Cardioselective anti-ischemic ATP-sensitive potassium channel (KATP) openers: Benzopyranyl indoline and indole analogues

Article abstract of DOI:10.1016/S0223-5234(03)00063-1

This paper describes the design, syntheses, and biological evaluations of novel ATP-sensitive potassium channel (K_ATP) openers, benzopyranyl indoline and indole derivatives. Among those, two enantiomers of indoline-2-carboxylic ethyl esters (14, 18) showed the best cardioprotective activities both in vitro and in vivo, while their vasorelaxation potencies were very low (concentration for 50% inhibition of vasorelaxation >30 μM). The cardioprotective effect of 14 was completely reversed by 5-hydroxydecanoate, a selective mitochondrial K_ATP blocker, indicating its provable protective mechanism through the mitochondrial K_ATP opening. In addition, we performed conformational analyses using ²D-NMR, X-ray crystallography and molecular modeling to study the structure-activity relationships in this series of compounds.

Full text of DOI:10.1016/S0223-5234(03)00063-1

European Journal of Medicinal Chemistry 38 (2003) 459ꢀ471
/
www.elsevier.com/locate/ejmech
Original article
Cardioselective anti-ischemic ATP-sensitive potassium channel
(KATP) openers: benzopyranyl indoline and indole analogues
Sunkyung Lee a, Kyu Yang Yi a, Soo-Kyung Kim a, Jeehee Suh a, Nak Jeong Kim a,
Sung-eun Yoo a,*, Byung Ho Lee a, Ho Won Seo a, Sun-Ok Kim b, Hong Lim b
a Medicinal Science Division, Korea Research Institute of Chemical Technology, 100 Jang-dong, Yoosung-gu, Taejon 305-600, South Korea
b Division of Life Science, AgroPharma Research Institute of Dongbu-Hannong Chemical Co., Taejon 305-600, South Korea
Received 2 February 2002; received in revised form 13 February 2003; accepted 13 February 2003
Abstract
This paper describes the design, syntheses, and biological evaluations of novel ATP-sensitive potassium channel (KATP) openers,
benzopyranyl indoline and indole derivatives. Among those, two enantiomers of indoline-2-carboxylic ethyl esters (14, 18) showed
the best cardioprotective activities both in vitro and in vivo, while their vasorelaxation potencies were very low (concentration for
50% inhibition of vasorelaxation ꢀ30 mM). The cardioprotective effect of 14 was completely reversed by 5-hydroxydecanoate, a
/
selective mitochondrial KATP blocker, indicating its provable protective mechanism through the mitochondrial KATP opening. In
addition, we performed conformational analyses using 2D-NMR, X-ray crystallography and molecular modeling to study the
structureꢀactivity relationships in this series of compounds.
/
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: KATP opener; Benzopyranyl indole; Cardioselectivity; Mitochondrial KATP; Conformational analyses
1. Introduction
action of numerous cellular signals, second messengers,
and end-effector mechanisms [5]. Many researchers have
identified the possible triggers, transducers, and end-
effectors involved in ischemic PC, which allows mimick-
ing of the physiological response by chemical agents [6],
known as ‘chemical PC’ [7]. ATP-sensitive potassium
channel (KATP) openers have shown to play a crucial
role in ischemic PC, and this hypothesis is supported by
studies showing the KATP blockers can attenuate this
protective effect, suggesting KATP to have an important
protective mechanism [8,9]. KATP channels are com-
posed of a complex of two distinct subunits, an inward
rectifying Kꢁ channel subunit (Kir 6.x) forming the
channel pore and a sulfonylurea receptor (SUR) belong-
ing to the ATP-binding cassette super family [10,11].
Diversity of KATP channels by the various combinations
of their subunit composition may provide the possibility
to target certain isoforms for the identification of tissue-
selective compounds [12]. In particular, the importance
of cardiac KATP channel opening in myocardial PC has
been demonstrated by many researchers [13,14], even
though it is still unclear how KATP activation can
mediate PC. Whatever the mechanism of PC, it is
Ischemic preconditioning (PC) is a powerful endo-
genous protective mechanism against ischemic injury
observed in a variety of organ systems [1], including
heart, brain, spinal cord, retina, liver, lung, and skeletal
muscle, which had been previously subjected to brief,
sub-lethal periods of ischemia [2ꢀ/4]. Ischemic PC is a
multifactorial physiological process, requiring the inter-
Abbreviations: PC,
potassium channel; Kir, inward rectifying Kꢁ channel; SUR,
sulfonylurea receptor; DDQ, 2,3-dichloro-5,6-dicyano-1,4-
preconditioning;
KATP
,
ATP-sensitive
benzoquinone; Boc, t-butyloxycarbonyl; LAH, lithium aluminum
hydride; IC50, concentration for 50% inhibition of vasorelaxation;
LVDP, left ventricular developing pressure; HR, heart rate; TTC, time
to contracture; i.v., intravenous; i.p., intraperitoneal; LDH, lactate
dehydrogenase; IZ/AAR, infarction zone to area at risk; 5-HD, 5-
hydroxydecanoate; M.p., melting point; THF, tetrahydrofuran; TFA,
trifluoroacetic acid; LVP, left ventricular pressure; EDP, end diastolic
pressure; LVEDP, left ventricular end diastolic pressure; LVSP, left
ventricular systolic pressure; TTC, 2,3,5-triphenyltetrazolium chloride.
* Corresponding author.
E-mail address: seyoo@krict.re.kr (S.-e. Yoo).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00063-1

460
S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ471
/
encouraging that KATP openers can mimic an endogen-
ous protective factor, which further increase the en-
thusiasm for developing novel cardioprotective KATP
openers [15].
gel column chromatography. The absolute stereochem-
istry was confirmed by using commercially available
(ꢂ)-(S)-indoline-2-carboxylic acid as a starting mate-
7 were
/
rial. Various indoline-2-carboxylic esters 5ꢀ
/
The first cardioselective KATP opener to be described
was BMS-180448 1 (Fig. 1), benzopyranyl cyanoguani-
prepared by acid-catalyzed esterification of indoline-2-
carboxylic acid. The indoline-2-carboxylic acid analogue
24 was obtained from the ester compound 14 by a
typical base hydrolysis.
dine [16,17]. From studies at BristolꢀMyers Squibb
/
using the analogues of urea, benzimidazolone and indole
derivatives [18], the imidazole analogue (BMS-191095,
2) has been discovered which displayed more than a 20-
fold improvement in selectivity for cardioprotective over
vasorelaxant effects in vitro compared with BMS-
180448 [19].
In our previous work, we found 6-aminobenzopyr-
anyl cyanoguanidine analogue (3), showing good car-
dio- and neuro-protective effects without vasorelaxation
activity [20] in contrast to SKP-450 (4) which is also a
K-channel activator but with a strong blood pressure
lowering activity [21,22]. Aiming at identifying cardio-
The N-(2-methoxycarbonyl)indolylbenzopyran has
been prepared from epoxide and indole in the presence
of sodium hydride in low yield [18]. The major product
in that reaction was reported to be a dehydrated
compound at 3,4-position of benzopyran ring and a
significant amount of a carboxylic acid product was also
obtained presumably due to saponification of an ester
group under the reaction condition. We tried the same
reaction with indole and an epoxide under various
conditions, but did not get any reasonable yield of the
desired product. Fortunately, we found that the oxida-
selective KATP openers and elucidating structureꢀ
/
activ-
tion of indoline derivatives (13ꢀ
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) [24]
provided corresponding indole derivatives 25ꢀ28 effi-
ciently (85ꢀ91% yields).
For the preparation of highly constrained lactone
analogues (29, 30) several intramolecular cyclization
reactions between the 3-hydroxyl group in the benzo-
pyran ring and the ester group at the 2-position of
indolines (21, 22) were examined to come up with the
method using aluminum diethylchloride in the presence
of a base catalysis [25] which provided the desired
products in good yields (85 and 81%).
/
16, 19ꢀ
/
22) with 2,3-
ity relationships, we prepared several structurally
constrained indole and indoline derivatives based on a
benzopyran scaffold having an acetal moiety at the 2-
position similarly, as the previously studied compound 3
and SKP-450 (4). Herein, we describe their design,
syntheses, structure analyses using NMR (NOESY),
X-ray crystallography, and molecular modeling as well
as their biological activities.
/
/
2. Chemistry
Indoline-2-ether analogues (31, 32) were prepared by
a series of reactions from indoline-2-carboxylic acid; (1)
t-butyloxycarbonyl (Boc) protection of the nitrogen
atom, (2) reduction of the acid group to the alcohol
using lithium aluminum hydride (LAH), (3) O-methyla-
tion of the alcohol to ether, (4) deprotection of the N-
Boc group under acidic condition, and finally the
coupling of methoxymethylindoline (10) with the ep-
oxide (12) using magnesium perchlorate in acetonitrile.
2.1. Synthesis
Optically pure benzopyranyl indoline and indole
derivatives substituted at the 2-position were prepared
according to the procedure described in Scheme 1.
Indoline-2-carboxylic ester analogues 13ꢀ18 were
/
prepared from previously described optically pure
benzopyranyl epoxides by epoxide ring opening with
the appropriate indolines in the presence of magnesium
perchlorate in acetonitrile [20,23]. The reaction of indo-
line with epoxide proceeded smoothly in good yield
(around 80%), and the resulting two separable diaster-
eoisomers at indoline-2-position were separated by silica
2.2. Structure analysis
Previously the structureꢀ/activity relationship on
KATP openers which have the aniline moiety
Fig. 1. KATP openers.

