Synthesis of water-soluble polyamine derivatives effective as N-methyl-D-aspartate receptor antagonists

Article abstract of DOI:10.1248/cpb.58.862

The novel water-soluble N-methyl-D-aspartate (NMDA) receptor antagonists, N-{4-[4-(4-Guanidinobutylamino) butylamino]butyl}-p-toluenesulfonamide trihydrochloride (1a, TsHSPMG), N-{4-[4-(4-Guanidinobutylamino) butylamino]butyl}butane-1-sulfonamide trihydro

Full text of DOI:10.1248/cpb.58.862

862
Note
Chem. Pharm. Bull. 58(6) 862—867 (2010)
Vol. 58, No. 6
Synthesis of Water-Soluble Polyamine Derivatives Effective as N-Methyl-D-
aspartate Receptor Antagonists
Takashi MASUKO, Shuhei YOSHIDA, Koichi METORI, Yasuo KIZAWA, Tadashi KUSAMA, and
Muneharu MIYAKE*
School of Pharmacy, Nihon University; 7–7–1 Narashinodai, Funabashi, Chiba 274–8555, Japan.
Received February 15, 2010; accepted March 25, 2010; published online March 29, 2010
The novel water-soluble N-methyl-D-aspartate (NMDA) receptor antagonists, N-{4-[4-(4-Guanidinobutyl-
amino)butylamino]butyl}-p-toluenesulfonamide trihydrochloride (1a, TsHSPMG), N-{4-[4-(4-Guanidinobutyl-
amino)butylamino]butyl}butane-1-sulfonamide trihydrochloride (1b, BsHSPMG), N-{3-[4-(3-Guanidinopropyl-
amino)butylamino]propyl}-p-toluenesulfonamide trihydrochroride (2a, TsSPMG) and N-{3-[4-(3-Guanidino-
propylamino)butylamino]propyl}butane-1-sulfonamide trihydrochroride (2b, BsSPMG), were synthesized, and
the effects of these polyamine derivatives on NMDA receptors were studied using voltage-clamp recordings of re-
combinant NMDA receptors expressed in Xenopus oocytes. Although spermine potentiates 153% and 310% of
NMDA (NR1A/NR2B) receptors in the presence of saturated and unsaturated glycine, respectively, all the novel
polyamine derivatives, TsHSPMG (1a), BsHSPMG (1b), TsSPMG (2a) and BsSPMG (2b), significantly inhibited
NR1A/NR2B receptors in both conditions. The degree of NMDA receptor inhibition by TsHSPMG (1a) and BsH-
SPMG (1b) was stronger than that by TsSPMG (2a) and BsSPMG (2b).
Key words N-methyl-D-aspartate receptor; polyamine; Xenopus oocyte; voltage-clamp recording
Three pharmacologically-defined classes of ionotropic glu-
tamate receptors were originally named according to their ag-
onist selectivity: N-methyl-D-aspartate (NMDA), a-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and
kainate. The NMDA receptor consists of at least two distinct
subunits, NR1 and NR21) (Fig. 1A). Each subunit has three
transmembrane domains (M1, M3, and M4) plus a cyto-
plasm-facing re-entrant membrane loop (M2). The M2 loop
region in the NR1 and NR2 subunits is a critical determinant
of divalent cation permeability and Mg2ꢀ block. NR1 is a
single gene product expressed as eight alternatively spliced
mRNAs, and NR2A, NR2B, NR2C and NR2D are distinct
gene products. NMDA receptors probably consist of
tetrameric subunit assemblies that have different physiologi-
cal and pharmacological properties depending on the specific
NR2 subunit. In the central nervous system, NMDA recep-
tors play critical roles in a variety of neurophysiological phe-
nomena, including neurodevelopment, synaptic plasticity,
and excitotoxicity, because of their high permeability to
Ca2ꢀ. Neurodegeneration associated with a variety of acute
and chronic disorders (e.g., ischemic stroke, Parkinson’s dis-
ease, Alzheimer’s disease and dementia) is due in part to
overactivation of NMDA receptors (Fig. 1B). Inhibitors of
NMDA receptors have thus been developed as anticonvul-
sants and neuroprotective agents.
Polyamines (putrescine, spermidine and spermine) are
ubiquitously present in prokaryotic and eukaryotic cells. In
the central nervous system, specific interactions of
polyamines with several structurally and functionally distinct
types of cation channels have been reported previously.2)
Among these, the most striking are the blockade of some
types of Kꢀ channels and the modulation of NMDA recep-
tors. Spermine has complex effects on NMDA receptors, in-
cluding two types of stimulation and one type of voltage-de-
Fig. 1. Structures of NMDA Receptor and Model for NMDA Receptor
Mediated Ca2ꢀ-Dependent Neurotoxicity
(A) Schematic illustration of the NR1 and NR2B subunits, which contain three trans-
membrane segments (M1, M3, M4), a re-entrant loop (M2), an extracellular N-terminal
domain, an extracellular loop between M3 and M4, an intracellular loop between M1
and M2, and an intracellular C-terminal domain. The glycine and glutamate binding
pockets involving residues from the N-terminal domain and from the M3-M4 loop of
NR1 and NR2 subunits, respectively. (B) Excitotoxicity is thought to be a major mecha-
nism contributing to neurodegeneration during central nervous system, ischemia,
trauma and other neurological disorders. Excitotoxicity is thought to result from an ex-
cessive synaptic release of glutamate. Also, it is generally accepted that the NMDA re-
ceptor plays a key role in mediating at least certain aspects of glutamate neurotoxicity,
possibly owing to their high Ca2ꢀ permeability. When Ca2ꢀ overloaded, such processes
including enzymes (e.g., proteases, lipases, endonucleases) and other metabolic ma-
chinery directly damage neurons or lead to the formation of toxic reaction products
which ultimately cause cell death.
