Synthesis and biological evaluation of memantine nitrates as a potential treatment for neurodegenerative diseases

Article abstract of DOI:10.1039/c6md00509h

A series of memantine nitrate derivatives, as dual functional compounds with neuroprotective and vasodilatory activity for neurodegenerative diseases, was designed and synthesized. These compounds combined the memantine skeleton and a nitrate moiety, and thus inhibited the N-methyl-d-aspartic acid receptor and released NO in the central nervous system. The biological evaluation results revealed that the new memantine nitrates were effective in protecting neurons against glutamate-induced injury in vitro. Moreover, memantine nitrates dilated aortic rings against phenylephrine-induced contraction. The structure-activity relationships of neuroprotection and vasodilation were both analyzed. In further studies, compound MN-05 significantly protected cortical neurons by inhibiting Ca²⁺ influx, reducing free radical production and maintaining the mitochondrial membrane potential. Further research on MN-05 is warranted.

Full text of DOI:10.1039/c6md00509h

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Z. Liu, S. Yang, X.
Jin, G. Zhang, B. Guo, H. Chen, P. Yu, Y. Sun, Z. Zhang and Y. Wang, Med. Chem. Commun., 2016, DOI:
10.1039/C6MD00509H.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/medchemcomm

Page 1 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
Scheme 3.cdx
Scheme 4.cdx
Scheme 5.cdx
Scheme 6.cdx
Scheme 7.cdx

MedChemComm
Page 2 of 38
View Article Online
DOI: 10.1039/C6MD00509H
MedChemComm
Synthesis and Biological Evaluation of Memantine Nitrates as
Potential Treatment for Neurodegenerative Diseases†
Zheng Liu, Si Yang, Xiaoyong Jin, Gaoxiao Zhang, Baojian Guo, Haiyun Chen, Pei Yu,
Yewei Sun,* Zaijun Zhang,* and Yuqiang Wang
Institute of New Drug Research and Guangzhou Key Laboratory of Innovative Chemical
Drug Research in Cardioꢀcerebrovascular Diseases, Jinan University College of Pharmacy,
Guangzhou, 510632, China.
*Corresponding author. Yewei Sun or Zaijun Zhang. Tel.: +86 20 8522 5030; Fax: +86 20
8522 4766.
Eꢀmail address: yxy0723@163.com (Y. Sun), zaijunzhang@163.com (Z. Zhang)
†The authors declare no competing interests.

Page 3 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
Abstract
A series of memantine nitrate derivatives as dual functional compounds of neuroprotection
and vasodilatory activity for neurodegenerative diseases was designed and synthesized. These
compounds combined the memantine skeleton and the nitrate moiety, thus inhibited the
NꢀmethylꢀDꢀaspartic acid receptor and released NO in central nervous system. The biological
evaluation results revealed that the new memantine nitrates were effective in protecting
neurons against glutamateꢀinduced injury in vitro. Moreover, memantine nitrates dilated the
aortic rings against phenylephrineꢀinduced contraction. The structureꢀactivity relationships of
neuroprotection and vasodilation were both analyzed. In further studies, compound MNꢀ05
significantly protected cortical neurons by inhibiting the Ca2+ influx, reducing free radical
production and maintaining the mitochondrial membrane potential. Further research on
MNꢀ05 is warranted.
Introduction
NꢀmethylꢀDꢀaspartic acid (NMDA) receptor, a subtype of glutamate receptor, plays a crucial
role in regulating a broad spectrum of processes in central neuron system (CNS).1,2 In
pathological conditions, the presence of excessive glutamate overstimulates NMDA receptors,
results in extensive cell damage,2,3 and leads to a number of chronic neurodegenerative
disorders, such as Alzheimer’s disease, Parkinson’s disease, vascular dementia (VaD) and
etc.4ꢀ7 Memantine (1ꢀaminoꢀ3,5ꢀdimethyladamantane, Fig. 1) is an openꢀchannel,
uncompetitive NMDA receptor blocker with lowꢀaffinity, high voltage dependency and fast
receptor kinetics.8ꢀ10 Memantine rapidly penetrates into brain to bind to NMDA receptors and
block the subsequent influx of calcium, affording neuronal protection.10ꢀ12 Memantine is well
tolerated clinically and was approved as a treatment for moderate to severe Alzheimer's
disease in the world.13 Recently, memantine was reported to reduce the blood supply of brain

MedChemComm
Page 4 of 38
View Article Online
DOI: 10.1039/C6MD00509H
possibly through nonꢀspecific blocking the nicotinic acetylcholine receptors in the CNS.14
This property of memantine may decrease its clinical efficacy since some CNS disorders,
such as ischemic stroke and VaD, are characterized by a reduced cerebral blood flow (CBF).
Nitric oxide (NO), a second messenger with high reactivity, plays an important role in
cardioꢀcerebrovascular system. It also participates in maintaining the normal function of CNS
and regulating neuronal death.15 Physiological concentration of NO elicits
antiꢀapoptotic/proꢀsurvival effects against various neurotoxic challenges and brain insults
through multiple mechanisms.16ꢀ18 It has been reported that NO donors, such as nitroglycerin,
significantly reduced NMDAꢀinduced neurotoxicity in cortical cultures and in vivo.18,19
Although nitroglycerin confers neuroprotective effects, it is not an ideal neuroprotectant
because of its systematic release of NO in peripheral vessels, resulting in a high risk of
hypotension. NO donors which release NO targeting brain tissue have a great potential for
treatment of vascular neurodegenerative disease.
Based on these findings, Wang et al. have previously designed and synthesized a number of
memantine nitrates to increase the neuroprotective effects of memantine and to avoid the
peripheral hypotensive effects of traditional NO donors.20 Their stability in serum,
electrophysiological properties as well as effects on blood vessels were characterized.20 It was
found that memantine nitrates exerted dual inhibitory effects on NMDA receptors, i.e., an
openꢀchannel blockage of NMDA receptor by the memantine moiety and inhibition via
Sꢀnitrosylation by NO released from the nitrate group. Further study demonstrated that the
memantine moiety was able to target NO to NMDA receptors in brain tissue, thus reduced the
risk of hypotension.21 Among the memantine nitrates, YQWꢀ036, bearing two ethyl side
groups, was extensively studied. YQWꢀ036 displayed improving efficacy and safety in

