A novel anti-ischemic ATP-sensitive potassium channel (KATP) opener without vasorelaxation: N-(6-aminobenzopyranyl)-N′-benzyl- N″-cyanoguanidine analogue

Article abstract of DOI:10.1021/jm010183f

This paper describes the design, synthesis, and biological evaluation of a novel anti-ischemic compound, (2S,3S,4R)-N-(6-amino-3,4-dihydro-2-dimethoxymethyl-3-h ydroxy-2-methyl-2H-benzopyranyl)-N′-benzyl-N″-cyanoguanidine (33), and the structure-activity

Full text of DOI:10.1021/jm010183f

J . Med. Chem. 2001, 44, 4207-4215
4207
A Novel An ti-Isch em ic ATP -Sen sitive P ota ssiu m Ch a n n el (KATP ) Op en er w ith ou t
Va sor ela xa tion : N-(6-Am in oben zop yr a n yl)-N′-ben zyl-N′′-cya n ogu a n id in e
An a logu e
Sung-eun Yoo,*,† Kyu Yang Yi,† Sunkyung Lee,† J eehee Suh,† Nakjeong Kim,† Byung Ho Lee,‡ Ho Won Seo,‡
Sun-Ok Kim,§ Dong-Ha Lee,§ Hong Lim,§ and Hwa Sup Shin
Bioorganic Division and Screening and Toxicology Research Center, Korea Research Institute of Chemical Technology,
Taejon 305-600, Korea, AgroPharma Research Institute of Dongbu-Hannong Chemical Co., Teajon 305-380, Korea, and
Division of Life Science, College of Natural Sciences, Konkuk University, Chungcheoungbuk-do 380-701, Korea
Received April 23, 2001
This paper describes the design, synthesis, and biological evaluation of a novel anti-ischemic
compound, (2S,3S,4R)-N-(6-amino-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-ben-
zopyranyl)-N′-benzyl-N′′-cyanoguanidine (33), and the structure-activity relationships leading
to the discovery of this compound. Compound 33 significantly reduced the myocardial infarct
zone to area at risk (IZ/AAR) in the ischemic myocardium rat model with high cardioselectivity.
Since the cardioprotective effect of compound 33 is reversed by ATP-sensitive potassium channel
(KATP) blockers, its anti-ischemic effect appears to be at least mediated by KATP opening. In
addition, compound 33 shows good protective activity on neuronal cells against oxidative stress,
and therefore it is suggested that compound 33 may have therapeutic potential both in cardio-
and in neuroprotection.
In tr od u ction
relationships,14-18 substantial efforts have been devoted
to find safe, cardioselective agents. In such an attempt,
scientists at Bristol-Myers Squibb discovered BMS-
180448 (1)19 and BMS-19109517 with enhanced selectiv-
ity toward the ischemic myocardium over vasorelax-
ation, which improved a narrow therapeutic window of
the first-generation KATP openers (e.g., cromakalim).20
Despite these efforts, more cardioselective compounds
are needed. Thus, we set out to find agents having
protective effects on both brain and cardiac ischemia
without vasorelaxant activity.
Ischemic cell injury may arise from complex interac-
tions which include disturbances in energy metabolism1
and modifications in synaptic transmission.2 Several
studies have shown that the membrane depolarization
is a trigger signal for the induction of neuronal hyper-
excitability, irreversible cellular dysfunction, and cell
death, which is mainly mediated by activation of the
glutamate receptor.3-5 Like brain ischemia, myocardial
ischemia initiates various cellular changes progres-
sively, ultimately leading to irreversible myocardial
injury, cell death, and tissue necrosis.6 Since complex
mechanisms are involved in the cellular changes caused
by ischemia, several strategies for protection against
ischemic injury have been proposed,7 including oxygen
free radical scavengers, adenosine agonists, calcium
channel blockers, and ATP-sensitive potassium channel
(KATP) openers.8
KATP openers9 have shown protective properties on
ischemia-reperfusion injury both in heart and brain,
presumably through “ischemic preconditioning”, an en-
dogenous protective mechanism.10,11 Certain similari-
ties12 for preconditioning between heart and brain
mediated by KATP channel have been reported,13 raising
the possibility of KATP openers having therapeutic
potential as cardio and neuroprotective agents. Since
the cardioprotective and vasorelaxant potencies of KATP
openers follow distinct and separable structure-activity
Previously, we discovered SKP-450 (2),21-23 a new
K-channel activator as a potential anti-hypertensive
drug. Structurally SKP-450 is different from croma-
kalim in a number of aspects, including an acetal moiety
at the 2-position which imparted a new chiral center
and resulted in improvement of efficacy and pharma-
cokinetic parameters as well.24,25 SKP-450 was orally
well absorbed in rats, of which bioavailablity calculated
on the basis of radioactivity was 68-97%.24 Since then,
we have turned our attention to discovering novel
compounds with protective properties for ischemic
diseases based on the KATP activation mechanism. For
this purpose, we have designed various chemical struc-
tures based upon the benzopyran scaffold having an
acetal moiety at the 2-position as in SKP-450. Onto this
backbone, we introduced a cyanoguanidine portion at
* To whom correspondence should be addressed: Bioorganic Div.,
Korea Research Institute of Chemical Technology, 100 J ang-dong,
Yoosung-gu, Taejon 305-600, Korea. Tel: 82-42-860-7140. Fax: 82-
42-861-1291. E-mail: seyoo@krict.re.kr.
† Bioorganic Division, Korea Research Institute of Chemical Tech-
nology.
‡ Screening and Toxicology Research Center, Korea Research In-
stitute of Chemical Technology.
§ Dongbu-Hannong Chemical Co.
Konkuk University.
10.1021/jm010183f CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/16/2001

4208 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Yoo et al.
Sch em e 1a
a
Reagents and conditions: (a) pyruvic aldehyde dimethylacetal, pyrrolidine, toluene, reflux; (b) DIBAL-H, toluene, -78 °C; (c) (R)-
(-)-R-methoxyphenylacetic acid, DCC, DMAP, EtOAc; (d) (1) separation by recrystallization (Et2O-hexane), (2) LiOH, MeOH-H2O, 0
°C; (e) (1) CH3SO2Cl, i-Pr2NEt, CH2Cl2; (2) DBU, toluene, reflux; (f) Mn(III)-Salen, aq NaOCl, CH2Cl2; (g) aq NH3, EtOH, 50 °C.
the 4-position, which seemed to be a crucial moiety for
cardioprotective activity. Then we identified a structur-
ally distinct analogue, (2S,3S,4R)-N-(6-amino-3,4-dihy-
dro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-benzo-
pyranyl)-N′-benzyl-N′′-cyanoguanidine (33), which has
both neuroprotective and cardioprotective effects with
negligible vasorelaxant activity. In this paper, we
describe the anti-ischemic and anti-oxidant effects of
this series of compounds and discuss the structure-
activity relationships leading to compound 33 (KR-
31378)
first in 28% yield, followed by the other stereoisomer
(2R,4R) 6 in 15% yield. The absolute stereochemistry
of the compounds 5 and 6 was determined by X-ray
crystallographic analysis.