S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ
/
471
461
Scheme 1. Reagents and conditions: (a) SOCl2, ROH (Rꢃ
NaH, THF, 0 8C0
CH2Cl2, r.t.
/
Me, Et, iPr), reflux; (b) (Boc)2O, NEt3, THF, r.t.; (c) BH3, THF, 0 8C0
/
r.t.; (d) MeI,
/
r.t.; (e) TFA, CH2Cl2, r.t.; (f) Mg(ClO4)2, CH3CN, r.t.; (g) NaOH, MeOH, r.t.; (h) DDQ, benzene, r.t.; (i) Et2AlCl, i-Pr2NH,
(PhNCH2COOEt) attached to C4 of the benzopyran
ring has been done and several key points found from
the study are as follows [19]; (1) The carboxylic acid
analogue is devoid of anti-ischemic activity. (2) Increase
in the size of ester groups causes attenuation of anti-
ischemic potency. (3) There are restrictions as to the size
of the pendent aryl/heterocyclic ring. (4) An sp3 carbon
adjacent to the aniline nitrogen is preferred. (5) Chan-
ging the ester to an imidazole retains full anti-ischemic
activity with excellent selectivity. (6) The SARs for the
vasorelaxant and anti-ischemic potencies are distinc-
tively different.
NOESY, Fig. 2), X-ray analyses (Fig. 3), and molecular
modeling (Fig. 4) on the compound 14 which has good
in vitro and in vivo cardioprotective activities with
selectivity.
To establish the pharmacophore of our series of
compounds and to compare with the pharmacophore
of other series of compounds, we carried out extensive
conformational analyses experimentally and theoreti-
cally including 2-D NMR experiments (COSY and
Fig. 2. NOESY cross peaks. 2D-NMR experiments were performed
using Bruker 600 MHz spectrometer at 25 8C. Mixing times of 400 ms
for NOESY were used.

462
S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ471
/
Fig. 3. X-ray crystallography of compound 14 and 15. ORTEP diagram showed the X-ray structures. Torsion angles and distances were calculated
using SYBYL (v. 6.7).
3. Pharmacology
myocardial infarction zone to area at risk (IZ/AAR),
using ischemic myocardium damage rat model at 0.3 mg
kgꢂ1 via intravenous (i.v.) injection [20,29].
The vasorelaxant potencies were determined by con-
centration for 50% inhibition of vasorelaxation (IC50)
values for relaxation of the methoxamine-contracted rat
aorta, as described previously [20,26]. Cardioprotective
effects in vitro were evaluated in globally ischemic,
isolated, perfused rat hearts at 10 mM concentration
[27,28]. Severity of ischemia was judged by following
parameters; the recovery of contractile function
4. Results and discussion
Besides the indole analogues described to have good
pharmacological profiles as a cardioprotective [18], we
investigated the indoline analogues based on the report
that an sp3 carbon adjacent to the aniline is preferred
rather than an sp2 carbon [19].
(LVDPꢄ
/
HR, left ventricular developing pressureꢄ
/
heart rate) at the end of the reperfusion period, the
time to contracture (TTC the time from the onset of
global ischemia in which the first 5 mmHg increase in
end diastolic pressure (EDP) is observed), and the
amount of cumulative LDH (lactate dehydrogenase)
release into the reperfusate. Anti-ischemic in vivo
potencies were determined by measuring a ratio of
Initially we synthesized four diastereomers of opti-
cally pure 6-nitobenzopyranylindoline-2-carboxylic acid
ethyl ester derivatives (13ꢀ16) with the same (2S)-
/
stereochemistry at the benzopyran as 6-aminobenzopyr-
anyl-N-cyanoguanidine analogue (3) and evaluated their
pharmacological activities [20]. We investigated the 6-

S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ
/
471
463
Fig. 4. Comparison of structures between X-ray and molecular modeling.
nitrobenzopyran analogues prior to the 6-amino com-
pounds, based on the report that an electron with-
drawing group at the 6-position of benzopyran ring
seems to be essential for KATP opening activity [26].
While all four compounds showed much weaker vasor-
elaxation activity compared to BMS-180448 (1), only
the compound 14 with (2S,3R,4S,2?S)-stereochemistry
showed a comparable cardioprotective effect both in
vitro and in vivo to BMS-180448 (Table 1). To
determine the importance of the absolute stereochem-
istry of the compounds, we synthesized the enantiomer
18 and found that the cardioprotective and vasorelaxa-
tion activities were almost the same as 14. Also, we
prepared the diastereomer 17 in which the stereochem-
istry at the 2-position of the indoline ring is opposite and
found that the cardioprotective activity was much
weaker than 18. These results indicate that the relative
stereochemistry of diastereoisomers is crucial for deter-
mining cardioprotective activity, but the absolute stereo-
chemistry is not in this case. While the compounds 14
and 18 showed the comparable cardioprotective effica-
cies to BMS-180448, the vasorelaxation potencies of
those compounds were more than 20 times weaker than
that of BMS-180448, demonstrating improved selectiv-
ity.
With the compound 14 as a standard, we then
examined the effect of ester group by preparing the
methyl and isopropyl analogues, and the acid itself, 20,
23, and 24, respectively. In general, in vitro cardiopro-
tective activities of the methyl 20 and ethyl 14 esters
were comparable but the ethyl ester derivative was much
weaker in the vasorelaxation activity to make the ethyl
ester analogue more desirable overall. Furthermore the
methyl ester analogue 20 did not demonstrate in vivo
cardioprotective efficacy. Isopropyl ester analogue 23
showed only marginal protective effect, and the acid
compound 24 completely lost the protective activity.
The indole analogues 25 and 27 with the same
(2S,3R,4S)-stereochemistry as the compound 14
showed good in vitro cardioprotective activities, but

464
S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ471
/
Table 1
Vasorelaxant potencies and cardioprotective effects of benzopyranyl indoline and indole analogues
cardioprotective effect was reversed by the action of 5-
hydroxydecanoate (5-HD), mitochondrial KATP
no in vivo efficacies. The activities among diastereomers
were also quite sensitive to the stereochemistry same as
the indoline case.
Structurally highly constrained lactone analogues (29,
30) did not show any significant cardioprotective
activities. The ether analogues (31, 32) showed excellent
in vivo cardioprotective activities in the rat model but
poor activities in the Langendorff model.
a
blocker. As can be seen in Table 2, the cardioprotective
effect of the compound 14 was completely reversed by 5-
HD, indicating that the compound 14 exhibits its
cardioprotective effect through the mechanism of open-
ing of KATP, especially a mitochondrial KATP channel.
The NMR spectra of the compounds 13 and 14
indicate the existence of two rotamers in a 3:2 ratio.
Other indoline-2-carboxylic esters exist as a mixture of
To elucidate the nature of the cardioprotective effects
of this series of compounds, we first checked whether the

