Design and synthesis of a novel water-soluble NMDA receptor antagonist with a 1,4,7,10-tetraazacyclododecane group

Article abstract of DOI:10.1248/cpb.53.444

Polyamines, especially spermine, inhibit N-methyl-D-aspartate (NMDA) receptors as open channel blockers. Two types of water-soluble NMDA receptor antagonist, ACCn (1) and TGCn (2), with a 1,4,7,10-tetraazacyclododecane cyclic polyamine group, were synthes

Full text of DOI:10.1248/cpb.53.444

444
Notes
Chem. Pharm. Bull. 53(4) 444—447 (2005)
Vol. 53, No. 4
Design and Synthesis of a Novel Water-Soluble NMDA Receptor
Antagonist with a 1,4,7,10-Tetraazacyclododecane Group
Takashi MASUKO, Koichi METORI, Yasuo KIZAWA, Tadashi KUSAMA, and Muneharu MIYAKE*
College of Pharmacy, Nihon University; Narashinodai, Funabashi, Chiba 274–8555, Japan.
Received October 28, 2004; accepted January 14, 2005
Polyamines, especially spermine, inhibit N-methyl-D-aspartate (NMDA) receptors as open channel blockers.
Two types of water-soluble NMDA receptor antagonist, ACCn (1) and TGCn (2), with a 1,4,7,10-tetraazacyclodo-
decane cyclic polyamine group, were synthesized and the effects of both compounds on NMDA receptors were
studied using voltage-clamp recordings of recombinant NMDA receptors expressed in Xenopus oocytes. These
compounds inhibited macroscopic currents in both NR1/NR2A and NR1/NR2B receptor subtypes in oocytes
voltage-clamped at ꢀ70 mV. Inhibition by the compounds of NR1/NR2A receptors were more prominent than
that of NR1/NR2B receptors. The inhibitory effects of ACCn (1) on both NMDA receptors were more potent than
those of TGCn (2).
Key words N-methyl-D-aspartate (NMDA) receptor; 1,4,7,10-tetraazacyclododecane; Xenopus oocytes
Glutamate receptors are classified into two major groups ence of K2CO3 furnished 4 in 93% yield, and then hydrolysis
termed ionotropic and metabotropic glutamate receptors. The of the methyl ester 4 with 5 N KOH in methanol provided di-
ionotropic receptors can be subdivided into three distinct carboxylic acid (5, 100%), which underwent smooth esterifi-
types of receptors, the receptors for N-methyl-D-aspartate cation upon treatment with N,Nꢀ-dicyclohexylcarbodiimide
(NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazolepropi- (DCC) and pentafluorophenol to give the pentafluorophenyl
onate (AMPA), and kainate, all of which contain glutamate- ester 6 in 92% yield. The pentafluorophenyl ester function in
gated, cation-specific ion channels. Among ionotropic recep- compound (6) was converted into carboxamide (7, 98%)
tors, the NMDA receptor subtype has been found to play a after treatment of pentafluorophenyl ester with 25% NH4OH
key role in glutamate effects promoting synaptic plasticity, in THF, followed sequentially by reduction with BH3 to
long-term potentiation, and neuronal cell death.1) The the corresponding primary amine (8, 85%), which was con-
NMDA receptor combines to form heteromeric complexes verted into 10 by treatment with 1-ethyl-3-(3-dimethyl-
containing NR1 and NR2 subunits. The NR1 subunit is ubiq- aminopropyl)cabodiimide hydrochloride (EDC) and 1-
uitous and assembles with a second family of subunits carboxymethyl-4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-
termed NR2, including NR2A, NR2B, NR2C and NR2D.
tetraazacyclododecane (9).6) Finally, deprotection of 10 with
Overactivation of these receptors can lead to neuronal cell concentrated HCl in THF resulted in the desired compound
death, and the receptors also play a role in seizure activity. ACCn (1) in 91% yield.
