopic mass values
were calculated using Data analysis software v 4.4 (Bruker Dal-
tonics, Germany). The analyses were conducted in the positive ion
mode at a scan range from m/z 50 to 1000, and nitrogen was used
as nebulizer gas at a pressure of 4 psi and flow of 3 L/ min for the
dry gas. The capillary voltage and temperature were set at 4500 V
Despite vastly studied cinnamic acid - based hybrids, compris-
ing ChEIs [24], those with NMDAR antagonist memantine are rare.
In this regard, Sozio et al. have designed memantine hybrids with
sulfur containing antioxidants (glutathione or (R)-α-lipoic acid)
[
25].
Moreover, polyamine-memantine hybrids were designed as
NMDA channel blockers. Amongst this series, the most active one
was the triamine derivative of memantine with the guanidine
group. Its antagonistic activity was found to be more potent even
than memantine [26].
●
and 220 °C, respectively. DPPH radical scavenging activity assay
was performed on microplate reader Stat Fax 303 Plus.
2.2. Synthesis and characterization
Other multifunctional agents based on memantine skeleton are
hybrids comprised of two pharmacophores - galantamine (AChE
inhibitor) and memantine through variable-length polymethylene
spacers. An outstanding neuroprotective profile was found for hy-
brid (named memagal), carrying a hexamethylene spacer [27].
2.2.1. Synthesis of substituted cinnamic acids by knoevenagel-doebner
condensation
3,4-Dimethoxy- and 4-dimethylaminocinnamic acids were syn-
thesized using Doebner-Knoevenagel modification condensation
2

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
[
29]. The corresponding substituted benzaldehydes (50 mmol),
0.4 g malonic acid (100 mmol), 30 mL pyridine (370 mmol), and
mL piperidine (20 mmol) were refluxed for 4 h at 70°C. The re-
(DMSO–d6, ppm) δ 167.9 (CO), 150.3 149.3, 138.2 (=CH), 128.3,
1
122.0 (CH), 121.7 (ArCH), 112.1 (ArCH), 110.1 (ArCH), 56.0 (OCH ),
3
2
55.8 (OCH ), 52.8, 50.7, 47.5 (CH ), 47.0 (2 x CH ), 42.8 (2 x CH ),
3
2
2
2
action progress was monitored over silica gel TLC plates using
40.2 (CH ), 32.3, 30.6 (2 x CH ), 30.0 (CH). HRMS (m/z): 370.2378
2 3
+
CH Cl :CH OH=3:0.2. After completing the reaction, pyridine was
(M + H) , calculated: 370.2377.
2
2
3
removed in vacuo and the residue was dissolved in a saturated
aqueous NaHCO3 solution and acidified to a pH = 3 with 6 M HCl.
The resulting precipitate was separated by filtration and washed
with water. The obtained crystals were recrystallized with a solu-
tion 96% ethanol/water and then separated by filtration and dried.
Physical properties and spectroscopic characterization of cin-
namic acids:
N-sinapoylamide of memantine (SA-Mem, 3): White solid
0
(51%). M.p: 193–195 C; UV (CH OH) λmax = 236, 318 nm. IR
3
(ATR)umax: 3298, 2941, 2897, 2840, 1658, 1612, 1511, 1453, 1422,
1205, 1109, 975, 826 сm 1. 1H (DMSO–d6, ppm) δ 8.77 (br 1H,
−
OH), 7.47 (s, 1H, NH), 7.20 (d, 1H, J = 15.6 Hz), 6.8 (s, 2H,), 6.51
(d, 1H, J = 15.6 Hz), 3.78 (s, 6H, 2 x OCH ), 2.09 (m, 1H), 1.81 (m,
3
2H,), 1.64 (d, 2H, J = 11.8 Hz), 1.61 (d, 2H, J = 12.1 Hz), 1.34 (d, 2H,
J = 12.0 Hz), 1.26 (d, 2H, J = 11.8 Hz), 1.12 (m, 2H), 0.83 (s, 6H, 2 x
ꢀ
ꢀ
(
E)−3-(3 ,4 -dimethoxyphenyl)
propenoic
acid
or
3,4-
1
3
dimethoxycinnamic acid was obtained as a white solid (75%).
CH ). C NMR (DMSO–d ppm) δ 164.8 (CO), 148.3, 138.6 (=CH),
3
6,
M.p: 182–184 °C (lit. 181–183 °C). UV (CH OH) λmax = 207, 234,
137.4, 125.8, 121.3 (=CH), 105.3 (Ar-CH), 56.2 (2 x OCH ), 52.7, 50.6
3
3
316 nm. IR (ATR)umax: 2930, 2836, 2596, 1686, 1627, 1593, 1515,
(CH ), 47.5 (2 x CH ), 42.7 (2 x CH ), 39.9 (CH ), 32.3, 32.2, 30.5
2 2 2 2
463,1448, 1417, 1240, 1208, 1140, 1025, 974, 841 сm 1. HRMS
−
(2 x CH ), 29.9 (CH). HRMS (m/z): 386.2326 (M + H) , calculated:
+
1
3
+
+
[
M+Н] : 209.0735 (calculated for: [C11 H13O ] : 209.0736).
386.2326.
4
ꢀ
(
E)−3-(4 -(N,N-dimethylamino)phenyl) propenoic acid or 4-
N-dihydro-p-coumaroylamide of memantine (4): White solid
0
dimethylaminocinnamic acid was obtained as an yellow solid
(45%). M.p:166–169 C. UV (CH OH) λmax = 204, 225, 279 nm. IR
3
(
62%). M.p: 226–228°С, (lit. 227–228°С). UV (CH OH) λmax = 204,
(ATR)umax: 3300, 2942, 2861, 2898, 1632, 1610, 1557, 1511, 1452,
3
2
39, 356 nm. IR (ATR)umax: 2967, 2805, 2588, 1664, 1582, 1523,
1349, 1336, 1288, 1102, 822, 719 сm 1. 1H (DMSO–d6, ppm) δ 9.14
(s, 1H), 7.26 (s, 1H), 6.96 (d, 2H, J = 8.4, CH-Ar), 6.63 (d, 2H, J = 8.4,
CH-Ar), 2.63 (2H, m, CH ), 2.22 (2H, m, CH ), 2.05 (m, 1H, CH),
−
435, 1419, 1308, 1260, 1200, 987, 940, 858, 812, 676 сm 1. HRMS
−
1
+
+
[
M+Н] : 192.1018 (calculated for: [C11 H14 NO ] : 192.1019).
