Stereoselective synthesis of 1-methoxyspiroindoline phytoalexins and their amino analogues

Article abstract of DOI:10.1016/j.tetasy.2014.07.006

The stereoselective synthesis of the spiroindoline phytoalexin (R)-(+)-1-methoxyspirobrassinin and its unnatural (S)-(-)-enantiomer were achieved by the bromine-induced spirocyclization of 1-methoxybrassinin using chiral auxiliaries (+)- and (-)-menthol,

Full text of DOI:10.1016/j.tetasy.2014.07.006

Tetrahedron: Asymmetry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Stereoselective synthesis of 1-methoxyspiroindoline phytoalexins
and their amino analogues
a
a
a,
b
ˇ a
⇑
ˇ
Aneta Salayová , Zuzana Kudlicková , Matej Baláz , Mariana Budovská , Martina Pilátová ,
b
c
d
d
ˇ
ˇ
Ján Mojziš , Kenji Monde , Ján Petrovaj , Tatána Gondová
a Department of Organic Chemistry, Institute of Chemical Sciences, Faculty of Science, P.J. Šafárik University, Moyzesova 11, 040 01 Košice, Slovak Republic
b Department of Pharmacology, Faculty of Medicine, P.J. Šafárik University, SNP 1, 040 66 Košice, Slovak Republic
c Division of Biological Sciences, Graduate School of Science, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University, Kita-ku, Sapporo 001-0021,
Japan
d Department of Analytical Chemistry, Institute of Chemical Sciences, Faculty of Science, P.J. Šafárik University, Moyzesova 11, 040 01 Košice, Slovak Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
The stereoselective synthesis of the spiroindoline phytoalexin (R)-(+)-1-methoxyspirobrassinin and its
unnatural (S)-(ꢀ)-enantiomer were achieved by the bromine-induced spirocyclization of 1-methoxy-
brassinin using chiral auxiliaries (+)- and (ꢀ)-menthol, followed by oxidation of the obtained menthyl
ethers. The TFA-catalyzed methanolysis of chiral 1-methoxyspirobrassinol menthyl ethers provided
(2R,3R)-(ꢀ)-1-methoxyspirobrassinol methyl ether and its other three unnatural stereoisomers. The
enantiomers of the 2-amino analogues of the indole phytoalexin (2R,3R)-(ꢀ)-1-methoxyspirobrassinol
methyl ether were prepared by the TFA-catalyzed replacement of the chiral alkoxy group with an amine.
The synthesized compounds were tested in vitro on cancer cell lines and examined with enantiopure 2-
amino analogues of the indole phytoalexin (2R,3R)-(ꢀ)-1-methoxyspirobrassinol methyl ether, which
showed in most cases, lower activity than the corresponding racemates.
Received 22 June 2014
Accepted 15 July 2014
Available online xxxx
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
N
N
N
N
SCH3
SCH3
SCH3
Indole phytoalexins are low molecular weight secondary
metabolites produced by plants of the family Cruciferae, after their
exposure to physical, biological, or chemical stress.1 Among them
several spiroindoline[3,5]thiazolidine-type phytoalexins, such as
(S)-(ꢀ)-spirobrassinin 1, from Japanese radish (Raphanus sati-
vus),2,3 (R)-(+)-1-methoxyspirobrassinin 2, from kohlrabi (Brassica
oleracea var. gongylodes)4,5, or (2R,3R)-(ꢀ)-1-methoxyspirobrassi-
nol methyl ether 3, from Japanese radish (Raphanus sativus)5,6 have
been isolated (Fig. 1), their absolute configuration determined. The
optically inactive spiroindoline phytoalexin 1-methoxyspirobrass-
inol 4 was isolated from Japanese radish and exists in solution as
a mixture of diastereoisomers 4a and 4b in a 1:4 ratio due to its
unstable hemiaminal structure.6 (+)-Erucalexin 5, with an as yet
unknown absolute stereochemistry, have been isolated as a phyto-
alexin of dog mustard.7
S
S
S
O
O
OCH3
N
H
N
N
OCH3
(R)-(+)-2
OCH3
(2R,3R)-(-)-3
(S)-(-)-1
N
SCH3
SCH3
O
S
S
S
SCH3
OH
OH
N
N
N
N
OCH3
OCH3
OCH3
( )-4a
(
)-4b
(+)-5
Figure 1. Selected indole phytoalexins.
nucleophile.8 A mixture of racemic trans- and unnatural cis-diaste-
reoisomer of 1-methoxyspirobrassinol methyl ether 3a and 3b in a
1:2 ratio was prepared using methanol as the nucleophile. Spiro-
cyclization in the presence of water afforded a mixture of 4a and
4b in a 2:1 ratio. The trans-diastereoisomer is regarded as the
one with the sulfur of the thiazoline ring and a group at the 2-posi-
tion being located on the opposite sides of the indoline ring,
In 2002, a spirocyclization strategy toward spiroindoline phyto-
alexins was developed based on the treatment of 1-methoxybrass-
inin 6 with dioxane dibromide in the presence of 5% of a
⇑
Corresponding author. Tel.: +421 552341196.
E-mail address: mariana.budovska@upjs.sk (M. Budovská).
http://dx.doi.org/10.1016/j.tetasy.2014.07.006
0957-4166/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Salayová, A.; et al. Tetrahedron: Asymmetry (2014), http://dx.doi.org/10.1016/j.tetasy.2014.07.006

2
A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
whereas the cis-diastereoisomer has the sulfur and the group at the
2-position on the same side of the indoline ring. Oxidation of the
mixture of isomers 4a and 4b with CrO3 afforded racemic ( )-2.8
Direct biomimetic oxidation of 1-methoxybrassinin 6 to racemic
( )-1-methoxyspirobrassinin 2 was carried out by treatment with
PCC in 40% yield.7 ( )-1-Methoxyspirobrassinin 2 and trans-( )-1-
methoxyspirobrassinol methyl ether 3b were resolved by chiral
HPLC and the absolute configurations of natural (R)-(+)-2 and
(2R,3R)-(ꢀ)-3 were determined by CD, VCD, and chemical correla-
tion.5 Analogues of the indole phytoalexin 1-methoxyspirobrassi-
nol methyl ether can be prepared by replacing its 2-methoxy
group with a 2-piperidyl,9 2-[N,N-bis(2-chloroethyl)amino]10 or
2-(substituted phenyl)amino group.11
their 2-amino analogues. Some of these results have already been
reported on.14 We studied the spirocyclization of 1-methoxybrass-
inin in the presence of bulky chiral secondary alcohols as nucleo-
philes, which were reacted with methoxyiminium ion
A
(Scheme 1). It was supposed that the chiral secondary alcohol
would approach the methoxyiminium ion A from the less hindered
CH2-side of the thiazoline ring in the direction of a Bürgi–Dunitz
trajectory15 with the alkoxy substituent being the most remote
from the reaction center. The (R)-methoxyiminium intermediate
(R)-A should be preferably attacked by the (S)-enantiomer of the
alcohol from the less hindered CH2-side of the thiazoline ring
(Fig. 2). In the case of (S)-methoxyiminium intermediate (S)-A,
the analogous attack of the (S)-alcohol should be unfavored. With
the (R)-enantiomer of the alcohol, we expected the opposite. By
analogy, the approach of the (S)-alcohol to the (S)-methoxyimini-
um ion, the (R)-alcohol to the (R)-methoxyiminium ion should be
more favored from the sulfur side of the thiazoline ring.15
Some indole phytoalexins also exhibit anticancer activity12 and
recently their antitrypanosomal effect was evaluated.13 The natural
isomers (R)-(+)-1-methoxyspirobrassinin
2
and (2R,3R)-(ꢀ)-1-
methoxyspirobrassinol methyl ether (ꢀ)-3 were obtained in their
individual enantiomeric forms and (2R,3R)-(ꢀ)-1-methoxyspiro-
brassinol methyl ether (ꢀ)-3 was found to be a more potent inhib-
itor of Jurkat cell proliferation than its (2S,3S)-(+)-enantiomer (+)-3
and racemic ( )-3. On the other hand, in the case of 1-methoxyspi-
robrassinin 2, there was no difference in potencies between the
enantiomers, and both displayed weak overall activity against Jur-
kat cells.5 Synthetic 2-amino derivatives of indole phytoalexins,
such as 1-methoxyspirobrassinol methyl ether, were synthesized
in order to achieve higher potency, as well as a better understand-
ing of the structure–activity relationship. The introduction of a
piperidyl moiety resulted in enhanced antiproliferative activity
with the most potent anticancer effect exhibited by the trans-( )-
isomer against leukemic cell line CCRF-CEM with IC50 = 0.0263 -
CH3
H
(S)
H
CH3
S
)
(
OH
H
HO
H
CH3
N
H3C
N
S
H
H
S
H
S
H
S
N
N
O
H3C
O
CH3
(R)-A
A
(S)-
l
mol ꢁ Lꢀ1 9
. The cis-diastereoisomers of the 2-amino analogues
of 1-methoxyspirobrassinol methyl ether with 4-methyl-, 3,4-
dimethyl-, 3,4-dichloro-, and 4-nitrophenylamino have potencies
higher than or comparable to cisplatin on Jurkat cells. The trans-
diastereoisomers of 4-chloro- and 3,4-dichlorophenylamino ana-
logues have higher potencies than the etoposide on MDA-MB-
231 cells, and the 4-trifluoromethylphenylamino analogue was
found to be the most potent among all of the compounds tested
on A-549 cells and was approximately twice as potent than
doxorubicin.11
Figure 2.
We investigated the spirocyclization in the presence of (S)-(ꢀ)-
and (R)-(+)-1-(2-naphthyl)ethanol, (1S,2R,5S)-(+)- and (1R,2S,5R)-
(ꢀ)-menthol, (R)-(+)-1-phenylethanol, [(1S)-endo]-(ꢀ)-borneol,
(S)-(+)-1-indanol, and (S)-cis-verbenol. In all cases, the formation
of all four possible diastereoisomers was observed. From analysis
of the reaction mixtures, it was found that the best diastereoselec-
tivity was obtained in the presence of chiral 1-(2-naphthyl)ethanol
(Table 1). Spirocyclization in the presence of (S)-(ꢀ)-1-(2-naph-
thyl)ethanol resulted in the formation of four diastereoisomers
with the product of nucleophilic addition of this alcohol to the
(R)-methoxyiminium ion from the Re-face as the major product
7a, whereas (R)-(+)-1-(2-naphthyl)ethanol attacked the (S)-inter-
mediate A mainly from the Si-face with the formation of diastereo-
isomer 8c as the major product. The second most abundant
isomers 7b and 8d correspond to the attack of the (S)-alcohol onto
2. Results and discussion
2.1. Chemistry
Herein we report the stereoselective synthesis of natural (+)-1-
methoxyspirobrassinin (R)-(+)-2, unnatural (S)-(ꢀ)-2 and diastere-
oisomers of 1-methoxyspirobrassinol methyl ether 3 as well as
S
N
N
SCH3
Br2
N
SCH3
+
SCH3
SCH3
NH
(R)
(S)
S
S
R*OH, Et3N
BrS
N
N
H3CO
(R)-
N
N
OCH3
6
H3CO
OCH3
(S)-A
A
N
N
N
N
SCH3
SCH3
SCH3
+
SCH3
(R)
(S)
(S)
(S)
(R)
(S)
S
S
S
S
+
+
(
)
R
(
R
)
OR
N
OR
OR
N
N
OR
N
OCH3
7 14d
OCH3
7-14a
OCH3
7 14b
OCH3
7 14c
-
-
-
Scheme 1. Spirocyclization of 1-methoxybrassinin 6 with bromine in the presence of a chiral alcohol.
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A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
3
Table 1
Stereochemistry and ratios of diastereoisomers 7-14 produced via Scheme 1
R⁄
Compound/ratioa (%)
N
N
N
N
SCH3
SCH3
SCH3
SCH3
(R)
S
(S)
(R)
( )
R
(S)
(S)
S
S
S
(R)
(S)
N
OR*
OR*
OR*
N
OR*
N
N
OCH3
OCH3
OCH3
OCH3
(S)-(ꢀ)-1-(2-Naphthyl)ethylb
(R)-(+)-1-(2-Naphthyl)ethylb
(1S,2R,5S)-(+)-Menthylb
(1R,2S,5R)-(ꢀ)-Menthylb
(R)-(+)-1-Phenylethylc
[(1S)-endo]-(ꢀ)-Borneylc
(S)-(+)-1-Indanylc
7a
69
8a
7
9a
57
10a
17
11a
51
12a
20
13a
39
14a
37
7b
22
8b
7
9b
18
10b
3
11b
12
12b
19
13b
15
14b
12
7c
2
8c
68
9c
20
10c
60
11c
14
12c
54
13c
28
14c
37
7d
7
8d
18
9d
5
10d
20
11d
23
12d
7
13d
18
14d
14
(S)-cis-Verbenylc
cis-Diastereoisomeric configuration was assigned to products that exhibited a cross peak in the NOESY spectra between the H-2 and Hb protons while a trans-diastereo-
isomeric configuration was assigned to products where this cross peak was absent.
a
The ratios of diastereoisomers of compounds 7–14 were determined by integration of the non-overlapping singlets of the H-2 protons and the doublets of the Ha or Hb
protons in the 1H NMR spectra of the crude product mixtures.
b
The absolute configurations of products 7–10 were determined in the literature.14
The configurations of products 11–14 are relative, and were identified by analogy with compounds 7–10.
c
the (S)-ion and the (R)-alcohol onto the (R)-ion from the sulfur side
of the thiazolidine ring. Although the expected diastereoselectivity
was achieved, the isolated yields of the major products of the 1-
methoxyspirobrassinol 1-(2-naphthyl)ethyl ethers were only 23%
for 7a or 29% for 8c. The second most abundant isomers were
11% for 7b and 12% for 8d. Using chiral alcohol (R)-(+)-1-phenyl-
ethanol resulted in lower stereoselectivity and yields than (R)-
(+)-1-(2-naphthyl)ethanol. Reaction with the more common chiral
auxiliary (1S,2R,5S)-(+)- and (1R,2S,5R)-(ꢀ)-menthol proceeded
with similar diastereoselectivity, although the corresponding 1-
methoxyspirobrassinol menthyl ethers 9a and 10c were obtained
in higher isolated yields. The ( )-trans- and ( )-cis-diastereoiso-
mers were isolated in 65% and 22% yield [for (+)-menthol] and
70% and 21% [for (ꢀ)-menthol] after chromatography on silica
gel (eluent: n-hexane/diethyl ether 4:1). Additional chromatogra-
phy with CH2Cl2 eluent provided the major diastereoisomers 9a
(38%) and 10c (40%) with 99% ee. The bicyclic chiral alcohol
[(1S)-endo]-(ꢀ)-borneol resulted in similar diastereoselectivity as
in the case of (+)-menthol; after the first chromatography, the
( )-trans- and ( )-cis-diastereoisomers were isolated in 50% and
32% yields, but the major diastereoisomers could not be separated
from the mixture of ( )-trans-diastereoisomers in all eluents
tested. Using other chiral alcohols, such as (S)-(+)-1-indanol and
(S)-cis-verbenol, did not improve the stereoselectivity, while the
trans- and cis-diastereoisomers were formed in similar ratios and
could not be separated.