Thus NMDA receptors are potential targets for neuroprotec-
On the other hand, TGCn (2) was synthesized as follows
tive agents and anticonvulsants.2,3) NMDA receptors have a (Chart 2). Treatment of pentafluorophenyl ester (11)7) with
complex pharmacology and are targets for antagonists acting 4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclodode-
at the glutamate and glycine coagonist sites, at a large num- cane (12)8) in the presence of triethylamine (TEA) gave 13 in
ber of modulatory sites, and at sites within the ion channel of 96% yield, and removal of the tert-butoxycarbonyl groups of
the receptor. A number of organic polycations, including the 13 by treatment with concentrated HCl in THF synthesized
endogenous polyamines spermine and spermidine, are antag- the desired compound TGCn (2) in quantitative yield.
onists at native and recombinant NMDA receptors.4) In addi-
The effects of ACCn (1) and TGCn (2) on NMDA recep-
tion, a number of polyamine-conjugated spider and wasp tox- tors were studied using voltage-clamp recordings of recombi-
ins are more potent antagonists than spermine at glutamate nant NMDA receptors expressed in Xenopus oocytes. We
receptors.5) These toxins, which include the philanthotoxins, measured the effects of ACCn (1) and TGCn (2) (10 mM) on
argiotoxins, and a-agatoxins, are characterized structurally responses to glutamate (10 mM, with 10 mM glycine) or
by the presence of an aromatic amino acid head group linked GABA 10 mM at NR1/NR2A, NR1/NR2B, or GABAc (r-1)
through a carbonamide bond to a polyamine tail such as sper-
mine or a pentamine or hexamine.
In this paper, we report the syntheses of two novel water-
soluble compounds ACCn (1) and TGCn (2) with two
1,4,7,10-tetraazacyclododecane groups to determine whether
these compounds have a role as NMDA receptor antagonists
(Fig. 1).
Results and Discussion
The host ACCn (1) was synthesized as shown in Chart 1.
Treatment of 4,4ꢀ-dihydroxydiphenylmethane (3) with methyl
bromoacetate in N,N-dimethylformamide (DMF) in the pres- Fig. 1. Structures of NMDA Receptor Antagonists
∗ To whom correspondence should be addressed. e-mail: miyake@pha.nihon-u.ac.jp
© 2005 Pharmaceutical Society of Japan

April 2005
445
Chart 1
Chart 2
CEZ-1250 (Nihon Koden, Tokyo, Japan). Electrodes were filled with KCl
3 M. Oocytes were continuously superfused (ca. 5 ml/min) with Mg2ꢂ-free
saline solution (NaCl 96 mM, KCl 2 mM, BaCl2 1.8 mM, HEPES 10 mM, pH
7.5). This solution contained BaCl2 rather than CaCl2, and, in most experi-
receptors in oocytes voltage-clamped at ꢁ70 mV. Both
ACCn (1) and TGCn (2) inhibited macroscopic currents at
both NMDA receptor subtypes (Fig. 2), but not at GABAc
(r-1) receptors (data not shown). These results indicate that ments, oocytes were injected with Kꢂ-1,2-bis(2-aminophenoxy)ethane-
N,N,Nꢀ,Nꢀ-tetraacetic acid (BAPTA; 100 nl of 40 mM solution at pH 7.5) on
the compounds specifically block NMDA receptors. The in-
hibition by these compounds of NR1/NR2A receptors were
more potent than those of NR1/NR2B receptors. An NMDA
the day of recording to eliminate Ca2ꢂ-activated Clꢁ currents.2,3) Glutamate
and glycine were purchased from Wako Pure Chemical Industries, Ltd.
(Osaka, Japan). BAPTA was purchased from Sigma (St. Louis, MO, U.S.A.).
glutamate receptor subtype is thought to play a predominant
role in triggering glutamate neurotoxicity in central nervous
system, it is possible that ACCn (1), TGCn (2) or these de-
rivatives have neuroprotective effects to glutamate neurotoxi-
city.
4,4ꢁ-Bis(methoxycarbonylmethoxy)diphenylmethane (4)
A mixture
of 4,4ꢀ-dihydroxydiphenylmethane (3) (1.0 g, 5 mmol), methyl bromoacetate
(1.53 g, 10 mmol), and K2CO3 (1.38 g, 10 mmol) in DMF (20 ml) was stirred
at room temperature. After 24 h, the reaction mixture was filtered. The fil-
trate was extracted with EtOAc (50 mlꢃ3), washed with brine, and dried
over MgSO4. After removal of the solvent under reduced pressure, the
residue was purified by column chromatography on silica gel with
EtOAc : CHCl3 (1 : 9) as an eluent to give 4 (1.6 g, 93%) as a colorless solid.