2
2
2
1
.72 (d, 2H, J = 2.7 Hz, CH ), 1.55 (d, 2H, J = 12 Hz, CH ), 1.51 (d,
2
2
2
.2.2. General synthesis method of memantine hybrids (1–5)
2H, J = 12 Hz, CH2), 1.29 (dd, 2H, J = 2.7 Hz), 1.22 (d, 2H, J = 12.5 Hz,
CH2), 1.08 (s, 2H, CH2), 0.80 (s, 6H, 2 x CH3). 13C NMR (DMSO–
d6, ppm) δ 171.4 (CO), 155.8 (Cquat), 132.0 (Cquat), 129.5 (CH-Ar),
115.4 (CH-Ar), 52.5 (Cquat), 50.7 (CH), 47.5(CH), 42.8 (CH), 39.9
(CH), 38.7 (CH), 32.3 (Cquat), 31.0 (CH), 30.6 (CH2), 29.9 (CH2).
The corresponding cinnamic/ dihydro-p-coumaric acids
(
15.4 mmol), 2.09 g (15.4 mmol) HOBt and 2.95 g (15.4 mmol)
EDC were dissolved in 25 mL CH Cl /DMF (1:1.5). The mixture was
2
2
stirred for 6–8 min on an ice bath, and then a solution of 2.77 g
15.4 mmol) memantine hydrochloride and 1.77 mL (15.4 mmol)
+
(
HRMS (m/z): 328.2272 (M + H) , calculated: 328.2271.
NMM in dry CH Cl2 (15 mL) was added. After one hour, the
N-(4-dimethylaminocinnamoyl) amide of memantine (5):
2
0
mixture was allowed to reach room temperature and stirred for an
additional 23 h. Then, the solvents were removed under reduced
pressure, and the residue was dissolved in ethyl acetate (100 mL).
The organic solution was subsequently washed with 5% NaHCO3
White solid (71%). M.p: 155–156 C; UV (CH3OH) λmax = 238,
350 nm; IR (ATR)umax: 3292, 2895, 2843, 1649, 1595, 1544, 1522,
1452, 1351, 1335, 1225, 1212, 1182, 1166, 980, 813 сm 1; 1H
(DMSO–d6, ppm) δ 7.41 (s, 1H, NH), 7.34 (d, 2H, J = 8.9 Hz, CH-
Ar), 7.18 (d, 1H, J = 15.7, =CH), 6.71 (d, 2H, J = 8.9 Hz, CH-Ar), 6.39
(d, 1H, J = 15.7, =CH), 2.94 (s, 6H, 2 x CH3), 2.08 (1H, m, CH2),
2.05 (m, 1H, CH), 1.81 (s, 2H, CH2), 1.65 (d, 2H, J = 12 Hz, CH2),
1.60 (d, 2H, J = 12 Hz, CH2), 1.33 (d, 2H, J = 11.7 Hz), 1.26 (d, 2H,
−
(
3 × 50 mL), 1 M HCl (3 × 50 mL), and brine (3 × 50 mL). After the
evaporation of ethyl acetate, the crude products were purified
by column chromatography (HE/ EtOAc) and recrystallized from
acetonitrile.
13
The complete spectral characterization of hybrids is shown be-
low:
J = 11.8 Hz, CH2), 1.12 (s, 2H, CH2), 0.82 (s, 6H, 2 x CH3) ppm;
C
NMR (DMSO–d6, ppm) δ 165.3 (CO), 151.3 (Cquat), 138.5 (CH),
129.0 (CH), 122.9 (Cquat), 118.6 (CH), 112.4 (CH), 52.6 (Cquat), 50.7
(CH2), 47.5(CH2), 42.7 (CH2), 40.2 (CH), 39.9 (CH2), 32.2 (Cquat),
N-feruloylamide of memantine (FA-Mem, 1) White solid
0
(
65%). M.p: 221–223 C. UV (CH OH) λmax = 218, 234, 293, 318 nm.
3
+
IR (ATR)umax: 3370, 2943, 2897, 2842, 1657, 1590, 1534, 1513, 1451,
30.5 (CH), 29.9 (CH) ppm; HRMS (m/z): 353.2589 (M + H) , calcu-
1
399, 1281, 1255, 1160, 1124, 1033, 973, 816 сm 1. 1H (DMSO–d6,
−
lated: 353.2591.
ppm) δ 9.41 (s, OH), 7.47(s, NH), 7.20 (d, 1H, J = 15.4 Hz), 7.08
(
d, 1H, J = 1.8 Hz), 6.94 (dd, 1H, J = 8.1 Hz, J = 1.8 Hz), 6.78 (d, 1H,
2
2
.3. X-ray diffraction analysis for compounds 2–5
J = 8.1 Hz), 6.48 (d, 1H, J = 15.5 Hz), 3.80, (s, 3H, OCH ), 2.09 (m,
3
1
H), 1.81 (s, 2H), 1.65 (d, 2H, J = 12.0 Hz), 1.61 (d, 2H, J = 12.0 Hz),
.3.1. Crystallization and cocktail soaking
1
.34 (d, 2H, J = 12.1 Hz), 1.26 (d, 2H, J = 12.1 Hz), 1.12 (s, 2H), 0.83 (s,
In order to obtain single crystals with suitable quality the dry
13
6
H). C NMR (DMSO–d6, ppm) δ 165.0 (CO), 148.5, 148.2, 138.5
powder substances of memantine hybrids (2–5) were dissolved in
(
=CH), 127.0, 121.9 (Ar-CH), 121.0 (=CH), 116.0 (Ar-CH), 110.8 (Ar-
ethanol, methanol and acetone at room temperature. Large crys-
CH), 55.9 (OCH ), 52.8, 50.8 (CH ), 47.6 (2 x CH ), 42.8 (2 x CH ),
3
2
2
2
3
tals (0.3 × 0.2 × 0.15 mm ) suitable for single crystal X-ray studies
4
0.0 (CH ), 32.3, 30.6 (2 x CH ), 30.0 (CH). HRMS (m/z): 356.2223
2 3
formed within 2–3 days, by slow evaporation. Single crystals were
obtained from the three solvents used, but the best quality crystal
grew in acetone except compound 5, where methanol conditions
produced crystals.
