5-Aminocoumarans: Dual inhibitors of lipid peroxidation and dopamine release with protective effects against central nervous system trauma and ischemia

Article abstract of DOI:10.1021/jm960411j

A series of 2,3-dihydro-5-benzofuranamines (5-aminocoumarans) were developed for the treatment of traumatic and ischemic central nervous system (CNS) injury. Compounds within this class were extremely effective inhibitors of lipid peroxidation in vitro an

Full text of DOI:10.1021/jm960411j

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J . Med. Chem. 1997, 40, 559-573
559
5-Am in ocou m a r a n s: Du a l In h ibitor s of Lip id P er oxid a tion a n d Dop a m in e
Relea se w ith P r otective Effects a ga in st Cen tr a l Ner vou s System Tr a u m a a n d
Isch em ia
Shigenori Ohkawa,* Kohji Fukatsu, Shokyo Miki, Tadatoshi Hashimoto, J unko Sakamoto, Takayuki Doi,
Yasuo Nagai, and Tetsuya Aono†
Pharmaceutical Research Laboratories I, Takeda Chemical Industries, Ltd., 17-85 J usohonmachi 2-chome,
Yodogawa-ku, Osaka 532, J apan
Received J une 10, 1996X
A series of 2,3-dihydro-5-benzofuranamines (5-aminocoumarans) were developed for the
treatment of traumatic and ischemic central nervous system (CNS) injury. Compounds within
this class were extremely effective inhibitors of lipid peroxidation in vitro and antagonized
excitatory behavior coupled with peroxidative injury induced by spinal intrathecal injection of
FeCl2 (mouse-FeCl2-it assay) in vivo. Selected compounds were tested for antagonistic activity
on methamphetamine (MAP)-induced hypermotility resulting from dopamine release in the
mouse brain. Among the compounds synthesized, compound 26n (2,3-dihydro-2,4,6,7-tetra-
methyl-2-[(4-phenyl-1-piperidinyl)methyl]-5-benzofuranamine) exhibited potent effects in these
assays (inhibition of lipid peroxidation, IC50 ) 0.07 µM; mouse-FeCl2-it assay, ID50 ) 10.4 mg/
kg, po; MAP-induced hypermotility, 98% inhibition, 10 mg/kg, ip). The S-(+)-form of compound
26n dihydrochloride (TAK-218), which has 30 times more potent antagonistic activity on MAP-
induced hypermotility than the R-(-)-form, improved more significantly the survival rate in
the cerebral ischemia model (rat, 1-3 mg/kg, ip) during the period of 1-14 days after ischemia
and decreased functional disorders in the traumatic brain injury model (rat, 0.1-1 mg/kg, ip)
3-14 days after injury. These results imply a role for dopamine in deterioration of CNS function
after ischemic and traumatic injury. TAK-218 is a promising compound for the treatment of
stroke and CNS trauma and is now under clinical investigation.
Delayed biochemical changes play an important role
in tissue damage resulting from central nervous system
(CNS) trauma and ischemia.1 Many possible secondary
factors have been proposed to contribute to this damage
including excitatory amino acids,2 monoamines,3 ions
such as calcium4 and sodium,5 arachidonate metabo-
lites,6 and oxygen radicals.7 Among these, the genera-
tion of oxygen radicals and subsequent lipid peroxida-
tion have become the focus of attention for investigators
in the field of CNS trauma and ischemia. Reactive
oxygen radicals are commonly formed in normal cell
metabolism. However, under normal conditions their
production is localized and the body’s natural defenses
including antioxidative vitamins (A, C, and E), glu-
tathione, superoxide dismutase (SOD), and catalase
protect against the damage caused by oxygen radicals.8
Under pathological conditions, these systems can be
overwhelmed and the formation of oxygen radicals is
enhanced.9 Lipid peroxidation initiated by oxygen
radicals results in membrane degradation and cell
death. Pharmacological strategies have therefore aimed
at inhibiting this degradative process. Several antioxi-
dative compounds10 including Tirilazad (1) are currently
in clinical trials for CNS trauma and ischemia.
dopamine by molecular oxygen results in the formation
of superoxide anions.11 The reaction between dopamine
and hydroxy radical generates the neurotoxin 6-hy-
droxydopamine.12 Experimental evidence suggests that
decreases in brain dopamine levels protect brain tissues
from ischemic damage.13
These factors, oxygen radicals and dopamine, are
components of an interactive cascade leading to mem-
brane damage and cell death. Consequently, com-
pounds capable of regulating these factors would po-
tentially be able to limit posttraumatic tissue damage
and enhance neurological recovery.
Here, we report the synthesis and biological evalua-
tion of 5-aminocoumarans. Compounds within this
class have shown excellent antioxidative activities and
antagonistic activity on methamphetamine (MAP)-
induced hypermotility resulting from dopamine release
in the CNS.
Dr u g Design
Tocopherols (e.g., R-tocopherol, 2) are known to be
efficient inhibitors of lipid peroxidation in vivo. Com-
pound 3 which has a coumaran (2,3-dihydrobenzofuran)
structure was reported to be a more efficient antioxidant
than compounds that have the chroman (tocopherol
type) structure.14 Since the oxygen lone pair in position
1 of the coumaran structure is perpendicular to the
aromatic plane, the coumaranoxyl radical is more
stabilized by conjugate delocalization.15 On this basis,
a number of coumaran type compounds were reported
as antioxidants for clinical purposes.16 However, gener-
ally these types of compounds are metabolically un-
In addition to oxygen radicals, dopamine is released
significantly after brain injury and ischemia. There are
a number of possible mechanisms through which dopa-
mine release could exacerbate the cell damage produced
by cerebral ischemia and trauma.3 The oxidation of
* Corresponding author. Phone: 0-0118163006078. Fax: 0-011816-
30-06306.
† Pharmaceutical Research Laboratories III, Takeda Chemical
Industries, Ltd.
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
S0022-2623(96)00411-6 CCC: $14.00 © 1997 American Chemical Society

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560 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ohkawa et al.
Ch a r t 1
Mannich reaction. Acid-catalyzed cyclization followed
by basic hydrolysis of acetamide groups provided 7-(ami-
nomethyl)coumarans 19a ,b.
As shown in Scheme 4, compounds with a hydrophobic
group on the benzene ring were synthesized from 2,2-
dimethallylphenol 21, which was obtained by O-meth-
allylation of 2-methallylphenol 6b followed by Claisen
rearrangement of 20. Cyclization of 21 under acidic
conditions gave coumaran 22 with deprotection of the
formamide group and double-bond isomerization. Com-
pound 22 was converted to 23 by catalytic hydrogena-
tion.
Compounds with a substituted methyl group in posi-
tion 2 were synthesized using 2-methallylphenol 6a as
a starting material (Scheme 5). Cyclization of 6a using
bromine afforded 2-(bromomethyl)coumaran 24. The
arylthio or alkylthio group-substituted derivatives were
obtained by condensation of 24 with various thiols in
DMF at 100 °C, using NaH as a base. Subsequent acidic
hydrolysis of the formamide group of 25a -j gave
5-aminocoumarans 26a -j. Since a series of 2-alkoxym-
ethyl or aminomethyl derivatives was obtained in very
low yields under the same conditions, condensation was
carried out by heating in sealed tubes at 180 °C, with
an excess of alcohols using NaH as a base, or an excess
of amines in the absence of NaH. When the excess of
nucleophilic reagents was used, the formamide group
was deprotected concurrently to give 5-aminocoumarans
26k -w . In the case of phenylpiperidine, the free base
of the 5-amino compound 27, prepared from 24 by acidic
hydrolysis, was used to avoid the formation of N-
formylated piperidine.
Oxidation of phenyl sulfide 25a to sulfoxide or sulfone
was carried out using sodium periodate in MeOH-water
(scheme not shown). Sulfoxide 28 was prepared from
25a at room temperature, and sulfone 29 was obtained
under refluxing conditions. The acidic hydrolysis of
formamide groups of both oxidized compounds (28, 29)
gave 5-aminocoumarans 30 and 31. Sulfoxide 30 was
obtained as a mixture of diastereoisomers.
As shown in Scheme 6, oxidation of 2-methallylphenol
6a using m-chloroperbenzoic acid in the presence of
aqueous NaHCO3 afforded alcohol 32 via epoxide.
Swern oxidation of 32 followed by Horner-Emmons
olefination provided acrylate 34, which was subse-
quently deprotected of the formamide group in MeOH
to give the methyl ester 35. Treatment of aldehyde 33
with benzylide gave Z olefin 36. Subsequent catalytic
hydrogenation of 36 gave phenethylcoumaran 37, and
this intermediate was then deprotected by acid hydroly-
sis.
stable in vivo because of the ease of formation of
O-glucuronide and O-sulfate on the phenolic hydroxy
group.17 To overcome this metabolic instability, we
introduced an amino group instead of the hydroxy group
in position 5 of coumaran. The amino group can donate
an electron to a reactive radical similarly to the hydroxy
group18 but has the advantages of hydrophilicity and
metabolical stability. Moreover, various substituents
including a cyclic amino moiety were introduced into
position 2 of 5-aminocoumaran to improve inhibition of
dopamine release.
Ch em istr y
The general synthetic pathways for preparation of
5-aminocoumarans listed in Table 1 are shown in
Schemes 1-9. The parent compound, 2,3-dihydro-
2,2,4,6,7-pentamethyl-5-benzofuranamine hydrochloride
(8a ), was synthesized as follows. The starting phenol
4a 19 was methallylated using methallyl chloride and
potassium carbonate as a base to give an ether (5a )
(Scheme 1). Claisen rearrangement of 5a provided
C-methallylated compound 6a in good yield. Cyclization
of 6a under acidic conditions gave coumaran 8a with
deprotection of the formamide group in quantitative
yield. The demethyl derivatives of 8a (8b-e) were
synthesized from phenols 4b-e20 in a similar manner
as for the synthesis of 8a .
Compounds with an additional amino group in posi-
tion 4 were synthesized as shown in Scheme 2. The
coumaran 7d was nitrated to give 9. Hydrolysis of the
acetamide group of 9 gave the 5-amino-4-nitro com-
pound 10, which was converted to diamine 11 by
catalytic hydrogenation. The nitro group of 9 was
reduced to afford an amino group by catalytic hydroge-
nation followed by methylation to give N,N-dimethyl-
amino compound 13. The hydrolysis of the acetamide
group of 13 was accomplished by refluxing with hydro-
chloric acid and methanol.
2-(2-Aminoethyl)coumarans 46a ,b were synthesized
as shown in Scheme 7. Bromide 40, prepared from
2,3,5-trimethylphenol by a sequence of reactions similar
to the synthesis of 24, was converted to carboxylic acid
42 by cyanation followed by basic hydrolysis. Com-
pound 42 was coupled with amines by using 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlo-
ride to provide amides 43a ,b, which were subsequently
reduced with LiAlH4 to give amines 44a ,b. Nitration
in position 5 of 44a ,b followed by catalytic hydrogena-
tion of the introduced nitro group gave 46a ,b.
Compounds with an aminomethyl group in position
7 were synthesized as shown in Scheme 3. Methal-
lylphenol 16, prepared from 3,5-dimethylphenol via 15,
was converted to (aminomethyl)phenols 17a ,b using the
Synthesis of compounds with an aryl or alkyl group
in position 3 was carried out as shown in Scheme 8.
Lithiation of bromoanisole (47)21 followed by addition