1 min to avoid substitution of the OH group of alcohol with bro-
mine in the presence of HBr formed during the spirocyclization.
The potential effect of Et3N on the spirocyclization was confirmed
with experiments with methanol (1.1 equiv) instead of menthol.
The ratio of trans- and cis-diastereoisomer 3a and 3b, when the spi-
rocyclization was carried out at 20 °C without Et3N was 52:48,
with Et3N 39:61 and at ꢀ70 °C without Et3N was 74:26, with
Et3N 17:83. It is possible that Et3N attacks the intermediate meth-
oxyiminium ion A from the less hindered thiazoline CH2 side with
the formation of an unstable triethylammonium ion. This further
reacts with the methanol that approaches from the sulfur side,
which results in the formation of the cis-diastereoisomer 3b. A
similar effect as with Et3N was observed when using dioxane
instead CH2Cl2 as the solvent, where the ratio of trans- and cis-dia-
stereoisomer 3a and 3b was 36:64. This can be explained by the
formation of an unstable oxonium ion after attacking the meth-
oxyiminium ion A from the less hindered thiazoline CH2 side with
dioxane and further reaction of the ion with the methanol from the
sulfur side.5 The alcohol is presumably less reactive at lower tem-
peratures and Et3N reacts in preference. When using menthol as
the secondary alcohol, it led to a remarkable decrease in the
reactivity.
Table 2
Influence of temperature on the stereoselectivity of the spirocyclization of 1-
methoxybrassinin 6 with (1S,2R,5S)-(+)-menthol
With the aim of improving the stereoselectivity of the spirocyc-
lization with (+)-menthol, the temperature effect on the diastere-
oselectivity was studied. The diastereoselectivity was improved
by raising the temperature, as can be seen in Table 2. It is interest-
ing to note that the major diastereoisomer 9a with a trans-config-
uration switched to 9b with a cis-configuration at ꢀ70 °C. This
could be due to a different mechanism at a low temperature. The
reaction begins by the addition of a solution of bromine to a solu-
tion of 1-methoxybrassinin whereby an iminium ion is formed. A
solution of chiral alcohol with 10 equiv of Et3N is then added after
t (°C)
Compound/ratio (%)
9a
9b
9c
9d
60a
20b
57
57
55
36
19
18
25
45
21
20
16
8
3
5
4
ꢀ20b
ꢀ70b
11
a
Solvent CHCl3, 1.1 equiv of menthol, 10 equiv Et3N.
Solvent CH2Cl2, 1.1 equiv of menthol, 10 equiv Et3N.
b
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4
A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
N
N
SCH3
SCH3
S
PCC
S
PCC
8c
7a
7b
CH2Cl2
72h
O
O
CH2Cl2
72h
N
N
OCH3
OCH3
2
(R)-(+)- (50%)
(S)-(-)-2 (52%)
92% ee
87% ee
PCC
PCC
8d
2
(R)-(+)- (57%)
(S)-(-)-2 (41%)
CH2Cl2
72h
CH2Cl2
72h
92% ee
84% ee
PCC
PHCH3CH3
(R)-(+)-2 (68%)
10c
10c
(S)-(-)-2 (70%)
9a
9a
CH2Cl2
24h
CH2Cl2
24h
97% ee
93% ee
PCC, MW
(S)-(-)-2 (63%)
PCC, MW
(R)-(+)-2 (60%)
[α]D25 = +105.7 (c 0.1, CHCl3)
CH2Cl2
1h
α
[
]
25 = -82.2 (c 0.1, CHCl3)
CH2Cl2
1h
D
99% ee
98% ee
Scheme 2. Synthesis of enantiomerically pure 1-methoxyspirobrassinin (R)-(+)-2 and (S)-(ꢀ)-2.
With the pure diastereoisomers in hand, the enantiomers of the
3 and (2R,3S)-(ꢀ)-3 isomers of 1-methoxyspirobrassinol methyl
ether in a 1:1 ratio. The ee of the synthesized enantiomers were
92–98%. The reaction was carried out in dry CH2Cl2 to avoid a com-
petitive reaction with water resulting in the formation of 1-meth-
oxyspirobrassinol 5.
indole phytoalexin 1-methoxyspirobrassinin (R)-(+)-2 and (S)-(ꢀ)-
2 were synthesized (Scheme 2). Oxidation of the main diastereo-
isomer of naphthyl ether 7a or 8c with PCC gave natural (R)-(+)-
1-methoxyspirobrassinin (R)-(+)-2 in 50% yield (92% ee) or (S)-
(ꢀ)-2 in 52% yield (87% ee). Oxidation of diastereoisomer 7b or
8d resulted in (S)-(ꢀ)-2 with 41% yield (84% ee) or (R)-(+)-2 with
57% yield (92% ee). The reaction time was 72 h, but could be short-
ened to 3.5 or 3 h by the application of microwave irradiation. The
same oxidation of the main and single isolated diastereoisomer of a
menthyl ether proceeded smoothly over 24 h from 9a to (R)-(+)-2
with 68% yield (97% ee) and from 10c to (S)-(ꢀ)-2 with 70% yield
(93% ee). By using microwave irradiation, the reaction time could
be shortened to 1 h [(R)-(+)-2 98% and (S)-(ꢀ)-2 99%].
Recently a set of racemic 2-amino analogues of 1-methoxyspiro-
brassinol methyl ether 15–18 was synthesized and their anticancer
activity determined.11 The preparation of enantiomerically pure
isomers of selected 2-amino analogues of 1-methoxyspirobrassinol
methyl ethers 15–18 was accomplished with the aim of establish-
ing and comparing anticancer activities of the racemates and its
enantiomers. The synthesis is based on the acid-catalyzed nucleo-
philic substitution of the chiral alcohol of menthyl ether 9a or 10c
with an amine (3,4-dichloroaniline, p-trifluoroaniline, p-toluidine
and p-anisidine, Scheme 4). In all cases, 2 equiv of amine and
1.1 equiv of TFA were used. The reactions were performed in dry
CH2Cl2. The reaction course was monitored by TLC and after con-
sumption of the starting material, triethylamine was added. The
reaction time depended on the nucleophilicity of the amine. Substi-
tution of the menthyl group with 3,4-dichloroaniline required
In order to prepare the natural (2R,3R)-(ꢀ)-1-methoxyspiro-
brassinol methyl ether (2R,3R)-(ꢀ)-3, the methanolysis of 9a was
investigated. It was found that TFA-catalyzed methanolysis affor-
ded a mixture of epimers (2R,3R)-(ꢀ)-3 and (2S,3R)-(+)-3 in a 1:1
ratio, and which were easily separable by column chromatography
(Scheme 3). The analogous reaction of 10c afforded the (2S,3S)-(+)-
N
N
N
SCH3
SCH3
SCH3
S
S
TFA
CH3OH
S
9a
- (+)-menthol
CF3
O
OCH3
OCH3
N
N
N
O
OCH3
OCH3
OCH3
3
(2S,3R)-(+)-3 (35%)
(2R,3R)-(-)- (43%)
[α]D25 = +39.1 (c 0.1, CHCl3)
98% ee
25
α
[
]
= -10.8 (c 0.1, CHCl3)
D
97% ee
N
N
N
SCH3
SCH3
SCH3
S
S
S
CH3OH
TFA
10c
CF3
O
OCH3
N
OCH3
N
- (-)-menthol
N
O
OCH3
OCH3
OCH3
3
3
(2R,3S)-(-)- (41%)
(2S,3S)-(+)- (36%)
[α]D25 = -38.5 (c 0.1, CHCl3)
92% ee
[α]D25 = +7.3 (c 0.1, CHCl3)
95% ee
Scheme 3. Synthesis of the enantiomerically pure diastereoisomers of 3.
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A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
5
N
N
SCH3
SCH3
S
S
R-Ph-NH2
9a
NH
NH
TFA
N
N
CH2Cl2
OCH3
OCH3
R
R
dr
R-Ph-NH2
cis
trans
(2S,3R)-(-)-15 (26%)
[α]D25 = -75.9 (c 0.3, CHCl3)
96% ee
cis:trans
15
(2R,3R)-(+)- (62%)
Cl
NH2
[α]D25 = +116.2 (c 0.4, CHCl3)
70:30
96% ee
Cl
16
(2S,3R)-(-)-16 (23%)
(2R,3R)-(+)- (61%)
F3C
H3C
NH2
NH2
25
74:26
84:16
[α]D = +20.2 (c 0.15, CHCl3)
[α]D25 = -72.5 (c 0.07, CHCl3)
98% ee
99% ee
17
(2S,3R)-(-)-17 (15%)
(2R,3R)-(+)- (60%)
25
α
[
]
25 = -74.7 (c 0.3, CHCl3)
[α]D = +211.7 (c 0.15, CHCl3)
D
96% ee
95% ee
18
(2S,3R)-(-)-18 (13%)
[α]D25 = -95.8 (c 0.15, CHCl3)
90% ee
(2R,3R)-(+)- (34%)
H3CO
NH2
[α]D25 = +226.2 (c 0.1, CHCl3)
84:16
96% ee
N
N
SCH3
SCH3
S
S
R-Ph-NH2
10c
NH
N
NH
N
TFA
CH2Cl2
OCH3
OCH3
dr
R-Ph-NH2
cis
trans
R
R
cis:trans
15
(2S,3S)-(-)-15 (69%)
[α]D25 = -131.6 (c 0.4, CHCl3)
99% ee
(2R,3S)-(+)- (21%)
Cl
NH2
[α]D25 = +118.7 (c 0.3, CHCl3)
77:23
>99% ee
Cl
16
(2S,3S)-(-)-16 (69%)
(2R,3S)-(+)- (18%)
F3C
H3C
NH2
NH2
25
[α]D25 = +42.7 (c 0.07, CHCl3)
99% ee
70:30
86:14
[α]D = -43.4 (c 0.15, CHCl3)
98% ee
17
(2S,3S)-(-)-17 (70%)
(2R,3S)-(+)- (15%)
[α]D25 = +90.1 (c 0.3, CHCl3)
98% ee
25
[α]D = -207.9 (c 0.15, CHCl3)
99% ee
18
(2S,3S)-(-)-18 (31%)
[α]D25 = -135.2 (c 0.1, CHCl3)
98% ee
(2R,3S)-(+)- (9%)
H3CO
NH2
74:26
[α]D25 = +116.3 (c 0.15, CHCl3)
94% ee
Scheme 4. Synthesis of enantiomerically pure amino analogues of 1-methoxyspirobrassinol methyl ether 15–18.
90 min while using p-trifluoroaniline required 150 min. The pres-
ence of an electron-donor substituent prolongs the reaction time;
in the case of p-toluidine to 24 h and in the case of p-anisidine
the reaction did not occur at room temperature and needed to be
heated at reflux for 14.5 h. The products of the substitution were
two epimers that differed at the C-2 carbon, in a ratio as indicated
in Scheme 4. The ratio of the epimers was determined by integra-
tion of the differentiated protons for Ha, Hb, NH, H-2, OCH3, and
SCH3. In all cases, the major products of the substitution of the men-
thyl group with an amine were the cis-diastereoisomers. Generally,
the SN1 reaction creates a racemic product (an equal amount of iso-
mers). An excess of the cis-isomer is caused by subsequent epimer-
ization of the products at the C-2 carbon under acidic condition,
resulting in the formation of a thermodynamically more stable dia-
stereoisomer.11 The cis- and trans-epimers were isolated by column
chromatography on silica gel. After the first chromatography, the
cis-isomer had a menthol impurity and was separated from the
trans-isomer, which was impure with amine. Further chromatogra-
phy in CH2Cl2 provides the pure epimers with ees of 90–99%.
For all of the isomers prepared, 7a, 8c, 9a, 10c, 2, 3, and 15–18,
the CD spectra were examined and the enantiomeric structures of
individual enantiomers were confirmed by absorption of the
inverse circular polarized light.
2.2. Antiproliferative activity
With the aim of comparing the antiproliferative activity of the
racemates and enantiomers of indole phytoalexins and their 2-
amino analogues selected human cancer cell lines were examined.
The results of this investigation are in Table 3.
