Discovery of (2S)-1-(4-Amino-2,3,5-trimethylphenoxy)-3-{4-[4-(4-fluorobenzyl)phenyl]-1-pi perazinyl}-2-propanol Dimethanesulfonate (SUN N8075): A dual Na+ and Ca2+ channel blocker with antioxidant activity

Full text of DOI:10.1021/jm000143w

3372
J . Med. Chem. 2000, 43, 3372-3376
Communications to the Editor
Ch a r t 1
Discover y of (2S)-1-(4-Am in o-2,3,5-
tr im eth ylp h en oxy)-3-{4-[4-(4-
flu or oben zyl)p h en yl]-1-p ip er a zin yl}-2-
p r op a n ol Dim eth a n esu lfon a te (SUN
N8075): A Du a l Na + a n d Ca 2+ Ch a n n el
Block er w ith An tioxid a n t Activity
Hirokazu Annoura,* Kyoko Nakanishi, Tetsuya Toba,
Naohiro Takemoto, Seiichi Imajo, Atsuko Miyajima,
Yoshiko Tamura-Horikawa, and Shigeki Tamura
Suntory Biomedical Research Limited, 1-1-1, Wakayamadai,
Shimamoto-cho, Mishima-gun, Osaka 618-8503, J apan
Received March 28, 2000
In tr od u ction . Stroke is a substantial public health
problem and one of the leading causes of death in major
industrialized countries. An effective and widely ap-
plicable treatment of cerebral ischemia would have an
enormous public health impact; however, no such treat-
ment has been found as yet.1 Pathogenesis of progres-
sive and delayed death of nerve cells that occurs in
cerebral injury and cerebrovascular diseases, such as
stroke and trauma, is considered to be caused by a rise
in intracellular Ca2+ concentration to a pathological
level due to ATP depletion following the failure of
intracellular energy-dependent ion homeostasis.2 This
phenomenon is known as Ca2+ overload which induces
functional disorders in the mitochondria, randomly
activates various Ca2+-dependent enzymatic reactions,
and invites further Ca2+ overload. It has been demon-
strated that aberrant activation of Na+ and Ca2+ chan-
nels is extensively involved in the Ca2+ overload path-
way and the accumulation of intracellular Na+ ions
results in rapid Ca2+ overload by the reverse operation
of Na+/Ca2+ exchange mechanism.2,3 In addition, the
Ca2+-dependent activation of nitric oxide (NO) synthase,
phospholipase A2, and xanthine oxidase causes a marked
increase in the generation of reactive oxygen species
such as superoxide anion radical (O2-•), hydrogen
peroxide (H2O2), hydroxy radical (OH•), and peroxyni-
trite (ONOO-) on reperfusion of ischemia, imparting
irreparable damage to the cell membrane through
extensive lipid peroxidation.4 These complex biochemi-
cal events mutually act as aggravating factors and
repeat in a vicious amplifying cycle that ultimately leads
to cell death.
combination of compounds, each possessing a different
mechanism of action in preventing cell death, exhibits
synergistic effects in ischemic animal models.10 Conse-
quently, a novel neuroprotectant with multiple mech-
anisms of action may provide clinically significant
efficacy. We previously reported a structurally novel
class of arylpiperidines, represented by SUN N5030, as
Na+ and Ca2+ channel blockers with reduced affinity
for dopamine D2 receptors11 (Chart 1). In this com-
munication, we report that our continuous endeavor for
the development of agents for acute ischemic stroke
based on molecular modification of flunarizine3b,c,12 as
a prototype has resulted in the discovery of a candi-
date: (2S)-1-(4-amino-2,3,5-trimethylphenoxy)-3-{4-[4-
(4-fluorobenzyl)phenyl]-1-piperazinyl}-2-propanol di-
methanesulfonate (1; SUN N8075). This compound
Although clinical trials of quite a few neuropro-
tectants, including N-methyl-D-aspartate (NMDA) re-
ceptor antagonists,5 AMPA receptor antagonists,6 Na+
and/or Ca2+ channel blockers,3c,7 and antioxidants,8 are
currently underway, most of them interfere with only
one of the pathways in the ischemic cascade and the
benefits of such therapies have not yet been estab-
lished.9 It has recently been demonstrated that a
possesses not only neuronal Na+ and T-type12 Ca2+
-
channel-blocking effects but also antioxidant activity,
which governs potent neuroprotective activity in an in
vivo transient middle cerebral artery occlusion (MCAO)