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5-Aminocoumarans for Treating CNS Injury
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 561
Sch em e 1a
a
(a) Methallyl chloride, K2CO3/DMF; (b) N,N-diethylaniline, 200 °C; (c) 35% aq HCl-MeOH (3:10, v/v); (d) 35% aq HCl-MeOH (1:1,
v/v).
Sch em e 2a
lowed by catalytic hydrogenation gave 5-amino com-
pounds 51a -h .
5-(Alkylamino)coumarans were synthesized as follows
(scheme not shown). The parent compound 8a was
acylated to give amides 52a -d , and LiAlH4 reduction
of 52a -d afforded N-alkylated compounds 53a -c. Sul-
famides 54a ,b were obtained by sulfonylation of 8a
using aryl- or alkylsulfonyl chlorides.
As shown in Scheme 9, 5-(arylamino)coumarans were
synthesized utilizing Weingarten’s method.22 Quinone
5523 and aromatic amines were condensed using TiCl4
as a dehydrating agent. The reaction proceeded per-
fectly regiospecifically in position 4. The quinonimines
56a -c thus obtained were reduced using sodium hy-
drosulfite, and subsequent acid-catalyzed cyclization of
aminophenols 57a -c gave N-arylated aminocoumarans
58a -c. When aminocoumaran 8e was used as an
aromatic amine, the compound isolated was aminophe-
nol 57d which was produced by reduction of quinon-
imine 56d . In this event, aminocoumaran 8e was
supposed to work as a reducing agent. Compound 57d
was cyclized to give bis-coumaran 58d . However, when
we used aminocoumaran 8a instead of aminocoumaran
8e, quinone 55 was recovered as its hydroquinone form
because of the high reducing activity of aminocoumaran
8a .24
a
(a) HNO3, Ac2O; (b) 35% aq HCl-MeOH (3:10, v/v); (c) H2,
Pd/C; (d) MeI, K2CO3.
Sch em e 3a
Optical resolution of 26n with (S)-(+)- and (R)-(-)-
mandelic acid afforded the two diastereomeric salts in
high optical purities. The (+)-isomer of 26n was shown
to have the S-configuration by X-ray crystallographic
analysis of (S)-(+)-mandelate.
P h a r m a cologica l Resu lts a n d Discu ssion
The compounds listed in Table 1 were initially tested
for their antioxidant activities in vitro and in vivo, and
selected compounds were evaluated for dopamine re-
lease inhibitory activity.
Lip id P er oxid a tion In h ibition . Inhibitory activi-
ties against lipid peroxidation were evaluated with
homogenized rat liver microsomes. Lipid peroxidation
was assessed by the formation of 2-thiobarbituric acid
reactive products.25 The compouds were tested at doses
up to 1 µM, and IC50 values are listed in Table 2. The
important factors related to in vitro lipid peroxidation
inhibitory activity were (1) lipophilicity of the compound,
(2) electron density of the benzene ring of the coumaran
structure, and (3) substitution manner of the amino
group in position 5.
a
(a) N,N-Diethylaniline, 200 °C; (b) (HCHO)n, R2NH; (c) 35%
aq HCl-MeOH (3:10, v/v); (d) NaOH/H2O.
to isopropyl ketones afforded tertiary alcohols 48a -h
in good yields, except for addition to diisopropyl ketone
(48h , 12%). Compounds 48a -h were converted to
coumarans 49a -h by treatment with boiling hydrobro-
mic acid via dehydration of the tertiary alcohol group,
hydrolysis of the methyl ether group, and cyclization
reaction. Nitration of 49a -h with acetyl nitrate fol-

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562 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ta ble 1. Physical Properties of 5-Aminocoumarans
Ohkawa et al.

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5-Aminocoumarans for Treating CNS Injury
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 563
Ta ble 1 (Continued)
a
b
No attempt was made to optimize yields. Numbers represent the yield for the last step. IPA ) 2-propanol. c IPE ) isopropyl ether.
(+0.5H2O) Calcd: C, 51.13; H, 6.56; N, 10.52. Found: C, 51.11; H, 6.49; N, 10.59. e (+0.5H2O) Calcd: C, 67.31; H, 7.63; N, 3.93. Found:
d
C, 67.34; H, 7.67; N, 4.19. f Calcd: C, 60.58; H, 7.82; N, 5.43. Found: C, 61.83; H, 8.25; N, 5.05. Without further purification.
g
h
i
Dihydrochloride. (+0.5H2O) Calcd: C, 62.77; H, 7.20; N, 7.32. Found: C, 62.89; H, 7.59; N, 7.64. (+H2O) Calcd: C, 60.72; H, 7.76; N,
j
k
6.74. Found: C, 60.57; H, 7.50; N, 6.79. (+0.5H2O) Calcd: C, 54.40; H, 6.85; N, 11.89. Found: C, 54.61; H, 6.70; N, 11.87. (+H2O)
l
Calcd: C, 54.39; H, 8.56; N, 7.93. Found: C, 54.41; H, 8.53; N, 7.92. Calcd: C, 76.56; H, 7.85; N, 9.92. Found: C, 76.12; H, 7.96; N,
9.74. Without further purification.
Sch em e 4a
a
(a) N,N-Diethylaniline, 200 °C; (b) 35% aq HCl-MeOH (1:1, v/v); (c) H2, Pd/C.
Substitution on the coumaran ring in positions 2, 3,
4, 6, and 7 by lipophilic groups such as methyl (com-
pound 8a ), isobutyl (compound 23), aryl (compounds
51a -f),26 and aralkyl (compound 38) resulted in an
increase in the activity. In a series of compounds which
has a heteroatom as a linker in position 2, no significant
differences were observed with regard to the kind of
heteroatom (compare 26g,k ,t). However, it is notewor-
thy that phenylpiperidinyl (compound 26n ), phenylpip-
erazinyl (compound 26q), and (diphenylmethyl)piper-
azinyl (compound 26r ) groups greatly enhanced the
activity. In contrast, introduction of hydrophilic groups
such as nitro (compound 10), (dimethylamino)methyl
(compound 19a ), ester (compound 35), heteroring (com-
pounds 26e,w and 51g), alcohol (26i), and carboxylic
acid (compound 26j) groups attenuated the activity.
Previously, we reported the linear dependence of lipid
peroxidation inhibition on the lipophilicity of the mol-
ecule for (pyridylmethyl)quinones.27
The 5-coumaranoxyl radical formed in the reaction
with oxygen radicals has been reported to be stabilized
by delocalization of the unpaired electron to the p-type
lone pair of oxygen in position 1 and π-electron of the
benzene ring.15 The same effect is expected in the case
of 5-coumaranaminyl radical. Therefore, electron-
donating substituents such as amino and methyl groups28
enhanced the activity due to stabilization of the aminyl
radical to exhibit good lipid peroxidation inhibition
(compounds 8a and 11). To enhance this stabilizing
effect, we designed 5-(arylamino)coumarans. With the

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564 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ohkawa et al.
Sch em e 5a
Sch em e 7a
a
(a) Br2, AcONa/AcOH; (b) R′SH, NaH/DMF, 100 °C; (c) 35%
a
(a) Br2, AcONa/AcOH; (b) NaCN/DMSO; (c) NaOH/H2O; (d)
R′2NH, WSC, HOBt/DMF; (e) LiAlH4/THF; (f) HNO3, Ac2O; (g) H2,
Pd/C.
aq HCl-MeOH (1:1, v/v); (d) R′OH, NaH, or R′R′′NH (10 equiv)
in sealed tube, 180 °C; (e) R′R′′NH (1.2 equiv), Et3N in sealed tube,
180 °C.
Sch em e 6a
dependent Haber-Weiss reaction. These highly reac-
tive oxidants injure the nerve cell membrane by perox-
idation of unsaturated fatty acids, and thus membrane
excitation increases as a result of the increase in
membrane permeability and brain edema is caused by
cell death. Following spinal intrathecal injection of
FeCl2, it is assumed from behavioral changes that the
increase in membrane permeability caused the excita-
tion of both the sensory and the motor nervous systems
in the spinal cord and that excessive degradation of
membrane structures caused the necrosis resulting in
the paralysis. Since only the compounds which have
high antioxidative activity and exhibit good perme-
ability across the blood-brain barrier (BBB) suppress
these phenomena, the mouse-FeCl2-it assay is a versa-
tile and sophisticated technique for screening of anti-
oxidative agents for use in the CNS. The compounds
tested were orally administered 30 min before FeCl2
injection. ID50 values were obtained by the score of
behavioral responses evaluated from 15 min to 1 h after
FeCl2 injection.
a
(a) m-CPBA, NaHCO3/CH2Cl2, H2O; (b) DMSO, (COCl)2/
The results of the mouse-FeCl2-it assay are also
shown in Table 2. In a previous experiment, aminopy-
rine which has antioxidative activity30 exhibited inhibi-
tory activity, and its ID50 value was determined as 108.0
mg/kg, po (95% confidence limits; 71.1-139.2). R-To-
copherol did not show any significant activity at a dose
of 100 mg/kg; however, the 5-hydroxycoumaran type
compound showed weak inhibitory activity (23% inhibi-
tion at a dose of 100 mg/kg). The parent aminocouma-
ran 8a had potent in vivo antioxidative activity with
an ID50 value of 7.8 mg/kg. Inhibitory activity decreased
in relation to the decrease in the number of methyl
groups. This result is in accordance with in vitro assay
data. Methyl groups were the best substituents for in
vivo activity among the substituents introduced in
positions 4, 6, and 7 on the benzene ring of the
coumaran structure. Introduction of aryl or alkyl sub-
stituents in position 3 decreased the activity. Among
the compounds that have a heteromethyl group at the
2 position, polar moieties such as hydroxy group and
carboxyl group reduced the activity (26i,j). Nitrogen
CH2Cl2; (c) (EtO)2P(O)CH2COOEt, NaH/THF; (d) 35% aq HCl-
MeOH (1:1, v/v); (e) Ph3PCH2Ar(Br), NaH/THF; (f) H2, Pd/C.
exception of 51g which has a polar pyridyl group,
enhancement of the activity was observed with these
compounds. As expected, bis-coumaran derivative 58d ,
in which two coumaran structures are able to contribute
to stabilization of the aminyl radical, was revealed to
be the most potent in this series.
Introduction of an alkyl group decreased activity most
likely by steric hindrance around the nitrogen atom in
position 5. Introduction of an acyl group diminished
activity by decreasing electron density on the nitrogen
atom in position 5.
Mou se-F eCl2-it Assa y (in Vivo). It is known that
the intracortical injection of FeCl2 causes acute focal
epileptiform discharges and formation of brain edema
along with lipid peroxidation, and these symptoms are
reduced by administration of R-tocopherol.29 Iron added
into cerebral cortex causes formation of active oxygen
species (e.g., superoxide, hydroxyl radical) via iron-

