In vitro and in vivo evaluation of polymethylene tetraamine derivatives as NMDA receptor channel blockers

Article abstract of DOI:10.1016/j.bmcl.2013.04.063

The biological activities of six symmetrically substituted 2-methoxy-benzyl polymethylene tetraamines (1-4) and diphenylethyl polymethylene tetraamines (5 and 6) as N-methyl-d-aspartate (NMDA) receptor channel blockers, were evaluated in vitro and in vivo

Full text of DOI:10.1016/j.bmcl.2013.04.063

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3901–3904
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro and in vivo evaluation of polymethylene tetraamine
derivatives as NMDA receptor channel blockers
Ryotaro Saiki a,b, Yuki Yoshizawa a, Anna Minarini c, Andrea Milelli d, Chiara Marchetti c,
Vincenzo Tumiatti d, Toshihiko Toida a, Keiko Kashiwagi e, Kazuei Igarashi a,b,
⇑
a Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
b Amine Pharma Research Institute, Innovation Plaza at Chiba University, Chiba, Japan
c Department of Pharmacy and Biotechnology, Alma Mater Studiorum, University of Bologna, Bologna, Italy
d Department for Life Quality Studies, Alma Mater Studiorum, University of Bologna, Rimini, Italy
e Faculty of Pharmacy, Chiba Institute of Science, Choshi, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The biological activities of six symmetrically substituted 2-methoxy-benzyl polymethylene tetraamines
(1–4) and diphenylethyl polymethylene tetraamines (5 and 6) as N-methyl- -aspartate (NMDA) receptor
Received 16 March 2013
Revised 20 April 2013
Accepted 23 April 2013
Available online 2 May 2013
D
channel blockers, were evaluated in vitro and in vivo. Although all compounds exhibited stronger channel
block activities in comparison to memantine in Xenopus oocytes voltage clamped at ꢀ70 mV, only com-
pound 2 (0.4 mg/kg intravenous injection) decreased the size of brain infarction in a photochemically
induced thrombosis model mice at the same extent of memantine (10 mg/kg intravenous injection).
Other compounds (1, 3, 4, 5 and 6) did not decrease the size of brain infarction significantly due to the
limited injection doses. The present study suggests that compound 2 could represent a valuable lead
compound to design low toxicity polyamines for clinical use against stroke.
Keywords:
Polymethylene teraamine derivative
NMDA receptor channel blocker
Ó 2013 Elsevier Ltd. All rights reserved.
N-Methyl-
D
-aspartate (NMDA) receptors are tetramers com-
nitrogen atoms, as well as appropriate spacers separating the
amine functions.8,9
posed of combinations of GluN1, GluN2, and GluN3 subunits, and
have been implicated in learning and memory through the func-
tion as ion channels.1,2 However, overexcitation of NMDA receptors
leads to excessive Ca2+ influx resulting in neuronal cell injury.
Therefore, NMDA receptor antagonists could be of therapeutic ben-
efit to some neurological disorders, such as stroke and Alzheimer’s
disease (AD). Memantine was the first NMDA receptor antagonist
approved as a medicine for dementia.
In this communication, a brief description of the synthesis of
the new polymethylene tetraamine 6, designed with the aim to in-
crease the lipophilicity, and the channel blocking activities of tetra-
amines 1–6, using Xenopus oocytes4,5 and thrombosis model
mice10 are presented. It is the first time to report the results of
in vivo experiments on these polymethylene tetraamine
derivatives.
In our continuing research of polyamines, especially spermine
and its derivatives, as channel blockers of NMDA receptors,
Synthesis of all compounds, except 6, (see Table 1 for chemical
structures) were already previously reported11,12 and used as tetr-
ahydrochloride salts to increase their solubility.
3–5
we thought of interest to study the channel block activity of NMDA
receptors using a series of polymethylene tetraamines (methoctr-
amine 1 and 2–6), differing in methylene chain lengths between
the nitrogen atoms and bearing 2-methoxy-benzyl (1–4) or
diphenylethyl moieties (5 and 6) on the terminal nitrogen atoms.
These compounds were originally reported as M2-muscarinic
receptor antagonists6 and some of them as muscular nicotinic
receptor non-competitive antagonists.7 With the discovery of com-
pound 1, we have demonstrated that it is possible to modulate
both affinity and selectivity of a polyamine structure for different
biological targets, by inserting appropriate groups onto the
The new diphenylethyl polyamine 6 has been synthesized fol-
lowing the procedure developed by our research group and re-
ported in Scheme 1.12 Briefly,
7 was obtained by reacting
N-[(benzyloxy)carbonyl]-6-aminocaproic acid with 1,12-diami-
nododecane; removal of the N-(benzyloxy)carbonyl group by
acidic hydrolysis gave diamine diamide 8, which was treated with
2,2-diphenylacetyl chloride to furnish derivative 9; reduction of 9
with BACH-EI (borane N-ethyl-N-isopropyl-aniline complex) in
dry diglyme led to the tetraamine 6.
The antagonistic effects on NMDA receptors of the synthetic
polyamines 1–6 (Table 1) were studied using recombinant NMDA
receptors expressed in Xenopus laevis oocytes. Most NMDA recep-
tors in the adult central nervous system contain a combination of
GluN1 and GluN2, with GluN2A and GluN2B predominating in
forebrain areas such as the cerebral cortex.1 Thus, IC50 values were
⇑
Corresponding author. Address: Graduate School of Pharmaceutical Sciences,
Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Tel.: +81 43 224
7500; fax: +81 43 379 1050.
E-mail address: iga16077@faculty.chiba-u.jp (K. Igarashi).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.063