pendent blockade. One of the effects of spermine is “glycine- only at receptors containing splice variants of NR1 that lack
independent” stimulation, observed in the presence of satu- the exon-5 insert, expressed together with the NR2B
rating concentrations of glutamate and glycine. With recom- subunit.3) Inhibition by extracellular spermine is strongly
binant NMDA receptors, this type of stimulation is observed voltage-dependent, being more pronounced at hyperpolarized
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: miyake.muneharu@nihon-u.ac.jp

June 2010
863
than depolarized membrane potentials. Single-channel patch- ronal cell death due to supranormal excitability of NMDA re-
clamp recording has been used to study the mechanism of ceptors is common under pathological depolarized condi-
this effect. Spermine (10—1000 mM) was found to decrease tions. The effects of these polyamine derivatives were inves-
both single-channel conductance and average channel open- tigated on NR1/NR2B receptors in the depolarized state in
time.
the presence of saturated or unsaturated concentrations of
We previously reported that N-{4-[4-(4-guanidinobutyl- glycine, because spermine stimulates NMDA receptor activ-
amino)butylamino]butyl}-p-toluenesulfonamide trihydrochlo- ity under depolarized conditions. Also, N4-benzylspermine
ride (TsHSPMG) (1a) and N-{3-[4-(3-guanidinopropyl- and N4-(3-phenylpropyl)spermine may stimulate NMDA re-
amino)butylamino]propyl}-p-toluenesulfonamide trihydro- ceptor activities, because the low concentration of these
chroride (TsSPMG) (2a) inhibited the activity of NMDA polyamine derivatives promoted quantity of [3H]MK-801
receptors expressed in Xenopus oocytes. Their antagonistic binding to NMDA receptor.7)
activities were more potent than that of memantine, an
NMDA antagonist. TsHSPMG (1a), which has a longer Results and Discussion
polyamine tail than TsSPMG (2a), showed more potent an-
The synthesis of polyamine compounds TsHSPMG (1a)
tagonistic activity. We therefore concluded that the homo- and BsHSPMG (1b) is outlined in Chart 1. 4-(4-Methoxy-
spermine moiety and guanidyl group play an important role benzyloxycarbonyl)amino-1-butanol (3) was synthesized
in neuroprotection against NMDA toxicity.4) Pöhler et from 4-methoxybenzyl S-(4,6-dimethylpyrimidin-2-yl)thio-
al. reported synthesis and pharmacological properties carbonate and 4-amino-1-butanol in CHCl3, which was con-
of polyamine derivatives and also exhibited that some verted to tosylate (4). Tosylate (4) was condensed with 4-
analogs of 5-(4-aminobutyl)-2-thiophene-octylamine inhib- amino-1-butanol in N,N-dimethylformamide (DMF) at 90 °C
ited NMDA receptor binding using [3H]MK-801 binding to yield 5. Protection of the amino nitrogen in 5 with a N-
assay methods.5)
tert-butoxycarbonyl (Boc) group, esterifcation of the result-
In this paper, we report the syntheses of TsSPMG (2a) and ing alcohol (6) as the tosylate, and condensation of the tosy-
N-{3-[4-(3-guanidinopropylamino)butylamino]propyl}bu- late (7) and 4-amino-1-butanol gave the corresponding pri-
tane-1-sulfonamide trihydrochroride (BsSPMG) (2b), which mary alcohol (8). After protecting the amino nitrogen of 8
have a guanidyl group on the spermine moiety, and with N-Boc, the terminal hydroxyl group was mesylated
TsHSPMG (1a) and N-{4-[4-(4-guanidinobutylamino)butyl- with methanesulfonyl chloride (MsCl) to yield 10. Mesylate
amino]butyl}butane-1-sulfonamide trihydrochloride (BsH- 10 was converted to azide compound (11) and subsequent
SPMG) (1b), which have a homospermine moiety instead of hydrogenation using palladium-activated carbon ethylenedi-
spermine. Through the addition of the guanidyl group or ho- amine complex (Pd–C(en)) gave primary amino compound
mospermine moiety we aimed at construct compounds which (12). Compound 13 was obtained by treatment of 12 with N,
are less sensitive to polyamine oxidases (spermine oxidase Nꢁ-bis(tert-butoxycarbonyl)-S-methylisothiourea in CH2Cl2.
and acetylpolyamine oxidase) and degrade spermine to pro- The removal of the 4-methoxybenzyloxycarbonyl group in
duce acrolein, a toxic unsaturated aldehyde.6) TsHSPMG 13 gave 14, then the terminal amino group in 14 was con-
(1a), TsSPMG (2a), BsHSPMG (1b) and BsSPMG (2b) (at verted to p-toluenesulfonamide compound 15 and butanesul-
concentrations up to 300 mM) were non-toxic to cells in the fonamide compound 16. Finally, the Boc protecting groups
presence of fetal bovine serum (FBS) (data not shown). Neu- of 15 and 16 were cleaved to give the desired compounds
Chart 1

864
Vol. 58, No. 6
Chart 2
TsHSPMG (1a) and BsHSPMG (1b).
The synthesis of polyamine compounds TsSPMG (2a) and
BsSPMG (2b) is outlined in Chart 2. Treatment of 4,9-diaza-
4,9-di-(tert-butoxycarbonyl)dodecane-1,12-diamine8) with p-
toluenesulfonyl chloride (TsCl) in the presence of triethy-
lamine (TEA) in CH2Cl2 gave p-toluenesulfonamide com-
pound 18 in 49% yield, and then the terminal primary amino
group in 18 was converted to guanidino compound (20)
by treating 18 with N,Nꢁ-bis(tert-butoxycarbonyl)-S-methyl-
isothiourea in CH2Cl2. Finally, the Boc protecting groups of
20 were removed using 36% HCl in tetrahydrofuran (THF) to
give the desired compound TsSPMG (2a). BsSPMG (2b)
was synthesized from butane-1-sulfonyl chloride (BsCl) by a
method similar to that used to synthesize TsSPMG (2a).