The antiproliferative activity of 1-methoxyspirobrassinin 2 was
noted only on Jurkat cells. The highest effect was observed with
(S)-(ꢀ)-2 with IC50 = 18.6
l
mol Lꢀ1. Natural racemic ( )-trans-
and unnatural racemic ( )-cis-1-methoxyspirobrassinol methyl
ether ( )-trans-3 and ( )-cis-3 showed comparable antiprolifera-
tive activities on Jurkat and Hela cells with (2S,3R)-(+)- being the
most effective with IC50 = 41
l
mol Lꢀ1 on Jurkat and with
IC50 = 33.6
l
mol Lꢀ1 on A-549 cells. Our data indicated that the
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6
A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
Table 3
Antiproliferative activities of racemates and enantiomers of indole phytoalexins and their 2-amino analogues
N
N
SCH3
SCH3
S
S
O
R
N
N
OCH3
OCH3
2
3,15-18
Compound
R
Cell line, IC50
MDA-MB-231
(l
mol ꢁ L-1
)
Jurkat
MCF-7
HeLa
CCRF-CEM
A-549
( )-2a
(R)-(+)-2
(S)-(ꢀ)-2
—
41.1
40.0
18.6
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
( )-trans-3b
(2R,3R)-(ꢀ)-3
(2S,3S)-(+)-3
( )-cis-3b
(2S,3R)-(+)-3
(2R,3S)-(ꢀ)-3
( )-cis-1511
(2R,3R)-(+)-15
(2S,3S)-(ꢀ)-15
( )-trans-1511
(2S,3R)-(ꢀ)-15
(2R,3S)-(+)-15
OCH3
30.2
100
100
57.4
41
100
100
100
100
100
100
100
100
100
100
100
100
48.9
100
100
53.2
100
100
100
100
100
100
100
100
100
78
100
100
33.6
100
100
3,4-Di-Cl-C6H3NH
4-CF3-C6H4NH
4-CH3-C6H4NH
4-CH3O-C6H4NH
10.0
24.0
50.0
21.0
100
100
48.0
100
22.6
100
30.0
35.5
67.0
100
20.7
100
15.4
100
48.0
78.5
22.6
100
27.0
24.7
100
100
21.1
100
30.0
41.0
82.0
21.8
100
22.0
30.2
36.0
( )-cis-1611
41.8
37.5
29
28.4
30.7
30.0
100
77.4
—
66.8
—
88
81.9
59.5
57.5
34.9
39.0
31.5
74.3
47.5
38.3
54.0
37.6
36.8
1.0
76.1
78.0
46.6
60.0
34.5
(2R,3R)-(+)-16
(2S,3S)-(ꢀ)-16
( )-trans-1611
(2S,3R)-(ꢀ)-16
(2R,3S)-(+)-16
47.2
51.7
31.1
44.0
39.2
30.5
( )-cis-1711
6.6
100
72.0
33.3
73.0
82.3
100
100
100
100
100
71.0
78.7
100
100
100
87.0
100
100
100
36.8
76.3
71.0
64.7
42.0
75.0
100
49.2
61.0
100
100
100
100
100
78.0
(2R,3R)-(+)-17
(2S,3S)-(ꢀ)-17
( )-trans-1711
(2S,3R)-(ꢀ)-17
(2R,3S)-(+)-17
100
( )-cis-1811
34.8
38.6
>100
69.4
65.2
70.5
100
>100
>100
100
96.8
>100
100
>100
>100
64.0
>100
>100
81.5
60.2
>100
100
63
46.7
10.0
54.7
>100
15.5
>100
90
73.9
>100
>100
100
>100
>100
(2R,3R)-(+)-18
(2S,3S)-(ꢀ)-18
( )-trans-1811
(2S,3R)-(ꢀ)-18
(2R,3S)-(+)-18
The potency of the compounds was determined using the MTT (Thiazolyl Blue Tetrazolium Bromide) assay after 72 h incubation of cells and presented as IC50 (concentration
of a given compound that decreased the amount of viable cells to 50% relative to untreated control cells, see Section 4.12.).
a
Synthesized according the literature precedure.8
b
Synthesized according the literature precedure.8
amino analogues of 1-methoxyspirobrassinol methyl ether showed
enhanced antiproliferative activity. The binding of dichloro-, meth-
oxy-, and methyl-groups to amino analogues of 1-methoxyspiro-
brassinol methyl ether significantly increased their activity with
the ( )-cis-diastereoisomers being more effective on Jurkat cells.
The addition of trifluoro substituents to methylaniline analogue
resulted in decreased antiproliferative activity except for the A-
549 cell line where the highest antiproliferative activity was noted
diastereoisomer of 1-methoxyspirobrassinol menthyl ether using
PCC and microwave irradiation provided natural (R)-(+)-1-meth-
oxyspirobrassinin (R)-(+)-2 in 60% yield and its unnatural isomer
(S)-(ꢀ)-2 in 63% yield, with enantiomeric excesses of 98% and
99%, respectively. The synthesis of natural (2R,3R)-(ꢀ)-1-methoxy-
spirobrassinol methyl ether (97% ee) as well its unnatural (2S,3S)-,
(2R,3S)-, and (2S,3R)-isomers (92–98% ee) was achieved by TFA-
catalyzed methanolysis of chiral 1-methoxyspirobrassinol menthyl
ethers. The TFA-catalyzed replacement of the chiral menthoxy
group with an amine afforded enantiomerically pure 2-amino ana-
logues of indole phytoalexin (2R,3R)-(ꢀ)-1-methoxyspirobrassinol
methyl ether.
with IC50 = 1 l
mol Lꢀ1. The (2R,3S)-(+)-diastereoisomer (2R,3S)-
(+)-16 showed the highest effect among the trifluoromethylaniline
diastereoisomers on all cell lines.
We also examined the cytostatic/cytotoxic activity of the syn-
thesized compounds against selected human solid tumor and leu-
kemia cell lines in vitro (Jurkat, MCF-7, MDA-MB-231, HeLa, CCRF-
CEM and A-549). The tested enantiomerically pure 2-amino ana-
logues of indole phytoalexin (2R,3R)-(ꢀ)-1-methoxyspirobrassinol
methyl ether showed lower activity compared to the correspond-
ing racemates in most cases.
3. Conclusion
In conclusion, we have developed a stereoselective approach to
spiroindoline phytoalexins and their unnatural enantiomers
involving the bromine-mediated spirocyclization of 1-methoxy-
brassinin in the presence of (+)- and (ꢀ)-menthol as the chiral aux-
iliary. Subsequent oxidation of the main and single isolated
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A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
7
J = 7.6, J = 1.1, J = 0.6 Hz, H-7), 5.17 (d, 1H, J = 15.2 Hz, Hb), 5.15 (s,
1H, H-2), 5.06 (quartet, 1H, J = 6.5 Hz, CH3CH), 3.94 (d, 1H,
J = 15.2 Hz, Ha), 3.67 (s, 3H, OCH3), 2.65 (s, 3H, SCH3), 1.62 (d, 3H,
J = 6,5 Hz, CH3CH). 13C NMR (100 MHz): d 164.0 (C@N), 140.2 (C-
20), 133.2 and 133.1 (C-80a, C-50a), 128.2, 128.0, 127.7, 126.1,
125.9, 125.7 and 124.5 (Cnaphthyl), 148.2 (C-7a), 129.7 (C-6), 126.9
(C-3a), 123.9 (C-4), 123.6 (C-5), 112.7 (C-7), 105.6 (C-2), 78.9 (CH3-
CH), 69.8 (C-3), 69.2 (CH2), 63.9 (CH3O), 23.9 (CH3CH), 15.2 (SCH3).
NOESY: Ha/H-4, Hb; CH3CH/H-2; H-7/H-6; H-5/H-6, H-4; H-30/H-40.
MALDI-TOF MS, m/z (%): 477.4 [M+K]+ (100), 458.4 [M+Na]+ (17),
436.7 [M+H]+ (42). Anal. Calcd for C24H24N2O2S2: C, 66.02; H,
5.54; N, 6.42. Found: C, 65.82; H, 5.73; N, 6.25. CD (CH3OH, nm) kext
4. Experimental
4.1. General
Melting points were determined on a Koffler micro melting
point apparatus and are uncorrected. IR spectra were recorded on
an IR-75 spectrometer (Zeiss Jena). 1H NMR (400 MHz) and 13C
NMR (100 MHz) spectra were measured on a Varian Mercury Plus
spectrometer. Chemical shifts (d) are reported in ppm downfield
from TMS as the internal standard and coupling constants (J) are
given in Hertz. Microanalyses were performed with a Perkin–
Elmer, Model 2400 analyzer. MALDI-TOF mass spectra were mea-
sured on a MALDI IV (Kratos Analytical). The samples were ionized
with a N2-laser (k = 337 nm). The progress of the chemical reac-
tions was monitored by thin layer chromatography, using Mache-
rey-Nagel plates Alugram_Sil G/UV254. Preparative column
chromatography was performed on Kieselgel 60 Merck Type
9385 (0.040–0.063 mm). The resolutions were performed with an
Agilent 1120 HPLC system (Agilent Technologies, Waldbronn, Ger-
many) equipped with a binary pump, an autosampler, a column
oven, and a variable wavelength detector. The collection and eval-
uation of data were performed using EZChrom Elite Compact soft-
(D
e
): 211 (ꢀ2.65), 217 (ꢀ2.07), 224 (ꢀ2.95), 241 (+0.07), 270
(ꢀ0.36), 294 (+0.41). >99% ee (CHIRALCELÒ OD (F 0.46 cm ꢁ
5 cm + F 0.46 cm ꢁ 25 cm, n-hexane/propan-2-ol = 77:23 at a flow
rate 1 mL/min); Rt = 5.69 min.
Enantiomeric product 8c (85 mg, 29%) was prepared by the
same procedure using (R)-(+)-1-(2-naphthyl)ethanol. Compound
8c: [a] (De): 211
25 = +183.1 (c 0.633, CHCl3). CD (CH3OH, nm) kext
D
(+5.60), 217 (+3.87), 224 (+4.94), 241 (ꢀ0.07), 270 (+0.58), 294
(ꢀ0.59). >99% ee (CHIRALCELÒ OD (F 0.46 cm ꢁ 5 cm + F
0.46 cm ꢁ 25 cm, n-hexane/propan-2-ol = 77:23 at
1 mL/min); Rt = 5.43 min.
a flow rate
ware. Analytical chiral HPLC was performed on
a column
CHIRALCELÒ OD (F 0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm), CHIR-
ALPAKÒ OD (F 0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm), CHIRALPAKÒ
AD (F 0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm), CHIRALPAK IA
[CARTRIDGE HOLDER F 0.4 ꢁ 1 cm + F 0.46 ꢁ 25 cm] and Larihc
RN-CF6 (F 0.46 ꢁ 25 cm). Optical rotations were measured at room
temperature in a 10 cm cell on a polarimeter P3002 (Kruess) and a
P-2000 Jasco polarimeter at the sodium D-line. CD spectra were
obtained in a 1 mm quartz cell on a JASCO J-810 spectrometer.
Microwave reactions were carried out on the focused microwave
system (CEM Discover). The temperature content of the vessel
was monitored using a calibrated infrared sensor mounted under
the vessel. At the end of all reactions, the contents of vessel were
cooled rapidly using a stream of compressed air.
4.2.2. (2R,3S)-1-Methoxyspirobrassinol[(S)-1-(2-naphthyl)ethyl]
ether 7b
[
a]
25 = ꢀ166.4 (c 0.382, CHCl3). 1H NMR (CDCl3, 400 MHz): d
D
7.88–7.82 (m, 4H, H-10, H-40, H-50, H-80), 7.60 (dd, 1H, J = 8.5,
J = 1.7 Hz, H-30), 7.50–7.44 (m, 2H, H–60, H-70), 7.27 (dd, 1H,
J = 7.6, J = 1.2 Hz, H-4), 7.21 (td, 1H, J = 7.6, J = 1.2 Hz, H-6), 6.98
(dt, 1H, J = 7.6, J = 1.0 Hz, H-5), 6.84 (dd, 1H, J = 7.6, J = 1.0 Hz, H-
7), 4.91 (quartet, 1H, J = 6.4 Hz, CH3CH), 4.81 (s, 1H, H-2), 4.50 (d,
J = 1H, 15.4 Hz, Ha), 4.44 (d, 1H, J = 15.4 Hz, Hb), 3.72 (s, 3H,
OCH3), 2.64 (s, 3H, SCH3), 1.64 (d, 3H, J = 6.4 Hz, CH3CH). 13C
NMR (100 MHz): d 164.0 (C@N), 140.2 (C-20), 133.2 and 133.1 (C-
80a, C-50a), 129.9, 128.0, 127.7, 124.5, 126.1, 125.9 and 125.7 (Cnaph-
thyl), 148.2 (C-7a), 129.7 (C-6), 126.9 (C-3a), 123.6 (C-4), 123.4 (C-
5), 112.4 (C-7), 102.3 (C-2), 78.5 (CH3CH), 73.3 (CH2), 70.8 (C-3),
63.8 (CH3O), 23.9 (CH3CH), 15.2 (SCH3). NOESY: Hb/H-2; CH3CH/
H-2; H-7/H-6; H-5/H-6, H-4. MALDI-TOF MS, m/z (%): 477.4
[M+K]+ (52), 458.6 [M+Na]+ (12), 436.5 [M+H]+ (32). Anal. Calcd
for C24H24N2O2S2: C, 66.02; H, 5.54; N, 6.42. Found: C, 65.71; H,
5.82; N, 6.63.
4.2. Spirocyclization of 1-methoxybrassinin 6 with bromine in
the presence of (S)-(ꢀ)-1-(2-naphthyl)ethanol
To
a stirred mixture of 1-methoxybrassinin 6 (180 mg,
0.676 mmol) and powdered molecular sieves (3 Å) in dry CH2Cl2
(12.5 mL) at room temperature was added a freshly prepared solu-
tion of Br2 (0.038 mL, 119 mg, 0.743 mmol) in dry CH2Cl2 (1.7 mL).