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5-Aminocoumarans for Treating CNS Injury
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 565
Sch em e 8a
Effects of TAK-218 a n d Its Op tica l Isom er on
Mor ta lity a fter Tr a n sien t Cer ebr a l Isch em ia (F ou r
Vessel Occlu sion ) Mod el in Ra ts.33 Transient global
ischemia was produced by 45 min occlusion of bilateral
common carotid arteries in male rats in which vertebral
arteries were cauterized bilaterally on the previous day.
TAK-218 and its optical isomer were given ip im-
mediately and then 2 and 24 h after reopening the
circulation at doses of 1 and 3 mg/kg. Survival of the
animals was monitored for 1, 3, 5, 7, 10, and 14 days
after reopening the circulation. As shown in Figure 1,
both TAK-218 and its R-isomer reduced the mortality;
the effect of TAK-218 was more prominent than that of
the R-isomer from day 1 to day 7 postischemia.
Effects of TAK-218 a n d Its Op tica l Isom er on th e
Fu n ction al Deficit after P en etr ation -In du ced Br ain
In ju r y in Ra ts. Male Wistar rats were used. Under
halothane anesthesia, the striatum was injured by
stereotactically inserting a glass rod, 1.5 mm in diam-
eter with a tapered tip, through a burr hole from the
cortex to striatum. TAK-218 and its R-isomer were
given ip immediately and 2 h after injury. Functional
changes were evaluated 3, 7, and 14 days after injury
utilizing APO-induced circling which has been reported
to be specific to animals with damage in the nigro-
striatal system. As shown in Figure 2, TAK-218 dose-
dependently decreased the functional deficit, and the
minimum effective dose was 0.1 mg/kg. Although the
R-isomer significantly decreased functional deficit at a
dose of 1 mg/kg on day 14 postinjury, it did not exhibit
any significant effect at doses ranging from 0.03 to 0.3
mg/kg during the observation period.
a
(a) n-BuLi/THF, then R′COMe2; (b) HBr/H2O; (c) HNO3, Ac2O;
(d) H2, Pd/C.
was the most efficacious linker in position 2. Both
optical isomers of 26n showed almost equivalent inhibi-
tory activity. Bulky substituents reduced in vivo activ-
ity (26r and 58d ). The reason for this might be
increased molecular weight because it is known that the
BBB permeability is related to lipophilicity of molecules
and molecular weight.31 The alkyl substituents on the
amino group in position 5 decreased the in vitro activity
markedly; however, in vivo activity was less attenuated.
In contrast, the aryl-substituted compounds 58a ,b,d
showed no significant activity in the in vivo assay
despite their potent activity in vitro. Acylation of the
amino group diminished both in vivo and in vitro
activity.
Su p p r ession of MAP -In d u ced Hyp er m otility in
Mice.32 Selected compounds were evaluated for an-
tagonizing hypermotility induced by MAP, a dopamine
releaser in mice. Thirty minutes after ip treatment with
compounds at a dose of 10 mg/kg, MAP was given ip to
male mice at a dose of 1 mg/kg. The results of this assay
are shown in Table 3. Basic molecules 8a and 27
exhibited significant effects against MAP-induced hy-
permotility. Except in these compounds, a phenyl group
adjacent to the methyl group in position 2 at a distance
seemed to be indispensable (compare compounds 26m ,o,p
with compounds 26n ,q,r ). Among these compounds,
26n which has a phenylpiperidinyl group in position 2
was found to be most effective. To investigate the
activities of both optical isomers, 26n was resolved using
(S)-(+)-mandelic acid. Both isomers inhibited lipid
peroxidation in vitro and antagonized excitatory behav-
ior induced by intrathecal injection of FeCl2 to the same
extent. However, the S-isomer (TAK-218) exhibited a
30-fold more potent antagonistic activity on MAP-
induced hypermotility than the R-isomer as shown in
Table 4. In addition, to clarify whether this effect of
TAK-218 was due to inhibition of dopamine release or
blockade of dopamine receptors, the effects of TAK-218
on apomorphine (APO)-induced hypermotility were also
studied. As shown in Table 4, APO (1 mg/kg, sc)-
induced hypermotility was not suppressed by TAK-218
at a dose of 1 mg/kg, ip, suggesting that TAK-218
suppresses aberrant dopamine release. To investigate
the role of dopamine in ischemic and traumatic brain
injury, we tested both isomers in the transient cerebral
ischemia model and penetration-induced head injury
model in rats.
As described above, TAK-218 decreased the functional
deficit in the brain injury model and mortality in the
cerebral ischemia model, and the effect of TAK-218 was
more potent than that of the R-isomer. Since the
antioxidant activities of both isomers were equipotent,
the inhibitory activity of TAK-218 on aberrant dopamine
release appeared to contribute to the protective effects
in the cerebral ischemia model and the brain injury
model. In other words, dopamine plays an important
role in deterioration of CNS function after ischemic and
traumatic injury in dopamine-rich regions such as the
striatum and nucleus of accubens. In the mouse-FeCl2-
it assay, both isomers antagonized excitatory behavior
to the same extent because the dopamine content in the
spinal cord is very low and only the antioxidative
activities of the compounds contribute to the protective
effect.
In conclusion, we reported here the discovery of
5-aminocoumarans which have highly potent inhibitory
activities on both lipid peroxidation and dopamine
release. The selected compound TAK-218 was effective
upon systemic administration in in vivo models of CNS
trauma and ischemia. The R-isomer of TAK-218, which
has the same level of antioxidative activity and less
inhibitory effect on dopamine release, was also effective
in in vivo models; however, its potency was less than
that of TAK-218. This provides evidence for an impor-
tant role of dopamine as well as lipid peroxidation in
traumatic and ischemic brain damage. Pharmacological
studies of TAK-218 are currently in progress in our
laboratory and will be reported in due course.