Page 5 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
animal model of cerebral ischemia.21 Although these memantine nitrates exert dual inhibitory
effects on NMDA receptors, they didn’t display stronger activities than memantine on
inhibiting NMDA current and have short halfꢀlives.20 To find compounds with improved
stability and better efficacy, in the current study, a series of new memantine nitrates were
designed and synthesized (Fig. 1). The structureꢀactivity relationships of the compounds were
analyzed. The neuroprotective and vasodilating effects were also investigated in vitro.
Results and discussion
Rational drug design and synthesis
The significant characteristics of previous synthesized memantine nitrates were that the
nitrate group was introduced at the 7ꢀposition of amantadine skeleton, and the hydrogens at 3
and 5ꢀpositions were substituted with methyl, ethyl or propyl groups (Fig. 1). Previous
studies of memantine distribution and binding of the receptor have revealed that: (1) The free
amino group is essential both for memantine to penetrate the bloodꢀbrain barrier 22,23 and bind
to the receptors;24,25 (2) The hydrophobicity group(s) on 3ꢀor (and) 5ꢀposition(s) of
memantine skeleton increase(s) binding affinity to NMDA receptors and decrease(s)
hydrophibicity.25 Based on these findings, the free amino group at the 1ꢀposition and the
hydrogen at the 7ꢀposition of memantine skeleton were preserved in the new memantine
nitrates (Fig. 1). The modifications on memantine skeleton were conducted on the 3ꢀ or (and)
5ꢀpositon(s). The side group on the 3ꢀposition of memantine varied from methyl to propyl
group. The nitrate moiety was introduced to the 5ꢀposition of memantine skeleton. The length
of the carbon chain linking the nitrate group and the memantine skeleton varied from 0ꢀ3. In
addition, the new compounds may have improved plasma stability due to the replacement of
a tertiary nitrate with a primary one. We also wondered whether increasing the number of
nitrate group would provide additional vasodilating and neuroprotective effects, thus

MedChemComm
Page 6 of 38
View Article Online
DOI: 10.1039/C6MD00509H
memantine derivatives MNꢀ12ꢀMNꢀ16 armed with two nitrate groups were designed and
synthesized.
Compounds MNꢀ01ꢀMNꢀ03 were prepared as illustrated in Scheme 1. The commercially
available corresponding precursors were brominated with Br2 and then underwent Ritter
reaction to afford acetamides 7ꢀ9, respectively. The hydroxyl group was introduced to
compounds 7ꢀ9 to afford compounds 10ꢀ12. The amide groups of compounds 10ꢀ12 were
hydrolyzed and the resulting amino groups were NꢀBOC protected to give compounds 13ꢀ15.
Compounds 13ꢀ15 were nitrated in the presence of fuming nitric acid to afford compounds
16ꢀ18. The BOC groups of compounds 16ꢀ18 were removed by treatment with anhydrous
hydrogen chloride in ethyl acetate to afford compounds MNꢀ01ꢀMNꢀ03.
Synthesis of compounds MNꢀ04ꢀMNꢀ06 was shown in Scheme 2. Compounds 1ꢀ3 were first
brominated followed by hydrolysis to afford adamantanols 19ꢀ21. Compounds 19ꢀ21
underwent KochꢀHaff reaction to afford compounds 22ꢀ24. The resulting compounds 22ꢀ24
were converted into compounds 25ꢀ27 via the Ritter reaction. Compounds 25ꢀ27 were treated
with ethyl chloroformate and then reduced to afford compounds 28ꢀ30. Compounds
MNꢀ04ꢀMNꢀ06 were synthesized from compounds 28ꢀ30 using similar methods to those as
described for synthesis of compound MNꢀ01 from compound 10.
Compounds MNꢀ07ꢀMNꢀ09 and MNꢀ11 were synthesized as illustrated in Scheme 3.
Compounds 4ꢀ6 were reacted with substituted acrylates to give intermediates 37ꢀ40, which
were then hydrolyzed to afford acids 41ꢀ44. The acids 41ꢀ44 were amidated and then reduced
to give compounds 49ꢀ52. Compounds MNꢀ07ꢀMNꢀ09 and MNꢀ11 were prepared from
compounds 49ꢀ52 using a similar method as described for the synthesis of MNꢀ01 from

Page 7 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
compound 10.
The synthesis of compound MNꢀ10 was described in Scheme 4. Compound 5 was treated
with isopropenyl acetate in the presence of AlBr3 to afford compound 61, which was then
reduced to afford compound 62. The alcohol 62 was esterified and then amidated to produce
compound 64. The target compound MNꢀ10 were prepared from compound 64 using a
similar method to that as described for the synthesis of MNꢀ01 from compound 10.
Synthesis of compounds MNꢀ12ꢀMNꢀ13 was shown in Scheme 5. Adamantane (67) was
dibrominated and then hydrolyzed to afford 1,3ꢀadamantandiol (69). Compound 69
underwent KochꢀHaff reaction to afford 1,3ꢀadamantanedicarboxylic acid (70). Compound 70
and 1,3ꢀadamantanediacetic acid (71) were reduced to adamantandiols 72 and 73, which were
then esterified to afford compounds 74ꢀ75. Acetamido was introduced to compounds 74ꢀ75
via the Ritter reaction to produce compounds 76ꢀ77. Compounds MNꢀ12ꢀMNꢀ13 were
prepared from compounds 76ꢀ77 using a similar method as that described for the synthesis of
compound MNꢀ01 from compound 10.
Compound MNꢀ15 was synthesized following the procedure as illustrated in Scheme 6.
1,3ꢀDibromoadamantan (68) was treated with isopropenyl acetate in the presence of AlBr3 to
afford compound 82. Compound 82 was reduced to afford compound 83, which was then
esterified and amidated to produce compound 85. The target compound MNꢀ15 was prepared
from compound 85 using a similar method to that as described for the synthesis of MNꢀ01
from compound 10.
Synthesis of compounds MNꢀ14 and MNꢀ16 was described in Scheme 7.