Alkaline hydrolysis of compounds 5 and 6 using LiOH
gave the corresponding alcohols 7 and 8, which were
readily converted to the mesylated derivatives (CH3SO2-
Cl, i-Pr2NEt, CH2Cl2). The elimination of C4-mesylate
by heating with 1,8-diazabicyclo[5.4.0]undec-7ene (DBU)
provided the olefin compounds 9 (2S) and 10 (2R). The
epoxidation of 9 and 10 with J acobsen’s reagent [(S,S)-
Mn(III)-salen complex] and NaOCl afforded two opti-
cally active epoxides 11 (2S,3S,4S) and 13 (2R,3S,4S)
in 83% and 85% yield, respectively.26 Similarly, the
other two chiral epoxides 12 (2S,3R,4R) and 14 (2R,3R,-
4R) were obtained from 9 and 10 using (R,R)-J acobsen’s
catalyst. Treatment of the four stereoisomeric epoxides
with aqueous ammonia in ethanol at 50 °C provided the
optically pure amino alcohols 15-18 (Scheme 1).
The cyanoguanidine derivatives with N-aryl groups
19-22 were prepared by the treatment of amino alco-
hols 15 or 17 with N-cyano-N-aryl-thioureas,27 prepared
from the corresponding isothiocyanates and preformed
monosodium cyanamide using a water soluble coupling
reagent (WSC), 1-[3-(dimethylamino)propyl]-3-ethylcar-
bodiimide hydrochloride (EDCI) in DMF (Scheme 2).
Similarly, the N-benzyl cyanoguanidine derivatives
27-32 were prepared with the amino alcohols 15-18
and diphenylcyanocarbonimidate to give intermediates
23-26, which were subsequently reacted with appropri-
ate benzylamines (Scheme 2).28
Reduction of the 6-nitro group was carried out either
by catalytic hydrogenation or by sodium borohydride-
copper(II) acetate in aqueous methanol to provide the
corresponding 6-amino compounds 33-37 (Scheme 3).
Ch em istr y
The stereochemistry of benzopyrans has been re-
ported to be critical for selectivity and cardioprotective
potency.17,18 Therefore, we prepared optically pure ben-
zopyranyl cyanoguanidine analogues via four stereo-
isomers of amino alcohols 15-18 as described in Schemes
1-4. First, the reaction of 6-hydroxy-3-nitroacetophe-
none with pyruvic aldehyde dimethylacetal in the pres-
ence of pyrrolidine provided the cyclized 4-chromanone
compound 3 in good yield. Next, we converted the keto
group to a hydroxyl group which will serve as a handle
for attaching an optically active auxiliary. The reduction
of chromanone 3 was examined with various reducing
agents to obtain preferentially one diastereoisomer
which would make a subsequent resolution step much
easier and effective. We found that the reduction with
diisobutylaluminum hydride (DIBAL-H) at -78 °C in
toluene was optimal to give a cis diastereoisomer
between the C4-hydroxy and C2-acetal groups as a
major product (cis:trans ) 7:1 ratio) in 96% yield. The
cis isomer of 4 was esterified with (R)-(-)-R-methoxy-
phenylacetic acid using 1,3-dicyclohexylcarbodiimide
(DCC). One stereoisomer (2S,4S) 5 was crystallized out

Anti-Ischemic ATP-Sensitive KATP Opener
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4209
Sch em e 2
Sch em e 3a
a
Reagents and conditions: (a) H2, 10% Pd/C, MeOH or NaBH4, Cu(OAc)2, MeOH-H2O; (b) for 38, Ac2O, Et3N, 4-DMAP, CH2Cl2; (c)
for 39, MeSO2Cl, Et3N, CH2Cl2; (d) for 40, PhNCO, CH2Cl2.
Sch em e 4
The amine 33 was elaborated to the acetamide 38,
sulfonamide 39, and urea 40 using straightforward
methods (Scheme 3).
To determine the pharmacological and pharmaco-
kinetical effects on acetal analogues at C2, cyclic acetal
compounds 41 and 42 were prepared by transacetaliza-
tions of olefin 10 with appropriate diols in the presence
of catalytic amount of p-toluenesulfonic acid (p-TsOH)
followed by the same procedures described for 33
(Scheme 4).
Resu lt a n d Discu ssion
We employed functional tests to determine cardio-
protective, neuroprotective, and vasorelaxant potencies
of new compounds: Vasorelaxant potencies were deter-
mined by measuring IC50 values for relaxation of the
methoxamine contracted rat aorta.15 Cardioprotective
effects were determined by measuring a ratio of myo-
cardial infarction zone to area at risk (IZ/AAR),29 using
an ischemic myocardium damage rat model. Neuropro-
tective effects of compounds against the iron-induced

4210 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Yoo et al.
Ta ble 1. Vasorelaxant Potencies and Cardio- and Neuroprotective Effects of Benzopyranyl Cyanoguanidine Analogues
a
b
Q ) CH(OCH3)2 for compounds 19-40. Vasorelaxant potency was assessed by measurement of IC50 for inhibition of methoxamine-
contracted rat aorta. IC50 value is presented as a mean with 95% confidence interval in parentheses, n ) 3. If IC50 is not available,
inhibition percentage at 30 µM (except 33) was given. Each value is an average of three determinations and within (10%. c Inhibition
d
percentage at 300 µM. Cardioprotective effect was determined by measuring a ratio of myocardial infarct zone to area at risk (IZ/AAR)
in ischemic myocardium damage rat model, n ) 3 or higher. Each value given is an average and within (10%. e Neuroprotective effect
was evaluated by the reduction rate of LDH release from neuronal cells injured by iron at 30 µM. Each value is an average of four
determinations and within (10%. IC50 value was determined on the compounds showing >70% reduction rate of LDH at 30 µM and
presented as a mean of 3 experiments (n ) 4 for each experiment) with 95% confidence interval in parentheses. f Not determined.
injury of neuronal cells were evaluated by the reduction
of lactate dehydrogenase (LDH) release into the me-
dium,30 primarily at 30 µM concentration. The com-
pounds showing greater than 70% neuroprotection at
30 µM were subjected to concentration-response studies
to determine IC50 values.
Initially, we synthesized a series of N-phenylcyano-
guanidine analogues (19-22) with the same (3S,4R)-
stereochemistry as BMS-18044815 and evaluated their
biological activities. Compounds 20 and 22 showed
comparable cardioprotective effects to BMS-180448 but
with 10 times weaker vasorelaxation potencies (Table

Anti-Ischemic ATP-Sensitive KATP Opener
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4211
Ta ble 2. Dose Dependency of Cardioprotective Effect and the
Effects of KATP Blockers
1), indicating better cardioselectivity. Compound 21
with (2S,3S,4R)-stereochemistry showed weaker voso-
relaxation effect than the stereoisomer 19 with (2R,3S,4R)-
stereochemistry (74% vs 18% at 30 µM), with somewhat
improved anti-ischemic potency. Thus, the stereochem-
istry at C2 seems to play a contribution to the pharma-
cological profile.