S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ
/
471
465
aVasorelaxant 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, nꢃ
3. bIn vitro cardioprotective effects were evaluated measuring the contractile function (LVDPꢄ
HR), TTC, and LDH in the globally ischemic rat heart. Each value is an average of three or higher determinations and within 9
10%. cIn vivo anti-
infarction effects were determined by measuring a ratio of myocardial IZ/AAR in ischemic myocardium damage rat model (0.3 mg kgꢂ1), nꢃ
3 or
higher. Each value given is an average and within 9
10%. dNot determined.
/
/
/
/
/
rotamers, too. Besides, there are few interesting points
to be mentioned. As shown in Fig. 2, NOEs were
observed between 7H (d, 5.53 ppm) in the indole ring
and 3H (d, 4.41), 5H (d, 8.80) in the benzopyran part in
the major conformer of 14 but not in the minor
conformer. The chemical shift of 7H in the indole ring
in the major conformer of 14 was 5.53 ppm, far shifted
to upfield, presumably due to the shielding effect of the
benzene ring in the benzopyran moiety. In the X-ray
structure, the distances between 5H in the benzopyran
and 7H in the indole ring of compound 14 and 15 were
indicating the shielding effect. From NMR and X-ray
analyses, two benzene rings of the benzopyran and
indole in both compounds 14 (d, 8.80) and 15 (d, 8.90)
seem to be close enough to show NOE and the shielding
effects, even though those minor conformer are not in
NMR (d, 7.76 for 14, 7.82 for 15). The torsion angle
between the benzopyran ring and indole ring in 14 was
ꢂ
/
78.28 in the X-ray structure, while it was ꢂ49.08 in the
/
compound 15 (Fig. 3). The structures from molecular
modeling study were matched well with the experimental
ones from NMR and X-ray (Fig. 4).
˚
3.15 and 3.68 A, respectively. The chemical shift of 7H
in the indole ring in NMR of 15 was 5.61 ppm,
In addition, there were NOEs between 5H (d, 8.80) in
the benzopyran and the CH3 protons (d, 1.35) in the
Table 2
The effects of KATP blockers on cardioprotection of compound 14
Compounds
LVDPꢄ/HR (%)
EDP (mmHg)
CFR (%)
TTC (min)
LDH (U gꢂ1
)
Vehicle
14 (10 mM)
15.5
67.9
17.7
55.3
25.7
52.0
83.4
91.6
93.5
19.8
23.7
18.6
33.6
20.3
33.7
14 (10 mM)ꢁ5-HD (100 mM)
/
In vitro cardioprotective effects were evaluated measuring the contractile function (LVDPꢄ
heart with or without KATP blocker, sodium 5-hydroxydecanoate (5-HD). Each value is an average of three determinations and within 9
/
HR), TTC, and LDH in the globally ischemic rat
10%.
/

466
S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ471
/
ester group of the major conformer of 14 (Fig. 2). The
distance between those carbons was estimated to be 4.0
in the Analytical Department of Korea Research
Institute of Chemical technology and C, H, and N
˚
A in the lowest energy conformer of compound 14 by
molecular mechanic calculation which is very close to
values were found to be within 9
values.
/
0.4% of the calculated
˚
3.95 A measured in the X-ray structure (Fig. 3). The
corresponding distance in the crystal structure of
˚
compound 15, an inactive compound, was 6.99 A. It is
6.2. General procedure for the syntheses of indoline-2-
carboxylic acid esters (5ꢀ8)
/
hard to know whether the major conformer of com-
pound 14 is the active one or not, but this distance
parameter could be used to differentiate the active
compounds from inactive compounds.
Due to the existence of rotamers, it is not appropriate
to correlate the conformational structures with the
biological activities from this study. But it is apparent
from NMR, X-ray, and molecular modeling analyses
that two aryl rings in the active compound 14 seem to be
perpendicular and closely located. We will continue the
To a solution of indoline-2-carboxylic acid (163 mg, 1
mmol) in an appropriate alcohol (40 mL) were added
SOCl2 (100 mL, 1.37 mmol) dropwise. The mixture was
heated at reflux for 5 h, and all volatiles were removed in
vacuo. The residue was diluted with water and extracted
with CH2Cl2 (10 mLꢄ3), and the organic layers were
/
combined. The organic layer was dried (MgSO4),
filtered, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatogra-
study to elucidate the structureꢀactivity relationships in
/
phy (hexane:ethyl acetateꢃ4:1).
/
this and related series.
6.2.1. Indoline-2-carboxylic acid ethyl ester (5)
1H-NMR (CDCl3) d; 1.29 (t, 3H, Jꢃ
7.3 Hz), 3.32
(m, 2H), 4.21 (q, 2H, Jꢃ7.2 Hz), 4.39 (dd, 1H, Jꢃ9.5,
6.1 Hz), 6.72 (m, 2H), 6.98ꢀ
7.12 (m, 2H); MS 191 [Mꢁ].
5. Conclusions
/
/
/
We prepared a series of optically active benzopyranyl
indoline and indole derivatives in which various esters,
acid, ethers and lactones are substituted at the 2-
position and evaluated their cardioprotective and vasor-
elaxation activities. Two enatiomers of indoline-2-ethyl
esters 14 and 18 showed the best cardioprotective
activities both in vitro and in vivo among those, while
/
6.2.2. Indoline-2-carboxylic acid methyl ester (6)
1H-NMR (CDCl3) d 3.38 (m, 1H), 3.74 (s, 3H), 4.36ꢀ
4.47 (m, 2H), 6.72 (m, 2H), 7.01ꢀ7.13 (m, 2H); MS 177
[Mꢁ].
/
/
their vasorelaxation potencies were very weak (IC50ꢀ30
/
mM). The cardioprotective effect of 14 was completely
reversed by 5-HD, a selective mitochondrial KATP
blocker, indicating its probable protective mechanism
through the mitochodrial KATP opening.
6.2.3. Indoline-2-carboxylic acid isopropyl ester (7)
1H-NMR (CDCl3) d 1.25 (s, 3H), 1.28 (s, 3H), 3.12ꢀ
3.54 (m, 2H), 4.48 (dd, 1H, Jꢃ10.1, 5.9 Hz), 5.08 (m,
1H), 6.83ꢀ6.96 (m, 2H), 7.08ꢀ7.16 (m, 2H);); MS 205
[Mꢁ].
/
/
/
/
6. Experimental
6.3. N-(t-butyloxycarbonyl)indoline-2-carboxylic acid
(8)
6.1. Chemistry
Melting points (m.p.) were determined on a capillary
m.p. apparatus and are uncorrected. Anhydrous sol-
vents were dried by conventional methods. Reagents of
commercial quality were used from freshly opened
containers unless otherwise stated. For purification of
products by column chromatography, Merck Silica gel
To the solution of indoline-2-carboxylic acid (5 g, 42.8
mmol) in tetrahydrofuran (THF) (50 mL) were added
triethylamine (12.8 mL, 91.8 mmol) and di-t-butyl
dicarbonate (13.4 g, 6.4 mmol). The mixture was stirred
at room temperature (r.t.) overnight, and concentrated
under reduced pressure. The residue was dissolved in 1
N NaOH (20 mL) and washed with ether. The water
layer was acidified with c-HCl, and then extracted with
60 (230ꢀ
400 mesh) was used. 1H-NMR spectra were
/
recorded on a Varian Gemini 200 with TMS as an
internal standard. Chemical shifts are reported in d
(ppm). 2-D NMR experiments were performed using
Bruker 600 MHz spectrometer at 25 8C. Mixing times of
400 ms for NOESY were used. Mass spectra were
obtained with a JEOL JMS-DX 303 instrument by
using electron impact or chemical ionization techniques.
Elemental analyses of key compounds were performed
ether (15 mLꢄ3). The organic layer was dried
/
(Na2SO4), filtered, and concentrated under reduced
1
pressure to give a white solid (7.6 g, 94.2%): H-NMR
(CDCl3) d 1.52 (s, 9H), 3.12 (dd, 1H, Jꢃ
3.52 (dd, 1H, Jꢃ14.8, 11.6 Hz), 3.81 (dd, 1H, Jꢃ
3.2 Hz), 6.93 (dd, 1H, Jꢃ7.8, 7.4 Hz), 7.15 (m, 2H),
7.88 (brd, 1H); MS 263 [Mꢁ].
/14.7, 3.2 Hz),
/
/11.6,
/