MedChemComm
Page 8 of 38
View Article Online
DOI: 10.1039/C6MD00509H
Dibromoadamantane (68) was reacted with corresponding acrylates to produce compounds
88 and 89, respectively. Compounds 88ꢀ89 underwent the Ritter reaction to produce
compounds 90ꢀ91, which were then reduced to compounds 92ꢀ93. MNꢀ14 and MNꢀ16 were
prepared from compounds 92ꢀ93 using a similar method to that as described for the synthesis
of MNꢀ01 from compound 10.
Biological evaluations
The target compounds were first evaluated for their protective effects against the
glutamateꢀinduced injury in primary cerebellar granule neurons (CGNs) using the MTT assay.
Compounds with promising protective effects were then chosen for further evaluations.
Protective effects against glutamate induced neurotoxicity.
Excessive amount of glutamate overstimulates NMDA receptors and results in a cascade of
cell damage. Increasing evidence demonstrates that memantine is effective in protecting
neurons against glutamate insult.26,27 In this project, we first investigated the protective
effects of the new memantine nitrates on primary culture of CGNs. CGNs were treated with
compounds at indicated concentrations for 2 h and were then exposed to glutamate (200 µM)
for 24 h in the presence of the compounds. Cell viability was measured by the MTT assay. As
shown in Fig. 2, all memantine nitrates showed moderate to strong protective effects against
glutamate induced damage (F(69, 851) = 124.5; P<0.001). Among these compounds, MNꢀ02,
MNꢀ03, MNꢀ05, MNꢀ08, MNꢀ12, MNꢀ14 and MNꢀ16 displayed stronger protective effects
than the others, and they were active at a concentration of 1 µM, as potent as the parent
memantine. Moreover, compounds MNꢀ02, MNꢀ03, MNꢀ05 and MNꢀ12 had lower EC50
values (the concentration that gives a half response of the maximum protection) than the
others (Table 1). The other compounds showed moderate protective effects with an initial

Page 9 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
effective concentration up to 10 µM. In addition, compounds MNꢀ06ꢀMNꢀ11 and
MNꢀ13ꢀMNꢀ16 displayed cytotoxicity at a concentration of 100 µM. By comparing the
structures and the activities of these compounds, we found that substitution at the 3ꢀposition
with an ethyl group was superior to a methyl or propyl group. The binding interactions of
memantine and NMDA receptor revealed that the methyl groups of memantine bind to the
hydrophobic pocket formed by the residues of V644, A645, and V656 in NMDA subunit
GluN1.25 The mutation of A645 (Ala) to L645 (Leu) significantly reduced the affinity of
memantine to receptor,25 which means the steric clash plays an important role in binding;
however, the ethyl group is smaller than propyl group. Thus, compounds substituted with a
ethyl group maybe more potency than that with a methyl or propyl group. In addition, we
found that the EC50 values and cytotoxic effects were both increased when lengthening the
linker between the nitrate group and memantine moiety.
Vasodilating effects on aortic rings
Organic nitrates are characterized by their abilities to dilate blood vessels via the release of
NO.28 In this work, we investigated the vasodilating activities of the new memantine nitrates
on aortic rings preꢀcontracted with phenylephrine. Since compounds MNꢀ02ꢀMNꢀ03, MNꢀ05,
MNꢀ08, MNꢀ12ꢀMNꢀ14 and MNꢀ16 displayed stronger protective effects in cultured CGNs
than the others, they were chosen for further evaluation in vasodilating assay. To determine
whether the vasodilating effect was endothelium dependent or not, investigations were
conducted on aortic rings with or without vascular endothelium (Fig. 3). The maximum effect
(Emax) and EC50 of compounds were listed in Table 2. As illustrated in Fig. 3A, compounds
MNꢀ02 and MNꢀ03 showed weak activities in dilating blood vessels without endothelium
with an Emax 42.03 ± 3.70 and 61.68 ± 1.63 ꢁM (Table 2), respectively. In contrast,
compounds MNꢀ05, MNꢀ08, MNꢀ12ꢀMNꢀ14 and MNꢀ16 were much more potent in dilating