+
(
M + H) , calculated: 356.2220.
N-(3,4-dimethoxycinnamoyl)amide of memantine (3,4-
0
(
ОМе)2СA-Mem, 2): White solid (78%). M.p: 133–134 C. UV
(
CH OH) λmax = 219, 234, 291, 315 nm.; IR (ATR)umax: 3290, 2941,
3
2
9
1
897, 2839, 1655, 1616, 1599, 1510, 1453, 1258, 1207, 1137, 1024,
78 сm 1
−
.
1H (DMSO–d6, ppm) δ 7.51 (s, 1H, NH), 7.24 (d,
2.3.2. X-ray diffraction data collection, processing and structure
refinement
H, J = 15.6 Hz), 7.10 (d, 1H, J = 2.0 Hz), 7.06 (dd, 1H, J = 8.4 Hz,
J = 2.2 Hz), 6.97 (d, 1H, J = 8.4 Hz), 6.53 (d, 1H, J = 15.6 Hz), 3.79 (s,
A single crystal of each compound was mounted on a glass cap-
illary and all data were collected at room temperature (290 K) on
an Oxford diffraction Supernova diffractometer using Mo-Kα radia-
tion (λ= 0.71013 A) from micro-focus source. The determination of
3
2
H, OCH ), 3.77(s, 3H, OCH ), 2.09 (m, 1H), 1.81 (m, 2H,), 1.65 (d,
3 3
H, J = 11.8 Hz), 1.61 (d, 2H, J = 12.0 Hz), 1.40 (d, 2H, J = 12.0 Hz),
13
˚
1
.27 (d, 2H, J = 11.8 Hz), 1.12 (m, 2H), 0.83 (s, 6H).
C NMR
3

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
unit cell parameters, data integration, scaling and absorption cor-
rections were carried out using the CrysalisPro [30]. The structures
were solved by direct methods ShelxS-2018 [31]. The refinement
of the structures involved several cycles of refinement using full-
matrix least-squares on F2 with the ShelxL-2018 package [31]. The
N hydrogen atoms were positioned from difference Fourier map
while all other hydrogen atoms were placed at idealized positions.
The non-hydrogen atoms were refined anisotropically and hydro-
gen atoms were refined using the riding model. Visual representa-
tions of the molecules present in the asymmetric unit were carried
out using ORTEP [32], while three-dimensional packing and hydro-
gen bonding interactions were drawn using Mercury [33]. The data
in the Crystallographic Information File (coordinates, structure fac-
tors, etc.) have been verified using IUCr checkCIF/ PLATON [34].
Crystallographic data (excluding structure factors) for the struc-
tural analysis were deposited with the Cambridge Crystallographic
Data centre, CCDC No. Deposition Number 1,956,759 of compound
two systems were used to specify the chelating or scavenging po-
tential of the tested substances [37]. After 30 min incubation at
37 °C in absence (control) and presence of increasing concentra-
tions of the tested substances, the reactions were stopped by the
addition of 0.2 mL 2.8% trichloroacetic acid, 0.1 mL 5 N HCl and
0.2 mL thiobarbituric acid (2% w/v in 50 mM NaOH). The samples
were incubated at 100 °C for 15 min for color development and af-
ter cooling the absorbance was read at 532 nm. The absorbance at
532 nm was read against blank sample (without substance); the
absorbance at 600 nm was considered a non-specific base-line drift
and was subtracted from A532
.
2.4.3. Superoxide radical generation system
•
−
The superoxide anion radicals (O2 ) were generated photo-
chemically (by using a mercury lamp) in a medium containing:
−6
50 mM phosphate buffer, pH 7.8; 1.17 × 10 M riboflavin; 0.2 mM
−
5
−5
methionine; 2 × 10 M KCN and 5.6 × 10 M nitro-blue tetra-
•
−
2
(Data Block Name: data_exp_487/ В120); Deposition Number
zolium (NBT) [38]. The generated O2 reduce the presented in the
medium nitro-blue tetrazolium (NTB) to produce blue colored for-
mazan. The absorbance decrease (at 560 nm) of the medium stain-
ing intensity in presence of the tested substances is a measure of
their antioxidant capacity. The antioxidant capacity of the test sub-
stances was expressed as percent inhibition vs. control.
1,957,027 of compound 3 (Data Block Name: data_exp_512/ В122);
Deposition Number 1,956,762 of compound 4 (Data Block Name:
data_exp_486/ В124); Deposition Number 1,956,761 of compound
5
(Data Block Name: data_exp_485/ В125).
A copy of this information may be obtained free of charge from:
The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK. Fax:
44 1223 336 033, e-mail:deposit@ccdc.cam.ac.uk, or www.ccdc.
cam.ac.uk.
+
2.5. In vitro evaluation of protective properties of memantine hybrids
Cell culture and treatment
The human neuroblastoma SH-SY5Y cells, PC12 cells, and EOC20
cells were obtained from ATCC (Manassas, VA, USA) and cultured
in 25 cm2 flask with DMEM (Invitrogen, Carlsbad, CA, USA) sup-
plemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad,
CA, USA) at 37 °C, in a humidified atmosphere with 5% CO2 until
the cells reached confluence between 80% and 90%. The SH-SY5Y
cells transfected with the Swedish mutant form of human APP (re-
ferred to as ‘‘APPswe cells’’) is a cell model imitating the neuro-
toxicity of Aβ triggered by copper [39, 40]. APPswe cells were cul-
tured in DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) sup-
plemented with 10% FBS and 200 μg/mL G418 maintained under
standard conditions of 37 °C and 5% CO2.
2
.3.3. Thermal analyses
Differential thermal analysis (DTA) and thermogravimetric mea-
surements (TG) were carried out simultaneously in a thermal an-
alyzer Stanton Redcroft STA780 at the following conditions: 30–
10 °C temperature range, a heating rate of 5 °C. min–1, dry argon
2
as a carrier gas (∼30 mL. min–1) and sample weight of 9–17 mg,
placed in corundum crucible. The stability of the compounds and
observed endo and exo effects were derived from these studies and
were employed in fixing the crystal growth conditions.