S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ
/
471
467
6.4. N-(t-butyloxycarbonyl)-2-hydroxymethylindoline
(9)
6.7. General procedure for the syntheses of compounds
(13-23)
The reaction mixture of an amine 5ꢀ7 (1 mmol),
/
To a solution of acid 8 (500 mg, 1.9 mmol) in THF (15
appropriate optically pure epoxide (282 mg, 1 mmol)
[20], and Mg(ClO4)2 (224 mg, 1 mmol) in CH3CN (0.5
mL) was stirred at r.t. for 6 h, and concentrated under
reduced pressure. Resulting two diastereomers were
separated by silica gel column chromatography (hex-
mL) was added 1 N BH3×
/
THF (7.6 mL) dropwise at
0 8C via syringe. The solution was stirred at r.t. over-
night, then all volatiles were removed in vacuo. The
residue was dissolved in ethyl acetate (15 mL) and
washed with 0.5 N NaOH (10 mLꢄ3). The organic
layer was dried (Na2SO4), filtered, and concentrated in
vacuo. The residue was purified by silica gel column
/
ane:ethyl acetateꢃ
(75ꢀ94%). Some foams were crystallized from an
appropriate solvent.
/4:1) to give a pale yellow foam each
/
chromatography (hexane:ethyl acetateꢃ
off-white solid (450 mg, 96%): 1H-NMR (CDCl3) d 1.58
(s, 9H), 2.83 (brd, 1H), 3.31 (dd, 1H, Jꢃ16.3, 10.1 Hz),
3.58ꢀ3.80 (m, 2H), 4.56 (m, 1H), 6.94 (dd, 1H, J
7.5, 7.5 Hz), 7.10ꢀ7.19 (m, 2H), 7.56 (br, 1H); MS
249 [Mꢁ].
/
1:1) to give an
6.7.1. 1-[(2S,3R,4S)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2R)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester
/
/
ꢃ
/
/
(13)
1
M.p.: 126ꢀ
MHz) d 0.94 (t, 1.2H, Jꢃ
Hz), 1.64 (s, 3H), 3.20 (d, 0.4H, Jꢃ
0.6H, Jꢃ11.1, 10.2 Hz), 3.33 (s, 1.8H), 3.40 (m, 1.6H),
3.49 (s, 1.2H), 3.51 (s, 1.8H), 3.58 (m, 0.4H), 3.65 (m,
0.4H), 3.72 (dd, 0.6H, Jꢃ14.6, 10.3 Hz), 4.28 (m, 0.4H),
4.34 (m, 1.2H), 4.38 (m, 0.6H), 4.55ꢀ4.63 (m, 1.8H),
4.72 (s, 0.6H), 4.90 (dd, 0.6H, Jꢃ12.0, 10.3 Hz), 4.98 (d,
0.4H, Jꢃ10.3 Hz), 5.25 (s, 0.6H), 5.79 (d, 0.6H, Jꢃ8.3
Hz), 6.56ꢀ
8.9 Hz), 6.94 (d, 0.6H, Jꢃ
/
127 8C (ethanol); H-NMR (CDCl3, 600
/
7.1 Hz), 1.36 (t, 1.8H, Jꢃ
/7.1
/10.0 Hz), 3.26 (dd,
6.5. N-(t-butyloxycarbonyl)-2-methoxymethylindoline
(10)
/
/
To a solution of alcohol 9 (400 mg, 1.6 mmol) in THF
(15 mL) was added NaH (60% in oil, 84 mg, 2.1 mmol)
at 0 8C. After stirring for 30 min, iodomethane (0.2 mL,
3.22 mmol) was added to the mixture. The reaction was
stirred at r.t. overnight, and all volatiles were removed
under reduced pressure. The residue was dissolved in
dichloromethane (15 mL), washed with water (15 mL),
dried (Na2SO4), filtered, and concentrated under re-
duced pressure. The residue was purified by silica gel
/
/
/
/
/
6.63 (m, 1H), 6.73 (m, 1H), 6.87 (d, 0.4H, Jꢃ
/
/
9.0 Hz), 7.05 (d, 0.6H, Jꢃ
/7.3
Hz), 7.12 (m, 0.8H), 7.61 (s, 0.6H), 7.95 (dd, 0.6H, Jꢃ
/
9.4, 2.2 Hz), 7.97 (d, 0.4H, Jꢃ10.8 Hz), 8.27 (s, 0.4H);
/
MS 472 [Mꢁ]; Anal. (C24H28N2O8) C, H, N.
6.7.2. 1-[(2S,3R,4S)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2S)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester
column chromatography (hexane:ethyl acetateꢃ
to give an off-white solid (260 mg, 61%): 1H-NMR
(CDCl3) d 1.57 (s, 9H), 2.98 (dd, 1H, Jꢃ10.9, 2.4 Hz),
3.19ꢀ3.33 (m, 2H), 3.35 (s, 3H) 3.60 (dd, 1H, Jꢃ10.3,
3.8 Hz), 4.57 (m, 1H), 6.97 (dd, 1H, Jꢃ7.3, 7.3 Hz),
7.11ꢀ
7.18 (m, 2H), 7.63 (br, 1H); MS 263 [Mꢁ].
/
10:1)
/
(14)
M.p.: 137ꢀ
/
/
1
/
138 8C (ethanol); H-NMR (CDCl3, 600
/
MHz) d 1.26 (m, 1.2H), 1.35 (m, 1.8H), 1.56 (s, 3H),
3.26 (br, 0.6H), 3.37 (s, 2.4H), 3.37 (m, 0.4H), 3.41 (m,
0.4H), 3.48 (s, 1.8H), 3.48 (m, 0.4H), 3.50 (s, 1.8H), 3.58
/
(m, 1H), 4.12 (q, 0.8H, Jꢃ6.8 Hz), 4.22 (br, 0.6H), 4.34
(m, 1.2H), 4.41 (m, 1.4H), 4.68 (m, 0.6H), 4.74 (s, 0.6H),
5.14 (br, 0.6H), 5.26 (br, 0.4H), 5.53 (brd, 0.6H), 6.60
/
6.6. 2-Methoxymethylindoline (11)
The solution of compound 10 (260 mg, 1 mmol) in
dichloromethane (7 mL) and TFA (0.8 mL) was stirred
for 20 h at r.t., and all volatiles were removed in vacuo.
The residue was dissolved in dichloromethane (10 mL)
and carefully washed with saturated NaHCO3 solution.
The organic layer was dried (Na2SO4), filtered, and
concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (hexa-
(m, 1H), 6.77 (m, 1H), 6.93 (d, 1H, Jꢃ9.1 Hz), 7.04 (m,
/
0.6H), 7.12 (m, 0.8H), 7.76 (br, 0.4H), 8.01 (br, 0.4H),
8.07 (m, 0.6H), 8.80 (s, 0.6H); MS 472 [Mꢁ]; Anal.
(C24H28N2O8) C, H, N.
6.7.3. 1-[(2S,3S,4R)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2R)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester
ne:ethyl acetateꢃ
/
4:1) to give an off-white solid (140 mg,
87%): H-NMR (CDCl3) d 2. 69 (dd, 1H, Jꢃ15.7, 6.7
Hz), 3.14 (dd, 1H, Jꢃ15.7, 9.0 Hz), 3.39ꢀ3.44 (m, 5H),
4.13 (m, 1H), 6.61ꢀ6.74 (m, 1H), 6.99ꢀ7.15 (m, 2H); MS
163 [Mꢁ].
(15)
1
1
/
M.p.: 196ꢀ
(CDCl3) d 1.23ꢀ
3.62 (s, 3H), 4.35 (q, 2H), 4.80 (m, 1H), 5.16 (m, 1H),
5.61 (d, 0.6H, Jꢃ8.3 Hz), 6.6ꢀ7.2 (m, 4.4H), 7.82 (d,
/
197 8C (ethyl acetateꢀ
/
hexane); H-NMR
/
/
/
1.47 (m, 6H), 3.28 (m, 2H), 3.55 (s, 3H),
/
/
/
/

