Spirocyclization reactions and antiproliferative activity of indole phytoalexins 1-methoxybrassinin and its 1-substituted derivatives

Article abstract of DOI:10.24820/ark.5550190.p009.958

The effect of the reaction temperature and the solvent on the diastereoselectivity of the spirocyclization of 1-methoxybrassinin leading to 1-methoxyspirobrassinol methyl ether was studied. 1-Acyl derivatives of 1-methoxyspirobrassinol and 1-methoxyspirobrassinol methyl ether were prepared by the bromine-mediated spirocyclization reactions of derivatives of brassinin bearing an acyl group on the indole nitrogen with water or methanol as nucleophilic agents. The cyclization of 1-acyl derivatives of brassinin afforded the trans-diastereoisomer as the major product, whereas using 1-methoxybrassinin afforded the cis- and trans-isomers in a ratio near to 1:1. Bromospirocyclization of brassinin and 1-methylbrassinin in the presence of methanol resulted in the formation of spirobrassinin and 1-methylspirobrassinin. The newly synthesized analogues of indole phytoalexins exhibited more significant antiproliferative activity against human leukemia cell lines than the natural phytoalexins.

Full text of DOI:10.24820/ark.5550190.p009.958

General Papers
ARKIVOC 2016 (vi) 198-234
Spirocyclization reactions and antiproliferative activity of indole
phytoalexins 1-methoxybrassinin and its 1-substituted derivatives
a
b
b
b
Mariana Budovská,* Martina Bago Pilátová, Viera Tischlerová, and Ján Mojžiš
aDepartment of Organic Chemistry, Institute of Chemical Sciences, Faculty of Science,
P. J. Šafárik University, Moyzesova 11, 040 01, Košice, Slovak Republic
bDepartment of Pharmacology, Faculty of Medicine, P. J. Šafárik University, SNP 1,
0
40 66 Košice, Slovak Republic
E-mail: mariana.budovska@upjs.sk
DOI: https://doi.org/10.24820/ark.5550190.p009.958
Abstract
The effect of the reaction temperature and the solvent on the diastereoselectivity of the
spirocyclization of 1-methoxybrassinin leading to 1-methoxyspirobrassinol methyl ether was
studied. 1-Acyl derivatives of 1-methoxyspirobrassinol and 1-methoxyspirobrassinol methyl
ether were prepared by the bromine-mediated spirocyclization reactions of derivatives of
brassinin bearing an acyl group on the indole nitrogen with water or methanol as nucleophilic
agents. The cyclization of 1-acyl derivatives of brassinin afforded the trans-diastereoisomer as
the major product, whereas using 1-methoxybrassinin afforded the cis- and trans-isomers in a
ratio near to 1:1. Bromospirocyclization of brassinin and 1-methylbrassinin in the presence of
methanol resulted in the formation of spirobrassinin and 1-methylspirobrassinin. The newly
synthesized analogues of indole phytoalexins exhibited more significant antiproliferative
activity against human leukemia cell lines than the natural phytoalexins.
Keywords:
Indole
phytoalexins,
spirocyclization,
diastereoselectivity,
1
-methoxyspirobrassinol methyl ether, antiproliferative activity
Introduction
1
In 1940 Müller first proposed the phytoalexin concept. Phytoalexins play a significant role in
the defence response of plants. These secondary metabolities, which are synthesized de novo in
2
response to biotic or abiotic stress, are part of the plant chemical defense mechanism. Indole
3
phytoalexins produced by crucifers were first reported in 1986 by Takasugi. To date, 44 indole
phytoalexins have been isolated from economically and dietary important plants of the family
Cruciferae (syn Brassicaceae), which are cultivated worldwide (e.g. cabbage, turnip, Chinese
4
cabbage, Japanese radish, wasabi, broccoli, rapeseed and arabidopsis). The 44 cruciferous
4
phytoalexins have been divided into six groups according to simple structural features. A
unique group of these natural products are spiroindoline structures containing a spirocyclic ring
5
6
in the C-3 position [(S)-()-spirobrassinin [()-1], (R)-(+)-1-methoxyspirobrassinin [(+)-2], 1-
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7