2
2
3
.4. In vitro radical scavenging assays
For neuroprotective properties of memantine hybrids studies,
●
.4.1. DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging assay
4
the cells were seeded in 96-well plates with density of 1 × 10
The DPPH assay was performed on microplate reader Stat Fax
cells per well. The SH-SY5Y cells were treated with meman-
tine structural derivatives and memantine hydrochloride (Sigma-
Aldrich, Cambridge, MA, USA) for 4 h prior to and during the pro-
cess of 100 μM NMDA [41, 42] [42] - and 200 μM H O - induced
03 Plus according to a modified method [35]. A methanolic stock
●
solution (0.1 mM) of the DPPH radical was freshly prepared and
protected from light by aluminum folio. Briefly, 10 ul of the 3.6 mM
methanolic solutions of the tested compounds (1–5) were added
2
2
excitotoxicity, respectively. The PC12 cells were used to establish
●
to DPPH solution (290 ul). The blank reference cuvette contained
a hypoxic condition and subjected to 150 μM CoCl . The tested
2
3
00 ul methanol.
The absorbance of DPPH solution at 515 nm was measured be-
memantine derivatives and memantine hydrochloride were added
●
4
h prior to and during NMDA, H O , and CoCl treatment. The
2 2 2
fore (Absstart) and 10 min after adding the solutions of the tested
compounds (Absend). Ferulic- and sinapic acids were used as posi-
tive controls.
APPswe cells were subjected to 300 μM copper to generate Aβ
overproduction. All tested compounds were added at the start of
a copper-initiated injury. Memantine derivatives and memantine
hydrochloride concentrations were used as follows: 0 (control),
The results were expressed in triplicate as the percentage of
radical scavenging activity (RSA%), calculated as follows:
RSA% = [(Absstart- Absend)/Absstart]x100
−4 −3 −2 −1
10
μM, 10 μM, 10 μM, 10 μM, 1 μM, 10 μM, and 100.0 μM.
Cytotoxicity assays
The cytotoxicity effect was investigated using 3-(4,5-
2
.4.2. Hydroxyl radicals generating system
dimethylthiazol-2-yl)−5-(3-carboxymethoxyphenyl)−2-(4-
sulfophenyl)−2H-tetrazolium (MTS, Promega, Madison, WI, USA)
assay, which determined mitochondrial metabolism in living cells.
In brief, cells were incubated for 2–4 h at 37 °C with an appropri-
ate amount of MTS according to the manufacturer’s instructions.
The soluble product formazan was detected using a Spark 20 M
multimode microplate reader (Tecan Group Ltd., Mannedorf,
Switzerland) at 490 nm.
●
For generation of hydroxyl radicals ( OH), the method of Hal-
liwell et al. (1988) was applied [36]. The method is based on the
measurement of the amount of thiobarbituric acid-reactive sub-
stances resulting from the deoxyribose degradation that serves as
a measure for the production of hydroxyl radicals. Hydroxyl radi-
cals were generated in a system containing either (a) 10 mM potas-
sium phosphate buffer, pH 7.4; 0.1 mM FeSO , 0.5 mM H O and
4
2
2
2
mM deoxyribose or (b) 10 mM potassium phosphate buffer, pH
ELISA for tumor necrosis factor alpha (TNF-α) and
interleukin-6 (IL-6)
.4; 0.1 mM EDTA-Fe2 , 0.5 mM H O2 and 2 mM deoxyribose. These
+
7
2
4

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
0
Scheme 1. Reagents and conditions: a) Mem · HCl, EDC/HOBt, NMM, CH2Cl2/ DMF, 1 h at 0 C, then 23 h at rt.
The EOC20 cells were stimulated by lipopolysaccharide (LPS),
X-ray studies
aiming to imitate the inflammatory and neurotoxic activation of
microglia. The EOC20 cells were subjected to 1 μg/ mL LPS for 24 h,
and the tested compounds were added 4 h prior to and during
LPS treatment. TNF-α and IL-6 concentrations in EOC20 cell super-
natant were evaluated by using ELISA kits (Invitrogen, Carlsbad, CA,
USA), following the manufacturer’s instructions. The results based
on absorbance values detected at 450 nm were expressed as pg/
mL.
Single crystal X-ray studies showed that the compounds 2 and 5
crystallize in a centrosymmetric manner both in monoclinic space
group (SG) P2 /c, (No 14) and in a non-centrosymmetric man-
1
ner for compounds 4 and 3, SG P2 2 2 , (No 19) and SG R3, (No
1
1 1
146) respectively. A summary of the most important crystal data
and refinement indicators is provided in Table 1. With the excep-
tion of compound 5, no solvent or water molecules are present
in the structures. The asymmetric units of the four studied com-
pounds are shown in Fig. 1. The bond lengths and angles of the
four molecules are comparable (Table S1 and Table S2, see sup-
plementary data). The aromatic ring system present in the four
3. Results and discussion
˚
˚
molecules is essentially planar (rmsd of 0.003 A - 0.015 A) and in
addition, the memantine geometry is highly conserved. Having in
mind the similarity (bond lengths and angles), one should expect
similar hydrogen bonding interactions as the side chain difference
provides the main difference between the structures. In the four
structures, intermolecular N–H…O hydrogen bonds (Table 2) stabi-
lizes the three-dimensional packing of the molecules. The crystal
structure of N-(4-dimethylaminocinnamoyl) amide of memantine
3
.1. Chemical synthesis
Following the afore-mentioned polypharmacological approach
for the discovery of small drug-like molecules directed to diverse
AD targets, herein, we obtained similar ligands. The desired me-
mantine hybrids 1–5 were designed as outlined in Scheme1.
Firstly, 3,4-dimethoxycinnamic acid (3,4-(OMe) CA) and 4-N,N-
2
dimethylaminocinnamic acid (4-NMe CA) were synthesized follow-
2
(
5) also reveals the presence of CH OH.
3
ing the Knoevenagel-Doebner condensation [29] in 75% and 62%
yields, respectively the other representative acids are commer-
cially available. Then, the merging of the corresponding substituted
cinnamic– or dihydro-p-coumaric acid with memantine (Mem) was
accomplished using standard EDC/ HOBt peptide method and in
Thermal behavior
The thermal behavior of the four compounds is investigated be-
tween room temperature and 210 °C using DTA-TG analysis (Fig. 2).