468
S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ471
/
0.4H), 8.08 (m, 1H), 8.90 (d, 0.6H, Jꢃ
/
2.0 Hz); MS 472
6.7.8. 1-[(2S,3R,4S)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2S)-2,3-dihydro-1H-indole-2-carboxylic acid methyl
ester (20)
[Mꢁ]; Anal. (C24H28N2O8) C, H, N.
M.p.: 99ꢀ
/
100 8C (ethyl acetateꢀ
hexane); 1H-NMR
(CDCl3) d 1.62 (s, 3H), 3.38 (m, 2H), 3.64 (s, 3H), 3.67
(s, 3H), 3.89 (s, 3H), 4.41 (m, 1H), 4.62 (m, 1H), 5.06 (s,
/
6.7.4. 1-[(2S,3S,4R)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2S)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester
1H), 5.52 (brd, 0.7H), 6.5ꢀ6.8 (m, 1.6H), 6.81 (d, 1H,
/
(16)
M.p.: 115ꢀ
Jꢃ8.0 Hz), 6.92 (m, 1H), 7.64 (m, 0.7H), 8.08 (m, 1H),
/
1
hexane); H-NMR
/
118 8C (ethyl acetateꢀ
/
8.26 (brd, 0.3H), 8.81 (brd, 0.7 H); MS 458 [Mꢁ]; Anal.
(C23H26N2O8) C, H, N.
(CDCl3) d 1.35 (m, 6H), 3.38 (m, 2H), 3.64 (s, 3H), 3.66
(s, 3H), 4.29 (q, 2H, Jꢃ7.1 Hz), 4.68 (m, 1H), 4.91 (m,
1H), 5.22 (s, 1H), 5.88 (d, 0.7H, Jꢃ7.3 Hz), 6.6ꢀ7.2 (m,
4.3H), 7.68 (d, 0.7H, Jꢃ1.6 Hz), 8.02 (m, 1H), 8.34 (d,
0.3H, Jꢃ
1.7 Hz); MS 472 [Mꢁ]; Anal. (C24H28N2O8)
C, H, N.
/
/
/
6.7.9. 1-[(2S,3S,4R)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2R)-2,3-dihydro-1H-indole-2-carboxylic acid methyl
/
/
ester (21)
M.p.: 174ꢀ
1
hexane); H-NMR
/
176 8C (ethyl acetateꢀ
/
(CDCl3) d 1.32 (s, 3H), 3.28 (m, 2H), 3.54 (s, 3H), 3.61
(s, 3H), 3.86 (s, 3H), 4.41 (m, 1H), 4.79 (d, 1H, Jꢃ9.1
Hz), 5.11 (m, 1H), 5.59 (d, 0.6H, Jꢃ7.8 Hz), 6.60ꢀ7.20
(m, 4.4H), 7.81 (d, 0.4H, Jꢃ2.1 Hz), 8.08 (m, 1H), 8.84
(d, 0.6H, Jꢃ
2.2 Hz); MS 458 [Mꢁ]; Anal.
6.7.5. 1-[(2R,3S,4R)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2S)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester
/
/
/
/
(17)
/
1
M.p.: 125ꢀ
(CDCl3) d 0.94 (t, 1.2H, Jꢃ
7.0 Hz), 1.64 (s, 3H), 3.1ꢀ3.3 (m, 1H), 3.33 (s, 1.8H),
3.41 (m, 1.6H), 3.49 (s, 1.2H), 3.51 (s, 1.8H), 3.55ꢀ3.8
(m, 1.4H), 4.25ꢀ5.0 (m, 5.6H), 5.25 (s, 0.6H), 5.78 (d,
0.6H, Jꢃ8.0 Hz), 6.5ꢀ7.2 (m, 4.4H), 7.61 (s, 0.6H),
7.9ꢀ
8.0 (m, 1H), 8.27 (br, 0.4H); MS 472 [Mꢁ]; Anal.
/
127 8C (ethyl acetateꢀ
/
hexane); H-NMR
(C23H26N2O8) C, H, N.
/
7.2 Hz), 1.36 (t, 1.8H, Jꢃ
/
/
6.7.10. 1-[(2S,3S,4R)-3,4-Dihydro-2-
/
dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-
benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-
/
/
/
carboxylic acid methyl ester (22)
1
hexane); H-NMR
/
M.p.: 108ꢀ
/
109 8C (ethyl acetateꢀ
/
(C24H28N2O8) C, H, N.
(CDCl3) d 1.57 (s, 3H), 3.26 (m, 1H), 3.46 (m, 1H), 3.64
(s, 3H), 3.68 (s, 3H), 3.89 (s, 3H), 4.30 (m, 1H), 4.60 (s,
1H), 4.69 (d, 1H, Jꢃ
1H), 6.72 (m, 1H), 6.94 (m, 1H), 7.08 (m, 2H), 7.68 (d,
0.3H, Jꢃ1.9 Hz), 8.01 (dd, 1H, Jꢃ7.9, 2.0 Hz), 8.39 (d,
0.7H, Jꢃ
2.1 Hz); MS 458 [Mꢁ]; Anal. (C23H26N2O8)
C, H, N.
/
10.1 Hz), 4.91 (m, 1H), 6.63 (m,
6.7.6. 1-[(2R,3S,4R)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2R)-2,3-dihydro-1H-indole-2-carboxylic acid ethyl ester
/
/
/
(18)
1
M.p.: 135ꢀ
(CDCl3) d 1.2ꢀ
9.4H), 4.1ꢀ4.5 (m, 4H), 4.6ꢀ
1H), 5.54 (br, 0.6H), 6.6ꢀ6.8 (m, 2H), 6.93 (d, 1H, Jꢃ
8.1 (m,
/
137 8C (ethyl acetateꢀ
/
hexane); H-NMR
6.7.11. 1-[(2S,3R,4S)-3,4-Dihydro-2-
/
1.4 (m, 3H), 1.56 (s, 3H), 3.2ꢀ
/3.6 (m,
dimethoxymethyl-3-hydroxy-2-methyl-6-nitro-2H-1-
benzopyran-4-yl]-(2S)-2,3-dihydro-1H-indole-2-
carboxylic acid isopropyl ester (23)
/
/
4.8 (m, 1H), 5.1ꢀ5.3 (br,
/
/
/
9.2 Hz), 7.0ꢀ
/
7.2 (m, 1.4H), 7.77 (br, 0.4H), 8.0ꢀ
/
M.p.: 95ꢀ
/
96 8C (ethyl acetateꢀ
hexane); 1H-NMR
(CDCl3) d 1.27 (m, 6H), 1.57 (s, 3H), 3.37 (m, 1H),
3.48 (s, 3H), 3.49 (s, 3H), 3.58 (m, 1H), 4.02 (m, 1H),
/
1H), 8.80 (brs, 0.6H); MS 472 [Mꢁ]; Anal.
(C24H28N2O8) C, H, N.
4.13 (d, 1H, Jꢃ
(d, 1H, Jꢃ6.9 Hz), 5.58 (m, 0.3H), 6.6ꢀ
6.94 (d, 1H, Jꢃ7.8 Hz), 7.06 (m, 1H), 7.6ꢀ
8.09 (dd, 1H, Jꢃ9.1, 2.6 Hz), 8.42 (d, 0.7H, Jꢃ
MS 486 [Mꢁ); Anal. (C25H30N2O8) C, H, N.
/
7.1 Hz), 4.44 (s, 1H), 4.68 (m, 1H), 5.12
6.8 (m, 2H),
7.7 (m, 1H),
2.6 Hz);
/
/
6.7.7. 1-[(2S,3R,4S)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2R)-2,3-dihydro-1H-indole-2-carboxylic acid methyl
ester (19)
/
/
/
/
M.p.: 96ꢀ
(CDCl3) d 1.64 (s, 3H), 3.10ꢀ
3H), 4.58 (m, 1H), 4.85ꢀ5.16 (m, 2H), 5.79 (d, 0.6H, Jꢃ
8.2 Hz), 6.4ꢀ7.2 (m, 4.4H), 7.64 (d, 0.4H, Jꢃ2.0 Hz),
8.04 (m, 1H), 8.31 (d, 0.6H, Jꢃ
1.9 Hz); MS 458 [Mꢁ];
Anal. (C23H26N2O8) C, H, N.
/
97 8C (ethyl acetateꢀ
/
hexane); 1H-NMR
6.8. 1-[(2S,3R,4S)-3,4-Dihydro-2-dimethoxymethyl-3-
hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-(2S)-
2,3-dihydro-1H-indole-2-carboxylic acid (24)
/
3.60 (m, 8H), 3.95 (s,
/
/
/
/
/
To the solution of KOH (29 mg, 0.5 mmol) in water
(30 mM) and ethanol (1.1 mL) was added, the compound