After stirring for 1 min, a suspension of (S)-(ꢀ)-1-(2-naphthyl)eth-
anol (128 mg, 0.743 mmol), triethylamine (685 mg, 0.943 mL,
6.76 mmol) and powdered molecular sieves (3 Å) in dry CH2Cl2
(12.5 mL) was added. Stirring was continued for 15 min, after
which the reaction mixture was diluted with CH2Cl2 (40 mL), and
then washed with water (2 ꢁ 40 mL) and brine (40 mL). The
organic layer was dried over anhydrous Na2SO4. The residue
obtained after evaporation of solvent was subjected to chromatog-
raphy on silica gel (35 g, petroleum ether/diethyl ether 4:1), afford-
ing 67 mg (23%) of diastereoisomer 7a and 32 mg (11%) of
diastereoisomer 7b as colorless oils. Diastereoisomers 7c and 7d
were not isolated.
4.3. Spirocyclization of 1-methoxybrassinin 6 with bromine in
the presence of (1S,2R,5S)-(+)-menthol
To
a stirred mixture of 1-methoxybrassinin 6 (133 mg,
0.5 mmol) and powdered molecular sieves (3 Å) in dry CH2Cl2
(7 mL) at room temperature was added a freshly prepared solution
of Br2 (0.028 mL, 88 mg, 0.55 mmol) in dry CH2Cl2 (1.25 mL). After
stirring for 1 min, a suspension of (1S,2R,5S)-(+)-menthol (86 mg,
0.55 mmol), triethylamine (509 mg, 0.702 mL, 5 mmol) and pow-
dered molecular sieves (3 Å) in dry CH2Cl2 (7 mL) was added. Stir-
ring was continued for 20 min, after which the reaction mixture
was diluted with CH2Cl2 (25 mL), and washed with 1 M HCl
(13 mL) and water (20 mL). The organic layer was dried over Na2-
SO4. The residue obtained after evaporation of the solvent was sub-
jected to chromatography on silica gel (13 g, petroleum ether/
diethyl ether 4:1), to afford 137 mg (65%) of a mixture of trans-dia-
stereoisomers 9a, 9c (80:20) and 46 mg (22%) of the mixture of cis-
diastereoisomers 9b, 9d (85:15). Subsequent chromatography of
the mixture of trans-diastereoisomers 9a and 9c on silica gel
(20 g, CH2Cl2) gave 9a (79 mg, 38%) and 9c (21 mg, 10%) as color-
less oils.
4.2.1. (2R,3R)-1-Methoxyspirobrassinol[(S)-1-(2-naphthyl)ethyl]
ether 7a
[a]
25 = ꢀ179.3 (c 0.569, CHCl3). IR (CHCl3):
m 3053, 2973, 2933,
D
2893, 1573, 1460, 1367, 1300, 1127, 1073, 993, 947 cmꢀ1. 1H NMR
(CDCl3, 400 MHz): d 7.87–7.81 (m, 4H, H-10, H-40, H-50, H-80), 7.58
(dd, 1H, J = 8.4, J = 1.6 Hz, H-30), 7.48–7.44 (m, 2H, H-60, H-70), 7.30
(ddd, 1H, J = 7.6, J = 1.2, J = 0.6 Hz, H-4), 7.20 (td, 1H, J = 7.6,
J = 1.2 Hz, H-6), 6.99 (td, 1H, J = 7.6, J = 1.1 Hz, H-5), 6.84 (ddd, 1H,
Please cite this article in press as: Salayová, A.; et al. Tetrahedron: Asymmetry (2014), http://dx.doi.org/10.1016/j.tetasy.2014.07.006

8
A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
4.3.1. (2R,3R)-1-Methoxyspirobrassinol-(1S,2R,5S)-menthyl
J = 6.9 Hz, H-70). 13C NMR (100 MHz, CDCl3): d 166.8 mj, 166.2 mn
(C@N), 148.1 mj, 147.4 mn (C-7a), 130.1 mj, 129.7 mn (C-3a),
129.6 (C-6), 124.1 mj, 123.9 mn (C-4), 123.0 mj, 122.9 mn (C-5),
113.1 mj, 112.6 mn (C-7), 102.6 mn, 99.3 mj (C-2), 81.4 mn, 78.8
mj (C-10), 73.6 mn, 72.6 mj (CH2), 71.3 mj, 70.8 mn (C-3), 64.0 mn,
63.5 mj (OCH3), 48.8 mn, 48.6 mj (C-50), 41.5 mn, 40.9 mj (C-60),
34.3 (C-40), 31.6 mj, 31.5 mn (C-20), 24.9 mj, 24.8 mn (C-80), 23.2
mj, 22.9 mn (C-30), 22.4 and 21.4 (C-90, C-100), 16.4 mj, 15.9 mn (C-
70), 15.1 (SCH3). NOESY: Hb/H-2; Ha/H-4; OCH3/H–7.
ether 9a
[
a]
25 = ꢀ8.9 (c 0.74, CHCl3). IR (CHCl3):
m
.
2953, 2893, 2863,
D
1567, 1453, 1120, 1033, 987, 933 cmꢀ1
1H NMR (CDCl3,
400 MHz): d 7.29 (dd, 1H, J = 7.5, J = 1.2 Hz, H-4), 7.23 (td, 1H,
J = 7.5, J = 1.2 Hz, H-6), 7.01 (td, 1H, J = 7.5, J = 1.0 Hz, H-5), 6.95
(dd, 1H, J = 7.5, J = 1.0 Hz, H-7), 5.16 (s, 1H, H-2), 5.02 (d, 1H,
J = 15.3 Hz, Hb), 3.93 (s, 3H, OCH3), 3.91 (d, 1H, J = 15.3 Hz, Ha),
3.59 (td, 1H, J = 10.5, J = 4.3 Hz, H-10), 2.56 (s, 3H, SCH3), 2.41 (quin-
tet, 1H, J = 6.9, J = 2.0 Hz, H-80), 2.24-2.19 (m, 1H, H-60), 1.68-1.63
(m, 2H, H-30, H-40), 1.42-1.26 (m, 2H, H-20, H-50), 1.06-0.86 (m,
3H, H-30, H-40, H-60), 0.94 (d, 3H, J = 6.9 Hz, H-70), 0.92 (d, 3H,
J = 6.9 Hz) and 0.83 (d, 3H, J = 6.9 Hz) [H-90, H-100]. 13C NMR
(100 MHz, CDCl3): d 163.3 (C@N), 148.5 (C-7a), 129.4 (C-6), 128.7
(C-3a), 124.0 (C-4), 123.8 (C-5), 112.8 (C-7), 104.1 (C-2), 79.5 (C-
10), 70.1 (CH2), 69.3 (C-3), 63.7 (OCH3), 48.7 (C-50), 41.6 (C-60),
34.3 (C-40), 31.5 (C-20), 24.8 (C-80), 23.1 (C-30), 22.4 (C-70), 21.4
and 16.3 (C-90, C-100), 15.0 (SCH3). NOESY: Ha/H-4, Hb; OCH3/H-
7; H-2/H-10, H-60. MALDI-TOF MS, m/z (%): 420.9 [M+H]+ (94). Anal.
Calcd for C22H32N2O2S2: C, 62.82; H, 7.67; N, 6.66. Found: C, 62.50;
4.4. Spirocyclization of 1-methoxybrassinin 6 with bromine in
the presence of (S)-(ꢀ)-1-phenylethanol
To
a stirred mixture of 1-methoxybrassinin 6 (118 mg,
0.442 mmol) and powdered molecular sieves (3 Å) in dry CH2Cl2
(6 mL) at room temperature was added a freshly prepared solution
of Br2 (1.12 mL, 0.478 mmol). The stock solution was obtained by
dissolving bromine (0.040 mL) in dichloromethane (1.76 mL). After
stirring for 1 min, a suspension of (S)-(ꢀ)-1-phenylethanol (59 mg,
0.487 mmol), triethylamine (447 mg, 0.615 mL, 4.42 mmol) and
powdered molecular sieves (3 Å) in dry CH2Cl2 (6 mL) was added.
Stirring was continued for 20 min, and then the reaction mixture
was diluted with CH2Cl2 (20 mL), washed with 1 M HCl (9 mL)
and water (20 mL). The organic layer was dried over Na2SO4. The
residue obtained after evaporation of the solvent was subjected
to chromatography on silica gel (20 g, n-hexane/ethyl acetate
8:1), to afford 29 mg (17%) of trans-diastereoisomer 11a, 9 mg
(5%) of cis-diastereoisomer 11b, 12 mg (7%) of trans-diastereoiso-
mer 11c, and 13 mg (8%) of cis-diastereoisomer 11d.
H, 7.90; N, 6.40. CD (CH3OH, nm) kext
(D
e
): 214 (ꢀ39.2), 238 (+8.1),
264 (ꢀ2.8), 293 (+4.6). >99% ee (CHIRALCELÒ OD F 0.46 cm ꢁ
5 cm + F 0.46 cm ꢁ 25 cm, n-hexane/propan-2-ol = 99.9:0.1 at a
flow rate 1 mL/min); Rt = 13.71 min.
Enantiomeric product 10c (85 mg, 40%) was prepared by the
same procedure using (1R,2S,5R)-(ꢀ)-menthol. 10c: [
a]
25 = +11.0
D
(c 0.88, CHCl3). CD (CH3OH, nm) kext (De): 214 (+38.3), 238
(ꢀ7.6), 264 (+2.8), 293 (ꢀ4.3). >99% ee (CHIRALCELÒ OD
F
0.46 cm ꢁ 5 cm + F
0.46 cm ꢁ 25 cm,
n-hexane/propan-2-ol =
99.9:0.1 at a flow rate 1 mL/min); Rt = 15.52 min.
4.4.1. trans-1-Methoxyspirobrassinol[(S)-1-(phenyl)ethyl]ether
11a
4.3.2. (2S,3S)-1-Methoxyspirobrassinol-(1S,2R,5S)-menthyl
[
a
]
25 = ꢀ160.8 (c 0.25, CHCl3). IR (CHCl3):
m 2956, 2925, 2859,
D
ether 9c
1572, 1463, 1316, 1121 cmꢀ1 1H NMR (CDCl3, 400 MHz): d 7.42
.
[
a]
25 = ꢀ50.7 (c 1.15, CHCl3). 1H NMR (CDCl3, 400 MHz): d 7.29
D
(d, 2H, J = 7.1 Hz, H-20, H-60), 7.35 (dd, 2H, J = 7.6, J = 7.1 Hz, H-30,
H-50), 7.29 (d, 1H, J = 7.6 Hz, H-4), 7.28 (d, 1H, J = 7.6 Hz, H-40),
7.20 (dt, 1H, J = 1.2, J = 7.6 Hz, H-6), 6.99 (dt, 1H, J = 1.2, J = 7.6 Hz,
H-5), 6.85 (d, 1H, J = 7.6 Hz, H-7), 5.12 (d, 1H, J = 15.3 Hz, Hb),
5.10 (s, 1H, H-2), 4.88 (quartet, 1H, J = 6.5 Hz, CH3-CH), 3.90 (d,
1H, J = 15.3 Hz, Ha), 3.68 (s, 3H, OCH3), 2.61 (s, 3H, SCH3), 1.54 (d,
3H, J = 6.5 Hz, CH3-CH). 13C NMR (100 MHz, CDCl3): d 163.4
(C@N), 148.2 (C-7a), 142.9 (C-10), 129.6 (C-6), 128.3 (C-30, C-50),
127.7 (C-40), 127.1 (C-3a), 126.6 (C-20, C-60), 123.9 (C-4), 123.6
(C-5), 112.7 (C-7), 105.6 (C-2), 78.8 (CH3-CH), 70.1 (CH2), 69.3 (C-
3), 63.8 (OCH3), 23.9 (CH3-CH), 15.0 (SCH3). NOESY: Ha/H-4;
OCH3/H–7. Anal. Calcd for C20H22N2O2S2: C, 62.15; H, 5.74; N,
7.25. Found: C, 62.22; H, 5.56; N, 7.44.
(dd, 1H, J = 7.6, J = 1.2 Hz, H-4), 7.23 (td, 1H, J = 7.6, J = 1.2 Hz, H-6),
7.01 (td, 1H, J = 7.6, J = 1.1 Hz, H-5), 6.94 (dd, 1H, J = 7.6, J = 1.1 Hz,
H-7), 5.19 (s, 1H, H-2), 5.15 (d, 1H, J = 15.5 Hz, Hb), 3.93 (s, 3H,
OCH3), 3.92 (d, 1H, J = 15.5 Hz, Ha), 3.58 (td, 1H, J = 10.5,
J = 4.3 Hz, H-10), 2.56 (s, 3H, SCH3), 2.43 (dddd, 1H, J = 12.2,
J = 4.3, J = 1.9, J = 1.6 Hz, H-60), 2.35 (quintet, J = 6.9, 2.3 Hz, 1H, H-
80), 1.70-1.64 (m, 2H, H-30, H-40), 1.43-1.13 (m, 2H, H-20, H-50),
1.10-0.86 (m, 3H, H-30, H-40, H-60), 0.94 (d, 3H, J = 6.9 Hz, H-70),
0.92 (d, 3H, J = 6.9 Hz) and 0.79 (d, 3H, J = 6.9 Hz) [H-90, H-100].
13C NMR (100 MHz, CDCl3): d 162.5 (C@N), 147.7 (C-7a), 129.9
(C-3a), 129.4 (C-6), 123.9 (C-4), 123.8 (C-5), 112.8 (C-7), 104.1
(C-2), 80.8 (C-10), 70.7 (CH2), 68.6 (C-3), 64.1 (OCH3), 49.0 (C-50),
41.3 (C-60), 34.3 (C-40), 31.5 (C-20), 25.1 (C-80), 22.8 (C-30), 22.4
(C-70), 21.5 and 15.8 (C-90, C-100), 15.0 (SCH3). NOESY: Ha/H-4,
Hb; OCH3/H-7; H-2/H-10, H-60. Anal. Calcd for C22H32N2O2S2: C,
62.82; H, 7.67; N, 6.66. Found: C, 62.62; H, 7.56; N, 6.44.