The effects of the polymines TsHSPMG (1a), BsHSPMG
(1b), TsSPMG (2a), BsSPMG (2b) and spermine on NMDA
(NR1A/NR2B) receptors were studied using voltage-clamp
recordings of recombinant NR1A/NR2B receptors expressed
in Xenopus oocytes. We measured the effects of TsHSPMG
(1a), BsHSPMG (1b), TsSPMG (2a), BsSPMG (2b) and
spermine (100 mM) on responses to glutamate (10 mM, with
0.1 or 10 mM glycine) by NR1A/NR2B receptors in oocytes
voltage-clamped at ꢂ20 mV. Spermine potentiates 153% and
310% of NMDA (NR1A/NR2B) receptors in the presence of
saturated or unsaturated glycine, respectively. Spermine po-
tentiates NMDA receptor currents in the presence of saturat-
ing concentrations of glycine (10 mM, glycine-independent
Fig. 2. Effects of Spermine (SPM) and Polyamine Derivatives on NR1/
NR2B Receptors at ꢂ20 mV
Representative traces showing the effects of 100 mM spermine or TsHSPMG (1a) on
the NR1/NR2B receptors. The effects of 100 mM spermine, TsHSPMG (1a), BsHSPMG
(1b), TsSPMG (2a) and BsSPMG (2b) were determined in oocytes expressing
NR1/NR2B receptors. Currents were evoked using 10 mM glutamate with 0.1 or 10 mM
glycine and voltage-clamped at ꢂ20 mV. Macroscopic currents in the presence of sper-
mine or polyamine derivatives were expressed as a percentage of the control response at
the NMDA receptors. Data represent the meanꢃS.E.M. from 4 or 5 oocytes.
stimulation), an effects that involves an increase in the fre- sized polyamine derivatives had voltage dependent inhibitory
quency of channel opening and decrease in the desensitiza- effects on NMDA receptor currents, but the facilitatory ef-
tion of NMDA receptors. A second effect for the potentiation fects disappeared. NMDA receptor subtypes are thought to
involves an increase in the affinity of NMDA receptors for play a predominant role in triggering glutamate neurotoxicity
glycine (0.1 mM, glycine-dependent stimulation). However, in the central nervous system. Thus, polyamine derivatives
all the novel derivatives, TsHSPMG (1a), BsHSPMG (1b), may have neuroprotective effects towards glutamate neuro-
TsSPMG (2a) and BsSPMG (2b) significantly inhibited toxicity. Further pharmacological data regarding BsHSPMG
NR1A/NR2B receptors in both conditions. The inhibition of and BsSPMG will be described elsewhere.
NMDA receptors by BsSPMG (2b), BsHSPMG (1b),
Experimental
TsSPMG (2a) and TsHSPMG (1a) in the presence of satu-
rated glycine (10 mM) was 26%, 83%, 62% and 82%, respec-
tively. Comparable results were obtained in the presence of
unsaturated glycine (0.1 mM) (Fig. 2). Inhibition by extracel-
lular spermine is strongly voltage-dependent and may be due
to fast open-channel block, similar to that by Mg2ꢀ. Synthe-
Melting points were determined using a Yanagimoto Yanaco MP melting
point apparatus and are uncorrected. 1H-NMR (500 MHz) and 13C-NMR
(125 MHz) spectra were recorded on a JEOL JNM-ECA 600 spectrometer
using tetramethylsilane as a standard. Mass spectra (MS) were measured on
a JEOL LMS-GC mate instrument. IR spectra were recorded on a JASCO
FT/IR300E spectrometer. Adult female Xenopus laevis were chilled on ice,

June 2010
865
(CDCl3) d: 1.41 (9H, s), 1.42—1.63 (8H, m), 2.45 (3H, s), 3.12 (4H, br s),
3.18—3.21 (2H, m), 3.80 (3H, s), 4.03 (2H, t, Jꢄ6.18 Hz), 5.02 (2H, s), 6.88
(2H, d, Jꢄ8.58 Hz), 7.29 (2H, d, Jꢄ8.58 Hz), 7.34 (2H, d, Jꢄ8.22 Hz), 7.78
(2H, d, Jꢄ8.22 Hz).
and the preparation and maintenance of oocytes were performed as de-
scribed previously.9) Capped cRNA was prepared from linearized cDNA
templates using mMessage mMachine kits (Ambion, Austin, TX, U.S.A.).
Oocytes were injected with NR1A and NR2B cRNAs at a ratio of 1 : 5
(0.2—1 ng of NR1A plus 1—5 ng of NR2B). Macroscopic currents were
recorded with a two-electrode voltage-clamp using Dual Electrode Voltage
Clamp Amplifier CEZ-1250 (Nihon Koden, Tokyo, Japan). Electrodes were
filled with 3 M KCl. Oocytes were continuously superfused (ca. 5 ml/min)
with a Mg2ꢀ-free saline solution (96 mM NaCl, 2 mM KCl, 1.8 mM BaCl2,
10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH
7.5). This solution contained BaCl2 rather than CaCl2, and in all experi-
ments, oocytes were injected with Kꢀ-1,2-bis(2-aminophenoxy)ethane-
N,N,Nꢁ,Nꢁ-bis-tetraacetic acid (BAPTA; Sigma, St. Louis, MO, U.S.A.;
100 nl of 40 mM solution at pH 7.5) on the day of recording to eliminate
Ca2ꢀ-activated Cl-currents. Glutamate and glycine were purchased from
Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
4-(4-Methoxybenzyloxycarbonyl)amino-1-butanol (3) A mixture of
4-amino-1-butanol (2.67 g, 30 mmol) and p-methoxybenzyl S-(4,6-di-
methylpyrimidin-2-yl)thiocarbonate (9.12 g, 30 mmol) in CHCl3 (160 ml)
was stirred at room temperature overnight. The reaction mixture was washed
successively with 4% aqueous NaHCO3 solution and H2O, dried over
Na2SO4, and concentrated under reduced pressure. The residue was chro-
matographed on a silica gel column. Elution with EtOAc : hexane (3 : 1) af-
forded a white solid (6.78 g, 89%). Recrystallization from EtOAc/hexane
gave colorless fine needles, mp 74—75 °C. HR-FAB-MS m/z: 254.1391
[MꢀH]ꢀ (Calcd for C13H20NO4: 254.1392). 1H-NMR (CDCl3) d: 1.42
(1H, t, Jꢄ5.16 Hz), 1.59 (4H, br s), 3.21—3.26 (2H, m), 3.65—3.68 (2H,
m), 3.81 (3H, s), 4.80 (1H, br s), 5.03 (2H, s), 6.88 (2H, d, Jꢄ8.58 Hz), 7.30
(2H, d, Jꢄ8.58 Hz). Anal. Calcd for C13H19NO4: C, 61,64; H, 7.56; N, 5.53.