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566 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ohkawa et al.
Sch em e 9a
a
(a) TiCl4, RNH2; (b) Na2S2O4; (c) 35% aq HCl-MeOH (3:10, v/v).
HCl (30 mL) with ice-water bath cooling, and the mixture
was refluxed under an argon atmosphere for 2 h. After cooling,
the reaction mixture was neutralized with aqueous NaHCO3
and extracted with CHCl3. The extract was washed with
brine, dried, and concentrated (6.4 g, 99% yield). A portion of
the residue was treated with 4 M HCl/EtOH followed by
recrystallization of the crude product from MeOH to give 8a :
mp 248-250 °C dec; NMR (DMSO-d6) δ 1.41 (6H, s), 2.02 (3H,
s), 2.20 (6H, s), 3.41 (2H, m), 9.65 (2H, br s).
Exp er im en ta l Section
Melting points were determined on a Yanagimoto mi-
cromelting point apparatus and are uncorrected. Proton
nuclear magnetic resonance (NMR) spectra were recorded on
a Varian Gemini-200 (200 MHz) spectrometer, with tetram-
ethylsilane as the internal standard. Optical rotations were
determined with a J ASCO DIP-370 polarimeter. TLC analyses
were carried out on Merck Kieselgel 60 F254 plates. Elemental
analyses were carried out by Takeda Analytical Laboratories,
Ltd., and are within (0.4% of the theoretical values unless
otherwise noted. THF and isopropyl ether (IPE) were distilled
over calcium hydride prior to use, and other solvents and
reagents were used without purification. Solutions in organic
solvents were dried over anhydrous magnesium sulfate unless
otherwise noted, and concentration of the organic solution was
carried out under reduced pressure. Chromatographic puri-
fication was carried out on silica gel columns (Kieselgel 60,
0.063-0.200 mm, Merck). Yields were not maximized.
Gen er a l P r oced u r e for O-Meth a llyla tion of P h en ols.
N-[2,3,6-Tr im eth yl-4-[(2-m eth yl-2-p r op en yl)oxy]p h en yl]-
for m a m id e (5a ). To a solution of N-(4-hydroxy-2,3,6-tri-
methylphenyl)formamide19 (86 g, 0.48 mol) and methallyl
chloride (45 g, 0.50 mol) in DMF (300 mL) was added K2CO3
(74 g, 0.54 mol), and the mixture was stirred under an argon
atmosphere at 80 °C for 3 h. The reaction mixture was poured
into ice-water, and the resulting solid was washed with water
and dried. The crude solid was recrystallized from IPE to yield
80 g (72% yield) of 5a : mp 144-145 °C; NMR (CDCl3) δ 1.84
(3H, m), 2.17 (3H, s), 2.19 (1.5H, s), 2.22 (3H, s), 2.26 (1.5H,
s), 4.40 (1H, s), 4.42 (1H, s), 4.99 (1H, m), 5.11 (1H, br s), 6.60
(1H, s), 6.75 (1H, m), 7.98 (0.5H, d, J ) 12.0 Hz), 8.41 (0.5H,
s).
With a similar procedure, 5b-e, 15, and 20 were prepared.
Gen er a l P r oced u r e for Cla isen Rea r r a n gem en t. N-[4-
Hyd r oxy-2,3,6-tr im eth yl-5-(2-m eth yl-2-p r op en yl)p h en yl]-
for m a m id e (6a ). A solution of 5a (80 g, 0.34 mol) in N,N-
diethylaniline (500 mL) was stirred under an argon atmosphere
at 200 °C for 3 h. After the reaction mixture had cooled,
hexane was added and the resulting solid was collected by
filtration, washed with hexane, and dried under reduced
pressure. The crude product was recrystallized from EtOAc-
IPE to yield 75 g (94% yield) of 6a : mp 163-164 °C; NMR
(CDCl3) δ 1.80 (3H, s), 2.16 (3H, s), 2.17 (1.5H, s), 2.19 (1.5H,
s), 2.20 (1.5H, s), 2.21 (1.5H, s), 3.38 (2H, br s), 4.65 (1H, m),
4.88 (1H, m), 5.16 (0.5H, s), 5.19 (0.5H, s), 6.70 (1H, m), 7.95
(0.5H, d, J ) 12.0 Hz), 8.42 (0.5H, d, J ) 1.8 Hz).
With a similar procedure, 7c,d , 8b,e, 18a ,b, 22, and 58a -d
were prepared.
Gen er a l P r oced u r e for Hyd r olysis of Aceta m id e or
F or m a m id e Gr ou p s u n d er Acid ic Con d ition s. 2,3-Dih y-
d r o-2,2,4,7-tetr a m eth yl-5-ben zofu r a n a m in e Hyd r och lo-
r id e (8c). To a solution of 7c (1.0 g, 4.3 mmol) in MeOH (15
mL) was added 35% aqueous HCl (15 mL) with ice-water bath
cooling. The mixture was refluxed under an argon atmosphere
for 2 h. After cooling, the reaction mixture was neutralized
with aqueous NaHCO3 and extracted with EtOAc. The extract
was washed with brine and dried, and the solvent was
removed. The residue was treated with 4 M HCl/EtOH (1.5
mL), and the HCl salt was recrystallized from EtOH-Et2O to
give 0.93 g (yield 95%) of 8c: mp 216-218 °C; NMR (DMSO-
d6) δ 1.47 (6H, s), 2.13 (3H, s), 2.38 (3H, s), 2.93 (2H, s), 7.18
(1H, s), 10.21 (2H, br s). Anal. C, H, N.
With a similar procedure, 8d , 10, 14, 26a -j, 27, 30, 31, 35,
and 38 were prepared.
Gen er a l P r oced u r e for Nitr a tion . N-(2,3-Dih yd r o-
2,2,6,7-tetr am eth yl-4-n itr o-5-ben zofu r an yl)acetam ide (9).
To a solution of 7d (15 g, 64 mmol) in acetic anhydride-acetic
acid (150 mL; 1:1) was added dropwise a solution of acetyl
nitrate (prepared from 69% nitric acid (7.7 mL) and acetic
anhydride (25 mL)) with ice-water bath cooling. The mixture
was stirred under the same conditions for 15 min and then
poured into ice-water. The aqueous mixture was extracted
with EtOAc, and the extract was washed with brine, dried,
and concentrated. The residue was purified by column chro-
matography (CHCl3) followed by recrystallization from CH2-
Cl2-IPE to afford 16 g (89% yield) of 9: mp 203-204 °C; NMR
(CDCl3) δ 1.48 (6H, s), 2.15 (3H, s), 2.18 (3H, s), 2.19 (3H, s),
3.29 (2H, s), 7.79 (1H, br s).
With a similar procedure, 45a ,b and 50a -h were prepared.
Gen er a l P r oced u r e for Red u ction of th e Nitr o Gr ou p .
2,3-D i h y d r o -2,2,6,7-t e t r a m e t h y l-4,5-b e n z o fu r a n d i -
a m in e Hyd r och lor id e (11). A mixture of 10 (4.9 g, 21 mmol)
and 10% Pd/C (1.4 g; 50% wet) in EtOH (100 mL) was stirred
at room temperature under a hydrogen atmosphere for 3 h.
The reaction mixture was filtered, and the filtrate was
concentrated. The residue was purified by column chroma-
tography (CHCl3) to afford 4.2 g (97% yield) of 11 (free base).
A portion of the free base obtained was treated with 4 M HCl/
EtOH, and the salt was recrystallized from EtOH to give 11:
With a similar procedure, 6b-e, 16, 21, and 39 were
prepared.
Gen er a l P r oced u r e for Acid -Ca ta lyzed Cycliza tion of
2-Meth a llylp h en ols. 2,3-Dih yd r o-2,2,4,6,7-p en ta m eth yl-
5-ben zofu r a n a m in e Hyd r och lor id e (8a ). To a solution of
6a (7.3 g, 36 mmol) in MeOH (100 mL) was added 35% aqueous

+
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5-Aminocoumarans for Treating CNS Injury
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 567
Ta ble 2. Effects of 5-Aminocoumarans on Lipid Peroxidation
and Mouse-FeCl2-it Assay
Ta ble 3. Effects of 5-Aminocoumarans on the
Methamphetamine-Induced Increase in Locomotor Activity in
Micea
mouse-FeCl2-it assayb
expt no. sample inducer
locomotor activity % increase
lipid peroxidationa
ID50
95%
no.
IC50(µM)
(mg/kg, po) confid limits
1
saline
saline
8a
saline
saline
26a
26b
26d
26e
26f
26k
51a
51g
saline
saline
26g
saline
MAPb
MAP
saline
MAP
MAP
MAP
MAP
MAP
MAP
MAP
MAP
MAP
saline
MAP
MAP
MAP
MAP
MAP
MAP
MAP
MAP
MAP
saline
MAP
MAP
MAP
saline
MAP
MAP
MAP
MAP
saline
MAP
MAP
MAP
saline
MAP
MAP
MAP
MAP
194 ( 53**
1195 ( 142
509 ( 153**
747 ( 268**
2168 ( 359
1098 ( 277*
1625 ( 398
892 ( 237*
2228 ( 403
1391 ( 367
1657 ( 506
1845 ( 190
1488 ( 330
447 ( 276**
2385 ( 548
1099 ( 288
1540 ( 455
1934 ( 461
482 ( 87**
1664 ( 440
1812 ( 333
1340 ( 398
1115 ( 339
495 ( 128**
3127 ( 420
2960 ( 295
3209 ( 416
336 ( 56**
1892 ( 232
3154 ( 738
2129 ( 636
3549 ( 552*
402 ( 118**
2689 ( 591
1484 ( 632
3950 ( 452
417 ( 38**
1376 ( 348
1185 ( 187
2156 ( 311
1229 ( 115
0
100
31.5
0
100
24.7
8a
8b
8c
8d
8e
10
11
14
0.76
(14)
(19)
(26)
(5)
7.8
16.3
32.3
24.4
6.2-10.5
10.9-23.6
20.6-42.7
17.2-33.9
2
>100
61.8
10.2
104.2
45.3
64.0
77.3
52.1
0
100
33.6
56.4
76.7
1.8
62.8
70.4
46.1
34.5
0
100
93.7
103.1
0
100
181.1
115.2
206.5
0
100
47.3
155.1
0
100
80.1
181.3
84.7
(0)
>100
>50
0.30
0.55
(4)
22.3
18.3-26.6
19a
19b
22
>50
(3)
>50
0.30
0.14
0.27
0.22
0.22
0.63
(11)
0.07
0.26
0.28
(11)
(0)
0.28
0.30
(18)
0.07
NTd
NT
0.41
(10)
0.07
0.07
0.26
0.29
0.29
0.08
(6)
0.30
0.66
0.80
(38)
0.08
(4)
21.9
16.0-28.0
7.5-19.6
20.0-30.3
12.6-22.5
23
13.3
24.7
17.9
3
26a
26b
26c
26d
26e
26f
26g
26h
26i
26j
26k
26l
26m
26n
>50
26h
19.9
37.1
33.7
32.0
24.6
14.1-25.3
32.0-44.0
30.7-36.7
9.1-49.0
26l
26n c
26r
26s
26t
27
saline
saline
26m
26o
saline
saline
26p
18.7-31.9
>50
43.0
18.6
20.1
11.7
10.4
10.8
11.6
19.2
25.3
13.1
>50
15.7
9.1
14.2
17.0
25.0
16.0
31.3
36.6
34.4-70.8
10.4-25.5
5.9-33.2
9.0-14.8
7.2-13.6
8.2-13.4
7.5-16.7
11.8-25.8
19.9-32.4
9.8-18.1
4
5
(S)-(+)-26n c
(R)-(-)-26n c
26o
26p
26q
26r
46a
46b
6
7
saline
saline
26q
26w
saline
saline
38
26s
26t
26u
26v
26w
27
30
31
35
38
46a
46b
51a
51b
51c
51d
51e
51f
51g
51h
52a
52b
52c
52d
53a
53b
53c
54a
54b
58a
58b
58c
58d
11.9-19.2
5.4-18.8
8.0-18.8
11.5-21.6
18.0-34.5
12.2-20.6
19.1-40.6
27.7-48.8
51f
53a
>100
a
Thirty minutes after ip injection of 5-aminocoumarans at a
11.5
19.0
18.4
24.8
8.4-15.1
16.8-21.8
14.3-23.4
14.5-41.9
dose of 10 mg/kg, methamphetamine at a dose of 1 mg/kg was
injected ip. The number of mice in each group was 8. Locomotor
activity was measured for 1 h after administration of metham-
phetamine. *p < 0.05 and **p < 0.01 compared to the respective
(8)
0.29
0.30
0.29
0.05
0.29
0.28
(22)
0.29
(0)
>50
>50
42.2
>50
26.7
32.1
14.0
>100
b
saline-methamphetamine-treated controls. Methamphetamine.
c Dihydrochloride was used.
26.3-80.2
mp 248-251 °C; NMR (DMSO-d6) δ 1.39 (6H, s), 1.93 (3H, s),
2.09 (3H, s), 2.82 (2H, s), 3.36 (4H, br s).
With a similar procedure, 46a ,b and 51a -h were prepared.
This method was utilized in hydrogenation of the olefin group
for the synthesis of 23 and 37.
20.2-36.6
24.1-41.6
9.3-19.2
(-2)
(1)
(1)
>100
>100
>100
N-[4-(Dim eth yla m in o)-2,3-d ih yd r o-2,2,6,7-tetr a m eth yl-
5-ben zofu r a n yl]a ceta m id e (13). To a solution of 12 (5.3 g,
21 mmol) in DMF (100 mL) were added K2CO3 (4.4 g, 32 mmol)
and iodomethane (4.0 mL, 21 mmol). The mixture was stirred
at room temperature under an argon atmosphere for 3 h. The
reaction mixture was poured into water, and the aqueous
mixture was extracted with EtOAc. The extract was washed
with brine, dried, and concentrated. The residue was purified
by column chromatography (CHCl3:MeOH ) 97:3), and the
product was recrystallized from CH2Cl2-IPE to yield 5.5 g
(94% yield) of 13: mp 185-186 °C; NMR (CDCl3) δ 1.44 (6H,
s), 2.09 (6H, s), 2.21 (3H, s), 2.67 (6H, s), 3.09 (2H, s), 7.17
(1H, br s).
Gen er a l P r oced u r e for Ma n n ich Rea ction of 2-Meth -
a llylp h en ols. N-[3-[(Dim eth yla m in o)m eth yl]-4-h yd r oxy-
2,6-d im eth yl-5-(2-m eth yl-2-p r op en yl)p h en yl]a ceta m id e
(17a ). To a suspension of paraformaldehyde (1.6 g, 43 mmol)
in EtOH (10 mL) was added 50% aqueous dimethylamine (6.5
mL, 64 mmol), and the mixture was stirred at room temper-
(18)
(16)
(24)
(-1)
(3)
0.28
0.30
(4)
16.6
6.6-23.9
31.3-46.2
24.2-116.4
38.9
51.1
>100
>100
>50
>50
>50
>25
0.03
a
The molar concentration of test compound required to reduce
by 50% the amount of lipid peroxide formed in rat liver mi-
crosomes. IC50 values were determined from four concentrations
by nonlinear regression analysis. Percent inhibition at the
b
concentration of 1 µM is shown in parentheses. The dose of test
compound required to reduce by 50% the score of excitatory
behavior induced by intrathecal injection of FeCl2. ID50 values
were generated from three or four doses; 10 animals were used
per dose. c Dihydrochloride was used. Not tested.
d