4.4.2. cis-1-Methoxyspirobrassinol[(S)-1-(phenyl)ethyl]ether 11b
[
a]
25 = ꢀ10.5 (c 0.20, CHCl3). 1H NMR (CDCl3, 400 MHz): d
D
7.44–7.28 (m, 5H, H-20, H-30, H-40, H-50, H-60), 7.25 (dt, 1H, J = 1.2,
J = 7.6 Hz, H-6), 7.22 (dd, 1H, J = 7.6, J = 1.2 Hz, H-4), 6.98 (dt, 1H,
J = 1.0, J = 7.6 Hz, H-5), 6.86 (d, 1H, J = 7.6, Hz, H-7), 4.76 (s, 1H,
H-2), 4.75 (quartet, 1H, J = 6.4, Hz, CH3-CH), 4.49 (d, 1H, J = 15.4,
Hz, Ha), 4.39 (d, 1H, J = 15.4 Hz, Hb), 3.72 (s, 3H, OCH3), 2.61 (s,
3H, SCH3), 1.56 (d, 3H, J = 6.4 Hz, CH3-CH). 13C NMR (100 MHz,
CDCl3): d 167.0 (C@N), 148.1 (C-7a), 142.6 (C-10), 129.9 (C-6),
128.5 (C-3a), 128.4 (C-30, C-50), 127.9 (C-40), 127.1 (C-20, C-60),
123.6 (C-5), 123.3 (C-4), 112.5 (C-7), 102.2 (C-2), 78.4 (CH3-CH),
73.4 (CH2), 70.7 (C-3), 63.8 (OCH3), 23.9 (CH3-CH), 15.1 (SCH3).
NOESY: Hb/H-2; Ha/H-4; OCH3/H-7. Anal. Calcd for C20H22N2O2S2:
C, 62.15; H, 5.74; N, 7.25. Found: C, 62.28; H, 5.76; N, 7.14.
4.3.3. cis-1-Methoxyspirobrassinol-(1S,2R,5S)-menthyl ethers
9b and 9d
1H NMR (CDCl3, 400 MHz): d 7.27–7.21 (m, 2H, H-4, H-6), 7.02
(dt, 0.85H, J = 0.7, J = 7.5 Hz, H-5 mj); 7.01 (dt, 0.15H, J = 1.1,
J = 7.6 Hz, H-5 mn), 6.96 (dd, 0.85H, J = 7.7, J = 0.7 Hz, H-7 mj), 6.93
(dd, 0.15H, J = 7.8, J = 0.7 Hz, H-7 mn), 4.92 (s, 0.85H, H-2 mj), 4.82
(s, 0.15H, H-2 mn), 4.48 (d, 0.15H, J = 15.2 Hz, Ha mn), 4.41 (d,
0.85H, J = 15.1 Hz, Ha mj), 4.35 (d, 0.15H, J = 15.2 Hz, Hb mn), 4.31
(d, 0.85H, J = 15.1 Hz, Hb mj), 3.92 (s, 0.45H, OCH3 mn), 3.91 (s,
2.55H, OCH3 mj), 3.65 (dt, 0.85H, J = 4.2, J = 10.5 Hz, H-10 mj), 3.48
(dt, 0.15H, J = 4.2, J = 10.5 Hz, H-10 mn), 2.55 (s, 3H, SCH3), 2.36 (quin-
tet, 1H, J = 7.1, J = 2.5 Hz, H-80), 2.17–2.12 (m, 1H, H-60), 1.68–1.63
(m, 2H, H-30, H-40), 1.41–1.26 (m, 2H, H-20, H-50), 1.06–0.86 (m, 3H,
H-30, H-40, H-60), 0.93 (d, 3H, J = 6.6 Hz) and 0.91 (d, 3H, J = 7.1 Hz,
H-90, H-100), 0.81 (d, 2.55H, J = 6.9 Hz, H-70), 0.75 (d, 0.45H,
4.4.3. trans-1-Methoxyspirobrassinol[(S)-1-(phenyl)ethyl]ether
11c
[a]
25 = +173.0 (c 0.1, CHCl3). 1H NMR (CDCl3, 400 MHz): 7.37–
D
7.21 (m, 5H, H-20, H-30, H-40, H-50, H-60), 7.22 (d, 1H, J = 7.6, H-6),
Please cite this article in press as: Salayová, A.; et al. Tetrahedron: Asymmetry (2014), http://dx.doi.org/10.1016/j.tetasy.2014.07.006

A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
9
7.16 (d, 1H, J = 7.5, H-4), 6.99–6.92 (m, H-5, H-7), 5.06 (quartet, 1H,
J = 6.5, Hz, CH3-CH), 4.63 (s, 1H, H-2), 4.18 (d, 1H, J = 14.3, Hz, Hb),
4.02 (s, 3H, OCH3), 3.69 (d, 1H, J = 14.3 Hz, Ha), 2.58 (s, 3H, SCH3),
1.61 (d, 3H, J = 6.5 Hz, CH3-CH). 13C NMR (100 MHz, CDCl3): d
167.7 (C@N), 147.6 (C-7a), 142.6 (C-10), 132.4 (C-3a), 130.8 (C-6),
128.6 (C-30, C-50), 128.1 (C-40), 127.0 (C-20, C-60), 123.9 (C-5),
122.9 (C-4), 112.7 (C-7), 100.6 (C-2), 79.0 (CH3-CH), 72.1 (CH2),
68.1 (C-3), 63.8 (OCH3), 23.9 (CH3-CH), 15.1 (SCH3). NOESY: Ha/
H-4; OCH3/H–7. Anal. Calcd for C20H22N2O2S2: C, 62.15; H, 5.74;
N, 7.25. Found: C, 62.18; H, 5.66; N, 7.15.
J = 15.3 Hz, Ha mj), 4.52 (d, 0.3H, J = 15.1 Hz, Ha mn), 4.38 (d,
0.3H, J = 15.1 Hz, Hb mn), 4.36 (d, 0.7H, J = 15.3 Hz, Hb mj), 4.18
(d, 1H, J = 8.7 Hz, H-10), 3.96 (s, 0.9H, OCH3 mn), 3.95 (s, 2.1H,
OCH3 mj), 2.56 (s, 2.1H, SCH3 mj), 2.54 (s, 0.9H, SCH3 mn), 2.27–
2.10 (m, 2H), 1.74–1.67 (m, 2H), 1.37–1.22 (m, 3H), 0.99–0.94
(m, 1H), 0.87 (s, 3H, CH3) and 0.85 (s, 3H, CH3). NOESY: Hb/H-2;
Ha/H-4.
4.6. Spirocyclization of 1-methoxybrassinin 6 with bromine in
the presence of (S)-(+)-1-indanol
4.4.4. cis-1-Methoxyspirobrassinol[(S)-1-(phenyl)ethyl]ether 11d
To a stirred mixture of 1-methoxybrassinin 6 (77 mg,
[a]
25 = ꢀ281.1 (c 0.09, CHCl3). NMR (CDCl3, 400 MHz): d 7.37–
0.289 mmol) and powdered molecular sieves (3 Å) in dry CH2Cl2
(5.1 mL) at room temperature was added a freshly prepared solu-
tion of Br2 (0.74 mL, 0.318 mmol). The stock solution was obtained
by dissolving bromine (0.040 mL) in dichloromethane (1.76 mL).
After stirring for 1 min, a suspension of (S)-(+)-1-indanol (43 mg,
0.318 mmol), triethylamine (292 mg, 0.40 mL, 2.89 mmol) and
powdered molecular sieves (3 Å) in dry CH2Cl2 (5.1 mL) was added.
Stirring was continued for 20 min, and then the reaction mixture
was diluted with CH2Cl2 (20 mL), washed with 1 M HCl (9 mL)
and water (20 mL). The organic layer was dried over Na2SO4. The
residue obtained after evaporation of the solvent was subjected
to chromatography on silica gel (20 g, n-hexane/diethyl ether
3:1), affording 23 mg (20%) of the mixture of trans-diastereoiso-
mers 13a, 13c (58:42) and 20 mg (17%) of diastereoisomer 13b
and 21 mg (18%) of diastereoisomer 13d
D
7.28 (m, 5H, H-20, H-30, H-40, H-50, H-60), 7.23 (dt, 1H, J = 7.6,
J = 1.1 Hz, H-6), 7.16 (dd, 1H, J = 7.6, J = 1.1 Hz, H-4), 6.98 (dt, 1H,
J = 7.6, J = 0.5 Hz, H-5), 6.93 (dd, 1H, J = 7.6, J = 0.5 Hz, H-7), 5.06
(quartet, 1H, J = 6.5 Hz, CH3-CH), 4.63 (s, 1H, H-2), 4.18 (d, 1H,
J = 15.3 Hz, Ha), 4.02 (s, 3H, OCH3), 3.70 (d, 1H, J = 15.3 Hz, Hb),
2.53 (s, 3H, SCH3), 1.61 (d, 3H, J = 6.5 Hz, CH3-CH). 13C NMR
(100 MHz, CDCl3): d 165.7 (C@N), 147.6 (C-7a), 142.6 (C-10),
129.7 (C-6), 129.4 (C-3a), 128.5 (C-30, C-50), 128.1 (C-40), 127.0
(C-20, C-60), 123.9 (C-5), 122.9 (C-4), 112.7 (C-7), 100.6 (C-2), 79.0
(CH3-CH), 72.1 (CH2), 69.8 (C-3), 63.9 (OCH3), 23.9 (CH3-CH), 15.1
(SCH3). NOESY: Hb/H-2; Ha/H-4; OCH3/H-7. Anal. Calcd for C20H22-
N2O2S2: C, 62.15; H, 5.74; N, 7.25. Found: C, 62.28; H, 5.76; N, 7.32.
4.5. Spirocyclization of 1-methoxybrassinin 6 with bromine in
the presence of [(1S)-endo]-(ꢀ)-borneol
4.6.1. trans-1-Methoxyspirobrassinol[(1S)-indanyl]ether 13a
and 13c
To
a stirred mixture of 1-methoxybrassinin 6 (90 mg,
0.338 mmol) and powdered molecular sieves (3 Å) in dry CH2Cl2
(6 mL) at room temperature was added a freshly prepared solution
of Br2 (0.86 mL, 0.372 mmol). The stock solution was obtained dis-
solving of bromine (0.040 mL) in dichloromethane (1.76 mL). After
stirring for 1 min, a suspension of [(1S)-endo]-(ꢀ)-borneol (57 mg,
0.372 mmol), triethylamine (342 mg, 0.472 mL, 3.38 mmol) and
powdered molecular sieves (3 Å) in dry CH2Cl2 (6 mL) was added.
Stirring was continued for 20 min, and then the reaction mixture
was diluted with CH2Cl2 (20 mL), washed with 1 M HCl (9 mL)
and water (20 mL). The organic layer was dried over Na2SO4. The
residue obtained after evaporation of the solvent was subjected
to chromatography on silica gel (20 g, n-hexane/diethyl ether
3:1), to afford 70 mg (50%) of the mixture of trans-diastereoiso-
mers 12a, 12c (24:76) and 45 mg (32%) of the mixture of cis-diaste-
reoisomers 12b, 12d (71:29).
1H NMR (CDCl3, 400 MHz): d 7.56 (d, 0.6H, J = 7.2 Hz, H-70 mj),
7.52 (d, 0.4H, J = 7.2 Hz, H-70 mn), 7.31 (dd, 1H, J = 7.5, J = 7.1 Hz,
H-6), 7.20–7.26 (m, 4H, H-4, H-40, H-50, H-60), 7.01 (dd, 1H,
J = 7.5 Hz, H-5), 6.94 (d, 1H, J = 7.1 Hz, H-7), 5.40 (dd, 0.6H, J = 5.1,
J = 5.5 Hz, H-10 mj), 5.36 (dd, 0.4H, J = 5.1, J = 5.5 Hz, H-10 mn),
5.35 (s, 0.6H, H-2 mj), 5.31 (s, 0.4H, H-2 mn), 5.09 (d, 0.4H,
J = 15.3 Hz, Hb mn), 5.01 (d, 0.6H, J = 15.3 Hz, Hb mj), 3.96 (s,
1.2H, OCH3 mn), 3.89 (s, 1.8H, OCH3 mj), 3.87 (d, 1H, J = 15.3 Hz,
Ha), 3.10 (m, 1H, H-30a), 2.83 (ddd, 1H, J = 14.9, J = 7.2, J = 6.5 Hz,
H-30b), 2.56 (s, 1.2H, SCH3 mn), 2.55 (s, 1.8H, SCH3 mj), 2.48
(ddd, 0.4H, J = 13.4, J = 5.5, J = 7.0 Hz, H-20a mn), 2.41 (ddd, 0.4H,
J = 13.4, J = 5.1, J = 6.9 Hz, H-20b mn), 2.31 (ddd, 0.6 H, J = 5.5,
J = 8.8, J = 12.9 Hz, H-20a mj), 2.19 (ddd, 0.6 H, J = 5.1, J = 8.0,
J = 12.9 Hz, H-20b mj). 13C NMR (100 MHz, CDCl3): d 163.2 mn,
162.8 mj (C@N), 148.3 mn, 148.1 mj (C-7a), 144.0 mj, 143.9 mn
(C-70a), 142.5 mj, 142.2 mn (C-40a), 129.6, 129.5 (C-6), 128.6,
128.5 (C-50), 127.7, 127.1 (C-3a), 126.4, 126.3 (C-60), 125.7, 125.2
(C-70), 124.8, 124.7 (C-40), 124.0 (C-5), 123.7, 123.6 (C-4), 112.7
(C-7), 106.9, 106.8 (C-2), 84.9 (C-10), 70.1 (CH2), 69.2 mn, 69.6 mj
(C-3), 64.3 mn , 64.1 mj (OCH3), 33.7 mj, 33.1 mn (C-20), 30.0 mn,
29.9 mj (C-30), 15.0. 14.9 (SCH3). NOESY: Ha/Hb; H-10/H-20a,b; H-
20a/H-20b; H-30a/H-30b.