Found: C, 61.62; H, 7.55; N, 5.57.
10-tert-Butoxycarbonyl-15-(4-methoxybenzyloxycarbonyl)-5,10,12-tri-
aza-1-pentadecanol (8) A solution of 7 (7.66 g, 13.2 mmol) and 4-amino-
1-butanol (1.42 g, 15.9 mmol) in DMF (8 ml) was stirred at 90 °C for 2.5 h.
The reaction mixture was poured into ice water. The solution was made al-
kaline (pH 12) by adding 5% aqueous KOH solution and then extracted with
CH2Cl2. The organic layer was washed with brine and dried over Na2SO4,
and then the solvent was removed under reduced pressure. The residue was
purified by column chromatography on silica gel with CHCl3 : MeOH : 25%
NH4OH (100 : 20 : 2) to give a colorless oil (3.86 g, 59%). HR-FAB-MS m/z:
496.3385 [MꢀH]ꢀ (Calcd for C26H45N3O6: 496.3386). 1H-NMR (CDCl3) d:
1.44 (9H, s), 1.45—1.54 (8H, m), 1.58—1.68 (4H, m), 2.61—2.65 (4H, m),
3.12—3.21 (6H, m), 3.55—3.57 (2H, m), 3.80 (3H, s), 5.02 (2H, s), 6.88
(2H, d, Jꢄ8.58 Hz), 7.29 (2H, d, Jꢄ8.58 Hz).
5,10-Di-tert-butoxycarbonyl-15-(4-methoxybenzyloxycarbonyl)-
5,10,15-triaza-1-pentadecanol (9) A mixture of 8 (1.3 g, 2.62 mmol) and
(Boc)2O (0.628 g, 2.88 mmol) in CH2Cl2 (15 ml) was stirred at 0—5 °C for
9 h. The mixture was concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel using EtOAc : hexane
(3 : 1) to give a colorless oil (1.48 g, 95%). HR-FAB-MS m/z: 596.3911
[MꢀH]ꢀ (Calcd for C31H54N3O8: 596.3910). 1H-NMR (CDCl3) d: 1.43 (9H,
s), 1.44 (9H, s), 1.45—1.56 (12H, m), 3.16—3.20 (10H, m), 3.62—3.67
(2H, m), 3.80 (3H, s), 5.02 (2H, s), 6.88 (2H, d, Jꢄ8.22 Hz), 7.29 (2H, d,
Jꢄ8.22 Hz).
5,10-Di-tert-butoxycarbonyl-15-(4-methoxybenzyloxycarbonyl)-
5,10,15-triaza-1-pentadecanol Methanesulfonate (10) To a solution of 9
(1.45 g, 2.43 mmol) and TEA (6.1 ml, 43.57 mmol) in CH2Cl2 (45 ml), MsCl
(0.334 g, 2.92 mmol) was added at 0—5 °C. The reaction mixture was stirred
for 2 h at 0—5 °C and then at room temperature overnight. The mixture was
washed with brine, dried over MgSO4, and concentrated under reduced pres-
sure. The residue was chromatographed on a silica gel column. Elution with
EtOAc : hexane (2 : 1) afforded a colorless oil (1.55 g, 95%). HR-FAB-MS
m/z: 674.3688 [MꢀH]ꢀ (Calcd for C32H56N3O10S: 674.3686). 1H-NMR
(CDCl3) d: 1.43 (9H, s), 1.44 (9H, s), 1.47—1.55 (8H, m), 1.60—1.65 (2H,
m), 1.71—1.74 (2H, m), 3.00 (3H, s), 3.15—3.20 (10H, m), 3.80 (3H, s),
4.24 (2H, t, Jꢄ6.54 Hz), 5.02 (2H, s), 6.88 (2H, d, Jꢄ8.58 Hz), 7.29 (2H, d,
Jꢄ8.58 Hz).
4-(4-Methoxybenzyloxycarbonyl)aminobutyl-1-p-toluenesulfonate (4)
To a solution of 3 (7.24 g, 28.58 mmol) and TEA (12 ml, 85.7 mmol) in
CH2Cl2 (150 ml), TsCl (5.43 g, 28.58 mmol) was added at 0—5 °C. The re-
action mixture was stirred for 5 h at 0—5 °C and then at room temperature
overnight. The mixture was washed with brine, dried over MgSO4, and con-
centrated under reduced pressure. The residue was chromatographed on
a silica gel column. Elution with EtOAc : hexane (1 : 1) afforded a colorless
oil (9.28 g, 80%). HR-FAB-MS m/z: 407.1402 [MꢀH]ꢀ (Calcd for
1
C20H25NO6S: 407.1402). H-NMR (CDCl3) d: 1.53—1.55 (2H, m), 1.65—
1.68 (2H, m), 2.44 (3H, s), 3.13—3.18 (2H, m), 3.80 (3H, s), 4.03 (2H, t,
Jꢄ6.18 Hz), 4.66 (1H, br s), 5.00 (2H, s), 6.88 (2H, d, Jꢄ8.58 Hz), 7.28 (2H,
d, Jꢄ8.58 Hz), 7.33 (2H, d, Jꢄ8.22 Hz), 7.77 (2H, d, Jꢄ8.22 Hz).