+
+
568 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ohkawa et al.
Ta ble 4. Effects of (S)-(+)- and (R)-(-)-26n Dihydrochloride on
the Methamphetamine- and Apomorphine-Induced Increase in
Locomotor Activity in Micea
dose
locomotor
activity
sample
saline
(mg/kg) inducer nb
% increase
saline
MAPc
MAP
MAP
MAP
saline
MAP
MAP
MAP
MAP
saline
APOe
APO
10
9
175 ( 46**
1878 ( 431
0
100
113.2
54.4
35.1
0
100
129.0
169.0
67.7
0
saline
(S)-(+)-26n d
0.1
0.3
1
10 2103 ( 451
9
9
1102 ( 317
773 ( 173*
245 ( 74**
saline
16
saline
14 1196 ( 314
15 1472 ( 257
13 1852 ( 493
16
13
11
11
(R)-(-)-26n d
1
3
10
889 ( 159
145 ( 46**
619 ( 160
841 ( 251
saline
saline
100
124.5
(S)-(+)-26n d
1
a
Thirty minutes after ip injection of (S)-(+)- and (R)-(-)-26n
dihydrochloride, methamphetamine at a dose of 1 mg/kg, ip, or
apomorphine at a dose of 1 mg/kg, sc, was injected. Locomotor
activity was measured for 1 h after administration of metham-
phetamine. *p < 0.05 and **p < 0.01 when compared to the
respective saline-methamphetamine-treated control group (two-
b
tailed Student’s t-test). Number of mice. c Methamphetamine.
d
Dihydrochloride was used. e Apomorphine.
F igu r e 2. Effects of (A) (S)-26n dihydrochloride (TAK-218)
and (B) (R)-26n dihydrochloride on the functional changes
after penetration-induced brain injury in rats; **p < 0.01 when
compared to the saline-treated control group (two-tailed Dun-
nett’s multiple range test following 2-way ANOVA).
With a similar procedure, 17b was prepared.
Gen er a l P r oced u r e for Hyd r olysis of th e Aceta m id e
Gr ou p u n d er Ba sic Con d ition s. 5-Am in o-2,3-d ih yd r o-
N ,N ,2,2,4,6-h e x a m e t h y l-7-b e n zo fu r a n m e t h a n a m in e
Eth a n ed ioa te (1:1) (19a ). A mixture of 18a (4.8 g, 17 mmol),
MeOH (3 mL), and 5 M aqueous NaOH (25 mL) was stirred
under an argon atmosphere in a sealed tube at 200 °C for 13
h. After cooling, the reaction mixture was diluted with water
and extracted with CHCl3. The extract was washed with
brine, dried, and concentrated. The residue was purified by
column chromatography (CHCl3:MeOH ) 88:12) to yield the
free base of 19a (1.7 g, 42% yield). A portion of the product
was treated with oxalic acid, and the salt obtained was
recrystallized from EtOH to give 19a : mp 178-180 °C; NMR
(DMSO-d6) δ 1.39 (6H, s), 2.02 (3H, s), 2.07 (3H, s), 2.74 (6H,
s), 2.93 (2H, s), 4.13 (2H, s).
F igu r e 1. Effects of (A) (S)-26n dihydrochloride (TAK-218)
and (B) (R)-26n dihydrochloride on the survival rates following
45-min transient cerebral ischemia in rats; *p < 0.05 when
compared to the saline-treated control group (two-tailed
Fisher’s exact probability test).
With a similar procedure, 19b was prepared.
Gen er al P r ocedu r e for Br om in ation of 2-Meth allylph e-
n ols. N-[2-(Br om om eth yl)-2,3-dih ydr o-2,4,6,7-tetr am eth yl-
5-ben zofu r a n yl]for m a m id e (24). To a suspension of 6a (30
g, 0.13 mol) and sodium acetate (21 g, 0.26 mol) in acetic acid
(500 mL) was added bromine (7.9 mL, 0.15 mol). After stirring
for 10 min at room temperature, the reaction mixture was
concentrated and water was added to the residue. Precipitates
formed were collected by filtration, washed with water and
IPE, and dried. The crude product was recrystallized from
ature for 30 min. Then the resulting solution was added to a
solution of 16 (5.0 g, 21 mmol) in EtOH (30 mL). The mixture
was refluxed under an argon atmosphere for 3.5 h and then
cooled and concentrated. The residue was used for the next
reaction without further purification.