S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ
/
471
469
14 (170 mg, 0.36 mmol) and the reaction mixture was
stirred at r.t. for 6 h. The reaction was diluted with water
(10 mL) and acidified with c-HCl, then extracted with
1H), 7.70 (m, 2H), 8.11 (dd, 1H, Jꢃ
/
7.9, 2.4 Hz); MS
456 [Mꢁ]; Anal. (C23H24N2O8) C, H, N.
ethyl acetate (10 mLꢄ
/
3). The organic layer was dried
6.9.4. 1-[(2S, 3S, 4R)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-
indole-2-carboxylic acid methyl ester (28)
(Na2SO4), filtered, and concentrated under reduced
pressure to give an off-white solid (150 mg, 94%):
m.p.: ꢀ
/
200 8C (decomposed); 1H-NMR (CD3OD) d
M.p.: 194ꢀ
1.49 (s, 3H), 3.36 (d, 1H, Jꢃ
3.62 (s, 3H), 3.98 (s, 3H), 4.48 (s, 1H), 4.76 (dd, 1H, Jꢃ
10.2, 2.2 Hz), 6.59 (d, 1H, Jꢃ8.0 Hz), 7.08 (m, 3H),
8.9, 2.1 Hz);
/
195 8C (ethanol); 1H-NMR (CDCl3) d
1.47 (s, 3H), 3.31 (s, 3H), 3.38 (s, 3H), 3.51 (m, 2H), 4.34
(m, 1H), 4.65 (s, 1H), 5.04 (m, 1H), 5.73 (m, 1H), 6.56
(m, 1H), 6.73 (m, 1H), 6.91 (d, 1H), 6.97 (m, 2H), 8.04
/2.3 Hz), 3.60 (s, 3H),
/
/
(dd, 1H, Jꢃ
/
7.6, 1.4 Hz), 8.66 (m, 1H); MS 444 [Mꢁ];
7.45 (s, 1H), 7.72 (m, 2H), 8.11 (dd, 1H, Jꢃ
/
Anal. C22H24N2O8) C, H, N.
MS 456 [Mꢁ]; Anal. (C23H24N2O8) C, H, N.
6.10. (6S, 6aR, 8aS, 13cS)-Dimethoxymethyl-6-
methyl-2-nitro-6a,8a,9,13c-tetrahydro-6H-5,7-dioxa-13b-
aza-indeno[2,1-c]phenanthren-8-one (29)
6.9. General procedure for the syntheses of compounds
(25ꢀ28)
/
To the solution of indoline compounds (0.25 mmol) in
benzene (10 mL) was added DDQ (100 mg, 0.44 mmol)
and the reaction was stirred at r.t. for 6 h. The reaction
mixture was diluted with ethyl acetate (10 mL) and
washed with 10% NaOH (10 mL) and water (10 mL).
The organic layer was dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by silica
To the solution of compound 20 (92 mg, 0.2 mmol) in
dichloromethane (4 mL) were added diisopropylamine
(30 mL, 0.22 mmol) and 1.8 M solution of diethyl
aluminum chloride in toluene (110 mL, 0.2 mmol) at ꢂ
/
20 8C. The reaction mixture was stirred at ꢂ20 8C for 2
/
h, then at r.t. for 4 h, and poured over ice-cold saturated
solution of NaHCO3 (15 mL). The mixture was ex-
gel column chromatography (hexane:ethyl acetateꢃ
/2:1)
tracted with ethyl acetate (10 mLꢄ3), dried (Na2SO4),
/
to give an off-white solid in 85ꢀ91% yields.
/
filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexa-
6.9.1. 1-[(2S, 3R, 4S)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-
indole-2-carboxylic acid ethyl ester (25)
ne:ethyl acetateꢃ
mg, 85%): m.p.: 117ꢀ
/
4:1) to yield a pale yellow solid (72
/
118 8C (ethyl acetateꢀ
/
hexane); 1H-
NMR (CDCl3) d 1.54 (s, 3H), 3.37 (s, 3H), 3.41 (m, 1H),
3.48 (s, 3H), 3.54 (m, 1H), 4.58 (m, 1H), 4.67 (m, 1H),
M.p.: 184ꢀ
1.44 (t, 3H, Jꢃ
6.0 Hz), 3.52 (s, 3H), 3.55 (s, 3H), 4.27ꢀ
4.61 (s, 1H), 6.46 (d, 1H, Jꢃ8.1 Hz), 6.98ꢀ
7.25 (d, 1H, Jꢃ8.4 Hz), 7.45 (s, 1H), 7.68 (m, 1H), 8.12
(dd, 1H, Jꢃ
7.3, 0.8 Hz); MS 470 [Mꢁ]; Anal.
/
185 8C (ethanol); 1H-NMR (CDCl3) d
/
7.1 Hz), 1.62 (s, 3H), 3.47 (d, 2H, Jꢃ
4.42 (m, 3H),
7.12 (m, 3H),
/
5.14 (d, 1H), 5.81 (m, 1H), 6.62 (d, 1H, Jꢃ
(m, 2H), 7.05 (d, 1H, Jꢃ9.1 Hz), 7.17 (m, 1H), 8.13 (dd,
1H, Jꢃ9.8, 2.3 Hz), 8.46 (d, 1H, Jꢃ2.2 Hz); MS 426
[Mꢁ]; Anal. (C22H27N2O7) C, H, N.
/
8.1 Hz), 6.82
/
/
/
/
/
/
/
/
(C24H26N2O8) C, H, N.
6.11. (6S, 6aS, 8aR, 13cR)-Dimethoxymethyl-6-
methyl-2-nitro-6a,8a,9,13c-tetrahydro-6H-5,7-dioxa-13b-
aza-indeno[2,1-c]phenanthren-8-one (30)
6.9.2. 1-[(2S, 3S, 4R)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-
indole-2-carboxylic acid ethyl ester (26)
The same reaction for the preparation of compound
29 was employed using compound 21 as a starting
material, to give a pale yellow foam (69 mg, 81%): m.p.:
M.p.: 185ꢀ
1.45 (t, 3H, Jꢃ
2.2 Hz), 3.59 (s, 3H), 3.60 (s, 3H), 4.40 (q, 2H, Jꢃ
Hz), 4.46 (s, 1H), 4.75 (dd, 1H, Jꢃ10.2, 2.2 Hz), 6.55
(d, 1H, Jꢃ7.5 Hz), 7.07 (m, 3H), 7.45 (s, 1H), 7.73 (m,
2H), 8.10 (dd, 1H, Jꢃ
9.4, 2.4 Hz); MS 470 [Mꢁ]; Anal.
/
186 8C (ethanol); 1H-NMR (CDCl3) d
/
7.3 Hz), 1.47 (s, 3H), 3.36 (d, 2H, Jꢃ
/
1
hexane); H-NMR (CDCl3)
/
7.1
128ꢀ
/
129 8C (ethyl acetateꢀ
/
/
d 1.66 (s, 3H), 3.24 (m, 1H), 3.38 (s, 3H), 3.48 (m, 1H),
3.54 (s, 3H), 3.82 (d, 1H, Jꢃ11.4 Hz), 4.55 (d, 1H, Jꢃ
11.2 Hz), 4.57 (s, 1H), 4.79 (d, 1H, Jꢃ11.6 Hz), 6.61 (d,
/
/
/
/
/
(C24H26N2O8) C, H, N.
1H, Jꢃ7.7 Hz), 6.94 (m, 2H), 7.23 (m, 2H), 8.09 (m,
/
2H); MS 426 [Mꢁ]; Anal. (C22H27N2O7) C, H, N.
6.9.3. 1-[(2S, 3R, 4S)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-1H-
indole-2-carboxylic acid methyl ester (27)
6.12. 1-[(2S,3R,4S)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2R)-2,3-dihydro-2-methoxymethyl-1H-indole (31)
M.p.: 178ꢀ
1.63 (s, 3H), 3.48 (d, 1H, Jꢃ
3.56 (s, 3H), 3.96 (s, 3H), 4.36 (m, 1H), 4.62 (s, 1H), 6.47
(d, 1H, Jꢃ7.3 Hz), 7.06 (m, 2H), 7.33 (m, 1H), 7.46 (s,
/
179 8C (ethanol); 1H-NMR (CDCl3) d
/10.5 Hz), 3.54 (s, 3H),
The compound 31 and 32 were prepared by the
/
general procedure for 13ꢀ23 using compound 11 as a
/