N-Arylcyanoguanidine analogues have been studied
extensively for cardioselectivity,17 but not with N-
benzylcyanoguanidines. Thus, we prepared a number
of optically pure N-benzylcyanoguanidinyl derivatives
(27-32). Replacement of the phenyl ring with a benzyl
group did not significantly change the cardioprotective
effects (Table 1), and likely the N-phenylcyanoguanidine
derivatives, compound 27 with (2S,3S,4R)-stereochem-
istry, showed the lowest vasorelaxant activity among
four stereoisomers, although these stereoisomers showed
only marginal neuroprotective activity.
Further work was concentrated on the modification
at the 6-position of the compound 27, since the modifi-
cation at this position was reported to have significant
effects on the pharmacological profiles.31 Compound 33
with an amino group at C6 showed significantly im-
proved anti-ischemic activity with much improved car-
dioselectivity (Table 1). While its anti-ischemic effect
(36.6%, IZ/AAR) was comparable with that of BMS-
180448 (39.1%, IZ/AAR), its vasorelaxant effect was
dramatically reduced (negligible vasorelaxation even at
300 µM). It was rather an unexpected result to us
because it has been reported that an electron withdraw-
ing group at C6 of the benzopyran ring seems to be
essential for the activity of KATP openers.15 Furthermore,
compound 33 showed a potent neuroprotective effect
(IC50, 2.2 µM) as well. Further modifications of amino
group at C6 to amide 38, sulfonamide 39, and urea 40
analogues, resulted in the decrease of both cardio- and
neuroprotective effects and selectivity, suggesting that
the amino group is optimal for cardioprotective and
neuroprotective potency.
With these results, we synthesized three other ste-
reoisomers of compound 33 and evaluated their biologi-
cal properties as shown in Table 1. The isomers with
(3S,4R)-stereochemistry showed consistently better car-
dioprotective activity than the isomers with (3R,4S)-
stereochemistry as in the case of BMS-180448. Among
four stereoisomers, compound 33 was confirmed to have
the best cardio- and neuroprotective properties.
Additionally we have modified the acetal moiety at
C2 of compound 33 to examine the possibility of further
improvement for pharmacological and particularly for
physicochemical properties. But, as seen in Table 1,
those modifications resulted in a loss of cardioprotective
effects while their neuroprotective effects were retained,
suggesting that structure-activity relationships for
cardioprotective effects and neuroprotective effects may
be different.
cardioprotection,a
IZ/AAR (%)/AAR/LV (%)
0.1
0.3
1
mg/kg (n)
mg/kg (n)
mg/kg (n)
vehicle
BMS-180448
33
33 + Gliben (1 mg/kg)
33 + 5-HD (10 mg/kg)
61/40 (6)
39/39 (5)
37/35 (7)
ndb
50/41 (5)
42/34 (5)
ndb
32/37 (4)
34/35 (7)
51/33 (3)
46/35 (3)
ndb
ndb
a
Cardioprotective effect was determined by measuring a ratio
of myocardial infarct zone to area at risk (IZ/AAR) in ischemic
myocardium damage rat model with or without a KATP blocker
(glibenclamide (Gliben) or sodium 5-hydroxydecanoate (5-HD)).
b
Each value is given as an average and within (10%. Not
determined.
However, the neuroprotective effect of compound 33
against FeSO4 toxicity was not reduced significantly by
glibenclamide (data not shown), suggesting that its
neuroprotective mechanism is not related to the activa-
tion of the KATP channel, as has been also demonstrated
with other KATP openers such as diazoxide and levcro-
makalim.32 Further studies will be needed to elucidate
its mechanism of action for neuroprotection as well as
cardioprotection. However, since ischemic cascades
proceed by complex interactions, it may be a useful
strategy to develop the compound acting at more than
one target site in ischemic cascade.
Con clu sion
We discovered a novel cardioprotective agent, (2S,3S,-
4R)-N-(6-amino-3,4-dihydro-2-dimethoxymethyl-3-hy-
droxy-2-methyl-2H-benzopyranyl)-N′-benzyl-N′′-cyano-
guanidine (33), which protected ischemic rat heart in a
dose-dependent manner by the mechanism of cardiac
KATP channel activation. Anti-ischemic effect of com-
pound 33 on ischemic rat heart was comparable with
that of BMS-180448, but with much improved cardi-
oselectivity than BMS-180448. In addition to the car-
dioprotective effect, compound 33 showed good neuro-
protective effect against oxidative stress. These activities
together make compound 33 (KR-31378) a new effective
protecting agent for brain and cardiac ischemia.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a capillary
melting point apparatus and are uncorrected. Anhydrous
solvents were dried by conventional methods. Reagents of
commercial quality were used from freshly opened containers
1
unless otherwise stated. H NMR spectra were recorded on a
Bruker AM-500 (500 MHz) or a Bruker AM-300 (300 MHz)
with TMS as an internal standard. Chemical shifts are
reported in δ (ppm) downfield from TMS. Mass spectra were
obtained with a J EOL J MS-DX 303 instrument by using
electron impact or chemical ionization techniques. Elemental
analyses were performed by Korea Research Institute of
Chemical Technology’s Analytical Department and C, H, and
N values are within (0.4% of the calculated values.
With compound 33 in hand, we carried out experi-
ments to determine whether the cardioprotective effect
of compound 33 was due to KATP activation using known
KATP blockers, glibenclamide and sodium 5-hydroxyde-
canoate (5-HD). As shown in Table 2, both glibenclamide
and 5-HD significantly blocked the cardioprotective
effect of compound 33, which suggests that at least the
anti-ischemic effect is mediated via KATP opening.