methoxyspirobrassinol (3) and trans-(2R,3R)-()-1-methoxyspirobrassinol methyl ether
7
[
(2R,3R)-()-4a, (Figure 1)]. In 1987, the first spiroindoline phytoalexin (S)-()-spirobrassinin
[
()-1] was isolated from Pseudomonas cichorii-inoculated Japanese radish (Raphanus
5
sativus). Natural ()-spirobrassinin [()-1] was assigned the absolute configuration (S) on the
base of X-ray crystallographic analysis and CD studies. (±)-Spirobrassinin [(±)-1] was
8
,9
synthesized by thionyl chloride- or methanesulfonyl chloride-mediated cyclization of (±)-
8
,9
dioxibrassinin. A stereoselective synthesis of (S)-()-spirobrassinin [()-1] was achieved by
bromine-induced spirocyclization of ()-1-(8-phenylmenthoxycarbonyl)brassinin with water,
1
0
followed by oxidation and removal of the chiral auxiliary. (R)-(+)-1-Methoxyspirobrassinin
6
[
(+)-2] was isolated in 1994 from kohlrabi after UV irradiation while trans-(2R,3R)-()-1-
methoxyspirobrassinol methyl ether [(2R,3R)-()-4a] and optically inactive 1-
methoxyspirobrassinol (3) were isolated in 1995 from Japanese radish after inoculation with
7
Pseudomonas cichorii. Compound 3 exists in solution as a mixture of two diastereoisomers
7
trans-3a and cis-3b in a 1:4 ratio, owing to its unstable hemiaminal structure. The enantiomers
of ()-1-methoxyspirobrassinin [(±)-2] and trans-()-1-methoxyspirobrassinol methyl ether
[
trans-(±)-4a] were resolved by chiral HPLC and the absolute configurations of natural (R)-(+)-
2
and (2R,3R)-()-4a were determined by electronic circular dichroism (ECD), vibrational
1
1
circular dichroism (VCD) and chemical correlation.
Figure 1. Selected indolic phytoalexins.
The first syntheses of 1-methoxyspirobrassinol (3) and 1-methoxyspirobrassinol methyl
ether (4) were achieved by the 1,4-dioxane-dibromide (DDB)-mediated spirocyclization of 1-
methoxybrassinin (5a) in 1,4-dioxane in the presence of water or methanol. The reaction
probably proceeds via sulfenyl bromide 6, which cyclizes to spiroindoleninium ion A. When
1
[
-methoxybrassinin (5a) was cyclized in the presence of water, 1-methoxyspirobrassinol
trans-(±)-3a] and [cis-(±)-3b] was produced. In the presence of methanol as a nucleophile, a
mixture of diastereoisomers, natural trans-(±)-4a and unnatural cis-diastereoisomer cis-(±)-4b
1
2
in a ratio 1:2, was obtained (Scheme 1). Oxidation of a mixture of diastereoisomers trans-
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(
±)-3a and cis-(±)-3b with CrO 12
3
or a mixture of diastereoisomers trans-(±)-4a and cis-(±)-4b
1
1
with PCC afforded racemic 1-methoxyspirobrassinin [(±)-2].
Indole phytoalexins have been previously shown to exert antibacterial, antifungal5-7,13,14 and
anticancer properties11,15-18 and can serve as lead compounds for anticancer drug design.
Brassinin (5b) and spirobrassinin [(±)-1] are effective in inhibiting the formation of 7,12-
dimethylbenz[a]anthracene (DMBA)-induced preneoplastic lesions in a mouse mammary
1
7
gland. In addition, brassinin (5b) has been reported to exhibit dose-dependent inhibition of
1
9
DMBA-induced and TPA-promoted skin carcinogenesis. Izutani et al. demostrated that
brassinin (5b) inhibits cell growth in human colon cancer cells by arresting the G phase via
inceased expression of p21 and p27. Spiroindoline phytoalexins and their derivatives exhibit
1
2
0
1
5,21-26
an antiproliferative effect against human cancer cell lines.
Brassinin (5b) and its synthetic
derivative 5-bromobrassinin (5c) act as bioavailable competitive inhibitors of indoleamine 2,3-
dioxygenase (IDO), a tryptophan-catabolizing enzyme that promotes tumor escape via a
2
7,28
mechanism of immune tolerance.
1-Methoxybrassinin (5a) displayed significant
antiproliferative effect on intramolecular amastigotes of Trypanosoma cruzi and demonstrated
a higher potency than shown by the currently used antichagasic agents (nifurtimox,
2
9
benznidazol). Kristofikova et al. documented the in vitro effect of anti-aggregation of
3
0
spirobrassinin [(±)-1] in the cerebrospinal fluid of patients with multiple sclerosis.