+
+
5-Aminocoumarans for Treating CNS Injury
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 569
EtOAc-IPE to yield 33 g (81% yield) of 24: mp 157-158 °C;
NMR (CDCl3) δ 1.61 (1.5H, s), 1.63 (1.5H, s), 2.09 (3H, s), 2.11
(3H, s), 2.13 (1.5H, s), 2.16 (1.5H, s), 2.93 (1H, d, J ) 15.8
Hz), 3.28 (0.5H, d, J ) 15.8 Hz), 3.29 (0.5H, d, J ) 15.8 Hz),
3.51 (1H, s), 3.53 (1H, s), 6.77 (0.5H, br s), 6.85 (0.5H, d, J )
12.0 Hz), 7.96 (0.5H, d, J ) 12.0 Hz), 8.40 (0.5H, d, J ) 1.4
Hz).
aqueous NaIO4 (20 mL), and the mixture was stirred at room
temperature for 3 h. The reaction mixture was diluted with
water and extracted with EtOAc. The extract was washed
with brine, dried, and concentrated. The residue was recrys-
tallized from EtOAc-IPE to afford 1.5 g (64% yield) of 28: mp
112-115 °C; NMR (CDCl3) δ 1.62 (3H, s), 2.08 (3H, s), 2.12
(1.5H, s), 2.14 (1.5H, s), 2.16 (1.5H, s), 2.18 (1.5H, s), 3.00-
3.40 (4H, m), 6.78 (1H, m), 7.45-7.70 (5H, m), 7.96 (0.25H, d,
J ) 12.0 Hz), 7.99 (0.25H, d, J ) 12.0 Hz), 8.40 (0.25H, d, J )
1.4 Hz), 8.42 (0.25H, d, J ) 1.4 Hz).
N -[2,3-Dih yd r o-2,4,6,7-t e t r a m e t h yl-2-[(p h e n ylsu lfo-
n yl)m eth yl]-5-ben zofu r a n yl]for m a m id e (29). To a solu-
tion of 26a (2.1 g, 6.2 mmol) in MeOH (20 mL) was added 2 M
aqueous NaIO4 (20 mL), and the mixture was refluxed for 3
h. The reaction mixture was diluted with water and extracted
with EtOAc. The extract was washed with brine, dried, and
concentrated. The residue was recrystallized from EtOAc-
IPE to afford 1.4 g (66% yield) of 29: mp 154-155 °C; NMR
(CDCl3) δ 1.70 (1.5H, s), 1.71 (1.5H, s), 1.81 (1.5H, s), 1.84
(1.5H, s), 2.05 (1.5H, s), 2.07 (1.5H, s), 2.12 (1.5H, s), 2.14
(1.5H, s), 3.01 (1H, d, J ) 15.6 Hz), 3.56 (1H, s), 3.58 (1H, s),
3.62 (0.5H, d, J ) 15.6 Hz), 3.67 (0.5H, d, J ) 15.6 Hz), 6.71
(0.5H, br s), 6.74 (0.5H, d, J ) 12.0 Hz), 7.15-7.70 (3H, m),
7.89 (2H, m), 7.96 (0.5H, d, J ) 12.0 Hz), 8.40 (0.5H, d, J )
1.6 Hz).
N-[2,3-Dih yd r o-2-(h yd r oxym eth yl)-2,4,6,7-tetr a m eth yl-
5-ben zofu r a n yl]for m a m id e (32). To a solution of 6a (2.0
g, 7.7 mmol) in CH2Cl2 (20 mL) were added saturated aqueous
NaHCO3 (10 mL) and m-chloroperbenzoic acid (3.2 g, 19 mmol)
with ice-water bath cooling. After the reaction mixture was
stirred at room temperature for 1 h, the solvent was removed.
The residue was dissolved in a mixture of EtOAc and THF
(3:1). The resulting solution was washed with water, 10%
aqueous sodium hydrosulfite, aqueous NaHCO3, and brine,
dried, and concentrated. The residue was recrystallized from
EtOAc-IPE to afford 1.4 g (64% yield) of 32: mp 149-150
°C; NMR (DMSO-d6) δ 1.33 (3H, s), 1.97 (3H, s), 1.98 (3H, s),
2.00 (3H, s), 2.73 (1H, d, J ) 15.4 Hz), 3.13 (1H, d, J ) 15.4
Hz), 3.42 (2H, d, J ) 5.8 Hz), 5.01 (1H, t, J ) 5.8 Hz), 7.83
(0.2H, d, J ) 11.6 Hz), 8.21 (0.8H, d, J ) 1.2 Hz), 9.05 (0.2H,
d, J ) 11.6 Hz), 9.20 (0.8H, br s).
N-(2-F or m yl-2,3-d ih yd r o-2,4,6,7-tetr a m eth yl-5-ben zo-
fu r a n yl)for m a m id e (33). To a solution of oxalyl chloride
(0.40 mL, 4.2 mmol) in CH2Cl2 (10 mL) was added DMSO (1
mL) at -78 °C. After the mixture was stirred for 10 min, a
solution of 32 (1.0 g, 4.0 mmol) in CH2Cl2 (2 mL) was added
and stirring was continued for a further 15 min. To the
reaction mixture was added Et3N (3.5 mL), and the mixture
was allowed to warm to room temperature, washed with 1 M
With a similar procedure, 40 was prepared.
Gen er a l P r oced u r e for Con d en sa tion of Th iols a n d 24.
N-[2,3-Dih ydr o-2,4,6,7-tetr am eth yl-2-[(ph en ylth io)m eth yl]-
5-ben zofu r a n yl]for m a m id e (25a ). To a solution of 24 (6.0
g, 19 mmol) and thiophenol (2.3 g, 21 mmol) in DMF (50 mL)
was added NaH (1.0 g, 21 mmol; 60% in oil), and the mixture
was stirred under an argon atmosphere at 80 °C for 1 h. The
reaction mixture was diluted with water, and the aqueous
mixture was extracted with EtOAc. The extract was washed
with brine, dried, and concentrated. The residue was purified
by column chromatography (IPE:EtOAc ) 1:1) followed by
recrystallization from IPE-hexane to afford 5.5 g (83% yield)
of 25a : mp 130-131 °C; NMR (CDCl3) δ 1.55 (1.5H, s), 1.56
(1.5H, s), 2.00 (3H, s), 2.06 (1.5H, s), 2.09 (1.5H, s), 2.11 (1.5H,
s), 2.14 (1.5H, s), 2.91 (1H, d, J ) 15.8 Hz), 3.23 (0.5H, d, J )
15.8 Hz), 3.43 (0.5H, d, J ) 15.8 Hz), 3.27 (2H, s), 6.74 (0.5H,
br s), 6.84 (0.5H, d, J ) 12.0 Hz), 7.15-7.40 (5H, m), 7.97
(0.5H, d, J ) 12.0 Hz), 8.40 (0.5H, d, J ) 1.4 Hz).
With a similar procedure, 25b-j were prepared.
Gen er a l P r oced u r e for Con d en sa tion of Alcoh ols a n d
24. 2,3-Dih yd r o-2,4,6,7-tetr a m eth yl-2-[(p h en ylm eth oxy)-
m eth yl]-5-ben zofu r a n a m in e Hyd r och lor id e (26k ). To a
mixture of NaH (1.0 g, 25 mmol; 60% in oil) and benzyl alcohol
(20 mL) was added 24 (2.0 g, 6.4 mmol) with cooling. The
mixture was stirred under an argon atmosphere at 180 °C for
18 h in a sealed tube. The reaction mixture was diluted with
water, and the aqueous mixture was extracted with EtOAc.
The extract was washed with brine, dried, and concentrated.
The residue was purified by column chromatography (IPE),
and the product was treated with 4 M HCl/EtOH (8.0 mL).
The crude salt was recrystallized from EtOH-IPE to afford
0.68 g (31% yield) of 26k : mp 195-200 °C; NMR (DMSO-d6)
δ 1.40 (3H, s), 2.05 (3H, s), 2.22 (6H, s), 2.88 (1H, d, J ) 15.8
Hz), 3.17 (1H, d, J ) 15.8 Hz), 3.51 (2H, s), 4.56 (2H, s), 7.31
(5H, m), 9.71 (2H, br s).
With a similar procedure, 26l was prepared.
Gen er a l P r oced u r e for Con d en sa tion of Am in es a n d
24 (In th e Absen ce of Tr ieth yla m in e). 2,3-Dih yd r o-
2,4,6,7-tetr am eth yl-2-(1-piper idin ylm eth yl)-5-ben zofu r an -
a m in e (26m ). A mixture of 24 (2.0 g, 6.4 mmol) and
piperidine (6.3 mL, 64 mmol) was stirred under argon atmo-
sphere at 180 °C for 18 h in a sealed tube. The reaction
mixture was diluted with water, and the aqueous mixture was
extracted with EtOAc. The extract was washed with brine,
dried, and concentrated. The residue was recrystallized from
IPE to afford 1.5 g (82% yield) of 26m : mp 60-61 °C; NMR
(CDCl3) δ 1.30-1.60 (6H, m), 1.42 (3H, s), 2.07 (6H, s), 2.10
(3H, s), 2.35-2.65 (6H, m), 2.80 (1H, d, J ) 15.9 Hz), 3.10
(2H, br s), 3.11 (1H, d, J ) 15.9 Hz).
aqueous HCl and saturated aqueous NaHCO3
, dried, and
concentrated. The residue was recrystallized from EtOAc-
IPE to afford 0.68 g (69% yield) of 33: mp 154-155 °C; NMR
(CDCl3) δ 1.55 (1.5H, s), 1.57 (1.5H, s), 2.08 (3H, s), 2.12 (3H,
s), 2.15 (3H, s), 2.94 (1H, d, J ) 15.4 Hz), 3.41 (0.5H, d, J )
15.4 Hz), 3.44 (0.5H, d, J ) 15.4 Hz), 7.00 (1H, m), 7.95 (0.5H,
d, J ) 12.0 Hz), 8.34 (0.5H, d, J ) 1.8 Hz), 9.73 (0.5H, s), 9.74
(0.5H, s).
With a similar procedure, 26o-w were prepared.
Eth yl (E)-3-[5-(F or m yla m in o)-2,3-d ih yd r o-2,4,6,7-tet-
r a m eth yl-2-ben zofu r a n yl]p r op en oa te (34). A mixture of
33 (1.0 g, 4.1 mmol), triethyl phosphonoacetate (0.91 g, 4.1
mmol), and NaH (0.16 g, 4.1 mmol; 60% in oil) in DMF (20
mL) was stirred at room temperature for 1 h. The reaction
mixture was diluted with water, and the aqueous mixture was
extracted with EtOAc. The extract was washed with brine,
dried, and concentrated. The residue was purified by column
chromatography (EtOAc:IPE ) 1:1) to afford 0.50 g (39% yield)
2,3-Dih yd r o-2,4,6,7-t et r a m et h yl-2-[(4-p h en yl-1-p ip e-
r id in yl)m eth yl]-5-ben zofu r a n a m in e (26n ). A mixture of
27 (36 g, 0.13 mol; free base), 4-phenylpiperidine (41 g, 0.25
mol), and triethylamine (53 mL, 0.38 mol) was stirred under
an argon atmosphere at 180 °C for 15 h in a sealed tube. The
reaction mixture was diluted with saturated aqueous NaHCO3,
and the aqueous mixture was extracted with CHCl3. The
extract was washed with brine, dried, and concentrated. The
residue was purified by column chromatography (CHCl3:MeOH
) 97:3) followed by recrystallization from CHCl3-IPE to yield
38 g (82% yield) of 26n : mp 142-144 °C; NMR (CDCl3) δ 1.46
(3H, s), 1.70-1.82 (4H, m), 2.08 (6H, s), 2.11 (3H, s), 2.18-
2.34 (2H, m), 2.37-2.49 (1H, m), 2.52 (1H, d, J ) 13.4 Hz),
2.61 (1H, d, J ) 13.4 Hz), 2.83 (1H, d, J ) 15.4 Hz), 2.94-
3.06 (1H, m), 3.14 (1H, d, J ) 15.4 Hz), 3.16 (2H, br s), 3.18-
3.28 (1H, m), 7.15-7.35 (5H, m).
of 34 as an oil: NMR (CDCl3
) δ 1.29 (3H, t, J ) 7.2 Hz), 1.60
(3H, s), 2.06 (1.5H, s), 2.11 (1.5H, s), 2.13 (1.5H, s), 2.15 (1.5H,
s), 2.17 (3H, s), 3.05 (1H, d, J ) 15.4 Hz), 3.15 (1H, d, J )
15.4 Hz), 4.19 (2H, d, J ) 7.2 Hz), 6.02 (1H, d, J ) 15.6 Hz),
6.92 (0.5H, br s). 6.95 (0.5H, d, J ) 12.0 Hz), 7.02 (1H, d, J )
15.6 Hz), 7.95 (0.5H, d, J ) 12.0 Hz), 8.39 (0.5H, d, J ) 1.6
Hz).
N-[2,3-Dih yd r o-2,4,6,7-tetr a m eth yl-2-[(p h en ylsu lfin yl)-
m eth yl]-5-ben zofu r a n yl]for m a m id e (28). To a solution of
26a (2.3 g, 6.7 mmol) in MeOH (20 mL) was added 1 M
N-[2-[2-(4-F lu or op h en yl)eth yl]-2,3-d ih yd r o-2,4,6,7-tet-
r a m eth yl-5-ben zofu r a n yl]for m a m id e (37). To a suspen-
sion of [(4-fluorophenyl)methyl]triphenylphosphonium bromide