model. A survey of literature has revealed that U-92032
endowed with similar multiple mechanisms of action
against ischemia was reported by the Upjohn group.7b
The pharmacophore for the blockade of ion channels and
* To whom correspondence should be addressed. Tel: +81-75-962-
8481. Fax: +81-75-962-6448. E-mail: Hirokazu_Annoura@suntory.co.jp.
10.1021/jm000143w CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/19/2000

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 18 3373
Sch em e 1a
a
Reagents and conditions: (a) piperazine (3 equiv), Et3N (2 equiv), MeCN, reflux, 12 h, 66%; (b) Et3SiH (2.3 equiv), concd H2SO4 (2.2
equiv), TFA, rt, 2 h, 79%; (c) (Boc)2O, THF, 80 °C, 2 h, 96%; (d) NaH, (S)-glycidyl 3-nitrobenzenesulfonate, DMF, rt, 2 h, 90%; (e) 4,
2-propanol, reflux, 6 h, 90%; (f) TFA, CH2Cl2, rt, 2 h, then MeSO3H (2.05 equiv), MeOH, rt, 65%.
Ta ble 1. Biological Activity of 1a
anticonvulsant effects on
IC50 (µM)
T-type Ca2+ currentsc
audiogenic seizures in DBA/2 mice
h
entry
anti-veratridineb
lipid peroxidationd
D2
>10
(n ) 6)i ED50 (mg/kg; ip)
1
0.42
0.29
0.36
NTj
2.0
2.2
0.31
>50e
6.5
11.4
14.5
NTj
flunarizine
U-92032
R-tocopherol
0.228
1.69
0.3
20.0f
NTj
>100g
NTj
a
b
Each value represents multiple determinations (g2) with a deviation of less than 20%. Determined as inhibitory effects upon the
veratridine-induced depolarization in rat cerebrocortical synaptosomes using a membrane potential sensitive fluorescent dye, rhodamine
6G.14 c Determined as inhibitory effects on low-threshold (T-type) Ca2+ currents in primary cultured rat cerebrocortical neurons using
whole-cell voltage-clamp recording technique.12a Determined as inhibitory effects on automatic oxidation in plasma membrane of rat
d
brain homogenates by measuring malondialdehyde and 4-hydroxyalkenals using peroxidized lipid colorimetric assay kit.17 e Results reported
in refs 7c and 17b are 64.1 and 85 µM, respectively. f Result reported in ref 7c is 13.5 µM. Result reported in ref 17b is >100 µM.
g
Determined in competition experiments with [3H]raclopride.20 Compounds were administered intraperitoneally to DBA/2 mice (n ) 6)
h
i
20 min prior to auditory stimulation of at least 90 dB for 1 min.22a NT, not tested.
j
the mechanism of action for the antioxidant activity of
1 are also discussed.
water (20:1). Its optical purity was determined by
normal phase HPLC using a Daicel Chiralpak OD
column (n-hexane:2-propanol:diethylamine ) 600:400:
1) and was found to increase to >99% ee upon repeated
recrystallization. J udging from the 19F NMR (500 MHz,
DMSO-d6), the signal attributable to trifluoroacetic acid
could not be detected. Therefore, complete salt exchange
occurred from the initially formed trifluoroacetate into
the methanesulfonate during the final deprotecting step,
presumably due to the higher acid dissociation constant
of methanesulfonic acid (pKa ) -1.2) as compared with
that of trifluoroacetic acid (pKa ) 0.2).