In this paper we describe our investigations into the diastereoselectivity of spirocyclization
of 1-methoxybrassinin (5a) in the presence of various alcohols as nucleophiles, in various
solvents and at various temperatures. We have also examined the bromine-initiated
spirocyclization of 1-acyl derivatives of brassinin in the presence of water and methanol and
studied the influence of acyl groups on the diastereoselectivity of bromospirocyclizations in
comparison with 1-methoxybrassinin (5a). To our knowledge, no spiroindoline derivatives had
been prepared by bromocyclization of brassinin (5b).
Scheme 1. Spirocyclization of 1-methoxybrassinin (5a) in the presence of water or methanol.
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Results and Discussion
First the effect of the solvent (dichloromethane, diethyl ether, diisopropyl ether,
tetrahydrofuran, 1,4-dioxane) was studied on the diastereoselectivity of the bromine-induced
spirocyclization reaction of 1-methoxybrassinin (5a) at rt. A distinct change in
diastereoselectivity was observed upon replacing 1,4-dioxane as a solvent with
dichloromethane. Bromine was used instead of 1,4-dioxane dibromide as a convenient
cyclization agent. The reaction mixture was stirred for 15 min at rt and then triethylamine was
added to trap the hydrogen bromide liberated during the reaction. Under these conditions a
mixture of isomers containing a small excess of the trans-diastereoisomer trans-(±)-4a was
obtained (Table 1, entry 1). This result highlights the influence of the solvent on the
diastereoselectivity of spirocyclization. When using 1,4-dioxane it is postulated that rather than
direct addition of methanol on the methoxyiminium ion A, which is favourable from both sides,
a solvent molecule attacks methoxyiminium ion A from the less hindered thiazoline CH
2
side
2
with the formation of an unstable oxonium ion B. Subsequently methanol attacks the oxonium
2
ion B from the sulfur side, which results in the formation of the cis-diastereoisomer cis-(±)-
4
b (Scheme 2).
The designations trans- and cis-diastereoisomers are used for differentiation of
diastereoisomers. The trans-diastereoisomer is regarded as the one with the sulfur of thiazoline
ring and methoxy group at C-2 located on the opposite sides of indoline ring, whereas the cis-
diastereoisomer has the sulfur and 2-methoxy group on the same side of indoline ring.
Scheme 2. The mechanism of the spirocyclization of 1-methoxybrassinin (5a) in 1,4-dioxane.
This mechanism was also supported by cyclizations with other ethers used as solvent
(diethyl ether, diisopropyl ether and tetrahydrofuran). The trans-diastereoisomer trans-(±)-4a
was obtained as the minor product (Table 1, entries 3 and 4) using diisopropyl ether and
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tetrahydrofuran, whereas the use of diethyl ether provided a 1:1 mixture of diastereoisomers
trans-(±)-4a and cis-(±)-4b (Table 1, entry 2). Cyclization reactions carried out in methanol
and n-heptane (Table 1, entries 6 and 7), in which methanol directly attacks the
methoxyiminium ion A, provided a 1:1 mixture of trans-(±)-4a and cis-(±)-4b diastereoisomers
as expected.
The effect of triethylamine on the diastereoselectivity of spirocyclization of 1-
methoxybrassinin (5a) was also determined. Performing the spirocyclization with bromine in
anhydrous dichloromethane and subsequent addition of a solution of Et
methoxyspirobrassinol methyl ethers were isolated in a 39:61 ratio in favor of cis-
diastereoisomer cis-(±)-4b (Table 1, entry 8). It is postulated that in this case the Et
preferably approaches the intermediate methoxyiminium ion A from the less hindered CH side
3
N in methanol, 1-
3
N
2
of thiazoline ring with the formation of an unstable triethylammonium ion analogous to that
produced from 1,4-dioxane.
Table 1. The effect of solvent on the diastereoselectivity of the spirocyclization of the 1-
methoxybrassinin (5a) at room temperature
Entry Conditions
Ratioa
trans-(±)-4a :
cis-(±)-4b
54:46
Yieldb
(%)
1
2
3
4
5
6
7
8
Br
Br
Br
Br
2
2
2
2
, CH
2
Cl
2
/MeOH (v:v 9:1)
65
67
73
67
60
76
67
, Et O/MeOH (v:v 9:1)
2
50:50
40:60
, diisopropyl ether/MeOH (v:v 9:1)
, THF/MeOH (v:v 9:1)
43:57
36:64
1
2
DDB, 1,4-dioxane/MeOH (v:v 9:1)
Br
Br
Br
2
2
2
, MeOH (v:v 9:1)
, n-heptane/MeOH (v:v 9:1)
50:50
50:50
, CH
2
Cl , after 1 min. 1.1 eq. MeOH, 10 eq.