3,4-Dih yd r o-2-d im et h oxym et h yl-2-m et h yl-6-n it r o-4-
oxo-2H-1-ben zop yr a n (3). To a solution containing a mixture
of 3- and 5-nito-6-hydroxyphenone (200 g, 1.10 mol; prepared
by nitration of 2-hydroxyacetophenone and used without
purification) in toluene (800 mL) were added pyruvic aldehyde
dimethylacetal (147 mL, 1.21 mol) and pyrrolidine (22.5 mL,
0.27 mol). The mixture was heated at reflux for 4 h using
Dean-Stark apparatus and cooled to room temperature, then
silica gel (100 g) was added to the reaction, which was stirred

4212 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Yoo et al.
for 10 min and filtered through a short silica gel column. The
filtrate was concentrated in vacuo, and the residue was
recrystallized from ethyl acetate-hexane to give 3 (120 g, 39%
based on 2-hydroxyacetophenone) as a yellow solid: mp 108
°C; 1H NMR (CDCl3) δ 1.46 (s, 3H), 2.70 (d, J ) 16.7 Hz, 1H),
3.10 (d, J ) 16.9 Hz, 1H), 3.44 (s, 3H), 3.52 (s, 3H), 4.31 (s,
1H), 7.07 (d, J ) 9.2 Hz, 1H), 8.31 (dd, J ) 9.4, 2.8 Hz, 1H),
8.71 (d, J ) 2.8 Hz, 1H); HRMS (M+) 281.0899 calcd for
zop yr a n (9). To a solution of 5 (95 g, 0.34 mol) in dichlo-
romethane (500 mL) was added N,N-diisopropylethylamine
(100.7 mL, 0.58 mol) followed by methansulfonyl chloride (37
mL, 0.47 mol). The mixture was stirred at room temperature
for 15 h, diluted with dichloromethane (300 mL), and then
washed with water (500 mL) and brine (500 mL). The organic
layer was dried (MgSO4), filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography to give 9 (75 g, 84%): mp 66-68 °C; 1H NMR
(CDCl3) δ 1.39 (s, 3H), 3.40 (s, 3H), 3.46 (s, 3H), 4.19 (s, 1H),
5.74 (d, J ) 10.1 Hz, 1H), 6.39 (d, J ) 10.1 Hz, 1H), 6.74 (d,
J ) 8.9 Hz, 1H), 7.75 (d, J ) 2.8 Hz, 1H), 7.89 (dd, J ) 8.9,
2.8 Hz, 1H); HRMS (M+) 265.0950 calcd for C13H15N1O5, found
265.0957.
Gen er a l P r oced u r e for th e Syn th esis of Ep oxid e 11-
14. To a solution of olefin (30 g, 113 mmol) and J acobson’s
reagent (3.59 g, 5.65 mol) in dichloromethane (150 mL) was
added a mixture of 0.55 M NaOCl (822 mL) and 0.05 M Na2-
HPO4 (342 mL) dropwise through an additional funnel at 0
°C. After vigorous stirring at room temperature overnight, the
reaction was passed through a pad of Celite and washed with
dichloromethane and water 3-4 times. The filtrate was
extracted with dichloromethane, and the organic layer was
dried (MgSO4), filtered, and concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography to give an oil, which was solidified with
standing.
C
13H15N1O6, found 281.0904.
3,4-Dih yd r o-2-d im eth oxym eth yl-4-h yd r oxy-2-m eth yl-
6-n itr o-2H-1-ben zop yr a n (4). The keto compound 3 (80 g,
285 mmol) was dissolved in toluene (800 mL) and cooled to
-78 °C. To the solution was added diisobutylaluminum
hydride (247 mL, 1.5 M in toluene) slowly over 2 h, and the
mixture was continuously stirred for an additional 1 h at -78
°C. An aqueous solution of 1 N HCl (1 L) was added to the
reaction, and the layers were separated. Water layer was
extracted with ethyl acetate (500 mL) and was combined with
the organic layer. The combined organic layers were washed
with water (500 mL), dried (MgSO4), filtered, and concentrated
under reduced pressure to give 4 (77 g, 96%) with 7:1 ratio of
cis and trans, which was carried to the next step without
further purification. Only small amounts of regioisomers were
separated by silica gel column chromatography for analysis.
Cis-compound: 1H NMR (CDCl3) δ 1.37 (s, 3H), 2.15 (dd, J )
14.1, 5.8 Hz, 1H), 2.23 (dd, J ) 14.1, 7.1 Hz, 1H), 3.46 (s, 3H),
3.54 (s, 3H), 4.28 (s, 1H), 4.38 (d, 1H), 4.77 (m, 1H), 6.87 (d, J
) 9.1 Hz, 1H), 8.00 (dd, J ) 9.1, 2.7 Hz, 1H), 8.32 (d, J ) 2.7
Hz, 1H); HRMS (M+) 283.1055 calcd for C13H17N1O6, found
283.1046. Trans-compound: 1H NMR (CDCl3) δ 1.43 (s, 3H),
1.78 (dd, J ) 14.1, 5.8 Hz, 1H), 2.59 (dd, J ) 14.1, 5.8 Hz,
1H), 3.39 (s, 3H), 3.49 (s, 3H), 4.01 (d, 1H), 4.18 (s, 1H), 5.03
(m, 1H), 6.85 (d, J ) 9.2 Hz, 1H), 7.98 (dd, J ) 9.2, 2.7 Hz,
1H), 8.34 (d, J ) 2.7 Hz, 1H).
(2S,3S,4S)-3,4-Dih yd r o-2-d im eth oxym eth yl-3,4-ep oxy-
2-m eth yl-6-n itr o-2H-1-ben zop yr a n (11). Epoxide 11 was
obtained from 9 and (S,S)-J acobson’s reagent: mp 67-69 °C;
1H NMR (300 MHz, CDCl3) δ 1.54 (s, 3H), 3.25 (s, 3H), 3.47
(s, 3H), 3.80 (d, J ) 4.8 Hz, 1H), 3.97 (d, J ) 4.8 Hz, 1H), 4.90
(s, 1H), 6.83 (d, J ) 9.9 Hz, 1H), 8.11 (dd, J ) 9.9, 3.0 Hz,
1H), 8.25 (d, J ) 3.0 Hz, 1H); HRMS (M+) 282.0977 calcd for
C
13H16N1O6, found 282.0967.
(2S ,4S )-3,4-Dih yd r o-2-d im e t h oxym e t h yl-4-(R -(-)-R-
m eth oxyp h en yla cetoxy)-2-m eth yl-6-n itr o-2H-1-ben zop y-
r a n (5). To a solution of 4 (320.0 g, 1.13 mol) in ethyl acetate
(2 L) were added R-(-)-R-methoxyphenylacetic acid (191.4 g,
1.15 mol), 1,3-dicyclohexylcarbodiimide (237.6 g, 1.15 mol), and
4-(dimethylamino)pyridine (6.9 g, 0.06 mol) slowly at 0 °C, then
the mixture was stirred at room temperature for 1 h. After
the completion of reaction, the reaction was filtered to remove
byproduct, mainly urea, and the filtrate was concentrated
under reduced pressure. The residue was purified by short
silica gel column chromatography, followed by recrystallization
(ethyl ether-hexane) twice to give 5 as a white solid (136 g,
(2S,3R,4R)-3,4-Dih yd r o-2-d im eth oxym eth yl-3,4-ep oxy-
2-m eth yl-6-n itr o-2H-1-ben zop yr a n (12). Epoxide 12 was
obtained from 9 and (R,R)-J acobson’s reagent: mp 61-63 °C;
1H NMR (CDCl3) δ 1.25 (s, 3H), 3.57 (s, 3H), 3.65 (s, 3H), 3.78
(d, J ) 4.5 Hz, 1H), 3.94 (d, J ) 4.5 Hz, 1H), 4.44 (s, 1H), 6.92
(d, J ) 8.9 Hz, 1H), 8.13 (dd, J ) 8.9, 2.8 Hz, 1H), 8.28 (d, J
) 2.8 Hz, 1H); HRMS (M+) 282.0977 calcd for C13H16N1O6,
found 282.0976.