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R. Saiki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3901–3904
Table 1
Determination of IC50 (nM) of compounds 1–6 on recombinant NMDA receptors expressed in Xenopus laevis oocytes
Compounda
Structure
GluN1/GluN2A channel
b
block at ꢀ70 mV, IC50
(nM)
10
OCH3
N1,N -(Octane-1,8-diyl)bis(N6-(2-
methoxybenzyl)hexane-1,6-
diamine)
H
N
H
N
1
2
3
4
138.0 10.5
N
H
N
H
OCH3
10
OCH3
OCH3
OCH3
N1,N -(Hexane-1,6-diyl)bis(N5-(2-
H
N
H
N
methoxybenzyl)pentane-1,5-
diamine)
315.0 31.6
89.3 23.8
256.7 69.5
N
H
N
H
OCH3
10
N1,N -(Decane-1,10-diyl)bis(N5-(2-
H
N
H
N
methoxybenzyl)pentane-1,5-
diamine)
N
H
N
H
OCH3
0
N1,N1 -(Hexane-1,6-diyl)bis(N8-(2-
H
N
H
N
methoxybenzyl)octane-1,8-
diamine)
N
H
N
H
OCH3
0
H
N
H
N1,N1 -(Octane-1,8-diyl)bis(N6-(2,2-
N
5
6
11.0 4.7
N
H
N
H
diphenylethyl)hexane-1,6-diamine)
0
N1,N1 -(Dodecane-1,12-diyl)bis(N6-
H
N
H
N
(2,2-diphenylethyl)hexane-1,6-
diamine)
4.2 2.3
N
H
N
H
a
Tetrahydrochloride salts.
b
Oocytes were injected with GluN1 plus GluN2A cRNA in a ratio of 1:5 (0.1–4 ng of GluN1 plus 0.5–20 ng of GluN2A).13 Inward currents were recorded at ꢀ70 mV. Values
are mean SEM of four oocytes. All animal experiments were approved by the Institutional Animal Care and Use Committee of Chiba University and carried out according to
the Guidelines for Animal Research of Chiba University.
O
O
O
O
O
H
N
H
N
i
H2N
NH2
O
N
H
N
H
O
O
N
H
COOH
10
10
10
3
3
3
3
3
O
O
O
O
7
9
H
N
H
N
H
N
H
N
ii
iii
N
H
N
H
H2N
NH2
10
3
3
O
O
8
iv
N
H
N
N
N
H
10
H
H
4
4
6
Scheme 1. Synthesis of compound 6. Reagents and conditions: (i) EtOCOCl, NEt3, dioxane, rt, 18 h, 84%; (ii) (a) HBr 33% in CH3COOH, rt, 4 h, quantitative yield; (b) 2 N NaOH,
CHCl3, 14 h; (iii) 2,2-diphenylacetylchloride, NEt3, CH2Cl2, rt, overnight, 20%; (iv) BACE-EI, diglyme, reflux, 12 h 36%.
obtained from concentration–inhibition curves with five concen-
trations of each compound at GluN1/GluN2A NMDA receptors.13
Representative traces of the effect of 1 lM compounds 2 and 6
on GluN1/GluN2A receptors are shown in Figure 1a, and concentra-
tion and inhibition curves of compounds 2 and 6 are shown in
Figure 1b.
Interestingly, the degree of inhibition (IC50) of NMDA receptors
by the six polymethylene tetraamines was stronger than that of
The IC50 values of compounds 1–6 on GluN1/GluN2A were found
in the following order: 6 (4.2 nM) > 5 (11 nM) > 3 (89 nM) > 1
(138 nM) > 4 (257 nM) > 2 (315 nM) (Table 1). These results clearly
indicate that within this series of compounds, the inhibitory po-
tency in in vitro experiments rises by increasing lipophilicity and
distance between the two inner nitrogen atoms of tetraamine
backbone. Indeed, the most active compound is 6 characterized
by a 12-methylene spacer between the inner nitrogen atoms and
a diphenylethyl moiety. In addition, comparing compounds 1 and
memantine (IC50 = 0.95 lM), chosen as the reference compound.