4.5.1. trans-1-Methoxyspirobrassinol[(1S)-endo-borneyl]ether
12a and 12c
IR (CHCl3):
m
2956, 2925, 2859, 1573, 1572, 1463, 1282, 1121,
1H NMR (CDCl3, 400 MHz): d 7.32 (d, 0.8H,
1073, 943 cmꢀ1
.
J = 7.5 Hz, H-4 mj), 7.30 (d, 0.2H, J = 7.5 Hz, H-4 mn), 7.24 (dd,
1H, J = 7.5, H-6), 7.01 (dd, 1H, J = 7.5 Hz, H-5), 6.92 (d, 1H,
J = 7.5 Hz, H-7), 5.10 (d, 0.2H, J = 15.3 Hz, Hb mn), 5.08 (d, 0.8H,
J = 15.0 Hz, Hb mj), 5.07 (s, 0.2H, H-2 mn), 5.01 (s, 0.8H, H-2 mj),
4.21 (d, 0.8H, J = 9.3 Hz, H-10 mj), 4.10 (d, 0.2H, J = 10.3 Hz, H-10
mn), 3.96 (s, 0.6H, OCH3 mn), 3.94 (s, 2.4H, OCH3 mj), 3.83 (d,
0.2H, J = 15.3 Hz, Ha mn), 3.79 (d, 0.8H, J = 15.0 Hz, Ha mj), 2.55
(s, 3H, SCH3), 2.30–2.15 (m,1H), 2.12–2.04 (m, 1H), 1.74–1.66 (m,
2H), 1.33–1.20 (m, 3H), 0.99–0.95 (m, 1H), 0.87 (s, 3H, CH3) and
0.86 (s, 3H, CH3). NOESY: Ha/H-4; OCH3/H-7.
4.6.2. cis-1-Methoxyspirobrassinol[(1S)-indanyl]ether 13b
[
a]
25 = +95.5 (c 0.29, CHCl3). IR (CHCl3):
m
2956, 2928, 1564,
D
1461, 1320, 1290, 1137, 1043 cmꢀ1
.
1H NMR (CDCl3, 400 MHz): d
7.56 (d, 1H, J = 7.2 Hz, H-70), 7.20–7.28 (m, 5H, H-4, H-40, H-50, H-
6, H-60), 7.01 (dd, 1H, J = 7.6 Hz, H-5), 6.95 (d, 1H, J = 7.9 Hz, H-7),
5.37 (dd, 1H, J = 5.4, J = 5.8 Hz, H-10), 5.05 (s, 1H, H-2), 4.48 (d,
1H, J = 15.3, Hz, Ha), 4.38 (d, J = 15.3 Hz, Hb), 3.86 (s, 3H, OCH3),
3.12 (ddd 1H, J = 15.9, J = 7.5, J = 5.6 Hz, H-30a), 2.82 (ddd, 1H,
J = 7.2, J = 7.6, J = 15.9 Hz, H-30b), 2.55 (s, 3H, SCH3), 2.43 (ddt, 1H,
J = 5.4, J = 6.7, J = 13.3 Hz, H-20a), 2.18 (ddt, 1H, J = 5.8, J = 7.6,
J = 13.3 Hz, H-20b). 13C NMR (100 MHz, CDCl3): d 166.9 (C@N),
147.8 (C-7a), 143.8 (C-70a), 142.4 (C-40a), 129.8 (C-6), 128.6
(C-50), 128.1 (C-3a), 126.4 (C-60), 125.2 (C-70), 124.8 (C-40), 123.7
4.5.2. cis-1-Methoxyspirobrassinol[(1S)-endo-borneyl]ether 12b
and 12d
1H NMR (CDCl3, 400 MHz): d 7.28–7.24 (m, 2H, H-4, H-6), 7.01
(dd, 1H, J = 7.7, J = 6.8 Hz, H-5), 6.92 (d, 1H, J = 7.7 Hz, H-7), 4.74
(s, 0.3H, H-2 mn), 4.71 (s, 0.7H, H-2 mj), 4.51 (d, 0.7H,
Please cite this article in press as: Salayová, A.; et al. Tetrahedron: Asymmetry (2014), http://dx.doi.org/10.1016/j.tetasy.2014.07.006

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A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
(C-5), 123.2 (C-4), 112.6 (C-7), 103.2 (C-2), 84.7 (C-10), 72.9 (CH2),
70.5 (C-3), 63.9 (OCH3), 33.5 (C-20), 30.0 (C-30), 15.1 (SCH3). NOESY:
Hb/H-2; Ha/H-4; H-2/H-10. Anal. Calcd for C21H22N2O2S2: C, 63.29;
H, 5.56; N, 7.03. Found: C, 63.28; H, 5.75; N, 7.22.
4.7.2. cis-1-Methoxyspirobrassinol[(S)-verbenyl]ether 14b and
14d
1H NMR (CDCl3, 400 MHz): d 7.25 (dt, 1H, J = 7.6, J = 1.1 Hz, H-6),
7.23 (d, 1H, J = 7.6 Hz, H-4), 7.03 (dt, 1H, J = 7.6, J = 0.8 Hz, H-5),
6.92 (dd, 1H, J = 7.6, J = 0.8 Hz, H-7), 5.55 (dt, 0.5H, J = 1.5,
J = 4.3 Hz, H-30), 5.42 (dt, 0.5H, J = 1.6, J = 4.2 Hz, H-30), 4.82 (s,
0.5H, H-2), 4.77 (s, 0.5H, H-2), 4.64 (d, 0.5H, J = 1.6 Hz, H-20), 4.59
(d, 0.5H, J = 1.5 Hz, H-20), 4.53 (d, 0.5H, J = 15.4 Hz, Ha), 4.52 (d,
0.5H, J = 15.3 Hz, Ha), 4.37 (d, 0.5H, J = 15.3 Hz, Hb), 4.29 (d, 0.5H,
J = 15.4 Hz, Hb), 3.98 (s, 1.5H, OCH3), 3.93 (s,1.5H, OCH3), 2.54 (s,
1.5H, SCH3), (2.53 s, 1.5H, SCH3), 2.44–2.57 (m, 1.5H, H-10, H-70),
2.33–2.37 (m, 0.5 H H-10), 1.98 (t, 1H, J = 5.4 Hz, H-50), 1.75 (t,
1.5H, J = 1.7 Hz, H-80), 1.74 (t, 1.5H, J = 1.7 Hz, H-80), 1.36 (s, 1.5H,
H-90), 1.35 (s, 1.5H, H-90), 1.33 (d, 1H, J = 8.7 Hz, H-70), 1.06 (s,
3H, H-100). 13C NMR (100 MHz, CDCl3): d 166.6, 166.5 (C@N),
148.6, 148.1 (C-7a), 148.0, 147.7 (C-40), 129.7 (C-6), 128.9, 128.5
(C-3a), 123.71, 123.69 (C-5), 123.1, 123.0 (C-4), 117.2, 116.3 (C-
30), 112.5 (C-7), 103.6, 103.1 (C-2), 82.8, 81.8 (C-20), 72.9 (CH2),
70.3, 69.9 (C-3), 64.0, 63.9 (OCH3), 47.9, 47.8 (C-50), 46.4, 46.1
(C-10), 39.6, 39.5 (C-60), 35.6, 35.5 (C-70), 26.9, 26.8 (C-90), 23.0,
22.8 (C-80), 22.7, 22.6 (C-100), 15.1, 15.0 (SCH3). NOESY: Hb/H-2;
Ha/H-4.
4.6.3. cis-1-Methoxyspirobrassinol[(1S)-indanyl]ether 13d
[
a]
25 = ꢀ292.5 (c 0.08, CHCl3). 1H NMR (CDCl3, 400 MHz): d
D
7.52 (d, 1H, J = 7.8 Hz, H-70), 7.28–7.26 (m, 3H, H-40, H-50, H-6),
7.25 (d, 1H, J = 7.5 Hz, H-4), 7.21(dd, 1H, J = 7.5, J = 7.8 Hz, H-60),
7.01 (dd, 1H, J = 7.5 Hz, H-5), 6.96 (d, 1H, J = 7.8 Hz, H-7), 5.28
(dd, 1H, J = 4.1, J = 5.2 Hz, H-10), 4.98 (s, 1H, H-2), 4.38 (d, 1H,
J = 15.3 Hz, Ha), 4.09 (d, 1H, J = 15.3 Hz, Hb), 3.99 (s, 3H, OCH3),
3.17 (dt, 1H, J = 7.2, J = 15.7 Hz, H-30a), 2.83 (dt, 1H, J = 6.6,
J = 15.7 Hz, H-30b), 2.52 (s, 3H, SCH3), 2.43-2.37 (m, 2H, H-20a, H-
20b). 13C NMR (100 MHz, CDCl3): d 166.4 (C@N), 148.1 (C-7a),
144.6 (C-70a), 141.8 (C-40a), 129.8 (C-6), 128.8 (C-50), 127.8 (C-
3a), 126.2 (C-60), 125.7 (C-70), 125.0 (C-40), 123.7 (C-5), 123.2 (C-
4), 112.6 (C-7), 102.6 (C-2), 84.6 (C-10), 72.7 (CH2), 70.3 (C-3),
64.1 (OCH3), 33.6 (C-20), 30.2 (C-30), 15.1 (SCH3). NOESY: Hb/H-2;
Ha/H-4; H-2/H-10. Anal. Calcd for C21H22N2O2S2: C, 63.29; H, 5.56;
N, 7.03. Found: C, 63.32; H, 5.59; N, 7.11.
4.7. Spirocyclization of 1-methoxybrassinin 6 with bromine in
the presence of (S)-cis-verbenol
4.8. Synthesis of (R)-(+)- and (S)-(ꢀ)-1-methoxyspirobrassinin
(R)-(+)-2 and (S)-(ꢀ)-2 from 7a/8c, 7b/8d
To
a stirred mixture of 1-methoxybrassinin 6 (90 mg,
0.338 mmol) and powdered molecular sieves (3 Å) in dry CH2Cl2
(6 mL) at room temperature was added a freshly prepared solution
of Br2 (0.86 mL, 0.372 mmol). The stock solution was obtained by
dissolving of bromine (0.040 mL) in dichloromethane (1.76 mL).
After stirring for 1 min, a suspension of (S)-cis-verbenol (56 mg,
0.372 mmol), triethylamine (342 mg, 0.472 mL, 3.38 mmol) and
powdered molecular sieves (3 Å) in dry CH2Cl2 (6 mL) was added.
Stirring was continued for 20 min, and then the reaction mixture
was diluted with CH2Cl2 (20 mL), washed with 1 M HCl (9 mL)
and water (20 mL). The organic layer was dried over Na2SO4. The
residue obtained after evaporation of solvent was subjected to
chromatography on silica gel (20 g, n-hexane/diethyl ether 4:1),
affording 41 mg (29%) of the mixture of trans-diastereoisomers
14a, 14c (50:50) and 27 mg (19%) of the mixture of cis-diastereoi-
somers 14b, 14d (47:53).
(a) To a vigorously stirred slurry of PCC (75 mg, 0.345 mmol)
and anhydrous MgSO4 (62 mg, 0.515 mmol) in dry CH2Cl2
(0.3 mL) a solution of 7a (50 mg, 0.115 mmol) in dry CH2Cl2
(1 mL) was added. The reaction mixture was stirred for 72 h
at room temperature, and then diluted with CH2Cl2 (2 mL).
After adding a small amount of silica gel, the solvent was
evaporated and the residue that was preabsorbed on silica
gel was purified by silica gel column chromatography (6 g,
petroleum ether/diethyl ether 4:1) to give 16 mg (50%) of
(R)-(+)-1-metoxyspirobrassinin (R)-(+)-2 as colorless nee-
dles, mp 129–131 °C (EtOAc/hexane), 92% ee. The spectro-
scopic data were fully identical with those of the natural
product.4 The absolute configuration was determined by
direct comparison of the ECD spectra with published data.5
(b) Application of the same procedure on 8c afforded 17 mg
(52%) of (S)-(ꢀ)-2, 87% ee.
4.7.1. trans-1-Methoxyspirobrassinol[(S)-verbenyl]ether 14a
and 14c
(c) Application of the same procedure on 7b afforded 13 mg
(41%) of (R)-(+)-2, 84% ee.
IR (CHCl3):
m
2987, 2923, 2869, 2845, 1566, 1463, 1149, 1130,
(d) Application of the same procedure on 8d afforded 18 mg
(57%) of (S)-(ꢀ)-2, 92% ee.
987 cmꢀ1 1H NMR (CDCl3, 400 MHz): d 7.31 (d, 0.5H, J = 7.5 Hz,
.
H-4), 7.28 (d, 0.5H, J = 7.5 Hz, H-4), 7.23 (dt,1H, J = 1.2, J = 7.5 Hz,
H-6), 7.00 (dt, 1 H, J = 7.5, J = 0.9 Hz, H-5), 6.92 (d, 1H, J = 7.5 Hz,
H-7), 5.54 (dd, 0.5H, J = 1.4, J = 2.7 Hz, H-30), 5.45 (dd, 0.5H,
J = 1.4, J = 2.5 Hz, H-30), 5.09 (s, 1H, H-2), 5.06 (s, 1H, H-2), 5.04
(d, 0.5H, J = 15.3 Hb), 5.03 (d, 0.5H, J = 15.2 Hb), 4.60 (d, 0.5H,
J = 1.4 Hz, H-20), 4.57 (d, 0.5 H, J = 1.4 Hz, H-20), 3.99 (s, 1.5H,
OCH3), 3,94 (s, 1.5H, OCH3), 3.85 (d, 0.5H, J = 15.3 Hz, Ha), 3.84 (d,
0.5H. J = 15.2 Hz, Ha), 2.55 (s, 1,5H, SCH3), 2.54 (s, 1.5H, SCH3),
2.44-2.55 (m, 1.5H, H-10, H-70), 2.41 (ddd, 0.5H, J = 2.4, J = 4.1,
J = 6.0 Hz, H-10), 1.97 (dd, 1H, J = 4.2, J = 9.3 Hz, H-50), 1.76 (t,
1.5H, J = 1.7 Hz, H-80), 1.74 (t, 1.5H, J = 1.6 Hz, H-80), 1.36 (s, 1.5H,
H-90), 1.35 (s, 1.5H, H-90), 1.33 (dd, 1H, J = 5.9, J = 8.7 Hz, H-70),
1.05 (s, 1.5H, H-100), 1.03 (s, 1.5H, H-100). 13C NMR (100 MHz.