MedChemComm
Page 10 of 38
View Article Online
DOI: 10.1039/C6MD00509H
blood vessels than the other compounds tested. The order of Emax was
MNꢀ13>MNꢀ05>MNꢀ14 ≈ MNꢀ16>MNꢀ12 ≈ MNꢀ08, and that of EC50 was
MNꢀ12>MNꢀ13>MNꢀ05>MNꢀ16 ≈MNꢀ14>MNꢀ08. All compounds showed a similar
pattern in dilating aortic rings with or without vascular endothelium, indicating that the
vasodilating effect of the memantine nitrate was endothelium independent. By comparing the
structures and activities of MNꢀ12ꢀMNꢀ14, MNꢀ16 with MNꢀ02ꢀMNꢀ03, we found that the
vasodilating efficacy is increased when the number of the nitrate groups was increased.
What’s more, lengthening the side groups significantly increased the potency of vasodilation
(EC50: MNꢀ14≈MNꢀ16<MNꢀ13<MNꢀ12, MNꢀ08<MNꢀ05).
MNꢀ05 protected cortical neurons against glutamate induced injury
Since compound MNꢀ05 showed better properties in protecting CGNs against glutamate
insult and dilating blood vessels, it was chosen for further investigations. Its neuroprotective
effect was further investigated on primary cortical neurons by the MTT assay and fluorescein
diacetate (FDA) and propidium iodide (PI) doubleꢀstaining. The parent memantine was used
as a positive control. Since the plasma concentration of memantine in patients is 1ꢀ10 µM, the
memantine concentration used in the present experiment was 5 µM. As shown in Fig. 4, cell
viability was decreased significantly in the glutamate aloneꢀtreated group compared with that
of control group. In contrast, MNꢀ05 treatment significantly protected cortical neurons
against glutamate insult at concentrations ranging from 5~45 µM in
a
concentrationꢀdependent manner (Fig. 4) (F(5, 61) = 34.87; P<0.001). FAD/PI doubleꢀstaining
can distinguish living (green) and dead (red) cells (Fig. 5A). The result shown in Fig. 5B
demonstrated that the percentage of cells (green) was decreased markedly in glutamate
aloneꢀtreated group compared with the control group. MNꢀ05 treatment improved neuronal
viability concentrationꢀdependently. MNꢀ05 is not as potent as the parent memantine at an

Page 11 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
equimolar concentration. The result was consistent with that seen in the glutamate induced
CGNs injury assay. Since the neuroprotective effect against glutamateꢀinduced neuronal
damage is attributed to the NMDA receptor inhibition, we think that MNꢀ05 is less potent
than memantine on NMDA receptor inhibition. However, the nitrate group provides MNꢀ05 a
new function, that is, release of NO to dilate blood vessel in vivo. Thus the effects of MNꢀ05
and memantine are worthy to be further compared in in vivo model.
MNꢀ05 blocked Ca2+ influx induced by glutamate
Overstimulation of NMDA receptors results in an excessive amount of calcium influx and
subsequently leads to an initiation of cell damage cascade.29,30 Compelling evidences
demonstrate that memantine binds to the NMDA receptor, blocks the Ca2+ influx, thus to
afford neuroprotective effects. We next investigated the effect of memantine derivative
MNꢀ05 on intracellular Ca2+ levels (F(3, 136) = 19.98; P<0.001). As shown in Fig. 6, glutamate
triggered a significant increase of intracellular Ca2+. In contrast, MNꢀ05 treatment markedly
inhibited the elevation of intracellular Ca2+ at a concentration of 15 ꢁM. In consistent with
the neuroprotection in vitro, the intracellular Ca2+ blockage effect of MNꢀ05 was not as
potent as memantine at an equal concentration, indicating that MNꢀ05 was weaker than
memantine in inhibiting NMDA receptor activity. The weaker activity of MNꢀ05 in
inhibiting NMDA receptors may be attributed to its lower affinity to NMDA receptors.
MNꢀ05 inhibited the production of reactive oxygen/nitrogen species induced by
glutamate
Overstimulated NMDA receptors mediated Ca2+ influx and triggered an overproduction of
reactive species, including reactive oxygen species (ROS) and reactive nitrogen species
(RNS). Experimental studies demonstrated that NMDA receptor blockade resulted in a

MedChemComm
Page 12 of 38
View Article Online
DOI: 10.1039/C6MD00509H
decrease in free radical production.31 To further characterize the neuroprotective effects of
MNꢀ05 against neurotoxicity induced by glutamate, the inhibitory effects of MNꢀ05 on ROS
and RNS production were evaluated. As shown in Fig. 7, MNꢀ05 could inhibit the production
of ROS and RNS, including hydrogen peroxide (H2O2, Fig. 7A) (F(5, 146) = 46.67; P<0.001),
hydroxyl radicals (•OH, Fig. 7B) (F(5, 86) = 18.43; P< 0.001), superoxide anion (O2•ꢀ, Fig. 7C)
(F(5, 106) = 32.63; P< 0.001), nitric oxide (NO, Fig. 7D) (F(5, 132) = 49.75; P<0.001) and
peroxynitrite (ONOO–, Fig. 7E) (F(5, 168) = 24.44; P<0.001), in a concentrationꢀdependent
manner. Consistently, the parent memantine was more effective in inhibiting the production
of ROS and RNS.
MNꢀ05 decreased the loss of mitochondrial membrane potential
Mitochondrial dysfunction results in an abnormal production of free radicals. Mitochondrial
membrane potential (ꢂψm) plays an important role in maintaining mitochondrial
physiological function and the loss of ꢂψm is an early event in the cell apoptotic process.32
To evaluate the mitochondrial protection of MNꢀ05, its effect on glutamateꢀinduced loss of
ꢂψm was investigated. Cortical neurons were incubated with MNꢀ05 or memantine at
indicated concentrations for 2 h, followed by exposure to 200 ꢁM of glutamate for another 12
h in the presence of compounds. The loss of ꢂψm was measured by fluorescence dye JCꢀ1.
As illustrated in Fig. 8, glutamate induced a significant reduction of ꢂψm compared with the
control group. In contrast, MNꢀ05 significantly inhibited the loss of ꢂψm at concentrations
ranging from 5ꢀ45 µM (F(5, 84) = 94.37; P<0.001), suggested that MNꢀ05 was effective in
preserving mitochondrial function. Consistently, memantine also showed a stronger activity
than MNꢀ05 in inhibiting the loss of ꢂψm at a concentration of 5 µM.
Compelling evidences suggest that inhibition of NMDA receptors affords therapeutic benefits