1-Azido-5,10-di-tert-butoxycarbonyl-15-(4-methoxybenzyloxycar-
bonyl)-5,10,15-triazapentadecane (11)
A
mixture of 10 (4.38 g,
6.5 mmol) and NaN3 (0.62 g, 9.5 mmol) in DMF (30 ml) was stirred at room
temperature overnight. The mixture was diluted with EtOAc, washed with
brine, dried over MgSO4, and concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel with
EtOAc : hexane (1 : 1) to give a colorless oil (3.91 g, 97%). HR-FAB-MS
m/z: 621.3973 [MꢀH]ꢀ (Calcd for C31H53N6O7: 621.3975). 1H-NMR
(CDCl3) d: 1.43 (9H, s), 1.44 (9H, s), 1.45—1.55 (12H, m), 3.16—3.20
(10H, m), 3.12—3.21 (10H, m), 3.29—3.30 (2H, m), 3.80 (3H, s), 5.02 (2H,
s), 6.88 (2H, d, Jꢄ8.88 Hz), 7.29 (2H, d, Jꢄ8.88 Hz).
5,10-Di-tert-butoxycarbonyl-15-(4-methoxybenzyloxycarbonyl)-
5,10,15-triazapentadecylamime (12) A solution of 11 (3.9 g, 6.28 mmol)
in THF (36 ml) was hydrogenated over 3.5—6.5% palladium-activated car-
bon ethylenediamine complex (Pd–C(en)) (0.65 g) at room temperature for
2 h under H2 atmosphere. The catalyst was filtered through a pad of celite,
and the filtrate was concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel with CHCl3 : MeOH : 25%
NH4OH (100 : 20 : 2) to give a colorless oil (3.14 g, 84%). HR-FAB-MS m/z:
595.4070 [MꢀH]ꢀ (Calcd for C31H55N4O7: 595.4070). 1H-NMR (CDCl3) d:
1.43 (9H, s), 1.44 (9H, s), 1.45—1.56 (12H, m), 2.70 (2H, t, Jꢄ7.26 Hz),
3.16—3.20 (12H, m), 5.02 (2H, s), 6.88 (2H, d, Jꢄ8.58 Hz), 7.29 (2H, d,
Jꢄ8.58 Hz).
(4-{[4-(4-Methoxybenzyloxycarbonylamino)butyl]-tert-butoxycar-
bonylamino}butyl)-{4-[Nꢀ,Nꢁ-bis(tert-butoxycarbonyl)guanidino-
butyl]}carbamic acid tert-Butyl Ester (13) A mixture of 12 (315 mg,
0.53 mmol) and N,Nꢁ-bis(tert-butoxycarbonyl)-S-methylisothiourea (154 mg,
0.53 mmol) in CH2Cl2 (8 ml) was stirred at room temperature for 2 d. The
mixture was concentrated under reduced pressure. The residue was purified
by column chromatography on silica gel with EtOAc : hexane (2 : 1) to give a
colorless oil (295 mg, 66%). HR-FAB-MS m/z: 837.5336 [MꢀH]ꢀ (Calcd
for C42H72N6O11: 837.5332). 1H-NMR (CDCl3) d: 1.43 (9H, s), 1.44 (9H, s),
1.47 (6H, br s), 1.49 (9H, s), 1.50 (9H, s), 3.16—3.22 (10H, m), 3.40—3.45
(2H, m), 3.80 (3H, s), 5.02 (2H, s), 6.88 (2H, d, Jꢄ8.58 Hz), 7.29 (2H, d,
10-(4-Methoxybenzyloxycarbonyl)-5,10-diaza-1-decanol (5) A solu-
tion of 4 (9.28 g, 22.8 mmol) and 4-amino-1-butanol (82.59 g, 29 mmol) in
DMF (14 ml) was stirred at 90 °C for 2.5 h. The reaction mixture was poured
into ice water. The solution was made alkaline (pH 12) by adding 5% aque-
ous KOH solution and extracted with CH2Cl2. The organic layer was washed
with brine and dried over Na2SO4, and then the solvent was removed under
reduced pressure. The residue was purified by column chromatography on
silica gel with CHCl3 : MeOH : 25% NH4OHꢄ100 : 10 : 1) to give a colorless
oil (4.43 g, 60%). HR-FAB-MS m/z: 325.2128 [MꢀH]ꢀ (Calcd for
C17H28N2O4: 325.2127). 1H-NMR (CDCl3) d: 1.53 (4H, br), 1.60—1.62
(4H, m), 1.65—1.67 (4H, m), 2.62—2.65 (4H, m), 3.18—3.19 (2H, m), 3.56
(2H, t, Jꢄ5.1 Hz), 3.80 (3H, s), 4.85 (1H, br s), 5.01 (2H, s), 6.88 (2H, d,
Jꢄ8.58 Hz), 7.29 (2H, d, Jꢄ8.58 Hz).
5-tert-Butoxycarbonyl-10-(4-methoxybenzyloxycarbonyl)-5,10-diaza-
1-decanol (6) A mixture of 5 (2.4 g, 7.4 mmol) and (Boc)2O (1.77 g,
8.1 mmol) in CH2Cl2 (36 ml) was stirred at 0—5 °C for 4 h and then at room
temperature overnight. The mixture was concentrated under reduced pres-
sure. The residue was purified by column chromatography on silica gel
eluted with CHCl3 : MeOHꢄ10 : 1) to give a colorless oil (3.11 g, 99%). HR-
FAB-MS m/z: 425.2653 [MꢀH]ꢀ (Calcd for C22H37N2O6: 425.2651). 1H-
NMR (CDCl3) d: 1.44 (9H, s), 1.45—1.57 (8H, m), 3.17—3.20 (6H, m),
3.64—3.66 (2H, m), 3.62 (3H, s), 5.02 (2H, s), 6.88 (2H, d, Jꢄ8.22 Hz),
7.29 (2H, d, Jꢄ8.22 Hz).