For each of the four compounds, DTA measurement shows an
endothermic effect around 120 – 180 °C without loss of mass on
the TG curve. Such kind of endo effect can be related to the melt-
ing point of the compounds. The single crystal structure of 5 has
shown the presence of a solvent molecule in the unit cell. Thus
the small effect observed just before the melting is probably due
to release of the solvent. For such organic compounds a second
endo effect is expected at higher temperatures over 210 °C, which
is associated with pronounced weight losses and is the possible
decomposition of the compounds.
CH Cl / DMF solution affording memantine hybrids 1–5 in 45–78%
2
2
yields [43].
3
.2. Structure elucidation
All memantine hybrids (1–5) were fully characterized by melt-
ing points and spectroscopic data (UV, IR, 1H NMR, 13C NMR,
HRMS). The double bond geometry of substituted cinnamic amides
1
–3, 5 was assigned as an (E)-configuration on the basis of J val-
DTA curves do not reveal other significant effects, below or
above the “endo” melting and thus no evident phase transitions or
molecular conformation changes should be expected.
ues (>15.0 Hz) of the vinylic protons in the 1H NMR spectra. IR
data were very informative. In the spectra of memantine hybrids
1
–5, the lack of very broad and intense O–H stretching absorption
in the 3100–2596 cm 1 region, indicates the absence of carboxylic
−
acid dimers in the structures. As expected, the C = O absorption of
3.3. Biological studies
−1
amides 1–5 occurs at lower frequencies (1658–1632 cm ) than
the carbonyl absorption of starting carboxylic acid due to the res-
onance effect.
3.3.1. Radical scavenging activities in vitro
Since the oxidative stress hypothesis has been assumed to play
an important role in the AD pathogenesis [44], a putative antiox-
idant potential of our target molecules could attenuate this im-
balance. There are a number of studies showing a positive ef-
fect of antioxidants in this disease [44]. The ambiguities in the
use of antioxidants in the treatment of AD especially in devel-
Detailed information for 1H NMR, qNMR, 13C NMR and HRMS
spectra of the synthesized compounds (1–5) are shown in supple-
mentary materials.
With the exception of compound 1, the others were additionally
characterized by single crystal X-ray studies.
5

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
Table 1
Crystallographic data-collection statistics for compounds 2–5.
Identification code
compound 2
compound 3
compound 4
compound 5
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a[ A˚ ]
C23H31NO3
369.49
C23H31NO4
385.49
290
C21H29NO2
327.45
C24H36N2O2
384.55
293(2)
290
293(2)
monoclinic
P21/c
trigonal
R3
orthorhombic
P212121
11.1077(7)
11.5555(7)
14.6783(10)
90
monoclinic
P21/c
18.8211(14)
12.1381(7)
9.5547(5)
90
20.5665(7)
20.5665(7)
13.1095(5)
90
24.2414(13)
7.7833(4)
12.2077(6)
90
b/A˚
c[ A˚ ]
α[°]
β[°]
γ[°]
Volume[ A˚ ]3
104.078(7)
90
90
90
94.262(5)
90
120
90
2117.2(2)
4
4802.2(4)
9
1884.0(2)
4
2297.0(2)
4
Z
ρ
calcg/cm3
1.159
1.2
1.154
1.112
-
1
μ/mm
F(000)
0.076
0.081
0.073
0.07
800
1872
712
840
Crystal size/mm3
0.3 × 0.25 × 0.12
MoKα
0.3 × 0.3 × 0.2
MoKα
0.3 × 0.25 × 0.12
MoKα
0.3 × 0.25 × 0.12
MoKα
Radiation
(
λ= 0.71073)
(λ= 0.71073)
5.53 to 56.966
(λ= 0.71073)
5.55 to 58.522
(λ= 0.71073)
5.498 to 57.052
2
ꢀ range for data
5.55 to 58.054
collection[°]
Index ranges
−24 ≤ h ≤ 24,
−26 ≤ h ≤ 24,
-26 ≤ k ≤ 27,
-16 ≤ l ≤ 17
16,783
−14 ≤ h ≤ 15,
-14 ≤ k ≤ 10,
-14 ≤ l ≤ 19
7699
−28 ≤ h ≤ 31,
-10 ≤ k ≤ 7,
-15 ≤ l ≤ 15
16,119
-
-
16 ≤ k ≤ 11,
12 ≤ l ≤ 12
Reflections
collected
12,670
Independent
reflections
4794 [Rint = 0.0373,
Rsigma = 0.0394]
4656 [Rint = 0.0289,
Rsigma = 0.0254]
4656/1/258
3931 [Rint = 0.0452,
Rsigma = 0.0576]
3931/0/227
5066 [Rint = 0.0432,
Rsigma = 0.0475]
5066/0/260
4
794/0/248
Data/restraints/parameters
Goodness-of-fit on
F2
1.018
1.058
1.078
1.036
Final R indexes
R1 = 0.0538,
wR2 = 0.1361
R1 = 0.0858,
wR2 = 0.1558
0.15/−0.19
R1 = 0.0484,
wR2 = 0.1091
R1 = 0.0673,
wR2 = 0.1193
0.16/−0.12
R1 = 0.0505,
wR2 = 0.1139
R1 = 0.0688,
wR2 = 0.1305
0.13/−0.19
R1 = 0.0671,
wR2 = 0.1649
R1 = 0.1334,
wR2 = 0.2029
0.17/−0.17
[
I>=2σ (I)]
Final R indexes
all data]
Largest diff.
[
peak/hole / e A˚ 3
−
Flack parameter
-
−0.2(4)
0.2(10)
-
opment of MTD are mainly regarding the search for appropriate
concentrations to achieve effectiveness. It is considered to be eas-
ier to introduce structurally non-specific activity, including antiox-
idant activity, in the multi-target molecule compared to the in-
clusion of a specific molecular fragment, ensuring binding to re-
ceptor or enzyme. However, in order to be effective, this moi-
ety requires a higher dose than specific receptor ligands or en-
zyme inhibitors [6]. Nevertheless, based on the involvement of OS
in the mechanism of AD provoking and progression, the presence
of antioxidant properties would be beneficial for the overall ef-
fect of the target substances. Therefore, to estimate the antioxi-
dant activity of target memantine hybrids 1–5, we applied differ-
ent scavenging tests. In this context, the chelating effects and rad-
ical scavenging ability of compounds were determined against 1,1-
Fig 3, the scavenging effect on the DPPH radical increased in the
following order: 4 ≈ 5 ≈ 2 < FA ≈ 1 < 3 < SA. Among the tested
memantine hybrids 1–5, compound 3 seems to be the most active.