An analytical sample was obtained by recrystallizing this material from
EtOAc–hexane, yielding colorless needles, mp 51—52 °C. 1H-NMR
(CDCl3) d: 3.80 (6H, s); 3.85 (2H, s); 4.60 (4H, s); 6.82 (4H, d, Jꢄ8.8 Hz);
7.08 (4H, d, Jꢄ8.8 Hz). EI-MS m/z: 344 [M]ꢂ. HR-EI-MS m/z: 344.1256
[M]ꢂ (Calcd for C19H20O6: 344.1259). Anal. Calcd for C19H20O6: C, 66.27;
H, 5.85. Found: C, 66.54; H, 5.91.
Experimental
Melting points were determined using the Yanagimoto melting point ap-
paratus Yanaco MP and are uncorrected. 1H-NMR spectra were recorded on
a JEOL JNM-LA400 spectrometer containing tetramethylsilane as the stan-
dard. Mass spectra (MS) were measured on a JEOL JMS-GC mate instru-
ment. Adult female Xenopus laevis were chilled on ice, and the preparation
and maintenance of oocytes were carried out as described previously.9,10)
Capped cRNAs were prepared from linearized cDNA templates using
mMessage mMachine kits (Ambion, Austin, TX, U.S.A.). Oocytes were in-
jected with NR1A and NR2 cRNAs at a ratio of 1 : 5 (0.2—4 ng of NR1A
plus 1—20 ng of NR2). Macroscopic currents were recorded with a two-
electrode voltage clamp using a Dual Electrode Voltage Clamp Amplifier
4,4ꢁ-Bis(carboxymethoxy)diphenylmethane (5)
A
mixture of
4
(1.19 g, 3.46 mmol) and 5 N KOH/MeOH (4 ml) in MeOH (40 ml) was re-
fluxed for 2 h. After removal of the solvent under reduced pressure, the
residue was dissolved in 100 ml of H2O. The solution was extracted with
EtOAc (100 ml). The aqueous solution was acidified to pH 1 with 10% HCl

446
Vol. 53, No. 4
(100 : 40 : 4) as an eluent to give 8 (243 mg, 85%) as a colorless amorphous
1
powder that was used in the next step without further purification. H-NMR
(CD3OD) d: 2.99 (4H, t, Jꢄ5.6 Hz); 3.82 (2H, s); 3.98 (4H, t, Jꢄ5.6 Hz);
6.84 (4H, d, Jꢄ8.4 Hz); 7.07 (4H, d, Jꢄ8.4 Hz). FAB-MS m/z: 287
[MꢂH]ꢂ. HR-FAB-MS m/z: 287.1757 [MꢂH]ꢂ (Calcd for C17H23N2O2:
287.1759). Anal. Calcd for C17H22N2O2: C, 71.30; H, 7.74; N, 9.78. Found:
C, 71.51; H, 7.78; N, 9.52.
4,4ꢁ-Bis{2-[4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclodo-
decane-1-yl]acethylaminoethoxy}diphenylmethane (10) A mixture of 8
(123 mg, 0.43 mmol), 9 (456 mg, 0.86 mmol) and EDC (197 mg, 1.03 mmol)
in CH2Cl2 (10 ml) was stirred at room temperature for 24 h. The reaction
mixture was diluted with CH2Cl2 (10 ml), washed with 2 N NaOH, and dried
over Na2SO4. The solvent was evaporated under reduced pressure, and the
residue was chromatographed on silica gel with CHCl3 : MeOH : 25%
NH4OH (100 : 10 : 1) as an eluent to afford 10 (314 mg, 56%) as a white
powder that was used in the next step without further purification, mp 118—
120 °C. 1H-NMR (CDCl3) d: 143 (36H, s); 1.46 (18H, s); 2.70 (4H, br); 3.19
(4H, s); 3.35—3.52 (28H, m); 3.58—3.62 (4H, m); 3.83 (2H, s); 4.00 (4H, t,
Jꢄ5.6 Hz); 6.81 (4H, d, Jꢄ8.5 Hz); 7.05 (4H, d, Jꢄ8.5 Hz). FAB-MS m/z:
1311 [MꢂH]ꢂ. HR-FAB-MS m/z: 1311.8190 [MꢂH]ꢂ (Calcd for
C67H111N10O16: 1311.8179). Anal. Calcd for C67H110N10O16: C, 61.35; H,
7.74; N, 8.45. Found: C, 61.20; H, 8.57; N, 10.46.