5-tert-Butoxycarbonyl-10-(4-methoxybenzyloxycarbonyl)-5,10-di-
azadecyl p-Toluenesulfonate (7) To a solution of 6 (6.14 g, 13.75 mmol)
and TEA (6.1 ml, 43.57 mmol) in CH2Cl2 (150 ml), TsCl (2.75 g,
14.42 mmol) was added at 0—5 °C. The reaction mixture was stirred for 5 h
at 0—5 °C and then at room temperature overnight. The mixture was washed
with brine, dried over MgSO4, and then concentrated under reduced pres-
sure. The residue was chromatographed on a silica gel column. Elution with
EtOAc : hexane (1 : 1) afforded a colorless oil (4.85 g, 61%). HR-FAB-MS
m/z: 579.2739 [MꢀH]ꢀ (Calcd for C29H43N2O8S: 579.2739). 1H-NMR

866
Vol. 58, No. 6
C27H49N4O6S: 557.3372). 1H-NMR (CDCl3) d: 1.39 (9H, s), 1.44 (9H, s),
1.53 (4H, br s), 1.64 (4H, quin, Jꢄ6.9 Hz), 2.41 (3H, s), 2.69 (2H, br s), 2.87
(2H, br s), 3.05 (2H, br s), 3.10—3.24 (6H, m), 7.26 (2H, br s), 7.74 (2H, d,
Jꢄ8.0 Hz).
Jꢄ8.58 Hz).
{4-[(4-Aminobutyl)-tert-butoxycarbonylamino]butyl}-{4-[Nꢀ,Nꢁ-bis-
(tert-butoxycarbonyl)guanidinobutyl]}carbamic Acid tert-Butyl Ester
(14) A solution of 13 (220 mg, 0.145 mmol) in THF (4 ml) was hydro-
genated over 10% Pd–C (60 mg) at room temperature for 2 d under H2 atmo-
sphere. The catalyst was filtered through a pad of celite, and the filtrate was
concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel with CHCl3 : MeOH : 25% NH4OH (100 :
10 : 1) to give a colorless oil (80 mg, 49%). HR-FAB-MS m/z: 673.4863
[MꢀH]ꢀ (Calcd for C33H65N6O8: 673.4864). 1H-NMR (CDCl3) d: 1.44—
1.59 (48H, m), 3.16 (10H, br s), 3.41—3.22 (2H, m).
4,9-Di-tert-butoxycarbonyl-13-(butane-1-sulfonyl)-4,9,13-triazatride-
cylamime (19) 4,9-Diaza-4,9-di-(tert-butoxycarbonyl)dodecane-1,12-di-
amine (630 mg, 1.57 mmol) and TEA (0.219 ml, 1.57 mmol) were dissolved
in CH2Cl2 (30 ml), and then a solution of BsCl (245 mg, 1.57 mmol) in
CH2Cl2 (15 ml) was added in 0.25-ml aliquots every 10 min while stirring at
room temperature. After the addition was complete, the mixture was stirred
at room temperature for 2 d, then the reaction mixture was washed with
water and dried over Na2SO4. Evaporation of the solvent yielded the crude
compound as an oil, which was purified by column chromatography on silica
gel with CHCl3 : MeOH : 25% NH4OH (100 : 20 : 2) to give a colorless oil
(406 mg 50%). HR-FAB-MS m/z: 523.3532 [MꢀH]ꢀ (Calcd for
C24H51N4O6S: 523.3528). 1H-NMR (CDCl3) d: 0.94 (3H, t, Jꢄ7.5 Hz),
1.45—1.46 (24H, m), 1.64 (2H, quin, Jꢄ6.9 Hz), 1.71—1.81 (4H, m), 2.69
(2H, m), 2.97 (2H, t, Jꢄ7.9 Hz), 3.06—3.34 (10H, m).
(
4-{[4-(p-toluenesulfonylamino)butyl]-tert-butoxycarbonylamino}butyl)-
{4-[Nꢀ,Nꢁ-bis(tert-butoxycarbonyl)guanidinobutyl]}carbamic Acid tert-
Butyl Ester (15) To a solution of 14 (67 mg, 0.1 mmol) and TEA (42 ml,
0.3 mmol) in CH2Cl2 (2 ml), TsCl (19 mg, 0.1 mmol) was added at 0—5 °C.
The reaction mixture was stirred for 2 h at 0—5 °C and then at room temper-
ature overnight. The mixture was diluted with CH2Cl2, washed with brine,
dried over MgSO4, and concentrated under reduced pressure. The residue
was chromatographed on a silica gel column. Elution with EtOAc : hexane
(1 : 1) afforded a colorless oil (66 mg, 80%). HR-FAB-MS m/z: 827.4967
[MꢀH]ꢀ (Calcd for C40H71N6O10S: 827.4951). 1H-NMR (CDCl3) d: 1.41—
1.59 (48H, m), 2.43 (3H, s), 2.94—2.96 (2H, m), 3.10—3.21 (8H, m),
3.41—3.44 (2H, m), 7.30 (2H, d, Jꢄ8.22 Hz), 7.75 (2H, d, Jꢄ8.22 Hz).