Resu lts a n d Discu ssion . The inhibitory effect of
compound 1 on Na+ channels stimulated by the neuro-
toxin veratridine was evaluated in rat cerebrocortical
synaptosomes using the voltage-sensitive fluorescent
dye rhodamine 6G.14 The effect of 1 on low-threshold
(T-type) Ca2+ currents in primary cultured rat cerebro-
cortical neurons was examined using a whole-cell volt-
age-clamp recording technique.12a The results are given
as IC50 values as shown in Table 1. Compound 1 blocks
both Na+ and T-type Ca2+ channels with potency equal
to that of the reference standards flunarizine and
U-92032.7b Compound 1 showed concentration-depend-
ent block of T-type Ca2+ currents induced by a depolar-
izing pulse to -40 mV from a holding potential (VH) of
Ch em istr y. Compound 1 was synthesized using the
pathway shown in Scheme 1. Treatment of commercially
available 4,4′-difluorobenzophenone with piperazine (3
equiv) in the presence of triethylamine in acetonitrile
gave 3 in 66% yield. Reduction of 3 with Et3SiH (2.3
equiv) in the presence of sulfuric acid (2.2 equiv) and
trifluoroacetic acid (TFA) produced the requisite 1-[4-
(4-fluorobenzyl)phenyl]piperazine (4) in 79% yield. Phe-
noxy-2,3-epoxypropane (7), the coupling partner of 4,
was synthesized from 2,3,5-trimethyl-4-aminophenol
(5).13 Thus, chemoselective protection of the amino
group in 5 with di-tert-butyl dicarbonate gave 6 as a
single product in 96% yield. None of the corresponding
O-butoxycarbonylated isomer was detected during this
reaction. Treatment of 6 with sodium hydride produced
the phenolate anion, which was quenched with (S)-
glycidyl 3-nitrobenzenesulfonate to give 7 in 90% yield.
Coupling reaction between 7 and 4 in 2-propanol
proceeded smoothly to give 8 in 90% yield, without
concomitant loss of optical purity. Deprotection of the
Boc group in 8 by exposure to trifluoroacetic acid
followed by treatment with methanesulfonic acid (2.05
equiv) furnished 1 as a dimethanesulfonate in 65% yield
with >98.5% ee after recrystallization from 2-propanol/

3374 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 18
Communications to the Editor
Sch em e 2
-100 mV. We also investigated the effect of 1 on
neurotoxin binding sites of Na+ channels. Compound 1
displaced the binding of the site 2 ligand, [3H]batra-
chotoxin,15b with high affinity (IC50 ) 0.75 µM) but had
no effect on the site 1 ligand, [3H]saxitoxin, binding
(IC50 > 10 µM).16 [Each value represents replicate
determinations, and tetrodotoxin was used as a refer-
ence compound: IC50 ) 0.0063 µM (site 1) and >10 µM
(site 2).] The neurotoxin receptor site 2 of Na+ channels
is believed to be localized on a region involved in voltage-
dependent activation and inactivation and is allosteri-
cally linked to the transmembrane pore of the chan-
nels.15a,c The Na+ channel site 2 blockers work in a
voltage-dependent manner, implying event- and site-
specific block of Na+ channels without primary hemo-
dynamic adverse effects in ischemic diseases.3b,c
F igu r e 1. Overlay of energetically allowed conformers of 1
(red) and flunarizine (white).
12. These results suggest that the aminophenoxy moiety
in 1 is the essential pharmacophore for antioxidant
activity and that the mechanism of action is similar to
that of R-tocopherol.18c The direct evidence for radical-
scavenging action was obtained on the ESR spectrum
where the intensity of the signal of DPPH radical
decreased dose-dependently upon the addition of 1. With
increasing evidence that the excess production of reac-
tive oxygen species affects ion homeostasis and is a
major causative factor in cell death,4 it is tempting to
speculate that the ion channel-blocking and antioxidant
properties of 1 may act synergistically against cerebral
ischemia.10 The dopamine D2 receptor binding affinity
was assessed in a competitive binding assay using [3H]-
racoplide in rat striatum membranes.19 In sharp con-
trast to flunarizine, compound 1 has no binding affinity
for dopamine D2 receptors (IC50 > 10 µM). These
differences clearly demonstrate that 1 not only has
structural features distinctly different from those of
flunarizine and related compounds but also may have
a decreased clinical risk for extrapyramidal side ef-
fects.20
To elucidate the common pharmacophore and/or the
special arrangement of essential parts for inhibition of
ion channels and influence for binding affinity at
dopamine D2 receptors, a systematic conformation
search and a similar structure search by root-mean-
square (rms) fitting were carried out for 1 and fluna-
rizine. Structures were minimized with MM2 param-
eters implemented by Macromodel.21 As depicted in
Figure 1, the corresponding pharmacophoric groups
between 1 (red) and flunarizine (white) closely overlap,
with the exception of each distal benzene ring. This
Antioxidant activity was measured by lipid peroxi-
dation-suppressing effect in rat cerebrocortical mem-
branes using a BIOXYTECH/LPO-586 peroxidized col-
orimetric assay kit.17 Surprisingly, compound 1 exhibits
lipid peroxidation-suppressing effects with a potency
greater than that of flunarizine, U-92032, or R-toco-
pherol. These results prompted us to investigate the
mechanism of action for antioxidant activity at the
molecular level by reacting 1 with 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical.18a,b Compound 1 dis-
solved in methanol/pH ) 6.86 standard buffer solution
(1:1, v/v) and when subjected to DPPH radical unex-
pectedly produced propanediol 11 and quinone mono-
imine 12. Both structures, 11 and 12, were elucidated
1
by H NMR, IR, and HRMS spectra. In the 1H NMR
(400 MHz, CDCl3) of 12, the signal assignable to the
hydrogen on the quinone monoimine moiety appeared
at δ 6.38 (singlet). In addition, the signal attributable
to the hydrogen of the trimethylphenoxy ring was at δ
6.64 (singlet), showing a shift to lower field than that
of the parent compound 1 (δ 6.57) due to the electron-
withdrawing effect of the quinone monoimine moiety.