4,4ꢁ-Bis[2-(1,4,7,10-tetraazacyclododecane-1-yl)acethylaminoethoxy]-
diphenylmethane Octahydrochloride (1, ACCn) 10 (255 mg, 0.19
mmol) was dissolved in THF (5 ml), to which concentrated HCl (0.5 ml) was
added. The reaction mixture was stirred at room temperature for 12 h. The
reaction mixture was diluted with THF (5 ml) and the precipitate was col-
lected by filtration, washed with THF, and dried to give 1 (ACCn) (173 mg,
Fig. 2. Effects of ACCn and TGCn on NMDA Receptors at ꢁ70 mV
(a) Representative traces showing blocking by ACCn and TGCn 10 mM at
NR1/NR2A and NR1/NR2B receptors at ꢁ70 mV. (b) Effects of ACCn and TGCn
10 mM were determined at NMDA receptors at ꢁ70 mV. Data are shown as percentage
of control measured in the absence of the compounds. Values are meanꢆS.E.M. from
four oocytes.
1
91%) as a white powder, mp 250 °C (decomp). H-NMR (D2O) d: 2.74—
2.89 (32H, m); 3.17 (4H, s); 3.37 (4H, t, Jꢄ5.4 Hz); 3.65 (2H, s); 3.94 (4H,
t, Jꢄ5.4 Hz); 6.73 (4H, d, Jꢄ8.6 Hz); 7.03 (4H, d, Jꢄ8.6 Hz). FAB-MS m/z:
711 [Mꢁ8HClꢂH]ꢂ. HR-FAB-MS m/z: 711.5029 [Mꢁ8HClꢂH]ꢂ (Calcd
for C37H63N10O4: 711.5033). Anal. Calcd for C37H62N10O4·8HCl: C, 44.32;
H, 7.04; N, 13.97. Found: C, 44.19; H, 7.27; N, 13.92.
and extracted with EtOAc (300 ml). The EtOAc layer was washed with
brine, dried over MgSO4, and concentrated under reduced pressure to give 5
as a colorless powder (1.09 g, 100%). An analytical sample was obtained by
recrystallizing this material from EtOAc–hexane, yielding colorless needles,
2,8-Bis{[4,7,10-tris(tert-butoxycarbonyl)-1,4,7,10-tetraazacyclodode-
cane-1-yl]oxomethyl}-6H,12H-5,11-mefhanodibenzo[b,f][1,5]diazocine
(13) A mixture of 11 (67 mg, 0.1 mmol), 12 (95 mg, 0.2 mmol), and TEA
(84 ml, 0.6 mmol) in CH2Cl2 (3 ml) was stirred at 60 °C for 24 h under a N2
atmosphere. The mixture was evaporated under reduced pressure. The
residue was purified by column chromatography on silica gel with EtOAc
and EtOAc : MeOH (9 : 1) to give 13 (120 mg, 96%) as a white powder that
1
mp 199—200 °C. H-NMR (DMSO-d6) d: 3.79 (2H, s); 4.59 (4H, s); 6.80
(4H, d, Jꢄ8.8 Hz); 7.10 (4H, d, Jꢄ8.8 Hz); 12.90 (2H, s). EI-MS m/z: 316
[M]ꢂ. HR-EI-MS m/z: 316.0944 [M]ꢂ (Calcd for C17H16O6: 316.0946).
Anal. Calcd for C17H16O6: C, 64.55; H, 5.10. Found: C, 64.60; H, 5.11.
4,4ꢁ-Bis(pentafluorophenoxycarbonylmethoxy)diphenylmethane (6)
A mixture of 5 (2.95 g, 9.3 mmol), pentafluorophenol (3.46 g, 18.8 mmol)
and DCC (3.88 g, 18.8 mmol) in THF (100 ml) was stirred at room tempera-
ture for 24 h. The mixture was filtered, and the filtrate was evaporated under
reduced pressure. The residue was purified by column chromatography on
silica gel with CH2Cl2 as an eluent to give 6 (5.56 g, 92%) as a colorless
solid. An analytical sample was obtained by recrystallizing this material
from EtOAc–hexane, yielding colorless needles, mp 135—136 °C. 1H-NMR
(CDCl3) d: 3.90 (2H, s); 4.97 (4H, s); 6.89 (4H, d, Jꢄ8.4 Hz); 7.13 (4H, d,
Jꢄ8.4 Hz). FAB-MS m/z: 648 [M]ꢂ. HR-FAB-MS m/z: 648.0628 [M]ꢂ
(Calcd for C29H14F10O6: 648.0630). Anal. Calcd for C29H14F10O6: C, 53.72;
H, 2.18. Found: C, 53.73; H, 2.14.