Page 13 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
in the treatment of CNS disorders.4,33 Researches have discovered a number of NMDA
receptor antagonists with neuroprotection since the 1980s. However, few of them succeeded
in patients.3 For example MKꢀ801 and phencyclidine, because of their too high affinity on
NMDA receptors, affect the normal physiological functions of NMDA receptors.34
Memantine is an uncompetitive NMDA receptor blocker with lowꢀaffinity and fast receptor
kinetics.8ꢀ10 At low micromolar concentrations (1ꢀ10 µM), it is quite specific for the NMDA
receptor compared to other ligandꢀgated and voltageꢀgated channels. Clinically achievable
plasma concentration of memantine (1ꢀ10 µM) does not attenuate longꢀterm potentiation in
hippocampal slices and affect the NMDA component of excitatory postsynaptic currents.35
Therefore, memantine is clinically well tolerated.13 In cultured neurons, MNꢀ05 significantly
protected the neurons from glutamate insults at a low concentration, but the inhibitory effects
of MNꢀ05 on neuronal damage, intracellular calcium influx and ROS/RNS overproduction
were not as potent as that of memantine, suggesting that MNꢀ05 may be effective in
neuroprotection and with fewer side effects (such as cognitive disruption and
psychoticꢀspectrum reactions) than memantine.
NO has a great activity on cardioꢀcerebrovascular system, it regulates the cerebral circulation
and promotes angiogenesis.36,37 A variety of structurally different NO donors have been
shown to reduce oxidative stress/inflammation, and increase blood supply.17 The traditional
organic nitrates often cause a side effect of hypotension due to the systematic release of NO,
which limited their use as neuroprotectants. However, memantine nitrates achieved target
releasing of NO in brain tissue since they have a quick distribution to brain tissue (data not
shown) and preferentially binds to NMDA receptors after administration. The target released
NO reacted with a thiol residue of cysteine (Sꢀnitrosylation) on NMDA receptor to obtain an
inhibition of NMDA receptor. In another way, the target releasing of NO can selectively

MedChemComm
Page 14 of 38
View Article Online
DOI: 10.1039/C6MD00509H
dilates brain vessels and increases CBF, which is important since some neurodegenerative
disorders are characterized by a decreased CBF due to vessel spasm. MNꢀ05 significantly
dilated the aortic rings in vitro and increased the CBF in vivo (data not shown), however,
there is no report that memantine has effect on contracted blood vessels. Taken together,
MNꢀ05 may be more effective and tolerable than memantine, providing a greater therapeutic
potential for the treatment of neurodegenerative disease. The comprehensive compare of
MNꢀ05 and memantine deserved an inꢀdepth in vivo study.
Experimental
The detailed procedures for the synthesis of memantine nitrates and the biological evaluation
experiments were found in the supplementary information.
Conclusions
In summary, we have designed and synthesized a serial of new memantine nitrates.
Compounds MNꢀ02, MNꢀ03, MNꢀ05, MNꢀ08, MNꢀ12–MNꢀ14 and MNꢀ16 displayed
protective effects in cultured cells and exhibited vasodilating effects on aortic rings. The SAR
investigation reveals that: in terms of cytoprotective effect, (1) substitution at the 3ꢀposition
of memantine skeleton with an ethyl group was superior to methyl and propyl groups; (2)
lengthening the linker between the nitrate group and memantine moiety decreased the
potency; (3) increasing the number of the nitrate groups didn’t enhanced the neuroprotective
effects. In terms of vasodilating effect, (1) the efficacy is increased by increasing the number
of the nitrate groups; (2) lengthening the side chains significantly increased the potency.
Among the new compounds, MNꢀ05 showed better activities in protecting cells from
glutamate insult and in dilating blood vessels. MNꢀ05 was effective in inhibiting the
elevation of intracellular calcium, the production of intracellular reactive species and the loss

Page 15 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
of mitochondrial membrane potential. MNꢀ05 may be a potential new treatment of
neurodegenerative diseases in including AD.
Supplementary Information
The detailed procedures for synthesis of the memantine nitrates and the biologic evaluation
experiments.
Acknowledgements
This work was partially supported by grants from the Science and Technology Planning
Project of Guangdong Province (2016A050503030), the Natural Science Foundation of
Guangdong Province (2014A030310174) and the National Natural Science Foundation of
China (NSFC 81603106), as well as the ITSPꢀGuangdongꢀHong Kong Technology
Cooperation Funding Scheme (GHP/012/16GD).
Notes and references
1
S. F. Traynelis, L. P. Wollmuth, C. J. McBain, F. S. Menniti, K. M. Vance, K. K. Ogden,
K. B. Hansen, H. Yuan, S. J. Myers and R. Dingledine, Pharmacol. Rev., 2010, 62,
405ꢀ496.
2
3
A. Lau and M. Tymianski, Pflugers Archiv, 2010, 460, 525ꢀ542.
J. C. PiñaꢀCrespo, S. SanzꢀBlasco and S. A. Lipton, Handbook of Neurotoxicity, 2014,
1015ꢀ1038.
4
5
6
E. E. Benarroch, Neurology, 2011, 76, 1750ꢀ1757.
F. H. Epstein, S. A. Lipton and P. A. Rosenberg, N. Engl. J. Med., 1994, 330, 613ꢀ622.
D. Lemoine, R. T. Jiang, A. Taly, T. Chataigneau, A. Specht and T. Grutter, Chem. Rev.,
2012, 112, 6285ꢀ6318.