2
3
9:61
89
Et N
3
a
The ratios of diastereoisomers trans-(±)-4a : cis-(±)-4b were determined by integration of
1
separate signals corresponding to H-2, H
a
and H
b
protons in the H NMR spectrum of the crude
product mixture.
b
Crude product.
With the aim of influencing the diastereoselectivity of the spirocyclization of 5a, the effect
of temperature on the reaction was examined. Performing experiments above and below rt
confirmed that temperature has a distinct effect on the diastereoselectivity of the
spirocyclization of 1-methoxybrasssinin (5a). Reactions performed at low temperature led
predominantly to the trans-diastereoisomer trans-(±)-4a (Table 2), whereas at rt or at 40-60 °C
in 1,4-dioxane a preference for the cis-diastereoisomer cis-(±)-4b was observed. The best ratio
was achieved at -70 °C in THF as solvent (Table 2, entry 16).
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Table 2. The effect of temperature on the diastereoselectivity of the spirocyclization of 1-
methoxybrassinin (5a)
Entry Conditions
Temperature
Ratioa
trans-(±)-4a : cis-(±)-4b
54:46
Yieldb
(%)
65
73
73
70
67
73
73
70
73
70
73
73
67
70
70
67
70
70
60
73
73
76
73
73
67
71
63
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
Br
Br
Br
2
2
2
, CH
2
Cl
2
/MeOH (v:v 9:1)
rt
0 °C
-20 °C
-60 °C
rt
0 °C
-20 °C
-60 °C
rt
0 °C
-20 °C
-60 °C
rt
0 °C
-20 °C
-70 °C
60 °C
40 °C
rt
62:38
65:35
74:26
, Et
2
O/MeOH (v:v 9:1)
50:50
61:39
68:32
75:25
, diisopropyl ether/MeOH
40:60
61:39
69:31
72:28
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
(v:v 9:1)
Br
2
, THF/MeOH (v:v 9:1)
43:57
68:32
70:30
85:15
DDB, 1,4-dioxane/MeOH (v:v
9:1)
27:73
28:72
36:64
1
2
0 °C
-20 °C
rt
-20 °C
-60 °C
rt
63:37
75:25
50:50
61:39
70:30
50:50
63:37
70:30
Br
2
, MeOH
Br
Br
2
2
, n-heptane/MeOH (v:v 9:1)
, n-heptane, after 1 min. 1.1
-60 °C
-60 °C
eq. MeOH
Br , CH Cl
1.1 eq. MeOH, 10 eq. Et
2
2
8
9
2
2
2
, after 1 min.
rt
39:61
17:83
89
88
3
N
-75 °C
a
The ratios of diastereoisomers trans-(±)-4a : cis-(±)-4b were determined by integration of
separate signals corresponding to H-2, H
1
a
and H
b
protons in the H NMR spectrum of the crude
product mixture.
b
Crude product.
Under these conditions a mixture of trans-diastereoisomer trans-(±)-4a and cis-
diastereoisomer cis-(±)-4b was obtained in an 85:15 ratio. It is postulated that at low
temperature, molecules of methanol form intermolecular hydrogen bonds with the solvent used
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as well as with each other to create bulky associates. Such associates reacts with the
methoxyiminium ion A from the less hindered thiazoline CH
formation of trans-diastereoisomer trans-(±)-4a.
2
side with the preferential
Finding the effect of solvent on the diastereoselectivity of the spirocyclization of 5a led us
to use methanol as a nucleophile in the form of a large complex. Therefore we performed the
bromospirocyclization of 1-methoxybrassinin (5a) in anhydrous dichloromethane with sodium
methoxide in the presence of 15-crown-5 ether. To a stirred mixture of 1-methoxybrassinin
(
5a) in anhydrous dichloromethane was added bromine. After stirring for one minute, a freshly
prepared solution of the complex CH ONa/15-crown-5 in anhydrous dichloromethane was
added. In the product misture, cis-diastereoisomer cis-(±)-4b predominated (Table 3, entry 1).