As shown in Scheme 2, the degradation pathway is
proposed to be triggered by abstraction of the hydrogen
atom from the amino group in the 2,3,5-trimethyl-4-
aminophenoxy moiety followed by disproportionation of
the generated anilino radical 9 and cyclohexadienyl
radical 9′ to give the dimerization product 10, which is
subsequently hydrolyzed to produce compounds 11 and

Communications to the Editor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 18 3375
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implies that the benzene ring directly connected to the
piperazine ring in 1 likely occupies the same binding
site of the ion channels as flunarizine, whereas the other
benzene ring of the diphenylmethane moiety likely
interferes with dopamine D2 receptor binding.
Next, we investigated the effect of 1 on audiogenic
seizures in DBA/2 mice to confirm in vivo activity and
brain permeability.22a,b Anticonvulsant and neuropro-
tective activities are mutually related in several voltage-
dependent Na+ channel blockers.22c Compound 1 exhib-
its potent anticonvulsant effects following systemic (ip)
administration with an ED50 of 6.5 mg/kg. The neuro-
protective activity of 1 was assessed in a transient
MCAO model.23 The middle cerebral artery (MCA)
occluded for 60 min and 24 h after reperfusion neuronal
damage was quantified by staining brains with 2,3,5-
triphenyltetrazolium chloride24 (TTC). The relative area
of infarction to the whole area of the corresponding
cerebrum is calculated using a computerized image
analysis system and was shown in percent (vide infra).
Compound 1 was administered intravenously immedi-
ately after both MCAO and reperfusion (each 3 mg/kg).
Consequently, 1 significantly reduced infarct size in all
treated groups compared to vehicle-treated rats
[4.77 ( 0.84% (n ) 14) vs 14.87 ( 1.24% (n ) 15)
(mean ( SEM), **p < 0.01 (Student’s t-tests)], amount-
ing to 67.9% reduction in mean infarct volume, whereas
U-92032 effected only miner reduction (<15%). In this
model, rectal temperature was found to increase during
transient MCAO to above 38.5 °C and 1 slightly reduced
the ischemic hyperthermia within 1 °C. Mild hypo-
thermia immediately after reperfusion reduces infarct
size and improves cerebral outcome.25 These results
indicate that 1 has a pronounced neuroprotective ef-
ficacy against neuronal damage induced by transient
focal ischemia in rats. Interestingly, 1 at the effective
doses had no effects on systemic blood pressure and
heart rate in anesthetized rats. These studies will be
described in more detail in subsequent papers.
In conclusion, we describe a novel neuroprotectant,
1, that blocks both neuronal Na+ and T-type Ca2+
channels but which does not affect dopamine D2 recep-
tors. Compound 1 is also a powerful antioxidant,
significantly more potent than the standard. Since
compound 1 (SUN N8075) has complementarily mul-
tiple beneficial effects against ischemia and remarkable
neuroprotective activity in the transient MCAO model,
this compound may have greater clinical efficacy for the
treatment of acute ischemic stroke compared to candi-
dates with a single mechanism of action in preventing
neuronal cell death or damage.9,10 The structure-
activity relationships (SAR) of this series of compounds
will be reported in due course.26
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his assistance in measuring ESR spectra.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
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This material is available free of charge via the Internet at
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