1
was used in the next step without further purification, mp 136—138 °C. H-
NMR (CDCl3) d: 1.41 (18H, s); 1.45 (18H, s); 1.47 (18H, s); 3.51—3.37
(36H, m); 4.12 (2H, d, Jꢄ16.8 Hz); 4.25 (2H, s); 4.66 (2H, d, Jꢄ16.8 Hz);
6.86 (2H, s); 7.06 (4H, s). FAB-MS m/z: 1247 [MꢂH]ꢂ. HR-FAB-MS m/z:
1247.7654 [MꢂH]ꢂ (Calcd for C65H103N10O14: 1247.7656). Anal. Calcd for
C65H102N10O14: C, 62.58; H, 8.24; N, 11.23. Found: C, 62.57; H, 8.06; N,
11.39.
2,8-Bis[(1,4,7,10-tetraazacyclododecane-1-yl)oxomethyl]-6H,12H-
5,11-mefhanodibenzo[b,f][1,5]diazocine Octahydrochloride (2, TGCn)
A mixture of 13 (80 mg, 0.064 mmol) and concentrated HCl (0.5 ml) in THF
(1 ml) was stirred at room temperature. After 36 h, the resulting precipitate
was collected by filtration, washed successively with THF and Et2O, and
1
dried to give (2, TGCn) (60 mg, 100%) as a white solid, mp ꢅ290 °C. H-
4,4ꢁ-Bis(carbamoylmethoxy)diphenylmethane (7) To a solution of 6
(4.0 g, 6.17 mmol) in THF (30 ml) was added 25% NH4OH (12 ml) at room
temperature. After stirring for 12 h, saturated NaHCO3 (200 ml) was added
to the reaction mixture. The precipitate was collected by filtration, washed
with H2O, EtOH, and Et2O, and dried under a vacuum at 60 °C to give 7
(1.9 g, 98%) as a colorless powder which was used in the next step without
further purification, mp 233—234 °C. 1H-NMR (DMSO-d6) d: 3.80 (2H, s);
4.36 (4H, s); 6.85 (4H, d, Jꢄ8.8 Hz); 7.11 (4H, d, Jꢄ8.8 Hz); 7.32 (2H, s);
7.43 (2H, s). FAB-MS m/z: 315 [MꢂH]ꢂ. HR-FAB-MS m/z: 315.1346
[MꢂH]ꢂ (Calcd for C17H19N2O4: 315.1344). Anal. Calcd for C17H18N2O4: C,
64.96; H, 5.77; N, 8.91. Found: C, 64.74; H, 5.67; N, 8.69.
4,4ꢁ-Bis(2-aminoethoxy)diphenylmethane (8) A mixture of 7 (314 mg,
1 mmol) and BH3·DMS (1.16 ml, 12 mmol) in THF (12 ml) was stirred for
24 h at 80 °C under a N2 atmosphere, then was cooled to room temperature.
Six milliliters of 0.7 M hydrogen chloride–MeOH solution was added, and
the mixture was refluxed for 0.5 h and evaporated under reduced pressure.
The residue was basified with 25% NH4OH. The mixture was extracted with
CH2Cl2, washed with brine, and dried over Na2SO4. Removal of the solvent
under reduced pressure afforded a pale yellow oil, which was purified by
column chromatography on silica gel with CHCl3 : MeOH : 25% NH4OH
NMR (D2O) d: 2.64—2.89 (24H, m); 3.35—3.37 (4H, m); 3.43—3.46 (4H,
m); 3.54 (4H, s); 4.00 (2H, d, Jꢄ16.8 Hz); 4.12 (2H, s); 4.45 (2H, d,
Jꢄ16.8 Hz); 6.69 (2H, d, Jꢄ1.44 Hz); 6.91 (2H, dd, Jꢄ8.28, 1.96 Hz); 6.98
(2H, d, Jꢄ8.44 Hz). FAB-MS m/z: 647 [Mꢁ8HClꢂH]ꢂ. HR-FAB-MS m/z:
647.4509 [Mꢁ8HClꢂH]ꢂ (Calcd for C37H63N10O4: 647.4509). Anal. Calcd
for C35H54N10O2·8HCl: C, 44.79; H, 6.66; N, 14.92. Found: C, 44.82; H,
6.75; N, 14.76.
Acknowledgments This work was supported by a grant from the Min-
istry of Education, Culture, Sports, Science and Technology of Japan to pro-
mote multidisciplinary research projects and a Joint Research Grant from
Nihon University College of Pharmacy (2003, 2004).
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