470
S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ471
/
starting material. Resulting two diastereisomers were
separated by silica gel column chromatography (hex-
amine-contracted rat aorta. Experimental details of
method are described [20].
1
ane:ethyl acetateꢃ
NMR (CDCl3) d 1.56 (s, 3H), 2.9ꢀ
3H), 3.49 (s, 3H), 3.52ꢀ3.9 (m, 5H), 4.1ꢀ
4.43 (s, 1H), 4.60 (m, 1H), 4.90 (dd, 1H, Jꢃ
Hz), 5.52 (dd, 1H, Jꢃ7.0, 1.8 Hz), 6.55 (m, 1H), 6.72
(m, 1H), 7.0 (m, 2H), 8.06 (dd, 1H, Jꢃ8.8, 1.9 Hz), 8.47
(d, 1H, Jꢃ
1.9 Hz); MS 444 [Mꢁ]; Anal. (C23H28N2O7)
C, H, N.
/
3:1): m.p.: 94ꢀ
/
95 8C (ethanol); H-
/
3.1 (m, 2H), 3.44 (s,
4.3 (m, 2H),
11.2, 1.9
6.15.2. Cardioprotective activity in isolated ischemic rat
heart model
The anti-ischemic effects of the compounds on
isolated rat hearts were determined, according to the
published methods [25,26] after some modification.
/
/
/
/
/
/
Male Spragueꢀ
/
Dawley rats (300ꢀ450 g) were anesthe-
/
tized with sodium pentobarbital (100 mg kgꢂ1, intra-
peritoneal (i.p.)). The jugular vein was injected with
heparin (1000 U kgꢂ1) and then the trachea was
incubated. While rats were mechanically ventilated
with a rodent ventilator (Model 7025, Ugobasile, Italy),
their hearts were perfused in situ with oxygenated
6.13. 1-[(2S,3R,4S)-3,4-Dihydro-2-dimethoxymethyl-
3-hydroxy-2-methyl-6-nitro-2H-1-benzopyran-4-yl]-
(2S)-2,3-dihydro-2-methoxymethyl-1H-indole (32)
M.p.: 117ꢀ
1.63 (s, 3H), 2.9ꢀ
3H), 3.54ꢀ3.7 (m, 4H), 4.1ꢀ
4.90 (m, 1H), 5.64 (dd, 1H, Jꢃ
1H), 6.94 (m, 1H), 7.0ꢀ7.2 (m, 2H), 7.67 (d, 0.6H, Jꢃ
1.9 Hz); MS 444
/
118 8C (ethanol); 1H-NMR (CDCl3) d
3.3 (m, 2H), 3.41 (s, 3H), 3.51 (s,
4.45 (m, 3H), 4.64 (m, 1H),
11.5, 1.9 Hz), 6.56 (m,
modified KrebsꢀHenseleit bicarbonate buffer (described
/
/
herein) via retrograde aortic cannulation. The hearts
were then excised and quickly moved to a Langendorff
apparatus (H.S.E., Germany), where they were perfused
/
/
/
/
/
with oxygenated modified KrebsꢀHenseleit bicarbonate
/
1.9 Hz), 8.00 (m, 1H), 8.34 (d, 1H, Jꢃ
/
buffer containing (in mM) NaCl 116, NaHCO3 24.9,
KCl 4.7, MgSO4 1.1, KH2PO4 1.17, CaCl2 2.52, glucose
8.32 and pyruvate 2.0 at a constant perfusion pressure
(85 mmHg). A latex balloon filled with solution
[Mꢁ]; Anal. (C23H28N2O7) C, H, N.
6.14. Molecular modeling
(ethanol: waterꢃ1:1 (v/v)) and attached to a metal
/
All molecular modeling techniques and conforma-
tional studies described herein were performed on
Silicongraphics workstation (Origin R1000, 256 Mbytes
memory, 2 CPU, 180 MHz IP27 processor) and PC,
using SYBYL (v. 6.7) (Tripos Associate Inc., 1699 S.
Hanley Road, Suite 303, St. Louis, MO 63144-2913,
USA) and CACHE (Compute-Aided Chemistry, Oxford
Molecular Group Inc., 11350 McLormick Road, Ex-
ecutive Plaza III, Suite 1100, Hunt Valley, MD 21030)
programs.
Initial geometry for conformational analysis was
obtained from Tripos force field using distance-depen-
dent dielectric function (The distance-dependent value
was used for a dielectric constant to simulate the effect
of solvent.), and 0.05 kcal molꢂ1 energy gradient
convergence. In order to obtain low energy conformers,
random search was performed. Options for random
search were 3000 iteration cycles, 100 kcal molꢂ1 energy
cut-off, and chirality checking. After we selected low
energy conformers, the geometry was re-optimized using
semi-empirical method with PM3 charge. And using
MM3 force field in CACHE program, optimized map was
calculated, rotating torsion angle, t1 between chromane
and indole ring, and t2 between indole ring and ester side
chain by 58 increments.
cannula was placed in the left ventricle through pul-
monary vein and connected to a Isotec pressure
transducer (H.S.E., Germany) for measurement of left
ventricular pressure (LVP). The hearts were allowed to
equilibrate for 15 min, at which time left ventricular end
diastolic pressure (LVEDP) was adjusted to 5 mm Hg
and this balloon volume was maintained throughout the
experiment. Then, baseline contractile function, HR,
and coronary flow (extra corporeal electromagnetic flow
probe, Narco Bio-System, USA) were measured. Car-
diac contractile function was determined by multiplying
HR by LVDP, calculated by subtracting LVEDP from
left ventricular systolic pressure (LVSP). Throughout
the experiment, all these parameters were measured, and
calculated before and 10 min after pretreatment with
each compound and 30 min after the onset of reperfu-
sion with buffer. Data on reperfusion were further
expressed as the percentage to pretreatment. After
stabilization for 15 min, the hearts were pretreated for
10 min with compounds (10 mM, 0.04% DMSO) or
vehicle (0.04% DMSO) before onset of global ischemia.
Test compounds included in the perfusate were admi-
nistered directly into the oxygenator of the Langendorff
apparatus. Then the hearts were subjected to global
ischemia by completely shutting off the perfusate for 30
min. Severity of ischemia was determined as the TTC
(min) during global ischemia in which the first 5 mmHg
increase in EDP was observed. Then, the hearts were
reperfused and, 30 min later, contractile function
6.15. Pharmacology
6.15.1. Relaxation of methoxamine contracted rat aorta
To measure the peripheral vasodilation potencies, we
determined IC50 values for relaxation of the methox-
(LVDPꢄ/HR) and cumulative reperfusion LDH release
were measured. LDH was measured as a sensitive index