CDCl3): d 162.7, 162.2 (C@N), 148.3, 148.0 (C-7a), 147.9, 147.6
(C-40), 129.5, 129.4 (C-6), 128.6, 127.6 (C-3a), 124.1, 124.0 (C-4),
123.6, 123.5 (C-5), 117.4 (C-30), 112.7, 112.6 (C-7), 108.0, 107.2
(C-2), 83.4, 82.8 (C-20), 70.5, 70.2 (CH2), 69.1, 68.7 (C-3), 64.1,
64.0 (OCH3), 47.7 (C-50), 46.7, 46.2 (C-10), 39.6, 39.3 (C-60), 35.6,
35.5 (C-70), 26.9, 26.8 (C-90), 23.0, 22.7 (C-80), 22.7, 22.6 (C-100),
14.9, 14.8 (SCH3). NOESY: Hb/H-2; Ha/H-4.
4.9. Synthesis of (R)-(+)- and (S)-(ꢀ)-1-methoxyspirobrassinin
(R)-(+)-2 and (S)-(ꢀ)-2 from 9a/10c
(a) To a vigorously stirred slurry of PCC (138 mg, 0.642 mmol)
and anhydrous MgSO4 (116 mg, 0.936 mmol) in dry CH2Cl2
(0.45 mL), a solution of 9a (90 mg, 0.214 mmol) in dry CH2-
Cl2 (1.8 mL) was added. The reaction mixture was stirred
for 24 h at room temperature, and then diluted with CH2Cl2
(2 mL). After adding a small amount of silica gel, the solvent
was evaporated and the residue that was preabsorbed on sil-
ica gel was purified by silica gel column chromatography
(4 g, petroleum ether/diethyl ether 4:1) to give 43 mg
(68%) of (R)-(+)-1-methoxyspirobrasinin (R)-(+)-2 as color-
less needles, mp 129–131 °C (EtOAc/hexane), 97% ee. The
spectroscopic data were fully identical with those of the nat-
ural product.4 The absolute configuration was determined
by direct comparison of the ECD spectra with published
data.5
Please cite this article in press as: Salayová, A.; et al. Tetrahedron: Asymmetry (2014), http://dx.doi.org/10.1016/j.tetasy.2014.07.006

A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
11
(b) Application of the same procedure on 10c afforded 42 mg
n-hexane/propan-2-ol = 95:5 at
Rt = 10.26 min.
a
flow rate 1 mL/min);
(70%) of (S)-(ꢀ)-2, 93% ee.
(c) Microwave-assisted oxidation: PCC (131 mg, 0.6 mmol) and
anhydrous MgSO4 (108 mg, 0.9 mmol) was weighed in a
10 mL glass pressure microwave tube equipped with a mag-
netic stirrer bar. A solution of 9a (85 mg, 0.2 mmol) in dry
CH2Cl2 (2.5 mL) was then added, the tube was closed with
a silicon septum, and the reaction mixture was subjected
to microwave irradiation for 1 h (power: 10 W, temperature:
60 °C). The reaction mixture was allowed to cool to room
temperature and then transferred to a round bottom flask
and worked-up as in the previous procedure to give 33 mg
4.11. General procedure for the synthesis of the diastereoisomers
of the 2-amino analogues of 1-methoxyspirobrassinol methyl
ethers 15–18
To the suspension of 9a (60 mg; 0.143 mmol) in dry CH2Cl2
(5 mL) with molecular sieves (3 Å) was added TFA (18 mg;
0.012 mL; 0.157 mmol) and (i) 3,4-dichloroaniline (46 mg,
0.286 mmol) (ii) p-trifluoromethylaniline (46 mg; 0.036 mL;
0.286 mmol), (iii) p-toluidine (31 mg; 0.286 mmol), (iv) p-anisidine
(35 mg, 0.286 mmol). Reaction mixture was stirred for (i) 1.5 h, (ii)
2.5 h, (iii) 24 h, or (iv) refluxed 14.5 h, and then Et3N (0.016 g;
0.022 mL; 0.157 mmol) was added, the solvent evaporated and
the residue preabsorbed on silica gel was subjected to chromatog-
raphy on silica gel.
(60%) of (R)-(+)-2, [a]
25 = +105.7 (c 0.1, CHCl3); 98% ee (CHI-
D
RALCELÒ OD F 0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm, n-hex-
ane/propan-2-ol = 85:15 at
a
flow rate 1 mL/min);
Rt = 12.03 min.
(d) Application of the same procedure on 10c afforded 35 mg
(63%) of (S)-(ꢀ)-2, [
a
]
25 = ꢀ82.2 (c 0.1, CHCl3); 99% ee (CHI-
D
RALCELÒ OD F 0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm, n-hex-
4.11.1. Synthesis of the isomers of 1-methoxy-2-(3,4-dichlorop-
henylamino)-20-(methylsulfanyl)spiro{indoline-3,50-[40,50]
dihydrotiazole} 15
ane/propan-2-ol = 85:15 at
a
flow rate 1 mL/min);
Rt = 13.86 min.
Following the general procedure, products (2R,3R)-(+)-15 and
(2S,3R)-(ꢀ)-15 were obtained using 3,4-dichloroaniline (46 mg,
0.286 mmol). Separation on silica gel (20 g, ethyl acetate/n-hexane
1:5) afforded product (2R,3R)-(+)-15 impure with menthol and
(2S,3R)-(ꢀ)-15 impure with 3,4-dichloroaniline. Pure diastereoiso-
mers were afforded after chromatography on silica gel (2R,3R)-(+)-
15 (15 g, CH2Cl2) and (2S,3R)-(ꢀ)-15 (8 g, CH2Cl2). All spectroscopic
data were fully identical with the described racemic products.11
(2R,3R)-(+)-15: 38 mg (62%); yellow oil; Rf = 0.41 (ethyl acetate/
4.10. Synthesis of diastereoisomers of 1-methoxyspirobrassinol
methyl ether (2R,3R)-3, (2S,3S)-3, (2R,3S)-3 and (2S,3R)-3
(a) To a solution of 9a (49 mg, 0.117 mmol) in dry methanol was
added TFA (15 mg, 0.01 mL, 0.13 mmol). The reaction mix-
ture was stirred for 12 h, then the solvent was evaporated
and the residue was subjected to chromatography on silica
gel (10 g, cyclohexane/diethyl ether 2:1) to afford 15 mg
(43%) of (2R,3R)-(ꢀ)-3 and 12 mg (35%) of (2S,3R)-(+)-3.
The spectroscopic data of (2R,3R)-(ꢀ)-3 were fully identical
with those of the natural product.6 The spectroscopic data
of (2S,3R)-(+)-3 were fully identical with those of the corre-
sponding racemic product.5
n-hexane 1:5); [
OH, nm) kext
a]
25 = +116.2 (c 0.4, CHCl3); CD (c 0.025 mM, CH3-
D
(D
e
): 210 (ꢀ21.8), 233 (16.6), 246 (6.3), 252 (7.9), 266
(ꢀ1.0), 292 (5.5). 96% ee (Larihc RN-CF6 F 0,46 ꢁ 25 cm, n-heptane/
ethanol = 95:5 at a flow rate 0.6 mL/min); Rt = 10.25 min.
(2S,3R)-(ꢀ)-15: 16 mg (26%); colorless crystals; mp 143–145 °C
(acetone/hexane); Rf = 0.23 (ethyl acetate/n-hexane 1:5);
First eluted diastereoisomer (2R,3R)-(ꢀ)-3: colorless oil;
[
(
a
D
]
e
25 = ꢀ75.9 (c 0.3, CHCl3); CD (c 0.025 mM, CH3OH, nm) kext
D
Rf = 0.51 (cyclohexane/diethyl ether 2:1); [
a
]
25 = ꢀ10.8 (c
): 216 (ꢀ38.5), 238 (ꢀ1.5), 249 (ꢀ6.6), 264 (4.9), 277 (0.2),
D
0.1, CHCl3); CD (CH3OH, nm) kext
(D
e): 214 (ꢀ26.5), 239
296 (2.8). 96% ee (CHIRALCELÒ OD
F
0.46 cm ꢁ 5 cm + F
flow rate
(+3.9), 263 (ꢀ0.7), 291 (+2.7). 97% ee (CHIRALCELÒ OD F
0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm, n-hexane/propan-2-
ol = 75:25 at a flow rate 1 mL/min); Rt = 15.31 min.
0.46 cm ꢁ 25 cm, n-hexane/propan-2-ol = 95:5 at
a
1 mL/min); Rt = 12.42 min.
Application of the same procedure on 10c afforded:
Second eluted diastereoisomer (2S,3R)-(+)-3: colorless crys-
(2S,3S)-(ꢀ)-15: 42 g (69%); yellow oil; Rf = 0.41 (ethyl acetate/n-
tals; mp 77–79 °C (hexane); Rf = 0.31 (cyclohexane/diethyl
hexane 1:5); [
a
]
D
25 = ꢀ131.6 (c 0.4, CHCl3); CD (c 0.025 mM, CH3OH,
ether 2:1); [
a
]
D
25 = +39.1 (c 0.1, CHCl3); CD (CH3OH, nm) kext
nm) kext
(
D
e
): 210 (21.9), 233 (ꢀ18.1), 246 (ꢀ7.2), 252 (ꢀ8.8), 266
(
D
e
): 206 (ꢀ17.2), 233 (+22.8), 250 (ꢀ2.1), 291 (+7.7). 98%
(0.8), 292 (ꢀ6.0). 99% ee (CHIRALCELÒ OD F 0.46 cm ꢁ 5 cm + F
ee (CHIRALCELÒ OD F 0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm,
0.46 cm ꢁ 25 cm, n-hexane/propan-2-ol = 80:20 at
1 mL/min); Rt = 6.84 min.
a flow rate
n-hexane/propan-2-ol = 95:5 at
Rt = 8.73 min.
a flow rate 1 mL/min);
(2R,3S)-(+)-15: 13 g (21%); colorless crystals; mp 143–145 °C
(acetone/hexane); Rf = 0.23 (ethyl acetate/n-hexane 1:5);
(b) Application of the same procedure on 10c afforded 13 mg
(36%) of (2S,3S)-(+)-3 and 15 mg (41%) of (2R,3S)-(ꢀ)-3.
The spectroscopic data of (2S,3S)-(+)-3 were fully identical
with those of the natural product.6 The spectroscopic data
of (2R,3S)-(ꢀ)-3 were fully identical with those of the corre-
sponding racemic product.5
[
(
a]
D
25 = +118.7 (c 0.3, CHCl3); CD (c 0.025 mM, CH3OH, nm) kext
D
e
): 216 (45.1), 238 (1.5), 249 (8.2), 264 (ꢀ6.0), 277 (0), 296
(ꢀ2.9). >99% ee (CHIRALCELÒ OD
F
0.46 cm ꢁ 5 cm + F
flow rate
0.46 cm ꢁ 25 cm, n-hexane/propan-2-ol = 95:5 at
a
1 mL/min); Rt = 10.75 min.
First eluted diastereoisomer (2S,3S)-(+)-3: colorless oil;
Rf = 0.51 (cyclohexane/diethyl ether 2:1); [a]
25 = +7.3 (c 0.1,
4.11.2. Synthesis of the isomers of 1-methoxy-2-(4-trifluoromet-
hylphenylamino)-20-(methylsulfanyl)spiro{indoline-3,50-[40,50]
dihydrotiazole} 16
Following the general procedure, products (2R,3R)-(+)-16 and
(2S,3R)-(ꢀ)-16 were obtained using p-trifluoromethylaniline
(46 mg; 0.036 mL; 0.286 mmol). Separation on silica gel (20 g, n-
hexane/ethyl acetate 5:1) afforded product (2R,3R)-(+)-16 impure
with menthol and (2S,3R)-(ꢀ)-16 impure with 4-trifluoromethyl-
aniline. Pure diastereoisomers were afforded after chromatography
on silica gel (2R,3R)-(+)-16 (15 g, CH2Cl2) and (2S,3R)-(ꢀ)-16 (7 g,
D
CHCl3); CD (CH3OH, nm) kext
(
D
e
): 214 (28.1), 239 (ꢀ3.9),
263 (1.0), 291 (ꢀ2.5). 95% ee (CHIRALCELÒ OD
F
0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm, n-hexane/propan-2-
ol = 75:25 at a flow rate 1 mL/min); Rt = 5.54 min.