MedChemComm
Page 16 of 38
View Article Online
DOI: 10.1039/C6MD00509H
7
8
9
S. A. Lipton, Nat. Rev. Drug Discov., 2006, 5, 160ꢀ170.
J. W. Johnson and S. E. Kotermanski, Curr. Opin. Pharmacol., 2006, 6, 61ꢀ67.
D. Wilkinson, Int. J. Geriatr. Psychiatry, 2012, 27, 769ꢀ776.
10 A. Witt, N. Macdonald and P. Kirkpatrick, Nat. Rev. Drug Discov., 2004, 3, 109ꢀ110.
11 H. Chen, J. Pellegrini, S. Aggarwal, S. Z. Lei, S. Warach, F. E. Jensen and S. Lipton, J.
Neurosci., 1992, 12, 4427ꢀ4436.
12 P. E. Stieg, S. Sathi, S. Warach, D. A. Le and S. A. Lipton, Eur. J. Pharmacol., 1999, 375,
115ꢀ120.
13 K. McKeage, CNS Drugs, 2009, 23, 881ꢀ897.
14 R. H. Lee, T. Y. Tseng, C. Y. Wu, P. Y. Chen, M. F. Chen, J. S. Kuo and T. J. Lee, PloS
One, 2012, 7, e40326. Doi: 10.1371/journal.pone.0040326.
15 J. R. Steinert, T. Chernova and I. D. Forsythe, Neuroscientist, 2010, 16, 435ꢀ452.
16 M. Willmot, L. Gray, C. Gibson, S. Murphy and P. M. Bath, Nitric oxide, 2005, 12,
141ꢀ149.
17 M. Khan, M. Jatana, C. Elango, A. S. Paintlia, A. K. Singh and I. Singh, Nitric oxide,
2006, 15, 114ꢀ124.
18 Y. B. Choi, L. Tenneti, D. A. Le, J. Ortiz, G. Bai, H. V. Chen and S. A. Lipton, Nat.
Neurosci., 2000, 3, 15ꢀ21.
19 D. Zurakowski, C. K. Vorwerk, M. Gorla, A. J. Kanellopoulos, N. Chaturvedi, C. L.
Grosskreutz, S. A. Lipton and E. B. Dreyer, Vision Res., 1998, 38, 1489ꢀ1494.
20 Y. Wang, J. Eu, M. Washburn, T. Gong, H. Vincent Chen, S, W. Larrick James, S. A.
Lipton, J. S. Stamler, G. T. Went and S. Porter, Curr. Alzheimer Res., 2006, 3, 201ꢀ204.
21 H. Takahashi, P. Xia, J. Cui, M. Talantova, K. Bodhinathan, W. Li, E. A. Holland, G.
Tong, J. PinaꢀCrespo, D. Zhang, N. Nakanishi, J. W. Larrick, S. R. McKercher, T.
Nakamura, Y. Wang and S. A. Lipton, Sci. Rep., 2015, 5, 14781.

Page 17 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
22 D. C. Mehta, J. L. Short and J. A. Nicolazzo, Mol. Pharm., 2013, 10, 4491ꢀ4498.
23 K. Higuchi, A. Kitamura, T. Okura and Y. Deguchi, Drug Metab. Pharmacokinet., 2015,
30, 182ꢀ187.
24 H. S. Chen and S. A. Lipton, J. Pharmacol. Exp. Ther., 2005, 314, 961ꢀ971.
25 W. Limapichat, W. Y. Yu, E. Branigan, H. A. Lester and D. A. Dougherty, ACS chem.
Neurosci., 2013, 4, 255ꢀ260.
26 C. Volbracht, J. van Beek, C. Zhu, K. Blomgren and M. Leist, Eur. J. Neurosci., 2006,
23, 2611ꢀ2622.
27 H. Ota, S. Ogawa, Y. Ouchi and M. Akishita, Exp. Gerontol., 2015, 72, 109ꢀ116.
28 D. Bonaventura, C. N. Lunardi, G. J. Rodrigues, M. A. Neto and L. M. Bendhack, Nitric
oxide, 2008, 18, 287ꢀ295.
29 A. Mehta, M. Prabhakar, P. Kumar, R. Deshmukh and P. L. Sharma, Eur. J. Pharmacol.,
2013, 698, 6ꢀ18.
30 M. Jia, S. A. Njapo, V. Rastogi and V. S. Hedna, CNS Drugs, 2015, 29, 153ꢀ162.
31 J. L. P. Velazquez, M. V. Frantseva and P. L. Carlen, J. Neurosci., 1997, 17, 9085ꢀ9094.
32 A. Perl and K. Banki, Antioxid. Redox Signal., 2000, 2, 551ꢀ573.
33 J. Gonzalez, J. C. JuradoꢀCoronel, M. F. Avila, A. Sabogal, F. Capani and G. E. Barreto,
Int. J. Neurosci., 2015, 125, 315ꢀ327.
34 J. Kornhuber and M. Weller, Bio. Psychiatry., 1997, 41, 135ꢀ144.
35 H. S. Chen and S. A. Lipton, J. Neurochem., 2006, 97, 1611ꢀ1626.
36 F. M. Faraci, Am. J. Physiol-Heart C, 1991, 261, 1038ꢀ1042.
37 K. Ranjbar, F. Nazem, A. Nazari, M. Gholami, A. R. Nezami, M. Ardakanizade, M.
Sohrabi, H. Ahmadvand, M. Mottaghi and Y. Azizi, EXCLI J., 2015, 14, 1104ꢀ1115.