As expected, since SA was found to be more active than FA,
the% RSA of N-sinapoylamide of memantine (compound 3) had
stronger antioxidant activity than N-feruloylamide of memantine
(compound 1). The observed results are in accordance with the
literature findings, indicating that the addition of two methoxy
groups at the ortho position increased the radical-scavenging ef-
fect of monophenols by stabilizing the aryloxyl radical [46]. The
N-feruloylamide of memantine (compound 1) just maintained the
activity of FA itself. Despite that dihydro-p-coumaroylamide of me-
mantine (compound 4) possesses para–hydroxyl group, it showed a
very weak radical-scavenging activity as most of the monophenols
with no substituents in the aromatic ring (e.g. coumaric acids) [35,
47].
●
●
diphenyl-2-picrylhydrazyl (DPPH ), hydroxyl ( OH) and superoxide
●▬
(
O2 ) radicals. For comparisons, well-known antioxidants such as
sinapic- and ferulic acids were used as positive controls.
Although, DPPH method is routinely practiced for evaluating
the free radical scavenging activity of any new drug [48], it has
many disadvantages [49]. The main ones are that DPPH is a syn-
thetic radical and possess limited similarities with peroxyl radical.
So, in this study assayed the antioxidant potential of the tested
memantine derivatives against naturally produced in cell ROS, as
superoxide and hydroxyl radicals.
●
3
.3.1.1. DPPH Radical
scavenging
assay. 1,1-Diphenyl-2-
●
picrylhydrazyl (DPPH ) test has been well known and extensively
applied as a primary index of studying in vitro the antioxidant
activity of phenolics [45]. It is a precise and quick method for
measuring the radical-scavenging ability of H-donating molecules.
Therefore, we applied DPPH method, as proposed by Nenadis and
Tsimidou [35] to estimate such activity of our hybrids, as well
as and the used positive controls, sinapic (SA) and ferulic (FA)
acids. Our results on the DPPH-scavenging activity are presented
in Fig 3 as% RSA (radical-scavenging activity). As can be seen from
3.3.1.2. Superoxide anion radical generation system. Although the
•
−
superoxide anion radical (O2 ) is reported to be less toxic than
the hydroxyl radical, it continues to be known as one of the most
deleterious reactive oxygen species, since is a precursor for genera-
6

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
Fig. 1. ORTEP view of asymmetric unit along with atom numbering scheme of a) compound 2; b) compound 3; c) compound 4; d) compound 5. Atomic displacement
parameters (ADP) are drawn at the 50% probability, while hydrogen atoms are shown as spheres with arbitrary radii.
tion of the other reactive oxygen and nitrogen species. In this assay
In the NBT test, at the higher tested concentration the most
powerful effect had SA, followed by FA and compound 3. The in-
hibitory effect of the ferulic and sinapic acid concentrations was:
at 20 μM about 30–40%, at 50 μM about 50–70% and at 100 μM -
respectively about 70% and 85%. Hybrid 3 displayed significant de-
crease in formazan production, especially at 50 μM (about 60%) and
at 100 μM about 75%. The other memantine hybrids (compounds 1,
•
−
the effects of the tested substances in the O
-generating system
2
•
−
are presented on Fig. 4. The O2 -scavenging effect increased in
the following order: 5 < 2 ≈ 1 < 4 < 3 ≈ FA < SA. Among the
memantine hybrids 1–5 examined, compound 3 emerges with the
strongest scavenging effect.
7

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
Table 2
Hydrogen Bonds geometry ( A˚ , °) for compounds 2–5.
Compound 2
D -
H…
A
d(D-H)[ A˚ ]
d(H…A)[ A˚ ]
d(D…A)[ A˚ ]
D-H…A[°]
N1 -
C2 -
C7 -
C23 -
C3 -
H1
O11
O1
0.86
0.97
0.97
0.96
0.97
2.26
2.64
2.55
2.64
2.44
3.109(17)
3.199(2)
3.127(2)
3.595(3)
3.378(3)
168.9
116.9
117.8
175.6
164
H2B
H7A
H23B
H3B
O1
O12
O33
Compound 3
D -
H…
A
d(D-H)[ A˚ ]
d(H…A)[ A˚ ]
d(D…A)[ A˚ ]
D-H…A[°]
N1 -
O3 -
O3 -
C7 -
C6 -
H1
O11
O12
O2
0.86
0.82
0.82
0.97
0.97
2.17
2.52
2.2
3.020(4)
3.228(5)
2.656(5)
3.107(4)
3.629(4)
171
H3
145.2
115
H3
H7A
H6B
O1
N13
2.55
2.69
116.9
162.5
Compound 4
D -
H…
A
d(D-H)[ A˚ ]
d(H…A)[ A˚ ]
d(D…A)[ A˚ ]
D-H…A[°]
C7 -
N1 -
O2 -
H7A
H1
O1
0.97
2.4
2.981(3)
2.972(3)
2.618(3)
118.4
O21
O12
0.88(3)
0.95(4)
2.09(3)
1.67(4)
174(3)
173(4)
H2
Compound 5
D -
H…
A
d(D-H)[ A˚ ]
d(H…A)[ A˚ ]
d(D…A)[ A˚ ]
D-H…A[°]
N1 -
O2 -
C2 -
C7 -
H1
O2
O11
O1
O1
0.86
0.82
0.97
0.97
2.12
1.86
2.64
2.58
2.965(3)
2.672(3)
3.192(4)
3.142(4)
165.7
174.2
116.5
116.9
H2
H2A
H7A
Fig. 2. DTA-TG curves: a) compound 2; b) compound 3; c) compound 4; d) compound5.
2
, and 4) inhibit weakly (less than 30% at concentration of 100 μM)
3.3.1.3. Hydroxyl radicals generating system. Hydroxyl radicals
•
−
●
the O2 - provoked NBT-reduction.
( OH) can be scrutinized as the most powerful oxidizing radicals
Superoxide anion radical scavenging assay, similarly to DPPH
assay are electron transfer reaction based assays [48]. They mea-
sure the reducing capacity of the compounds via redox reaction,
where antioxidant compounds oxidizing themselves reduce the
free radicals. Therefore it was not surprising that the obtained re-
sults are similar to that from the DPPH assay, showing good an-
tioxidant potential for SA, FA and compound 3. It was unexpected
the low inhibitory potential against formazan production of com-
that can interact with the biological important molecules (pro-
teins, DNA, polyunsaturated fatty acids, amino acids), and to cause
oxidative damage [51].