Gen er a l P r oced u r e for th e Syn th esis of Am in o Alco-
h ol 15-18. To a solution of epoxide (29 g, 103 mmol) in ethanol
was added 25% NH4OH (288 mL, 2.06 mol), and the mixture
was stirred at 50 °C overnight. After evaporation of ethanol
under reduced pressure, the residue was treated with NH4Cl
and extracted with ethyl acetate. The organic layer was
washed with water, dried (MgSO4), filtered, and concentrated
under reduced pressure to give a foam (26.4 g, 86%).
(2S,3S,4R)-4-Am in o-3,4-d ih yd r o-2-d im eth oxym eth yl-3-
h yd r oxy-2-m eth yl-6-n itr o-2H-1-ben zop yr a n (15): mp 108-
1
28%): mp 111 °C; H NMR (CDCl3, 300 MHz) δ 1.34 (s, 3H),
1.99 (dd, J ) 13.4, 6.3 Hz, 1H), 2.23 (dd, J ) 14.4, 6.3 Hz,
1H), 3.47 (s, 3H), 3.48 (s, 3H), 3.53 (s, 3H), 4.20 (s, 1H), 4.91
(s, 1H), 6.06 (dd, J ) 6.3, 6.3 Hz, 1H), 6.90 (d, 1H), 7.37-7.50
(m, 5H), 8.03-8.10 (m, 2H); HRMS (M+) 432.1658 calcd for
C
22H26N1O8, found 432.1648.
(2R,4R)-3,4-Dih ydr o-4-(R-(-)-R-m eth oxyph en ylacetoxy)-
1
2-m eth yl-6-n itr o-2H-1-ben zop yr a n (6). Further crystalliza-
tion of mother liquor gave 6 as a 15% yield: mp 78 °C; 1H
NMR (CDCl3, 300 MHz) δ 1.32 (s, 3H), 2.10 (dd, J ) 13.4, 9.9
Hz, 1H), 2.23 (dd, J ) 13.4, 6.3 Hz, 1H), 3.43 (s, 3H), 3.51 (s,
6H), 4.21 (s, 1H), 4.81 (s, 1H), 5.96 (dd, J ) 9.9, 6.3 Hz, 1H),
6.84 (d, 1H), 7.37-7.48 (m, 5H), 7.59 (d, 1H), 7.98-8.02 (m,
1H); HRMS (M+) 432.1658 calcd for C22H26N1O8, found 432.1659.
113 °C; H NMR (CDCl3) δ 1.23 (s, 3H), 3.52 (s, 3H), 3.53 (s,
3H), 3.71 (d, J ) 9.75 Hz, 1H), 3.74 (d, J ) 9.75 Hz, 1H), 4.34
(s, 1H), 6.78 (d, J ) 9.10 Hz, 1H), 7.93 (dd, J ) 9.10, 2.75 Hz,
1H), 8.43 (d, J ) 2.75 Hz, 1H); HRMS (M+) 298.1165 calcd for
C
13H19N2O6, found 298.1165.
(2S,3R,4S)-4-Am in o-3,4-d ih yd r o-2-d im eth oxym eth yl-3-
h yd r oxy-2-m eth yl-6-n itr o-2H-1-ben zop yr a n (16): mp 101-
104 °C; 1H NMR (CDCl3) δ 1.56 (s, 3H), 3.38 (m, 4H), 3.45 (s,
3H), 3.93 (d, J ) 9.8 Hz, 1H), 3.74 (d, J ) 9.75 Hz, 1H), 4.36
(s, 1H), 6.85 (d, J ) 9.0 Hz, 1H), 8.02 (dd, J ) 9.1, 2.8 Hz,
1H), 8.49 (s, 1H); HRMS (M+) 298.1165 calcd for C13H19N2O6,
found 298.1148.
Gen er a l P r oced u r e for th e Syn th esis of N-P h en yl
Cya n ogu a n id in e 19-22. To a solution of of an appropriate
N-cyano-N′-phenylthiourea sodium salt (1.68 mmol) and amino
alcohol 15-18 (500 mg, 1.68 mmol) in DMF (5 mL) was added
1-[3-(dimethylamino)propyl]-2-ethylcarbodiimide hydrochlo-
ride (418 mg). The mixture was stirred for 5 h at room
temperature and acidified with 10 mL of 1 N HCl, then
extracted with ethyl acetate (30 mL × 2). The organic layer
was washed with water and brine, dried (MgSO4), filtered, and
(2S,4S)-3,4-Dih yd r o-2-d im eth oxym eth yl-4-h yd r oxy-2-
m eth yl-6-n itr o-2H-1-ben zop yr a n (7). To a solution of 5 (156
g, 0.36 mol) in THF (870 mL) was added an aqueous solution
of 1 N LiOH (434 mL) at 0 °C, and the mixture was stirred for
30 min. After the completion of reaction, the mixture was
extracted with ethyl acetate (500 mL × 3). The organic layer
was dried (MgSO4), filtered, and concentrated under reduced
pressure to give 7 (101 g, 99%) as a solid: 1H NMR (CDCl3) δ
1.37 (s, 3H), 2.15 (dd, J ) 14.1, 5.7 Hz, 1H), 2.23 (dd, J )
14.1, 7.1 Hz, 1H), 3.47 (s, 3H), 3.55 (s, 3H), 4.28 (s, 1H), 4.42
(brs, 1H), 4.77 (m, 1H), 6.87 (d, J ) 9.0 Hz, 1H), 8.01 (dd, J )
9.0, 2.8 Hz, 1H), 8.34 (d, J ) 2.8 Hz, 1H); HRMS (M+) 283.1055
calcd for C13H17N1O6, found 283.1049.
(2S)-2-Dim et h oxym et h yl-2-m et h yl-6-n it r o-2H -1-b en -

Anti-Ischemic ATP-Sensitive KATP Opener
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4213
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (n-hexane:ethyl acetate
) 1:1) to afford the phenylcyanoguanidine 19-22 as a foam.