Second eluted diastereoisomer (2R,3S)-(ꢀ)-3: colorless crys-
tals; mp 77-79 °C (hexane); Rf = 0.31 (cyclohexane/diethyl
ether 2:1); [
a
]
25 = ꢀ38.5 (c 0.1, CHCl3); CD (CH3OH, nm) kext
D
(D
e
): 206 (15.9), 233 (ꢀ21.3), 250 (2.2), 291 (ꢀ7.0). 92% ee
(CHIRALCELÒ OD
F
0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm,
Please cite this article in press as: Salayová, A.; et al. Tetrahedron: Asymmetry (2014), http://dx.doi.org/10.1016/j.tetasy.2014.07.006

12
A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
CH2Cl2). All spectroscopic data were fully identical with the
described racemic products.11
(0.1). 98% ee (CHIRALPAK IA [CARTRIDGE HOLDER
F
0.4 ꢁ 1 cm + F 0.46 ꢁ 25 cm], n-hexane/propan-2-ol = 95:5 at a
(2R,3R)-(+)-16: 37 mg (61%); yellow oil; Rf = 0.28 (n-hexane/
flow rate 1 mL/min); Rt = 14.22 min.
ethyl acetate 5:1); [
a]
D
25 = +20.2 (c 0.15, CHCl3); CD (c 0.023 mM,
CH3OH, nm) kext
(
D
e
): 210 (ꢀ12.3), 233 (16.9), 245 (5.1), 253
4.11.4. Synthesis of the isomers of 1-methoxy-2-(4-methoxy-
phenylamino)-20-(methylsulfanyl)spiro{indoline-3,50-
[40,50]dihydrotiazole} 18
(9.5), 267 (ꢀ0.1), 294 (6.6). 99% ee (CHIRALPAKÒ OD
F
0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm, n-hexane/ethanol = 99:1 at a
flow rate 1 mL/min); Rt = 10.67 min.
Following the general procedure, products (2R,3R)-(+)-18 and
(2S,3R)-(ꢀ)-18 were obtained using p-anisidine (35 mg,
0.286 mmol). Separation on silica gel (20 g, n-hexane/ethyl acetate
5:1) afforded product (2R,3R)-(+)-18 impure with menthol
and (2S,3R)-(ꢀ)-18 impure with p-anisidine. Pure diastereoisomers
were afforded after chromatography on silica gel (2R,3R)-(+)-18
(10 g, CH2Cl2/MeOH 60:1) and (2S,3R)-(ꢀ)-18 (4 g, CH2Cl2/ethyl
acetate 30:1). All spectroscopic data were fully identical with the
described racemic products.11
(2S,3R)-(ꢀ)-16: 14 mg (23%); white crystals, mp 114–116 °C
(acetone/hexane), Rf = 0.13 (n-hexane/ethyl acetate 5:1);
[
(
a
D
]
e
25 = ꢀ72.5 (c 0.07, CHCl3); CD (c 0.025 mM, CH3OH, nm) kext
D
): 213 (ꢀ50.4), 236 (0.4), 249 (ꢀ10.4), 264 (5.9), 276 (0.9),
292 (4.0). 98% ee (CHIRALPAKÒ OD
F
0.46 cm ꢁ 5 cm + F
0.46 cm ꢁ 25 cm, n-hexane/ethanol = 99:1 at a flow rate 1 mL/
min); Rt = 19.94 min.
Application of same procedure on 10c afforded:
(2S,3S)-(ꢀ)-16: 42 mg (69%); yellow oil; Rf = 0.28 (n-hexane/
(2R,3R)-(+)-18: 19 mg (34%); white crystals; mp 178–180 °C
(acetone/hexane); Rf = 0.24 (n-hexane/ethyl acetate 5:1);
ethyl acetate 5:1); [
a
e
]
25 = ꢀ43.4 (c 0.15, CHCl3); CD (c 0.023 mM,
D
CH3OH, nm) kext
(D
): 210 (14.2); 233 (ꢀ18.5); 245 (ꢀ5.4); 253
[
(
a]
25 = +226.2 (c 0.1, CHCl3); CD (c 0.025 mM, CH3OH, nm) kext
): 210 (ꢀ14.0), 233 (13.5), 263 (0.2), 292 (5.9). 96% ee (Larihc
D
(ꢀ10.1); 267 (0.9); 294 (ꢀ6.4). 98% ee (CHIRALPAKÒ OD
F
D
e
0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm, n-hexane/ethanol = 99:1 at a
RN-CF6 F 0,46 ꢁ 25 cm, n-heptane:ethanol = 95:5 at a flow rate
flow rate 1 mL/min); Rt = 12.51 min.
0.6 mL/min); Rt = 10.96 min.
(2R,3S)-(+)-16: 11 mg (18%); white crystals; mp 114–116 °C
(acetone/hexane); Rf = 0.13 (n-hexane/ethyl acetate 5:1);
(2S,3R)-(ꢀ)-18: 7 mg (13%); white crystals; mp 143–145 °C
(acetone/hexane); Rf = 0.13 (n-hexane/ethyl acetate 5:1);
[a
]
D
25 = +42.7 (c 0.07, CHCl3); CD (c 0.025 mM, CH3OH, nm) kext
[a]
25 = ꢀ95.8 (c 0.15, CHCl3); CD (c 0.025 mM, CH3OH, nm) kext
D
(
D
e
): 213 (38.1), 236 (ꢀ0.5), 249 (8.5), 264 (ꢀ4.3), 276 (ꢀ0.3),
(
D
e
): 214 (ꢀ8.6), 232 (ꢀ1.3), 244 (ꢀ2.2), 261 (1.8), 280 (1.1), 297
292 (ꢀ2.6). 99% ee (CHIRALPAKÒ OD
F
0.46 cm ꢁ 5 cm + F
(1.5). 90% ee (CHIRALPAKÒ AD
F
0.46 cm ꢁ 5 cm + F
flow rate
0.46 cm ꢁ 25 cm, n-hexane/ethanol = 99:1 at a flow rate 1 mL/
min); Rt = 17.42 min.
0.46 cm ꢁ 25 cm, n-hexane/propan-2-ol = 95:5 at
a
1 mL/min); Rt = 26.00 min.
Application of the same procedure on 10c afforded:
4.11.3. Synthesis of the isomers of 1-methoxy-2-(4-methyl-
phenylamino)-20-(methylsulfanyl)spiro{indoline-3,50-[40,50]
dihydrotiazole} 17
(2S,3S)-(ꢀ)-18: 17 mg (31%); white crystals; mp 178–180 °C
(acetone/hexane); Rf = 0.24 (n-hexane/ethyl acetate 5:1);
[a]
25 = ꢀ135.2 (c 0.1, CHCl3); CD (c 0.025 mM, CH3OH, nm) kext
D
Following the general procedure, products (2R,3R)-(+)-17 and
(2S,3R)-(ꢀ)-17 were obtained using p-toluidine (31 mg;
0.286 mmol). Separation on silica gel (20 g, n-hexane/ethyl acetate
8:1) afforded product (2R,3R)-(+)-17 impure with menthol and
(2S,3R)-(ꢀ)-17 impure with 4-methylaniline. Pure diastereoiso-
mers were afforded after chromatography on silica gel (2R,3R)-
(+)-17 (15 g, CH2Cl2) and (2S,3R)-(ꢀ)-17 (4 g, CH2Cl2). All spectro-
scopic data were fully identical with the described racemic
products.11
(D
e
): 210 (11.1), 233 (ꢀ11.0), 263 (1.0), 293 (ꢀ3.3). 98% ee (CHIR-
ALPAKÒ AD F 0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm, n-hexane/pro-
pan-2-ol = 95:5 at a flow rate 1 mL/min); Rt = 15.48 min.
(2R,3S)-(+)-18: 5 mg (9%); white crystals; mp 143–145 °C (ace-
tone/hexane); Rf = 0.13 (n-hexane/ethyl acetate 5:1); [
a]
25 = +116.3
D
(c 0.15, CHCl3); CD (c 0.025 mM, CH3OH, nm) kext (De): 214 (8.2),
232 (1.3), 244 (3.0), 261 (ꢀ0.9), 280 (0.4), 297 (0.1). 94% ee (CHIR-
ALPAKÒ AD F 0.46 cm ꢁ 5 cm + F 0.46 cm ꢁ 25 cm, n-hexane/pro-
pan-2-ol = 95:5 at a flow rate 1 mL/min); Rt = 31.26 min.
(2R,3R)-(+)-17: 32 mg (60%); colorless oil; Rf = 0.38 (n-hexane/
ethyl acetate 8:1); [
CH3OH, nm) kext
(6.7). 95% ee (CHIRALCELÒ OD
a
]
25 = +211.7 (c 0.15, CHCl3); CD (c 0.025 mM,
4.12. Antiproliferative activity
D
(
D
e
): 210 (ꢀ16.1), 233 (16.2), 262 (ꢀ0.4), 293
F
0.46 cm ꢁ 5 cm + F
flow rate
4.12.1. Cell culture—Tumor cell lines
0.46 cm ꢁ 25 cm, n-hexane/propan-2-ol = 95:5 at
a
Jurkat (acute T-lymphoblastic leukemia), HeLa (cervical adeno-
carcinoma), MCF-7 (mammary gland adenocarcinoma), MDA-MB-
231 (mammary gland adenocarcinoma), A-549 (non-small cell
lung cancer), and CCRF-CEM cell line (acute T-lymphoblastic leuke-
mia) cells were maintained in RPMI 1640 medium supplemented
with Glutamax-I or in Dulbecco’s modified Eagle’s medium with
Glutamax-I and glucose. Both of these media were supplemented
with 10% (v/v) fetal calf serum, penicillin (100 IU ꢁ mLꢀ1), and
1 mL/min); Rt = 12.36 min.
(2S,3R)-(ꢀ)-17: 8 mg (15%); colorless crystals, mp 155–157 °C
(acetone/hexane); Rf = 0.18 (n-hexane/ethyl acetate 8:1);
[
(
a
D
]
e
25 = ꢀ74.7 (c 0.3, CHCl3); CD (c 0.025 mM, CH3OH, nm) kext
D
): 213 (ꢀ14.5), 233 (ꢀ1.3), 244 (ꢀ3.8), 261 (3.1), 278 (1.2),
294 (1.8). 96% ee (CHIRALPAK IA [CARTRIDGE HOLDER
U
0.4 ꢁ 1 cm +
U
0.46 ꢁ 25 cm], n-hexane/propan-2-ol = 95:5 at a
flow rate 1 mL/min); Rt = 10.20 min.
streptomycin (100
l
g ꢁ mLꢀ1) (all from Invitrogen, UK), in humid-
Application of same procedure on 10c afforded:
ified air with 5% CO2 at 37 °C. Before each cytotoxicity assay, cell
viability was determined by the trypan blue (Invitrogen) exclusion
method and found to be greater than 95%.
(2S,3S)-(ꢀ)-17: 37 mg (70%); colorless oil; Rf = 0.38 (n-hexane/
ethyl acetate 8:1);
[
a
]
25 = ꢀ207.9 (c 0.15, CHCl3); CD (c
D
0.025 mM, CH3OH, nm) kext
(D
e
): 210 (15.9), 233 (ꢀ16.3), 262
(1.7), 293 (ꢀ5.3). 99% ee (CHIRALCELÒ OD F 0.46 cm ꢁ 5 cm + F
4.12.2. Cytotoxicity assay
0.46 cm ꢁ 25 cm, n-hexane/propan-2-ol = 95:5 at
a
flow rate
The cytostatic/cytotoxic effects of the compounds were studied
using the colorimetric microculture assay with the MTT end-
point.16 Briefly, 5 ꢁ 103 cells were plated per well in 96-well poly-
1 mL/min); Rt = 7.05 min.
(2R,3S)-(+)-17: 8 mg (15%); colorless crystals; mp 155–157 °C
(acetone/hexane); Rf = 0.18 (n-hexane/ethyl acetate 8:1);
styrene microplates (Sarstedt, Germany) in 100 lL of the culture
[a
]
D
25 = +90.1 (c 0.3, CHCl3); CD (c 0.025 mM, CH3OH, nm) kext
medium containing tested chemicals at final concentrations of
(D
e
): 213 (16.9), 233 (3.1), 244 (5.1), 261 (ꢀ1.9), 278 (0.6), 294
10ꢀ6–10ꢀ4 mol ꢁ Lꢀ1
. After 72 h incubation, 10 lL of MTT
Please cite this article in press as: Salayová, A.; et al. Tetrahedron: Asymmetry (2014), http://dx.doi.org/10.1016/j.tetasy.2014.07.006

A. Salayová et al. / Tetrahedron: Asymmetry xxx (2014) xxx–xxx
13
(5 mg ꢁ mLꢀ1) (Sigma–Aldrich) was added into each well. After an
additional 4 h at 37 °C, during which insoluble formazan was pro-
duced, 100 lL of 10% (m/m) sodium dodecylsulfate (SDS, Sigma–
Aldrich) was added into each well and another 12 h were allowed
for the dissolution of the formazan. The absorbance was measured
at 540 nm and 630 nm–reference wavelength by the automated
uQuant™ Universal Microplate Spectrophotometer (Biotek Instru-
ments Inc., Winooski, VT USA). The blank corrected absorbance of
the control wells was taken as 100% and the results were expressed
as a percentage of the control.
Properties; Attanasi, O. A., Spinelli, D., Eds.; Italian Society of Chemistry:
Urbino, Italy, 2009; Vol. 12, pp 120–148.
2. Takasugi, M.; Monde, K.; Katsui, N.; Shirata, A. Chem. Lett. 1987, 1631–1632.
3. Suchy´, M.; Kutschy, P.; Monde, K.; Goto, H.; Harada, N.; Takasugi, M.; Dzurilla,
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Dzurilla, M.; Miklošová, M.; Mezencev, R.; Mojziš, J. Tetrahedron Lett. 2002, 43,
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Acknowledgments
10. Mezencev, R.; Kutschy, P.; Salayová, A.; Updegrove, T.; McDonald, J. F.
Neoplasma 2009, 56, 321–330.
We would like to thank the Slovak Grant Agency for Science
(Grant No. 1/0954/12 and No. 1/3553/06) for financial support of
this work. This work was also supported by the State NMR Pro-
gramme No. 2003SP200280203.
ˇ
ˇ
ˇ
11. Kutschy, P.; Salayová, A.; Curillová, Z.; Kozár, T.; Mezencev, R.; Mojziš, J.;
Pilátová, M.; Balentová, E.; Pazdera, P.; Sabol, M.; Zburová, M. Bioorg. Med.
Chem. 2009, 17, 3698–3712.
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Please cite this article in press as: Salayová, A.; et al. Tetrahedron: Asymmetry (2014), http://dx.doi.org/10.1016/j.tetasy.2014.07.006