Herein, the newly synthesized memantine hybrids were eval-
●
uated in a system generating OH, containing H O , ferrous ions
2
2
and a chelator (EDTA). The chelator will interact with the metal
ion and thus prevent it from binding to the deoxyribose molecule
●
(as detector molecule). Thus, the OH generated by the reaction
•
−
2+
pound 1, since Mem alone is unable to scavenge O2
[50] and its
between Fe -EDTA and H O2 will have equal access to all compo-
2
antioxidant moiety is similar to FA.
nents of the reaction medium, including deoxyribose. The reduc-
tion of the degree of degradation of the detector molecule will be
8

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
Fig 3. DPPH-scavenging abilities of memantine hybrids 1–5.
O2•
−
scavenging effect of newly hybrids 1–5.
Fig. 4.
based on scavenger properties of the tested substance [36]. The ef-
fect of the tested compound concentration on the extent of de-
oxyribose degradation is shown in Fig 5.
acids-ferulic and sinapic acid, used as references. In contrast to the
phenolic acid, the established augmented activities of both amides
1, 3 might be due to the presence of additional hydrogen-donating
amide group, since the antioxidant capacity of substances against
As can be seen, we detected changes in the rate of degra-
dation of DR....... N-sinapoylamide of memantine (3) and 3,4-
●
OH radicals is based on hydrogen atom transfer reaction [48].
dimethoxycinnamoyl amide (2) at
a
concentration of 100 μM
Dimethoxy hybrid molecule 2 was highly effective as hydroxyl rad-
ical scavenger as compound 3 itself (about 80%) although possess-
ing no -OH group. The found antioxidant activity of compound 2
might be ascribed to its ability to chelate iron ion, stronger than
DR.......
showed the strongest protective effect - about 80% inhibition
●
of formed OH hydroxyl radicals, followed by compound 1 (N-
feruloylamide of memantine)– about 50% at the same concentra-
tion.
●
●
Concerning the OH scavenging ability of hybrids 1 and 3 it
The rest of hybrids 4 and 5 had OH - scavenging abilities lower
seemed reasonable that the observed in vitro radical inhibition
of both hybrids 1 and 3 can be attributed to their methoxy–
substituted phenol structure. Moreover, these amides displayed
a higher antioxidant capacity compared to their parent phenolic
than 50% at concentration of 100 μM, however they seem to be
more active even than used positive controls. It has been show
●
that reaction of OH radicals with SA in aqueous solutions leads
to adduct formation to both the ring and the olefinic group as the
9

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
●
Fig 5. OH- scavenging effect of newly hybrids 1–5.
addition to the side chain is favored [52]. Hydroxyl radical adducts
with FA were also identified [53]. Thus, in addition to the amide
bond, the presence of double bond in the chain contributes to the
cluding Aβ deposits, tau protein hyperphosphorylation, metal ion
dyshomeostasis, excitotoxicity, oxidative stress, neuroinflammation,
and neurotransmitter system dysfunction [58]. Considering the
complex pathogenesis of AD, multifunctional agents that simulta-
neously combine two or more pharmacological effects in a single
molecule on resisting the deficits of AD may achieve better thera-
peutic efficacy with complementary mechanisms of action [59].
In this study, the series of new memantine hybrids were eval-
uated for multifunctional activities related to AD pathophysiology,
involving the effects on the resistance to Aβ toxicity, excitotoxicity,
oxidative stress, hypoxia, and neuroinflammation.
●
overall OH - scavenging effect of the compounds.
3
.3.1.4. Chelating effects. It is well known that the generation of
●
OH in vivo required redox active metal (Fenton reaction) [36].
Therefore, the presence of chelating agents could prevent oxidative
damage.
In order to specify the chelating potential of the tested hybrids
●
1
–5, a OH generating systems with deoxyribose (DR...…) as a de-
tector molecule, Fe2 and H O2 (without metal chelator) were ap-
+
2
plied [35]. This system comprises the possibility of DR...… to bind
3
.3.2.1. Neuroprotective effects of memantine hybrids in APPswe cells
●
ferrous ions. Hence, the site-specific generation of OH radicals led
due to copper-induced aβ toxicity (A). It is well-evidenced that Aβ
play a pivotal role in the pathogenesis of AD. The APPswe cells es-
tablished in this study overexpress the Swedish mutant form of
human APP. Studies have shown that Swedish-type APPswe (mu-
tant of APP695) can produce more Aβ than wild-type APP, es-
pecially the efficiency of BACE1 cleaving Swedish-type APPswe is
about 10 times higher than that of wild type APP, causing neuro-
toxicity [60, 61]. Furthermore, some studied have found that the
level of the BACE1 increase due to oxidative stress [62]. The transi-
tion metals, such as copper, are involved in the assembly and neu-
rotoxicity of Aβ species [63]. In our study, the APPswe cells injured
by copper were established as an AD cell model by using copper
to trigger the neurotoxicity of Aβ, based on the literature that APP
and Aβ have the copper-binding domain (CuBD) through which Cu
to subsequent oxidative fragmentation of DR....... If the tested sub-
stance has a higher affinity for binding to iron than the detector
(
exhibits stronger chelating properties than those of deoxyribose),
it can protect the detector molecule.
The effects of increasing concentrations (0.01, 0.02, 0.05 and
●
0
.1 mM) of hybrids 1–5, ferulic and sinapic acids in the OH-
generating system containing, are presented on Fig. 6.
Our results indicate that with exception of compound 2 (about
35% inhibitory effect) the rest of tested hybrids did not exert
protective potential at concentrations up to 100 μM. It has been
demonstrated that many phenolic compounds including ferulic
acid, did not show complex formation with Fe2 [54]. In compound
+
2
, the electron delocalization and electron donation of the un-
shared pair of electrons from OCH3 groups in the p-orbital [55] in
addition to ethylene group likely contribute to it chelator poten-
tial. It has been suggested that the presence of ethylene group may
have an influence on the chelation capacity [56]. The inclusion of
this group between the benzene ring and carboxylic group in the
hydroxycinnamic acids, make them better ligand for iron than hy-
droxybenzoic acids [54]. Furthermore the metal cations can bind to
the phenyl ring by cation-π-type interactions mediated by electron
delocalization [57].