S. Lee et al. / European Journal of Medicinal Chemistry 38 (2003) 459ꢀ
/
471
471
[7] C. Napoli, A. Pinto, G. Cirino, Pharmacol. Therapeut. 88 (2000)
311ꢀ331.
[8] G.J. Gross, R.M. Fryer, Circ. Res. 84 (1999) 973ꢀ
[9] E. Kevelaitis, J. Peynet, C. Mouas, J.M. Launay, P. Menasche,
Circulation 99 (1999) 3079ꢀ3085.
[10] N. Inagaki, T. Gonoi, J.-P. Clement, C.-Z. Wang, L. Aguilar-
Bryan, J. Bryan, S. Seino, Neuron 16 (1996) 1011ꢀ1017.
[11] L. Aguilar-Bryan, J.-P. Clement, G. Gonzalez, K. Kunjilwar, A.
Babenko, J. Bryan, Physiol. Rev. 78 (1998) 227ꢀ245.
[12] M.J. Coghlan, W.A. Carroll, M. Gopalakrishnan, J. Med. Chem.
44 (2001) 1627ꢀ1653.
[13] G. Edewards, A.H. Weston, Expert Opin. Invest. Drugs 5 (1996)
1453ꢀ1464.
[14] M. Gopalakrishnan, R.A. Janis, D.J. Triggle, Drugs Dev. Res. 28
(1993) 95ꢀ127.
[15] K.S. Atwal, G.J. Grover, Curr. Pharm. Design 2 (1996) 585ꢀ
for loss of cell viability with a kit supplied by Boerhinger
Mannheim.
/
/
979.
6.15.3. In vivo cardioprotective activity in ischemic
myocardium rat model
/
/
To measure cardioprotective potencies in vivo, we
determined the % ratio of myocardial IZ/AAR (%) in
ischemic myocardium rat model [27]. Experimental
details of method are described [20]. Briefly, male rats
/
/
(350ꢀ450 g) were anesthetized with pentobarbital (75
/
mg kgꢂ1, i.p.). After a left thoracotomy operation, the
rats were allowed to stabilize for 20 min. Vehicles or test
compounds were administered into the rats via the
catheter inserted into the femoral vein at 30 min before
the occlusion. Left ventricle occlusion was maintained
for 45 min, following reperfusion for 90 min. The
coronary artery was reoccluded and 2 mL of a 1%
Evans blue was i.v. injected. Rats were then sacrificed by
i.v. injection of pentobarbital, and the hearts were
removed. The slices of the left ventricles were analyzed
using a Hi-scope installed with an image analyzing
program (Image Pro Plus) to calculate the AAR, and
then 1,2,3-triphenyltetrazolium chloride staining to
obtain the infarct zone area.
/
/
/
595.
[16] K.S. Atwal, G. J. Grover, S.Z. Ahmed, F.N. Ferrara, T.W.
Harper, K.S. Kim, P.G. Sleph, S. Dzwonczyk, D.E. Normandin,
J. Med. Chem. 36 (1993) 3971ꢀ
[17] G.J. Grover, J.R. McCullough, A.J. D’Alonzo, C.A. Sargent,
K.S. Atwal, J. Cardiovasc. Pharmacol. 25 (1995) 40ꢀ50.
[18] K.S. Atwal, G.J. Grover, P.G. Sleph, S. Dzwonczyk, A.J. Baird,
D.E. Normandin, J. Med. Chem. 38 (1995) 3236ꢀ3245.
/
3974.
/
/
[19] G.C. Rovnyak, S.Z. Ahmed, C.Z. Ding, S. Dzwonczyk, F.N.
Ferrara, W.G. Humphreys, G.J. Grove, D. Santafianos, K.S.
Atwal, A.J. Baird, L.G. McLaughlin, D.E. Normandin, P.G.
Sleph, S.C. Traeger, J. Med. Chem. 40 (1997) 24ꢀ34.
/
[20] S. Yoo, K.Y. Yi, S. Lee, J. Suh, N. Kim, B. Lee, H.W. Seo, S.-O.
Kim, D.-H. Lee, H. Lim, H.S. Shin, J. Med. Chem. 44 (2001)
4207ꢀ
[21] H.S. Shin, H.W. Seo, J.H. Oh, B.H. Lee, Arzneim.-Forsch./Drug
Res. 48 (II) (1998) 969ꢀ978.
[22] H.S. Shin, H.W. Seo, S.E. Yoo, B.H. Lee, Pharmacology 56
(1998) 111ꢀ124.
[23] M. Chini, P. Crotti, F. Macchia, Tetrahedron Lett. 31 (1990)
4661ꢀ4664.
[24] T.A. Engles, S.P. Meduna, K.O. Latessa, W. Chai, J. Org. Chem.
61 (1996) 8598ꢀ8603.
[25] C.-M. Yu, M.-S. Choi, J.K. Lee, S.-K. Yoon, J. Org. Chem. 62
(1997) 6687ꢀ6689.
[26] K.S. Atwal, G.J. Grover, F.N. Ferrara, S.Z. Ahmed, P.G. Sleph,
S. Dzwonczyk, D.E. Normandin, J. Med. Chem. 38 (1995) 1966ꢀ
/
4215.
Acknowledgements
/
/
We are grateful to the Ministry of Science and
Technology of Korea for financial support of this
research.
/
/
/
References
/
[1] C.E. Murry, R.B. Jennings, Circulation 74 (1986) 1124ꢀ
[2] J. Chen, R. Simon, Neurology 48 (1997) 306ꢀ311.
[3] A. Hawaleshka, E. Jacobson, Can. J. Anaesth. 45 (1998) 670ꢀ
[4] S.B. Roth, Li, P.S. Rosenbaum, H. Gupta, I.H. Goldstein, K.M.
Maxwell, J.M. Gidday, Invest. Ophthalmol. Vis. Sci. 39 (1998)
/
1136.
1973.
[27] G.J. Grover, J. Newburger, P.G. Sleph, S. Dzwonczyk, S.C.
Taylor, S.Z. Ahmed, K.S. Atwal, J. Pharmacol. Exp. Ther. 257
/
/682.
(1991) 156ꢀ162.
/
[28] A.J. D’Alonzo, R.B. Darbenzio, J.C. Sewter, T.A. Hess, G.J.
777ꢀ
[5] M. Ettaiche, C. Heurteaux, N. Blondeau, M. Borsotto, N. Tinel,
M. Lazdunski, Brain Res. 890 (2001) 118ꢀ129.
[6] M.W. Riepe, A.C. Ludolph, Mol. Cell Biochem. 174 (1997) 249ꢀ
254.
/
785.
Grover, P.G. Sleph, D.E. Normandin, N.J. Lodge, Eur. J.
Pharmacol. 294 (1995) 271ꢀ
[29] J.Y. Lee, R.B. Warner, A.L. Adler, T.J. Opgenorth, Pharmacol-
ogy 49 (1994) 319ꢀ324.
/
280.
/
/
/