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570 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ohkawa et al.
(9.3 g, 23 mmol) in THF (100 mL) was added 1.6 M n-BuLi
(14 mL, 23 mmol; hexane solution) at -78 °C, and the mixture
was stirred for 15 min. To the mixture was added 33 (6.0 g,
23 mmol), and stirring was continued at room temperature
for 30 min. The reaction mixture was diluted with water, and
the aqueous mixture was extracted with EtOAc. The extract
was washed with brine, dried, and concentrated. The residue
(36, 4.2 g, 59% yield) was diluted with EtOH (50 mL), and 5%
Pd/C (0.2 g) was added. The mixture was stirred under a
hydrogen atmosphere at room temperature for 2 h. The
reaction mixture was filtered, and the filtrate was concen-
trated. The residue was diluted with EtOAc, and insoluble
material was removed by filtration. After concentration of the
filtrate, the residue was recrystallized from MeOH to yield 3.6
g (86% yield) of 37: mp 139-140 °C; NMR (CDCl3) δ 1.48
(1.5H, s), 1.50 (1.5H, s), 2.00 (2H, m), 2.10 (1.5H, s), 2.11 (1.5H,
s), 2.12 (4.5H, s), 2.17 (1.5H, s), 2.70 (2H, m), 2.91 (1H, d, J )
15.4 Hz), 3.05 (1H, d, J ) 15.4 Hz), 6.66 (0.5H, br s), 6.71 (0.5H,
s), 6.95 (2H, t, J ) 8.6 Hz), 7.13 (2H, dd, J ) 5.6, 8.6 Hz), 7.98
(0.5H, d, J ) 12.2 Hz), 8.42 (0.5H, d, J ) 1.6 Hz).
2,3-Dih yd r o-2,4,6,7-tetr a m eth yl-2-ben zofu r a n a ceton i-
tr ile (41). A mixture of 40 (6.5 g, 19 mmol) and sodium
cyanide (1.4 g, 19 mmol) in DMSO (30 mL) was stirred at 80
°C for 18 h. The reaction mixture was diluted with water, and
the aqueous mixture was extracted with EtOAc. The extract
was washed with brine, dried, and concentrated. The residue
was purified by column chromatography (hexane:IPE ) 2:1)
followed by recrystallization from MeOH to afford 4.1 g (80%
yield) of 41: mp 58-59 °C; NMR (CDCl3) δ 1.66 (3H, s), 2.07
(3H, s), 2.16 (3H, s), 2.20 (3H, s), 2.68 (1H, d, J ) 10.8 Hz),
2.75 (1H, d, J ) 10.8 Hz), 3.00 (1H, d, J ) 15.8 Hz), 3.12 (1H,
d, J ) 15.8 Hz), 6.54 (1H, s).
2,3-Dih yd r o-2,4,6,7-t et r a m et h yl-2-b en zofu r a n a cet ic
Acid (42). To a solution of 41 (6.9 g, 32 mmol) in MeOH (30
mL) was added 10 M aqueous NaOH (30 mL), and the mixture
was refluxed for 18 h. The reaction mixture was made acidic
with 6 M aqueous HCl and extracted with EtOAc. The extract
was washed with brine, dried, and concentrated. The residue
was recrystallized from EtOAc-hexane to afford 6.0 g (80%
yield) of 42: mp 139-140 °C; NMR (DMSO-d6) δ 1.61 (3H, s),
2.07 (3H, s), 2.16 (3H, s), 2.21 (3H, s), 2.78 (1H, d, J ) 10.8
Hz), 2.85 (1H, d, J ) 10.8 Hz), 2.97 (1H, d, J ) 15.4 Hz), 3.21
(1H, d, J ) 15.4 Hz), 6.52 (1H, s), 8.50 (1H, br s).
Gen er a l P r oced u r e for Con d en sa tion of Am in es a n d
42. 2,3-Dih yd r o-N,N,2,4,6,7-h exa m et h yl-2-b en zofu r a n -
a ceta m id e (43a ). To a solution of 42 (3.0 g, 13 mmol) in DMF
(30 mL) were added 1-hydroxybenzotriazole monohydrate (2.1
g, 14 mmol) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodi-
imide hydrochloride (3.7 g, 19 mmol), and the mixture was
stirred at room temperature for 1 h. To the mixture was added
50% aqueous dimethylamine (3 mL), and stirring was contin-
ued for a further 30 min. The reaction mixture was diluted
with water and extracted with EtOAc. The extract was
washed with brine, dried, and concentrated under reduced
pressure. The residue was purified by column chromatography
(IPE) to yield 3.1 g (93% yield) of 43a as an oil: NMR (CDCl3)
δ 1.59 (3H, s), 2.07 (3H, s), 2.14 (3H, s), 2.20 (3H, s), 2.77 (1H,
d, J ) 15.8 Hz), 2.88 (1H, d, J ) 15.8 Hz), 2.94 (3H, s), 3.00
(1H, d, J ) 15.8 Hz), 3.03 (3H, s), 3.27 (1H, d, J ) 15.8 Hz),
6.50 (1H, s).
With a similar procedure, 44b and 53a -c were prepared.
Gen er a l P r oced u r e for Syn th esis of 48. 2-Meth oxy-
3,4,6-tr im eth yl-r-(1-m eth yleth yl)-r-p h en ylben zen em eth -
a n ol (48a ). To a solution of 4715
(3.0 g, 13 mmol) in THF (20
mL) was added 1.6 M n-BuLi (8.2 mL, 13 mmol; hexane
solution) at -78 °C, and the mixture was stirred for 15 min.
To the mixture was added a solution of isobutyrophenone (1.9
g, 13 mmol) in THF (5 mL), and the mixture was stirred at
room temperature for an additional 30 min. The reaction
mixture was diluted with water and extracted with IPE. The
extract was washed with brine, dried, and concentrated. The
residue was crystallized from hexane to afford 3.1 g (80% yield)
of 48a . This product was used without further purification:
mp 80-81 °C; NMR (CDCl3) δ 0.88 (3H, d, J ) 6.6 Hz), 1.05
(3H, d, J ) 6.4 Hz), 2.07 (3H, s), 2.18 (3H, s), 2.58 (3H, s),
2.82 (1H, qq, J ) 6.4, 6.6 Hz), 2.90 (3H, s), 6.18 (1H, br s),
6.75 (1H, s), 7.10-7.30 (3H, m), 7.40-7.50 (2H, m).
With a similar procedure, 48b-h were prepared.
Gen er a l P r oced u r e for P r ep a r a t ion of 49 fr om 48.
2,3-Dih yd r o-2,2,4,6,7-p en t a m et h yl-3-p h en ylb en zofu r a n
(49a ). A suspension of 48a (3.1 g, 10 mmol) in 47% hydro-
bromic acid (20 mL) was refluxed under an argon atmosphere
for 18 h. After cooling, the reaction mixture was extracted
with IPE, and the extract was washed with brine, dried, and
concentrated. The residue was recrystallized from EtOH to
afford 2.4 g (88% yield) of 49a : mp 86-87 °C; NMR (CDCl3) δ
1.02 (3H, s), 1.51 (3H, s), 1.84 (3H, s), 2.15 (3H, s), 2.24 (3H,
s), 4.13 (1H, s), 6.49 (1H, s), 6.70-7.40 (5H, m).
With a similar procedure, 49b-h were prepared.
N-(2,3-Dih yd r o-2,2,4,6,7-p en ta m eth yl-5-ben zofu r a n yl)-
for m a m id e (52a ). A solution of 8a (1.0 g, 4.9 mmol; free base)
in formic acid (20 mL) was refluxed under an argon atmo-
sphere for 48 h. After cooling, the reaction mixture was
concentrated, and saturated aqueous NaHCO3 was added to
the residue. The aqueous mixture was extracted with CHCl3,
and the extract was washed with brine, dried, and concen-
trated. The residue was purified by column chromatography
(CHCl3:MeOH ) 97:3) to afford 1.1 g (93% yield) of 52a . An
analytical sample of 52a was recrystallized from CH2Cl2-
IPE: mp 177-179 °C; NMR (CDCl3) δ 1.46 (3H, s), 1.48 (3H,
s), 2.09-2.16 (9H, m), 2.94 (2H, s), 6.62-6.80 (1H, m), 7.97
(0.5H, d, J ) 12.0 Hz), 8.42 (0.5H, d, J ) 1.4 Hz).
Gen er a l P r oced u r e for P r ep a r a tion of N-Acyla ted
Com p ou n d s Usin g Acyl Ha lid e. N-(2,3-Dih yd r o-2,2,4,6,7-
p en ta m eth yl-5-ben zofu r a n yl)a ceta m id e (52b). To a solu-
tion of 8a (1.0 g, 4.9 mmol; free base) and Et3N (0.64 g, 6.3
mmol) in THF (20 mL) was added acetyl chloride (0.46 g, 5.8
mmol) with cooling. The mixture was stirred at room tem-
perature for 4 h and then poured into water. The aqueous
mixture was extracted with CHCl3, and the extract was
washed with saturated aqueous NaHCO3 and brine, dried, and
concentrated. The residue was purified by column chroma-
tography (CHCl3:MeOH ) 97:3) followed by recrystallization
from CH2Cl2-IPE to give 0.92 g (76% yield) of 52b: mp 190-
191 °C; NMR (CDCl3) δ 1.46 (3H, s), 1.50 (3H, s), 1.73 (1.5H,
s), 2.06 (3H, s), 2.09 (3H, s), 2.14 (3H, s), 2.21 (1.5H, s), 2.93
(2H, s), 6.58 (0.5H, br s), 6.63 (0.5H, br s).
With a similar procedure, 52c,d were prepared. This
method was utilized in sulfonylation of 8a for the synthesis of
54a .
N-(2,3-Dih yd r o-2,2,4,6,7-p en ta m eth yl-5-ben zofu r a n yl)-
4-m eth ylben zen esu lfon a m id e (54b). A solution of 8a (2.0
g, 9.7 mmol; free base) and p-toluenesulfonyl chloride (2.0 g,
11 mmol) in pyridine (30 mL) was stirred at 50 °C for 1 h.
The reaction mixture was concentrated, and the residue was
diluted with CHCl3. The organic solution was washed with 1
M aqueous HCl and brine, dried, and concentrated. The
residue was purified by column chromatography (hexane:
EtOAc ) 97:3) followed by recrystallization from CH2Cl2-IPE
to afford 2.4 g (69% yield) of 54b: mp 219-220 °C; NMR
(CDCl3) δ 1.46 (6H, s), 1.80 (3H, s), 1.93 (3H, s), 2.01 (3H, s),
2.43 (3H, s), 2.87 (2H, s), 5.81 (1H, s), 7.24 (2H, d, J ) 8.4
Hz), 7.60 (2H, d, J ) 8.4 Hz).
With a similar procedure, 43b was prepared.
Gen er a l P r oced u r e for Red u ction of Am id es. 2,3-
D ih y d r o -N ,N ,2,4,6,7-h e x a m e t h y l-2-b e n zo fu r a n e t h a -
n a m in e (44a ). To a solution of 43a (3.1 g, 12 mmol) in THF
(50 mL) was gradually added lithium aluminum hydride (0.45
g, 12 mmol) with cooling. After stirring at room temperature
for 30 min, the mixture was poured into ice-water. The
aqueous mixture was extracted with EtOAc, and the extract
was washed with brine and dried. The organic solution was
concentrated, and the residue was purified by column chro-
matography (CHCl3:MeOH ) 95:5) to give 2.2 g (82% yield) of
44a as an oily product: NMR (CDCl3) δ 1.42 (3H, s), 1.90 (2H,
m), 2.06 (3H, s), 2.12 (3H, s), 2.19 (3H, s), 2.23 (6H, s), 2.40
(2H, m), 2.82 (1H, d, J ) 15.4 Hz), 3.00 (1H, d, J ) 15.4 Hz),
6.47 (1H, s).
Gen er a l P r oced u r e for P r ep a r a tion of Qu in on im in es
56a -c. 4-[(4-Ch lor op h en yl)im in o]-2,3,5-tr im eth yl-6-(2-
m eth yl-2-p r op en yl)-2,5-cycloh exa d ien -1-on e (56c). To a