(
II) serves as cofactors to Aβ peptides and APP protein, facilitating
the formation of Aβ aggregates, influencing their conformational
transformation, and exacerbating the toxicity of Aβ [64].
The results revealed that the target compounds 1–5 (A)
showed moderate and similar neuroprotective activities against
Aβ-induced cytotoxicity triggered by copper, with the EC50 val-
ues ranging from (18.23 ± 1.08) μM to (35.24 ± 1.84) μM (1–5 (A),
Table 3).
These observations suggested that memantine hybrids 1–5 had
the neuroprotective activities against Aβ-mediated neurotoxicity.
3
.3.2. Neuroprotective in vitro evaluation and structure–activity
relationship studies
In the etiology of AD, there are diverse factors that seem
to play pivotal roles in the pathophysiological progression, in-
3.3.2.2. Neuroprotective effects of memantine hybrids against NMDA-
induced toxicity (B). It is well-documented that a substantial in-
10

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
Fig. 6. Chelating effect of newly hybrids 1–5.
Table 3
The EC50 and IC50 values of hybrids 1–5, and positive drug evaluated for neuroprotective effects.
Copper—
EC50(μM)
(A)
NMDA—
EC50(μM)
(B)
H2O2—
EC50(μM)
(C)
CoCl2—
EC50(μM)
(D)
TNF-α—
IC50(μM)
(E)
IL-6—
IC50(μM)
(F)
Cmpd
1
20.14 ± 0.21
35.24 ± 1.84
22.17 ± 1.80
27.43 ± 7.10
18.23 ± 1.08
16.81 ± 3.12
31.98 ± 1.06
14.90 ± 0.25
> 100
> 100
22.59 ± 0.29
35.71± 2.96
46.18 ± 1.90
39.64 ± 8.24
26.18 ± 3.49
0.12 ± 0.05
15.26 ± 1.79
39.21 ± 2.01
0.16 ± 0.02
20.17 ± 0.86
13.28 ± 4.85
0.08 ± 0.02
19.46 ± 3.57
41.69 ± 2.39
0.19 ± 0.02
25.87 ± 9.14
17.46 ± 0.76
0.14 ± 0.01
2
10.94 ± 0.10
16.96 ± 0.61
> 100
3
4
5
> 100
> 100
Mem·HCl
0.56 ± 0.11
1.07 ± 0.36
.34 × 10− ± 0.51 × 10−3
3
8
Note: The 50% effective concentration (EC50) and the 50% inhibitory concentration (IC50) of the compounds were calculated by GraphPad software. The data are presented as
the mean ± SD from three independent experimental titrations. Mem · HCl is the abbreviation for memantine hydrochloride.
crease in extracellular glutamate is toxic to neurons in a patho-
logical process called excitotoxicity that is involved in Alzheimer’s
deficits, due to prolonged activation and post-synaptic signaling
components of ionotropic glutamate receptors, of which NMDA re-
ceptors play the major role [65]. The SH-SY5Y cells were used to
induce excitotoxicity treated with NMDA at 100 μM concentrations
3.3.2.3. Neuroprotective effects of memantine derivatives against
H O -induced toxicity (C). Oxidative stress is known to be closely
2
2
related to age-related neurodegeneration associated with AD [68],
which also causes a remarkable accumulation of Aβ [44]. An in-
crease in oxidative stress has been suggested to be one of the ear-
liest pathological changes in the brain in cognitive impairment due
to AD [69]. Since memantine derivatives are effective in preventing
glutamate-induced toxicity, we considered that the effects of me-
mantine analogues may reduce oxidative damage to neuronal cells
that is in correlation with over-activation of NMDA receptors to a
certain extent.
[
66]. The MTS assay was applied to examine the neuroprotective
effects against NMDA-induced toxicity and EC50 value was used to
evaluate the efficacy of memantine derivatives. The results demon-
strated that amongst the tested memantine hybrids 1–5, com-
pounds 1–3 demonstrated protective effects due to NMDA toxicity,
showing the EC50 ranging from (14.90 ± 0.25) μM to (31.98 ± 1.06)
μM (1–5 (B), Table 3), and there was no effect of compounds 4,
As shown in Table 3 (1–5 (C)), hybrids 2 and 3 demonstrated
moderate neuroprotective effects against H O -induced toxicity
2
2
5
on increasing cell viability against NMDA-induced injury within
with the EC50 values of (10.94 ± 0.10) μM to (16.96 ± 0.61) μM, re-
spectively, which were similar to their EC50 values got in NMDA-
damaged SH-SY5Y cells.
the tested concentrations. Based on the MTS assays, this series of
1–3 displayed moderate neuroprotection due to NMDA toxicity.
It is known that NMDA - induced excitotoxicity is mainly
caused by an excessive influx of calcium through the over-
activated NMDA receptors [67]. Thus, this preliminary evalua-
tion is suggested that compounds 1–3 might provide neuropro-
tective effects against NMDA - induced neurotoxicity by block-
3
.3.2.4. Neuroprotective effects of memantine derivatives against
hypoxia-induced toxicity (D). During the progression of AD, a grad-
ual decline in the supply of oxygen to the brain is manifested
[
70]. There is abundant literature revealing that exposures to hy-
ing the NMDA receptor and inhibiting the Ca2 influx. But fur-
ther target docking and mechanism needs to be confirmed by
patch-clamp technology, molecular docking simulation, and gene
silencing.
+
poxia/ischemia increasing the overexpression increase the amy-
loidogenic processing of amyloid-β precursor protein (APP) and
leading to the imbalance in the production and clearance of Aβ
[
71]. CoCl2 is a well-known hypoxia reagent and has been widely
11

M. Chochkova, H. Jiang, R. Kyoseva et al.
Journal of Molecular Structure 1234 (2021) 130147
used in PC12 cells to establish a chemical hypoxia in vitro cell
model [72]. The protective effect of memantine analogues against
hypoxia was studied on this cell model [73].
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130147.
As shown in Table 3, compounds 1–5 (D) displayed relatively
weak anti-hypoxic properties in comparison with the positive con-
trol memantine hydrochloride, with the EC50 values ranging from
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