(2R,3S,4R)-N′-(4-Ch lor op h en yl)-N′′-cya n o-N-(3,4-d ih y-
d r o-2-d im eth oxym eth yl-3-h yd r oxy-2-m eth yl-6-n itr o-2H-
ben zop yr a n -4-yl)gu a n id in e (19): mp 117-121 °C; 1H NMR
(CDCl3, 300 MHz) δ 1.38 (s, 3H), 3.45 (s, 3H), 3.51 (s, 3H),
4.15 (d, J ) 7.6 Hz, 1H), 4.35 (s, 1H), 5.04 (m, 1H), 6.24 (br,
1H), 6.90 (d, J ) 9.0 Hz, 1H), 7.26-7.36 (m, 4H), 8.06 (dd, J
) 2.2, 9.0 Hz, 1H), 8.31 (br, 1H), 8.44 (br, 1H); HRMS
(M+1)+ calcd 476.1337, found 476.1343; Anal. (C21H22ClN5O6)
C, H, N.
(2R,3S,4R)-N′-(3-Ch lor op h en yl)-N′′-cya n o-N-(3,4-d ih y-
d r o-2-d im eth oxym eth yl-3-h yd r oxy-2-m eth yl-6-n itr o-2H-
ben zop yr a n -4-yl)gu a n id in e (20): mp 118-121 °C; 1H NMR
(CDCl3, 300 MHz) δ 1.39 (s, 3H), 3.48 (s, 3H), 3.54 (s, 3H),
4.21 (d, J ) 8.1 Hz, 1H), 4.37 (s, 1H), 5.02 (m, 1H), 6.23 (br,
1H), 6.92 (d, J ) 9.0 Hz, 1H), 7.18-7.40 (m, 4H), 8.10 (dd, J
) 2.4, 9.0 Hz, 1H), 8.39 (br, 2H); HRMS (M+1)+ calcd
476.1337, found 476.1333; Anal. (C21H22ClN5O6) C, H, N.
(2S,3S,4R)-N′-(4-Ch lor op h en yl)-N′′-cya n o-N-(3,4-d ih y-
d r o-2-d im eth oxym eth yl-3-h yd r oxy-2-m eth yl-6-n itr o-2H-
ben zop yr a n -4-yl)gu a n id in e (21): mp 129-132 °C; 1H NMR
(CDCl3, 300 MHz) δ 1.29 (s, 3H), 3.40 (s, 3H), 3.41 (s, 3H),
4.08 (m, 1H), 4.40 (s, 1H), 5.04 (dd, J ) 8.1, 8.4 Hz, 1H), 5.48
(br, 1H), 6.85 (d, J ) 9.0 Hz, 1H), 7.23-7.40 (m, 3H), 7.95 (dd,
J ) 2.4, 9.0 Hz, 1H), 8.09 (br, 1H), 9.22 (br, 1H); HRMS
(M+1)+ calcd 476.1337, found 476.1339; Anal. (C21H22ClN5O6)
C, H, N.
(2S,3S,4R)-N′′-Cya n o-N-(3,4-d ih yd r o-2-d im et h oxym e-
th yl-3-h yd r oxy-2-m eth yl-6-n itr o-2H-ben zop yr a n -4-yl)-N′-
(4-m eth oxyp h en yl)gu a n id in e (22): mp 105-108 °C; 1H
NMR (CDCl3, 300 MHz) δ 1.37 (s, 3H), 3.47 (s, 3H), 3.49 (s,
3H), 3.77 (s, 3H), 4.09 (d, J ) 7.8 Hz, 1H), 4.16 (br, 1H), 4.34
(s, 1H), 5.10 (dd, J ) 8.1, 9.3 Hz, 1H), 5.69 (br, 1H), 6.89 (m,
3H), 7.26 (m, 2H), 7.95 (br, 1H), 8.01 (dd, J ) 2.0, 4.5 Hz, 1H),
8.16 (s, 1H); HRMS (M+1)+ calcd 472.1832, found 472.1833;
Anal. (C22H25N5O7) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of N-Ben zyl
Cya n ogu a n id in e 27-32. To a solution of an appropriate
amino alcohol 15-18 (26 g, 87 mmol) in 2-propanol (400 mL)
and DMF (100 mL) were added triethylamine (16 mL, 113
mmol) and diphenylcyanocarbonimidate (22.8 g, 95.7 mmol)
portionwise. After the mixture was stirred at room tempera-
ture overnight, all volatiles were removed under reduced
pressure. To the residue was added water, which was extracted
with ethyl acetate. The organic layer was dried (MgSO4),
filtered, and concentrated in vacuo. The residue was purified
by silica gel chromatography (hexane:ethyl acetate ) 1:2) to
give a pale yellow foam (24.7 g, 64%) 23-26. Phenoxymethyl
imine 23-26 (24.7 g, 55.8 mmol) was dissolved in hot 2-pro-
panol (100 mL), to which was added an appropriate benzyl-
amine (139.5 mmol). After the mixture was stirred overnight,
the volatiles were removed under reduced pressure. The
residue was purified by silica gel column chromatography
(hexane:ethyl acetate ) 1:1) to give benzyl cyanoguanidine 27-
32 as a pale yellow foam.
(2R,3S,4R)-N′-Be n zyl-N′′-cya n o-N-(3,4-d ih yd r o-2-d i-
m et h oxym et h yl-3-h yd r oxy-2-m et h yl-6-n it r o-2H -b en zo-
p yr a n -4-yl)gu a n id in e (29): 1H NMR (CDCl3, 300 MHz) δ
1.43 (s, 3H), 3.43 (s, 3H), 3.46 (s, 3H), 3.60 (m, 1H), 3.74 (d, J
) 8.6 Hz, 1H), 4.47 (m, 3H), 5.17 (m, 1H), 5.30 (br, 1H), 6.57
(m, 1H), 6.87 (d, J ) 9.3 Hz, 1H), 7.26-7.38 (m, 5H), 8.01 (m,
2H); HRMS (M+1)+ calcd 456.1883, found 456.1868; Anal.
(C22H25N5O6) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 6-Am in oben -
zop yr a n s 33-37. To a solution of 6-nitrobenzopyrans 27-31
(43.7 mmol) in methanol (300 mL) was added 61 mL of 0.36
M Cu(OAc)2 (21.8 mmol). After portionwise addition of NaBH4
during 1 h at room temperature, the reaction was stirred for
an additional 1 h, then filtered through a pad of Celite to
remove black precipitate. Saturated NaHCO3 (50 mL) was
added to the filtrate, which was extracted with ethyl acetate
(500 mL). The organic layer was washed with brine, dried
(MgSO4), filtered through a pad of silica gel, and concentrated
under reduced pressure. The residue was recrystallized from
hexanes-ethyl acetate (1:3) to give a solid.
(2S,3S,4R)-N-(6-Am in o-3,4-dih ydr o-2-dim eth oxym eth yl-
3-h yd r oxy-2-m et h yl-2H -b en zop yr a n -4-yl)-N′-b en zyl-N′′-
1
cya n ogu a n id in e (33): mp 160-162 °C; H NMR (CDCl3) δ
1.20 (s, 3H), 3.56 (s, 6H), 4.09 (d, 1H), 4.31 (s, 1H), 4.42 (dd, J
) 15.1, 5.8 Hz, 1H), 4.50 (dd, J ) 15.1, 5.8 Hz, 1H), 5.64 (d,
1H), 6.50 (d, J ) 8.7 Hz, 1H), 6.63 (d, J ) 8.7 Hz, 1H), 7.18
(br, 1H), 7.32 (m, 6H); HRMS (M+) calcd 425.2063, found
425.2059; Anal. (C22H27N5O4) C, H, N.