MedChemComm
Page 18 of 38
View Article Online
DOI: 10.1039/C6MD00509H
Figs., Tables and Scheme Legends.
Fig. 1 Structures of Memantine and New Memantine Nitrates.
Fig. 2 Protective effects against glutamateꢀinduced excitotoxicity. Cerebellar granule
neurons were preꢀincubated with memantine nitrates or memantine for 2 h, and then exposed
to 200 µM glutamate for another 24 h in the presence of compounds. Cell viability was
*
measured by the MTT assay. ###P<0.001 versus control (Ctrl) group; P<0.05, **P<0.01 and
***P<0.001 versus glutamate alone (Glu) group, respectively.
Fig. 3 Vasodilation effects on aortic rings. The aortic rings with or without endothelium
were preꢀcontracted with phenylephrine, and then incubated with memantine nitrates or the
positive ISDN. The relaxation responses of arterial rings were expressed as the percentage of
maximal contraction induced by phenylephrine. (A) Effect on aortic rings with endothelium
(E+). (B) Effect on aortic rings without endothelium (Eꢀ).
Fig. 4 Protective effects on cortical neurons induced by glutamate. Rat primary cortical
neurons were treated with drugs for 2 h, and then exposed 200 µM glutamate for another 24 h
without removal of compounds. Cell viability was tested using the MTT assay. ###P<0.001
versus control (Ctrl) group; *P<0.05, **P<0.01 and ***P<0.001 versus glutamate (Glu) group.
Fig. 5 MNꢀ05 enhanced cortical neurons viability in glutamate induced injured. Rat
primary cortical neurons were treated with drugs for 2 h, and then exposed to 200 µM
glutamate for another 24 in the presence of compounds. Cell viability was tested using
FDA/PI double staining. (A) Captured fluorescence images (green labeling, FDA (live cells)

Page 19 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
and red labeling, PI (dead cells)). (B) Statistical data of (FDA/PI). ###P<0.001 versus control
(Ctrl) group; *P<0.05, **P<0.01 and ***P<0.001 versus glutamate (Glu) group.
Fig. 6 MNꢀ05 inhibited calcium influx. Cortical neurons were loaded with Fluoꢀ3/AM (5
o
ꢁM) for 30 min at 37 C, and then incubated with HBSS buffer for 30 min at room
temperature. After washed Fluoꢀ3/AM, cortical neurons were treated with MNꢀ05 or
memantine for 5 min. The cells were imaged using a 20× objective lens. ***P<0.001 versus
glutamate (Glu) group.
Fig. 7 MNꢀ05 inhibitied the production of intracellular ROS and RNS. Cortical neurons
were pretreated with MNꢀ05 or memantine at indicated concentrations for 2 h, followed by
exposure to 200 ꢁM of glutamate for another 12 h in the presence of compounds. H2DCFꢀDA
(5 ꢁM), HPF (10 ꢁM), DHE (5 ꢁM), DAFꢀFM (5 ꢁM) and DHR123 (10 ꢁM) was added and
incubated for another 30 min. MNꢀ05 significantly inhibited the production of reactive
species induced by glutamate in cortical neurons. (A) H2O2; (B) •OH; (C) O2•ꢀ; (D) NO and
*
(E) ONOO–. ###P<0.001 versus control (Ctrl) group, P<0.05, **P<0.01 and ***P<0.001
versus glutamate (Glu) group.
Fig. 8 MNꢀ05 inhibited the loss of mitochondrial membrane potential (ꢁψm). Cortical
neurons were treated with MNꢀ05 or memantine at indicated concentrations for 2 h, followed
by exposure to 200 ꢁM of glutamate for 12 h without removal of compounds. Cells were
washed with JCꢀ1 buffer and stained with 2 ꢁM JCꢀ1 for 20 min. Removed the supernatant
and washed cells with JCꢀ1 buffer for three times, and then added 100 ꢁL of HBSS solution
to each well. Fluorescence intensity was measured on a microplate reader at 488 nm

MedChemComm
Page 20 of 38
View Article Online
DOI: 10.1039/C6MD00509H
excitation and 529 nm/590 nm dual emissions. ###P<0.001 versus control (Ctrl) group,
***P<0.001 versus glutamate (Glu) group.
Table 1 The EC50 values of new memantine nitrates in cultured CGNs.
Table 2 The EC50 and Emax values of memantine nitrates in dilating aortic rings.
Scheme 1 Synthesis of compounds MNꢀ01ꢀMNꢀ03. Reagents and conditions: (a) (1) Br2,
reflux, 4 h; (2) r.t., overnight; (b) CH3CN, conc. H2SO4, r.t., 12 h; (c) (1) conc. HNO3, conc.
H2SO4, 0 oC, 2 h; r.t., 30 h; (2) NaOH, H2O; (d) (1) NaOH, diethylene glycol, 175 oC, 15 h; (2)
(Boc)2O, THF, TEA, DMAP, r.t., 5 h; (e) fuming HNO3, Ac2O, CH2Cl2, 0 oC; (f) HCl, ethyl
acetate.
Scheme 2 Synthesis of compounds MNꢀ04ꢀMNꢀ06. Reagents and conditions: (a) (1) Br2,
o
o
reflux, 4 h; (2) (COONa)2, H2O, 75 C, 1.5 h; (b) HCOOH, conc. H2SO4, 0 C, 4 h; (c)
o
CH3CN, conc. HNO3, conc. H2SO4, 0 C, 1 h; (d) (1) ClCOOC2H5, TEA, THF, r.t., 4 h; (2)
o
NaBH4, H2O, 1 h; (e) (1) NaOH, diethylene glycol, 175 C, 15 h; (2) (Boc)2O, THF, TEA,
DMAP, r.t., 5 h; (f) fuming HNO3, Ac2O, CH2Cl2, 0 oC; (g) HCl, ethyl acetate.
Scheme 3 Synthesis of compounds MNꢀ07ꢀMNꢀ09 and MNꢀ11. Reagents and conditions:
(a) ethyl acrylate for 37ꢀ39, ethyl methacrylate for 40, AIBN, nꢀBu3SnH, toluene, reflux, 2 h;
o
(b) KOH, MeOH, H2O, r.t., overnight; (c) CH3CN, conc. HNO3, conc. H2SO4, 0 C, 1 h; (d)
(1) ClCOOC2H5, TEA, THF, r.t., 4 h; (2) NaBH4, H2O, 1 h; (e) (1) NaOH, diethylene glycol,
175 oC, 15 h; (2) (Boc)2O, THF, TEA, DMAP, r.t., 5 h; (f) fuming HNO3, Ac2O, CH2Cl2, 0 oC;
(g) HCl, ethyl acetate.