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5-Aminocoumarans for Treating CNS Injury
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 571
solution of pyridine (7.1 mL, 88 mmol) in 1,2-dichloroethane
(40 mL) was added TiCl4 (2.4 mL, 22 mmol) with ice-water
bath cooling, and the mixture was refluxed under an argon
atmosphere for 20 min. After the mixture was cooled in an
ice-water bath, a solution of 5523 (3.0 g, 15 mmol) and
p-chloroaniline (5.6 g, 44 mmol) in 1,2-dichloroethane (20 mL)
was added and the resulting mixture was stirred at 90 °C for
45 min. The reaction mixture was filtered through Celite, and
the filtrate was washed with brine, dried, and concentrated.
The residue was purified by column chromatography (hexane:
EtOAc ) 93:7) to afford 4.3 g (96% yield) of 56c as an oil: NMR
(CDCl3) δ 1.53-2.20 (12H, m), 3.21 (2H, s), 4.51 (1H, s), 4.74
(1H, s), 6.68 (2H, d, J ) 8.8 Hz), 7.30 (2H, d, J ) 8.8 Hz).
Compounds 56a ,b were prepared by a similar procedure.
Gen er a l P r oced u r e for R ed u ct ion of Qu in on im in es
56a -c. 4-[(4-Ch lor op h en yl)a m in o]-2,3,5-tr im eth yl-6-(2-
m eth yl-2-p r op en yl)p h en ol (57c). To a solution of 56c (4.4
g, 14 mmol) in THF (20 mL) was added 2.8 M aqueous sodium
hydrosulfite (50 mL), and the mixture was stirred at room
temperature for 30 min. The organic layer was separated, and
the aqueous layer was extracted with EtOAc. The combined
organics were washed with brine, dried, and concentrated. The
residue was purified by column chromatography (hexane:
EtOAc ) 95:5) to afford 4.3 g (97% yield) of 57c as an oil: NMR
(CDCl3) δ 1.80 (3H, s), 2.11 (3H, s), 2.12 (3H, s), 2.19 (3H, s),
3.40 (2H, s), 4.68 (1H, s), 4.87 (1H, s), 5.04 (1H, s), 5.14 (1H,
br s), 6.34 (2H, d, J ) 8.8 Hz), 7.06 (2H, d, J ) 8.8 Hz).
With a similar procedure, 57a ,b were prepared.
4-[(2,3-Dih yd r o-2,2-d im et h yl-5-b en zofu r a n yl)a m in o]-
2,3,5-tr im eth yl-6-(2-m eth yl-2-p r op en yl)p h en ol (57d ). To
a solution of pyridine (7.1 mL, 88 mmol) in 1,2-dichloroethane
(40 mL) was added TiCl4 (2.4 mL, 22 mmol) with ice-water
bath cooling, and the mixture was stirred under an argon
atmosphere at 90 °C for 30 min. After the reaction mixture
was cooled, a solution of 55 (3.0 g, 15 mmol) in 1,2-dichloro-
ethane (5 mL) and a solution of 8e (12 g, 74 mmol; free base)
in 1,2-dichloroethane (20 mL) were added to the reaction
mixture. The resulting mixture was stirred under an argon
atmosphere at 90 °C for 30 min. The reaction mixture was
allowed to cool to room temperature, then Celite and CHCl3
were added, and the resulting mixture was filtered through
Celite. The filtrate was washed with brine, dried, and
concentrated. The residue was purified by column chroma-
tography (hexane:EtOAc ) 9:1) followed by recrystallization
from IPE-pentane to give 2.3 g (45% yield) of 57d : mp 160-
162 °C; NMR (CDCl3) δ 1.44 (6H, s), 1.80 (3H, s), 2.15 (6H, s),
2.19 (3H, s), 2.90 (2H, s), 3.41 (2H, s), 4.65-5.00 (4H, m), 6.18-
6.27 (2H, m), 6.53 (1H, d, J ) 8.4 Hz).
Op tica l Resolu tion of 26n . To a solution of 26n (35 g, 97
mmol) in CHCl3 (500 mL) was added a solution of (S)-(+)-
mandelic acid (15 g, 97 mmol) in MeOH (300 mL), and the
mixture was concentrated. To the residue was added Et2O
(500 mL), and the precipitates formed were collected by
filtration, washed with Et2O, and dried to afford 35 g of crude
salt. The crude salt was recrystallized as follows: The salt
was dissolved in MeOH, the solution was concentrated to a
volume of about 100 mL, and crystals formed. To the mixture
was added Et2O (500 mL), and the crystals were collected by
filtration, washed with Et2O, and dried (22 g). This solid was
recrystallized once again from the same solvent system to
afford 20 g (40% yield) of diastereomeric salt: [R]D +57.1° (c
1.23, MeOH); mp 186-190 °C. X-ray crystallographic analysis
showed this salt to have the S-configuration after recrystal-
lization from 2-propanol. (S)-Mandelate of (S)-26n (20 g, 39
mmol) was dissolved with EtOAc-0.5 M aqueous NaOH. The
organic phase was separated, washed with 0.5 M aqueous
NaOH, saturated aqueous NaHCO3, and brine, and then dried
over anhydrous Na2CO3 and concentrated. The residue was
dissolved in MeOH (140 mL), and 4 M HCl/EtOAc (23 mL)
was added to the solution. After the solution was concen-
trated, the residue was crystallized from EtOAc, and the crude
solid was recrystallized from MeOH-EtOAc to afford 14 g
(89% yield) of (S)-26n dihydrochloride: [R]D +27.8° (c 1.05,
MeOH); mp 226 °C dec.
dissolved with EtOAc-0.5 M aqueous NaOH. The organic
phase was separated, washed with 0.5 M aqueous NaOH,
saturated aqueous NaHCO3, and brine, dried over anhydrous
Na2CO3, and concentrated. From the residue (20 g) and (R)-
(-)-mandelic acid (8.4 g), (R)-26n (R)-mandelate (20 g, 40%
yield) was obtained in the same manner as for the preparation
of (S)-26n (S)-mandelate: [R]D -57.0° (c 1.09, MeOH); mp
186-191 °C. (R)-26n dihydrochloride was obtained after
recrystallization from MeOH-EtOAc of the crude dihydro-
chloride, which was prepared from (R)-26n (R)-mandelate in
the same manner as for the preparation of (S)-26n dihydro-
chloride (92% yield): [R]D -27.9° (c 1.28, MeOH); mp 226 °C
dec.
Lip id P er oxid a tion In h ibition . To rat liver microsomes
(S-9) (0.3 mg of protein/40 mM Tris-malate buffer (pH 7.4),
2.4 mL) (2.4 mL) was added a mixture (1:1; 0.1 mL) of aqueous
FeCl2 solution (0.25 mM) and NADPH (3 mM). After incuba-
tion of the homogenate for 1 h at 37 °C, peroxide production
was determined by the thiobarbituric acid method.25 Before
incubation, test compounds dissolved in DMSO were added
to the rat liver microsomes so that their final concentration
became 10-6 M. The inhibitory activities on lipid peroxidation
are expressed as IC50 values or percent inhibition as compared
with the amount of production in the vehicle (DMSO) group.
Mou se-F eCl2-it Assa y. Male Slc:ICR mice (5 weeks) were
used. Each group consisted of 10 mice; 5 µL of 50 mM FeCl2
in saline was injected into the spinal subarchnoid space
between the 1st sacral and the 6th lumbar segments. The
behavioral responses were observed from 15 min to 1 h after
intrathecal infection of FeCl2 and scored as follows:
score
behavioral responses
0
1
normal (no abnormal behavior)
vigorously biting the lower abdomen or
lower extremities
2
(a) vigorously biting the lower body with
rolling, (b) hyperreactivity and aggressive
response to external stimuli, (c) tremor,
(at least one of the above three behavioral
changes were observed)
3
4
5
clonic convulsion
tonic convulsion or paralysis of lower extremities
death
Water-soluble test compounds were dissolved in distilled
water, and water-insoluble ones were suspended in a 0.5% gum
arabic solution. Test compounds were orally administered 30
min prior to FeCl2 injection in a volume of 0.2 mL/10 g. The
inhibitory activities on mouse-FeCl2-it assay are expressed as
ID50 values as compared with the score in the vehicle (saline)
group.
Su p p r ession of MAP -In d u ced Hyp er m otility in Mice.
Five-week old male ICR mice (25-35 g) were used. Following
a 90-min acclimation period, the test compound suspended in
5% gum arabic was injected intraperitoneally in a volume of
20 mL/kg. Thirty minutes after treatment with test com-
pound, methamphetamine dissolved in saline was injected
intraperitoneally at a dose of 1 mg/kg in a volume of 20 mL/
kg. Immediately after methamphetamine injection, spontane-
ous motor activity was monitored for 90 min using Animex
Auto. In another experiment, subcutaneous apomorphine at
a dose of 1 mg/kg was used as an inducer of hypermotility
instead of methamphetamine. Two-tailed Student’s t-test was
used for statistical analysis.
Tr a n sien t Cer ebr a l Isch em ia (F ou r -Vessel Occlu sion )
Mod el in Ra ts. The surgical procedure to induce cerebral
ischemia in rats was almost identical with that reported by
Pulsinelli and Brierly.34 Eight-week old male Wistar rats in
which the vertebral arteries had been cauterized bilaterally
with a bipolar electrocoagulator and common carotid arteries
had been wrapped with thread bilaterally on the previous day
were used. Under light halothane anesthesia, the common
carotid arteries were occluded bilaterally with clips for 45 min.
Drugs were given intraperitoneally immediately and 2 and 24
h after reopening the circulation. Survival of the animals was
monitored for 1, 3, 5, 7, 10, and 14 days after reopening the
The combined filtrate obtained in preparation of (S)-26n (S)-
mandelate was concentrated, and the residue (28 g) was

+
+
572 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ohkawa et al.
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Ack n ow led gm en t. We thank Dr. Shinji Terao and
Dr. Naohisa Fukuda for their helpful discussions and
Mr. Akira Fujishima and Ms. Keiko Higashikawa for
X-ray crystallographic analysis. We also thank Dr.
Shin-ichi Yoshikubo for statistical analysis and Mr.
Hisao Nishikawa for technical assistance.
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graphic data of (S)-mandelate of (S)-26n (7 pages). Ordering
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