(2R,3S,4R)-N-(6-Am in o-3,4-dih ydr o-2-dim eth oxym eth yl-
3-h yd r oxy-2-m et h yl-2H -b en zop yr a n -4-yl)-N′-b en zyl-N′′-
cya n ogu a n id in e (35): mp 184-186 °C; 1H NMR (DMSO-d6)
δ 1.13 (s, 3H), 3.38 (s, 3H), 3.39 (s, 3H), 4.72 (m, 3H), 4.59 (s,
2H), 4.82 (br, 1H), 4.98 (br, 1H), 6.35 (br, 1H), 6.45 (m, 2H),
7.11 (br, 1H), 7.26-7.38 (m, 5H), 7.51 (br, 1H); HRMS (M+)
calcd 425.2063, found 425.2059; Anal. (C22H27N5O4) C, H,
N.
(2R,3R,4S)-N-(6-Am in o-3,4-dih ydr o-2-dim eth oxym eth yl-
3-h yd r oxy-2-m et h yl-2H -b en zop yr a n -4-yl)-N′-b en zyl-N′′-
1
cya n ogu a n id in e (36): H NMR (CDCl3) δ 1.20 (s, 3H), 3.55
(s, 6H), 4.08 (d, 1H), 4.31 (s, 1H), 4.42 (d, J ) 15.2, 5.3 Hz,
1H), 4.50 (d, J ) 15.2, 5.3 Hz, 1H), 5.78 (br, 1H), 6.51 (br,
1H), 6.61 (d, 1H), 7.14 (br, 1H), 7.29 (m, 6H); HRMS (M+) calcd
425.2063, found 425.2057; Anal. (C22H27N5O4) C, H, N.
(2S,3S,4R)-N-(6-Am in o-3,4-dih ydr o-2-dim eth oxym eth yl-
3-h yd r oxy-2-m eth yl-2H-ben zop yr a n -4-yl)-N′′-cya n o-N′-(4-
1
m eth oxyben zyl)gu a n id in e (37): mp 159-162 °C; H NMR
(CDCl3) δ 1.26 (s, 3H), 3.57 (s, 6H), 3.80 (s, 3H), 4.10 (d, 1H),
4.15 (br, 1H), 4.30 (s, 1H), 4.41 (m, 3H), 5.53 (br, 1H), 6.69
(m, 2H), 6.88 (d, J ) 8.3 Hz, 3H), 7.10 (br, 1H), 7.24 (d, J )
8.37 Hz, 2H); HRMS (M+) calcd 455.2171, found 455.2171;
Anal. (C23H29N5O5) C, H, N.
(2S ,3S ,4R )-N ′-B e n zy l-N ′′-c y a n o -N -(3,4-d ih y d r o -2-
d im eth oxym eth yl-3-h yd r oxy-6-m eth a n esu lfon yla m in o-
2-m eth yl-2H-ben zop yr a n -4-yl)gu a n id in e (39). To a solu-
tion of 33 (91 mg) in dichloromethane (2 mL) were added 45
µL of triethylamine and 20 µL of methanesulfonyl chloride.
The reaction mixture was stirred for 2 h at room temperature
and diluted with 10 mL of water and 20 mL of ethyl acetate.
The layers were separated, and the organic layer was dried
(MgSO4), filtered, and concentrated under reduced pressure.
The residue was purified by silica gel chromatography (n-
hexane:ethyl acetate ) 1:2) to afford 39 (90 mg, 85%): mp
128-130 °C;1H NMR (CDCl3) δ 1.23 (s, 3H), 2.91 (s, 3H), 3.56
(s, 6H), 4.13 (m, 1H), 4.23 (br, 1H), 4.32 (s, 1H), 4.39 (m, 1H),
4.54 (m, 2H), 5.73 (br, 1H), 6.78 (d, 1H), 7.30 (m, 7H), 8.15
(br, 1H); HRMS (M+) calcd 503.1839, found 503.1834; Anal.
(C23H29N5O6S) C, H, N, S.
(2S,3S,4R)-N-[6-Am in o-3,4-d ih yd r o-2-([1,3]d ioxola n -2-
yl)-3-h yd r oxy-2-m et h yl-2H -b en zop yr a n -4-yl]-N′-b en zyl-
N′′-cya n ogu a n id in e (41). The compound 41 was prepared
from an appropriate epoxide by the general procedure for 27-
32, followed by the reduction using the general procedure for
33-37: 1H NMR (CDCl3) δ 1.24 (s, 3H), 3.54 (br, 3H), 4.08
(m, 5H), 4.48 (m, 2H), 4.96 (s, 1H), 5.48 (br, 1H), 6.71 (br, 1H),
(2S ,3S ,4R )-N ′-Be n zyl-N ′′-cya n o-N -(3,4-d ih yd r o-2-d i-
m et h oxym et h yl-3-h yd r oxy-2-m et h yl-6-n it r o-2H -b en zo-
pyr an -4-yl)gu an idin e (27): mp 117-120 °C; 1H NMR (CDCl3,
300 MHz) δ 1.33 (s, 3H), 3.53 (s, 3H), 3.58 (s, 3H), 4.16 (d, J
) 8.4 Hz, 1H), 4.38-4.52 (m, 3H), 4.82 (br, 1H), 5.85 (br, 1H),
6.25 (br, 1H), 6.72 (br, 1H), 6.90 (d, J ) 8.7 Hz, 1H), 7.30 (m,
5H), 8.06 (d, J ) 8.7 Hz, 1H), 8.29 (br, 1H); HRMS (M+) calcd
455.1805, found 455.1802; Anal. (C22H25N5O6) C, H, N.
(2S ,3R ,4S )-N ′-Be n zyl-N ′′-cya n o-N -(3,4-d ih yd r o-2-d i-
m et h oxym et h yl-3-h yd r oxy-2-m et h yl-6-n it r o-2H -b en zo-
p yr a n -4-yl)gu a n id in e (28): mp 99-100 °C; 1H NMR (CDCl3)
δ 1.40 (s, 3H), 3.41 (s, 3H), 3.43 (s, 3H), 3.80 (dd, J ) 7.8, 8.0
Hz, 1H), 4.30 (d, J ) 5.7 Hz, 1H), 5.12 (m, 1H), 5.57 (br, 1H),
6.61 (dd, J ) 5.5, 5.5 Hz, 1H), 6.84 (d, J ) 9.0 Hz, 1H), 7.21-
7.35 (m, 5H), 7.98 (dd, J ) 2.4, 7.2 Hz, 1H), 8.03 (br, 1H);
HRMS (M+) calcd 455.1805, found 455.1827; Anal. (C22H25N5O6)
C, H, N.