Page 21 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
Scheme 4 Synthesis of compound MNꢀ10. Reagents and conditions: (a) isopropenyl acetate,
AlBr3, CH2Cl2, 0 oC, 2 h; (b) NaBH4, EtOH, r.t., 4 h; (c) Ac2O, HClO4, r.t., 3 h; (d) CH3CN,
o
o
conc. HNO3, conc. H2SO4, 0 C, 1 h; (e) (1) NaOH, diethylene glycol, 175 C, 15 h; (2)
(Boc)2O, THF, TEA, DMAP, r.t., 5 h; (f) fuming HNO3, Ac2O, CH2Cl2, 0 oC; (g) HCl, ethyl
acetate.
Scheme 5 Synthesis of compounds MNꢀ12ꢀMNꢀ13. Reagents and conditions: (a) Br2, Fe, 0
oC, 2 h; (b) CH3COCH3:H2O = 1:1, reflux, 3 h; (c) HCOOH, conc. H2SO4 , 0 oC, 2 h; r.t.,10 h;
(d) (1) ClCOOC2H5, TEA, THF, 4 h; (2) NaBH4, H2O, 1 h; (e) Ac2O, HClO4, r.t., 3 h; (f)
CH3CN, conc. HNO3, conc. H2SO4, 0 oC, 1 h; (g) (1) NaOH, diethylene glycol, 175 oC, 15 h;
(2) (Boc)2O, THF, TEA, DMAP, r.t., 5 h; (h) fuming HNO3, Ac2O, CH2Cl2, 0 oC; (i) HCl,
ethyl acetate.
Scheme 6 Synthesis of compound MNꢀ15. Reagents and conditions: (a) isopropenyl acetate,
AlBr3, CH2Cl2, 0 oC, 2 h; (b) NaBH4, EtOH, r.t., 4 h; (c) Ac2O, HClO4, r.t., 3 h; (d) CH3CN,
o
o
conc. HNO3, conc. H2SO4, 0 C, 1 h; (e) (1) NaOH, diethylene glycol, 175 C, 15 h; (2)
(Boc)2O, THF, TEA, DMAP, r.t., 5 h; (f) fuming HNO3, Ac2O, CH2Cl2, 0 oC; (g) HCl, ethyl
acetate.
Scheme 7 Synthesis of compounds MNꢀ14 and MNꢀ16. Reagents and conditions: (a) ethyl
acrylate for 88, ethyl methacrylate for 89, AIBN, nꢀBu3SnH, toluene, reflux, 2 h; (b) CH3CN,
o
conc. HNO3, conc. H2SO4, 0 C, 1 h; (c) NaBH4, AlCl3, THF, reflux, 3 h; (d) (1) NaOH,
o
diethylene glycol, 175 C, 15 h; (2) (Boc)2O, THF, TEA, DMAP, r.t., 5 h; (e) fuming HNO3,
Ac2O, CH2Cl2, 0 oC; (f) HCl, ethyl acetate.

MedChemComm
Page 22 of 38
View Article Online
DOI: 10.1039/C6MD00509H
Table 1 The EC50 values of new memantine nitrates in cultured cerebellar granule
neurons.
EC50 (ꢂM)
Mean ± SD
24.60 ±0.04
15.36 ±3.01
17.08±3.23
25.20±2.33
10.68±4.59
38.06±7.43
39.82±5.68
35.24±9.84
EC50 (ꢂM)
Mean ± SD
57.55±3.20
58.43±10.62
55.23±9.25
18.91±6.43
25.88±9.41
39.82±5.68
43.01±3.87
53.22±4.45
2.48 ± 0.43
Compounds
Compounds
MNꢀ01
MNꢀ02
MNꢀ03
MNꢀ04
MNꢀ05
MNꢀ06
MNꢀ07
MNꢀ08
MNꢀ09
MNꢀ10
MNꢀ11
MNꢀ12
MNꢀ13
MNꢀ14
MNꢀ15
MNꢀ16
Memantine

Page 23 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H
Table 2 The EC50 and Emax values of memantine nitrates in dilating aortic rings.
Eꢀ
E+
Compounds
EC50 (ꢂM)
Emax (ꢂM)
EC50 (ꢂM)
Emax (ꢂM)
146.33±32.80
42.03±3.70
39.47±2.41
ꢀ
MNꢀ02
MNꢀ03
MNꢀ05
MNꢀ08
MNꢀ12
MNꢀ13
MNꢀ14
MNꢀ16
94.82±10.11
50.57±4.38
26.85±7.23
62.81±8.45
57.05±1.23
34.40±10.38
36.46±6.84
61.68±1.63
98.19±1.01
89.76±3.31
90.50±4.94
99.37±0.77
95.36±2.62
95.79±4.05
51.99±1.44
101.26±2.46
94.96±0.42
96.75±2.15
101.47±1.04
97.74±2.16
100.21±1.48
ꢀ
46.35±1.56
24.38±0.97
54.97±6.87
36.04±2.02
24.60±3.37
31.97±9.55

MedChemComm
Page 24 of 38
View Article Online
DOI: 10.1039/C6MD00509H

Page 25 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H

MedChemComm
Page 26 of 38
View Article Online
DOI: 10.1039/C6MD00509H

Page 27 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H

MedChemComm
Page 28 of 38
View Article Online
DOI: 10.1039/C6MD00509H

Page 29 of 38
MedChemComm
View Article Online
DOI: 10.1039/C6MD00509H