(4-{[3-(p-Toluenesulfonylamino)propyl]-tert-butoxycarbonylamino}-
butyl)-{3-[Nꢀ,Nꢁ-bis(tert-butoxycarbonyl)guanidinopropyl]}carbamic
Acid tert-Butyl Ester (20) A mixture of 18 (126 mg, 0.23 mmol) and
N,Nꢁ-bis(tert-butoxycarbonyl)-S-methylisothiourea (66 mg, 0.23 mmol) in
CH2Cl2 (5 ml) was stirred at room temperature for 2 d. Evaporation of the
solvent yielded the crude compound as an oil, which was purified by column
chromatography on silica gel with EtOAc : hexane (1 : 1) to give a colorless
oil (136 mg, 74%). HR-FAB-MS m/z: 799.4639 [MꢀH]ꢀ (Calcd for
(
4-{[4-(Butane-1-sulfonylamino)butyl]-tert-butoxycarbonylamino}butyl)-
{4-[Nꢀ,Nꢁ-bis(tert-butoxycarbonyl)guanidinobutyl]}carbamic Acid tert-
Butyl Ester (16) To a solution of 14 (76 mg, 0.11 mmol) and TEA (46 ml,
0.33 mmol) in CH2Cl2 (2 ml), BsCl (17 mg, 0.11 mmol) was added at 0—
5 °C. The reaction mixture was stirred for 2 h at 0—5 °C and then at room
temperature overnight. The mixture was diluted with CH2Cl2, washed with
brine, dried over MgSO4, and concentrated under reduced pressure. The
1
C38H67N6O10S: 799.4638). H-NMR (CDCl3) d: 1.38 (9H, s), 1.43 (18H, s),
1.49 (9H, s), 1.62 (6H,s), 1.78 (2H, quin, Jꢄ7.5 Hz), 2.40(3H, s), 2.87 (2H,
br s), 3.04 (2H, br s), 3.18—3.24 (6H, m), 3.41 (2H, q, Jꢄ6.9 Hz), (6.14, 1H,
br), 7.26 (2H, br s), 7.74 (2H, d, Jꢄ8.0 Hz), 8.35 (1H, br), 11.48 (1H, s).
(4-{[3-(Butane-1-sulfonylamino)propyl]-tert-butoxycarbonylamino}-
butyl)-{3-[Nꢀ,Nꢁ-bis(tert-butoxycarbonyl)guanidinopropyl]}carbamic
Acid tert-Butyl Ester (21) A mixture of 19 (230 mg, 0.44 mmol) and
N,Nꢁ-bis(tert-butoxycarbonyl)-S-methylisothiourea (128 mg, 0.44 mmol) in
CH2Cl2 (5 ml) was stirred at room temperature for 2 d. Evaporation of the
solvent yielded the crude compound as an oil, which was purified by column
chromatography on silica gel with EtOAc : hexane (1 : 1) to give a colorless
oil (293 mg, 87%). HR-FAB-MS m/z: 765.4794 [MꢀH]ꢀ (Calcd for
C35H69N6O10S: 765.4795). 1H-NMR (CDCl3) d: 0.94 (3H, t, Jꢄ7.5 Hz), 1.44
(20H, m), 1.49 (18H, m), 1.62—1.71 (6H, m), 1.76—1.81 (4H, m), 2.97
(2H, t, Jꢄ7.5 Hz), 3.06—3.20 (8H, m), 3.33 (2H, br s), 3.40—3.44 (2H, m),
5.82 (1H, br s), 8.36 (1H, br s), 11.48 (1H, s).
residue was chromatographed on
a silica gel column. Elution with
EtOAc : hexane (1 : 1) afforded a colorless oil (70 mg, 80%). HR-FAB-MS
m/z: 793.5109 [MꢀH]ꢀ (Calcd for C37H72N6O10S: 793.5108). 1H-NMR
(CDCl3) d: 0.95 (3H, t, Jꢄ7.56 Hz), 1.44—1.60 (50H, m), 1.78—1.80 (2H,
m), 2.99—3.02 (2H, m), 3.12—3.25 (12H, m).
N-{4-[4-(4-Guanidinobutylamino)butylamino]butyl}-p-toluenesulfon-
amide Trihydrochloride (1a, TsHSPMG) A mixture of 15 (116 mg,
0.14 mmol) and 36% HCl (0.5 ml) in THF (2 ml) was stirred at room tem-
perature. After 36 h, the mixture was concentrated under reduced pressure.
The residue was washed successively with THF and Et2O, and dried to give
a white solid (75 mg, 100%). HR-FAB-MS m/z: 427.2854 [Mꢂ3HClꢀH]ꢀ
(Calcd for C20H39N6O2S: 427.2855). 1H-NMR (D2O) d: 1.31—1.36 (2H, m),
1.44—1.57 (10H, m), 2.24 (3H, s), 2.73 (2H, t, Jꢄ6.9 Hz), 2.80—2.82 (2H,
m), 2.85—2.88 (6H, m), 3.02 (2H, t, Jꢄ6.9 Hz), 7.28 (2H, d, Jꢄ8.22 Hz),
7.56 (2H, d, Jꢄ8.22 Hz). 13C-NMR (D2O) d: 20.85, 22.82, 22.93, 22.98,
23.06, 25.29, 25.86, 40.67, 42.08, 46.82, 46.99, 47.14, 47.34, 126.90,
130.26, 135.08, 145.23, 156.94. IR (KBr) cmꢂ1: 3386, 3162, 2956, 2800,
1675, 1644, 1452, 1328, 1155. Anal. Calcd for C20H38N6O2S·3HCl: C,
44.81; H, 7.71; N, 15.68. Found: C, 45.08; H, 7.93; N, 15.81.
N-{4-[4-(4-Guanidinobutylamino)butylamino]butyl}butane-1-sulfon-
amide Trihydrochloride (1b, BsHSPMG) A mixture of 16 (50 mg,
0.063 mmol) and 36% HCl (0.2 ml) in THF (0.5 ml) was stirred at room tem-
perature. After 36 h, the mixture was concentrated under reduced pressure.
The residue was washed successively with THF and Et2O, and dried to give
a white solid (31 mg, 100%). HR-FAB-MS m/z: 393.3012 [Mꢂ3HClꢀH]ꢀ
(Calcd for C17H41N6O2S: 393.3011). 1H-NMR (D2O) d: 0.72 (3H, t, Jꢄ
7.56 Hz), 1.25 (2H, sex, Jꢄ7.56 Hz), 1.40—1.49 (4H, m), 1.50—1.59 (10H,
m), 2.86—2.89 (8H, m), 2.92 (2H, t, Jꢄ6.9 Hz), 3.00—3.04 (4H, m). 13C-
NMR (D2O) d: 12.97, 20.98, 22.82, 22.95, 22.98, 23.05, 25.05, 25.29,
26.57, 40.66, 41.93, 46.87, 47.00, 47.23, 47.33, 51.03, 156.95. IR (KBr)
cmꢂ1: 3307, 3160, 2950, 2871, 2794, 1664, 1444, 1305, 1133. Anal. Calcd
for C17H41N6O2S·3HCl: C, 40.67; H, 8.63; N, 16.74. Found: C, 40.70; H,
8.88; N, 16.80.