R. Saiki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3901–3904
3903
(a)
(b)
GluN1/GluN2A
1µM Compound 2
120
100
80
60
40
20
0
Gly + Glu
0.01
0.1
1
10
100 nA
1 min
Compound 2 (µM)
1µM Compound 6
100
80
60
40
20
0
Gly + Glu
0.0001 0.001 0.01 0.1
1
100 nA
1 min
Compound 6 (µM)
Figure 1. Effect of compounds 2 and 6 on GluN1/GluN2A NMDA receptors. Channel block activity was measured using Xenopus oocyte voltage clamped at ꢀ70 mV. NMDA
receptors were activated by superfusing glycine and glutamate (10 M). Inward currents were recorded with a two-electrode voltage clamp using a Gene Clamp 500 amplifier
l
(molecular devices). (a) Representative traces are shown to illustrate the effect of 1
lM compounds 2 and 6. (b) Concentration–inhibition curves were determined using 5
different concentrations of compounds 2 and 6.
1.25
1.00
*
0.75
*
0.50
0.25
0.00
2
3
4
5
6
Memantine
(10 mg/kg)
1
Saline
(0.4 mg/kg)
(1.5 mg/kg) (6.0 mg/kg) (3.0 mg/kg)
(10 mg/kg)
(Methoctramine,
1.0 mg/kg)
Figure 2. Effect of compounds 1–6 on the size of brain infarction in photochemically induced thrombosis model mice. Experiments were performed using 8 mice in each
group as described previously.10 Each compound was injected intravenously with the dose of approximately one-third of LD50, so that all mice survived during experiments.
Infarct volume with saline (control; relative size: 1) was between 30 and 35 mm3 in these experiments. Data are shown as mean SE ⁄p <0.05 compared with control mice.
5, both characterized by a 6-8-6 tetraamine backbone, it is evident
that the replacement of the 2-methoxy-benzyl functions of 1 with
diphenylethyl moieties as in 5, increased the potency almost 13
times (IC50: 138 and 11 nM, respectively).
tetraamine derivatives for clinical use against stroke, by choosing
the appropriate methylene chain lengths and substituents on
nitrogen atoms. Experiments are in progress to develop this point
using compound 2 as lead compound. We also found recently that
butanesulfonyl-homospermine guanidine decreased the size of
brain infarction in mice as memantine did.15 Interestingly, the size
of methylane chain lengths of this compound was equal to that of
compound 2.
Then, it was tested whether these compounds were able to de-
crease the size of brain infarction, using photochemically induced
thrombosis model mice.10 We found that the biological profile
trend in in vivo studies on compounds 1–6 was different from that
observed in in vitro studies. As shown in Figure 2, with the dose
(0.4 mg/kg) of approximately one-third of LD50, compound 2
greatly decreased the size of brain infarction as memantine did
at higher dose (10 mg/kg). Compounds 1 and 3–6 did not reduce
the size of brain infarction. This is mainly due to the limited injec-
tion doses because of their toxicity.14 These results suggest that it
will be crucial to design and synthesize low toxicity polymethylene
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science
and Technology, Japan and by a Grant from MIUR, Rome (PRIN
2009ESXPT2_001).

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9. Melchiorre, C.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V. J. Med.
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