4214 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Yoo et al.
6.74 (m, 1H), 7.00 (br, 1H), 7.36 (m, 6H); HRMS (M+) calcd
423.1907, found 423.1905; Anal. (C22H25N5O4) C, H, N.
the infarct zone (IZ) was evaluated by tetrazolium staining,
and its size was calculated as a percent of AAR. The heart
slices were incubated for 15 min in 2,3,5-triphenyltetrazolium
chloride (TTC) phosphate buffer (pH 7.4) at 37 °C and fixed
for 20-24 h in a 10% formalin solution. The viable portion of
the slice is stained red by TTC, whereas the TTC stain is
absent in the area of infarct. The infarct zone area of each
slice was summed for all slices, and the resulting summed
infarct zone area was divided by total AAR weight to yield %
IZ to AAR (IZ/AAR, %).
P r otective Activity in Neu r on a l Cells. From the brains
of 17-18-day-old rat embryos, cerebral cortical neurons were
isolated and cultured at 37 °C for 7-9 days in a 5% CO2
incubator. The cortical cell cultures were washed twice with
a minimum essential medium (MEM) to reduce the serum
concentration to 0.2% and pretreated for 30 min with varying
concentrations of compound. The test compounds were dis-
solved in DMSO and diluted in a medium, of which final
concentration of DMSO was not allowed to exceed 0.2%. After
the pretreatment with test compounds or vehicle, FeSO4 was
added to a final concentration of 50 µM, and the cultures were
maintained for 24 h in a CO2 incubator. During incubation,
lactate dehydrogenase (LDH) was released into the medium
upon neuronal death by the oxidative toxicity of iron. The
extent of neuronal damage was assessed by measuring the
amount of LDH secreted into the media. The protective effect
of the compounds on neurons was evaluated by calculating the
reduction rate of LDH release in the treatment group com-
pared with the control group.
(2S,3S,4R)-N-[6-Am in o-3,4-d ih yd r o-2-([1,3]-5,5-d im eth -
yld ioxa n -2-yl)-3-h yd r oxy-2-m eth yl-2H-ben zop yr a n -4-yl]-
N′-ben zyl-N′′-cya n ogu a n id in e (42). The compound 42 was
prepared from an appropriate epoxide by the general procedure
for 27-32, followed by the reduction using the general
1
procedure for 33-37. H NMR (CDCl3) δ 0.74 (s, 3H), 1.19 (s,
3H), 1.24 (s, 3H), 3.52 (t, J ) 11.4 Hz, 2H), 3.72 (t, J ) 11.4
Hz, 2H), 4.14 (d, 3H), 4.45 (m, 2H), 4.55 (br, 1H), 4.56 (s, 1H),
5.77 (d, 1H), 6.48 (d, J ) 8.7 Hz, 1H), 6.53 (br, 1H), 6.64 (d, J
) 8.7 Hz, 1H), 7.28 (m, 6H); HRMS (M+) calcd 465.2376, found
465.2369; Anal. (C25H31N5O4) C, H, N.
Biology. Rela xa tion of Meth oxa m in e Con tr a cted Ra t
Aor ta . Sprague-Dawley rats (350-450 g) were sacrificed by
cervical dislocation and underwent thoracotomy. The thoracic
aorta was deprived of the adipose tissue and cut into aortic
rings of 3 mm width. The aorta was mounted in an organ bath
containing an oxygenated physiological salt solution (PSS, a
modified Krebs Henseleit buffer) at 37 °C, and was allowed to
equilibrate under a resting tension of 2 g, then stand for 1 h
for stabilization. The vascular tissue was constricted with 10-5
M of phenylephrine and washed several times with PSS, and
this procedure was repeated again to ensure the stable
reactivity of vascular smooth muscle on repetitive constriction/
dilatation. Then, 3 × 10-6 M of methoxamine was added to
induce an intensive constriction in the vascular smooth muscle.
When the vasoconstriction induced by methoxamine reached
and maintained a maximum, test compound was cumulatively
added to the organ bath in concentrations of 1, 3, 10, and 30
µM. Following the addition of the test compounds, the change
in the maximal constriction induced by methoxamine was
calculated to plot a concentration-relaxation response curve.
Through a linear regression analysis, IC50, the drug concentra-
tion at which the methoamine induced vasoconstriction is 50%
relaxed, was obtained for each compound.
Ack n ow led gm en t. We thank the Ministry of Sci-
ence and Technology of Korea for financial support of
this research.
Su p p or tin g In for m a tion Ava ila ble: Crystallographic
data for compound 5. This material is available free of charge
via the Internet at http://pubs.acs.org.
Ca r d iop r otective Activity in Isch em ic Myoca r d iu m
Ra t Mod el. Male Sprague-Dawley rats (350-450 g) were
anesthetized with pentobarbital (75 mg/kg, i.p.). Artificial
respiration was managed at a rate of 60/min with a stroke
volume of 10 mL/kg. A catheter for drug administration was
inserted into the femoral vein and another was inserted into
the femoral artery for blood pressure measurement. Body
temperature was maintained at 37 °C using a homeothermic
blanket control unit. The left coronary artery was occluded
according to the Selye H. method.33 The rats underwent a left
thoracotomy operation. Immediately after the left anterior
descending coronary artery (LAD) was carefully stitched with
silk ligature, the heart was then repositioned in the thoracic
cavity with the ligature ends exteriorized. The opposite
ligature ends were passed through a PE tube (PE100, 2.5 cm),
and the rats were allowed to stabilize for 20 min. Via the
catheter inserted into the femoral vein, vehicles or test
compounds were administered into the rats for 30 min. The
PE tube was pressed on the surface of the heart directly above
the coronary artery, and a hemostatic pincette was applied to
clamp the tube and ligature for 45 min, resulting in coronary
artery occlusion. Reperfusion was allowed for 90 min by the
removal of the hemostatic pincette. The coronary artery was
reoccluded, and 2 mL of a 1% Evans blue was injected
intravenously to negatively mark the risk zone. Rats were then
sacrificed by i.v. injection of pentobarbital, and the heart was
removed. The left ventricle (LV) was cut horizontally to the
heart apex into five or six slices, which were weighed. These
slices were analyzed using a Hi-scope installed with an image
analyzing program (Image Pro Plus). The area of the normal
blood stream tissue region appeared blue in a computer
monitor, while the ischemic area appeared colorless. The
percentage of the colorless area to the total area of each slice
was calculated and multiplied by the weight of each slice to
determine the area at risk (AAR) of each slice. AARs obtained
from each slice were summed for all slices, and the total AAR
was divided by the total weight of the LV to yield % AAR in
the entire LV. The AAR/LV (%) ranged from 31% to 47%. Then,
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