N-{3-[4-(3-Guanidinopropylamino)butylamino]propyl}-p-toluenesul-
fonamide Trihydrochroride (2a, TsSPMG) To a solution of 20 (80 mg,
0.1 mmol) in THF (2 ml), 36% HCl (1 ml) was added at room temperature.
The reaction mixture was stirred for 48 h, and then concentrated under re-
duced pressure to give a white solid which was washed with Et2O to give a
white solid (50 mg, 100%), mp 188—192 °C. HR-FAB-MS m/z: 399.2542
[Mꢂ3HClꢀH]ꢀ (Calcd for C18H35N6O2S: 399.2541). 1H-NMR (D2O) d:
1.55—1.58 (4H, m), 1.64—1.69 (2H, m), 1.76—1.81 (2H, m), 2.24 (3H, s),
2.80 (2H, t, Jꢄ6.9 Hz), 2.86—2.93 (8H, m), 3.10 (2H, t, Jꢄ6.9 Hz), 7.28
(2H, d, Jꢄ8.22 Hz), 7.56 (2H, d, Jꢄ8.22 Hz). 13C-NMR (D2O) d: 20.86,
22.90, 22.97, 25.19, 25.84, 38.38, 39.92, 45.09, 45.18, 47.01, 47.13, 126.92,
130.31, 134.86, 145.34, 156.98. IR (KBr) cmꢂ1: 3357, 3160, 3064, 2964,
2803, 1654, 1459, 1321, 1155. Anal. Calcd for C18H34N6O2S·3HCl: C,
42.56; H, 7.34; N, 16.55. Found: C, 42.29; H, 7.44; N, 16.48.
N-{3-[4-(3-Guanidinopropylamino)butylamino]propyl}butane-1-sul-
fonamide Trihydrochroride (2b, BsSPMG) To a solution of 21 (108 mg,
0.14 mmol) in THF (2 ml), 36% HCl (1 ml) was added at room temperature.
The reaction mixture was stirred for 48 h, and then concentrated under re-
duced pressure to give a white solid which was washed with Et2O to give a
white solid (66 mg, 100%), mp 178—180 °C. HR-FAB-MS m/z: 365.2696
[Mꢂ3HClꢀH]ꢀ (Calcd for C15H37N6O2S: 365.2698). 1H-NMR (D2O) d:
0.74 (3H, t, Jꢄ7.5 Hz), 1.26 (2H, sex, Jꢄ7.5 Hz), 1.52—1.60 (6H, m),
1.74—1.77 (2H, m), 1.78—1.82 (2H, m), 2.92—2.99 (8H, m), 3.00—3.06
(4H, m), 3.12 (2H, t, Jꢄ7.5 Hz). 13C-NMR (D2O) d: 12.96, 20.96, 22.93,
22.99, 25.01, 25.20, 26.53, 38.38, 39.73, 45.10, 45.15, 47.06, 47.14, 51.05,
157.01. IR (KBr) cmꢂ1: 3301, 3153, 2952, 2796, 1664, 1454, 1309, 1135.
Anal. Calcd for C15H36N6O2S·3HCl: C, 38.01; H, 8.29; N, 17.73. Found: C,
38.00; H, 8.51; N, 17.62.
4,9-Di-tert-butoxycarbonyl-13-(p-toluenesulfonyl)-4,9,13-triazatride-
cylamime (18) 4,9-Diaza-4,9-di-(tert-butoxycarbonyl)dodecane-1,12-di-
amine (17) (201 mg, 0.5 mmol) and TEA (0.07 ml 0.5 mmol) were dissolved
in CH2Cl2 (10 ml), and then a solution of TsCl (95 mg, 0.5 mmol) in CH2Cl2
(5 ml) was added in 0.25-ml aliquots every 10 min while stirring at room
temperature. After the addition was complete, the mixture was stirred at
room temperature for 2 d, and then the reaction mixture was washed with
water and dried over Na2SO4. Evaporation of the solvent yielded the crude
compound as an oil, which was purified by column chromatography on silica
gel with CHCl3 : MeOH : 25% NH4OH (100 : 20 : 2) to give a colorless oil
(138 mg, 49%). HR-FAB-MS m/z: 557.3376 [MꢀH]ꢀ (Calcd for
References
1) Dingledine R., Borges K., Bowie D., Traynelis S. F., Pharmacol. Rev.,
51, 7—61 (1999).

June 2010
867
2) Williams K., Biochem. J., 325, 289—297 (1997).
(2001).
3) Herin G. A., Aizenman E., Eur. J. Pharmacol., 500, 101—111 (2004).
4) Masuko T., Namiki R., Nemoto Y., Miyake M., Yasuo Kizawa Y.,
Suzuki T., Kashiwagi K., Igarashi K., Kusama T., J. Pharmacol. Exp.
Ther., 331, 522—530 (2009).
5) Pöhler T., Schadt O., Niepel D., Rebernik P., Berger M. L., Noe C. R.,
Eur. J. Med. Chem., 42, 175—197 (2007).
7) Berger M. L., Bitar A. Y., Waitner M. J., Bioorg. Med. Chem. Lett., 16,
2837—2841 (2006).
8) Ilies M. A., Seitz W. A., Johnson B. H., Ezell E. L., Miller A. L.,
Thompson E. B., Balaban A. T., J. Med. Chem., 49, 3872—3887
(2006).
9) Masuko T., Metori K., Kizawa Y., Kusama T., Miyake M., Chem.
Pharm. Bull., 53, 444—447 (2005).
6) Sharmin S., Sakata K., Kashiwagi K., Ueda S., Iwasaki S., Shirahata
A., Igarashi K., Biochem. Biophys. Res. Commun., 282, 228—235