Probably, the complex CH ONa/15-crown-5-ether was decomposed by the influence of
3
3
hydrogen bromide liberated during reaction and 15-crown-5-ether liberated from the complex
had the same effect on the diastereoselectivity as did 1,4-dioxane. To prevent decomposition
of this complex, triethylamine (2 eq.) was added to the reaction mixture to trap the hydrogen
bromide and then a solution of complex CH ONa/15-crown-5-ether in anhydrous
3
dichloromethane was added. As can be seen from Table 3 (entry 2), this spirocyclization of 5a
led to formation of the trans-diastereoisomer trans-(±)-4a preferentially. Performing the
spirocyclization with anhydrous K
2
CO (2 eq., Table 3, entry 3), the natural diastereoisomer of
3
1
-methoxyspirobrassinol methyl ether trans-(±)-4a was prepared with improved
diastereoselectivity in a 69:31 ratio. Probably, the complex of sodium methoxide with 15-
crown-5-ether as nucleophile approaches the intermediate methoxyiminium ion A
preferemtially from the less hindered CH side of thiazoline ring and leads to formation trans-
2
diastereoisomer trans-(±)-4a.
Table 3. Spirocyclization of the 1-methoxybrassinin (5a) in the presence of the complex
MeONa/15-crown-5 ether at room temperature
Entry Conditions
Ratioa
trans-(±)-4a : cis-(±)-4b
Yieldb
(%)
68
1
2
3
Br
Br
Br
2
2
2
, CH
, CH
, CH
2
Cl
Cl
Cl
2
2
2
, MeONa/15-crown-5
, Et N, MeONa/15-crown-5
, K CO , MeONa/15-crown-5
39:61
64:36
69:31
2
3
71
1
1
2
2
3
71
a
The ratios of diastereoisomers trans-(±)-4a : cis-(±)-4b were determined by integration of
1
separate signals corresponding to H-2, H
a
and H
b
protons in the H NMR spectrum of the crude
b
product mixture. Crude product.
We also investigated the effect of the bulkiness of alcohols (ethanol, isopropyl alcohol, tert-
butanol, phenol and naphth-2-ol) on the diastereoselectivity of the spirocyclization of 1-
methoxybrassinin (5a, Scheme 3). Diastereoisomers trans-(±)-7a, cis-(±)-7b and trans-(±)-9a,
cis-(±)-9b were obtained in a 57:43 ratio (Table 4, entries 2 and 4). The use of isopropyl alcohol
provided a 62:38 mixture of isomers trans-(±)-8a, cis-(±)-8b (Table 4, entry 3). The
bromocyclization reaction of 1-methoxybrassinin (5a) in the presence of phenol and naphth-2-
ol as nucleophiles afforded mixtures of diastereoisomers (±)-10a-(±)-10b and (±)-11a-(±)-11b
with a slight excess of the cis-isomer (Table 4, entries 5 and 6).
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S
N
N
SCH3
SCH3
SCH3
NH
S
S
i) Br , ROH,
2
CH Cl , rt
OR
OR
N
OCH3
2
2
N
N
OCH3
ii) Et N
3
OCH3
5a
R = CH CH3 trans-(±)-7a
R = CH(CH3)2 trans-(±)-8a
R = C(CH3)3 trans-(±)-9a
cis-(±)-7b
cis-(±)-8b
cis-(±)-9b
cis-(±)-10b
cis-(±)-11b
2
R = Ph
trans-(±)-10a
R = 2-naphthyl trans-(±)-11a
Scheme 3. Cyclization reactions of 1-methoxybrassinin (5a) with alcohols.
Table 4. The effect of the bulkiness of alcohols (ethanol, isopropyl alcohol, tert-butanol,
phenol and naphth-2-ol) on the diastereoselectivity of spirocyclization of 1-methoxybrassinin
(
5a) at room temperature
Entry
Ratioa
trans-(±)- : cis-(±)-
54:46
Yieldb
(%)
65
Compound
R
1
2
3
4
5
6
()-4a, ()-4b
()-7a, ()-7b
()-8a, ()-8b
()-9a, ()-9b
()-10a, ()-10b
()-11a, ()-11b
Me
Et
57:43
68
i-Pr
62:38
77
t-Bu
Ph
57:43
71
8823
32:68
2-naphthyl
32:68
65
a
The ratios of diastereoisomers (±)-7a:(±)-7b-(±)-11a:(±)-11b were determined by integration
1
of separate signals corresponding to H-2, H
crude product mixture. Crude product.
a
and H
b
protons in the H NMR spectrum of the
b
We also decided to study the influence of an acyl group on the diastereoselectivity of the
bromospirocyclization reactions of 1-acylderivatives, 12, and 23-25, of brassinin. For the
experiments we selected tert-butoxycarbonyl, acetyl, benzoyl and methoxycarbonyl groups.
The key intermediate 1-Boc-brassinin (12) was prepared by the previously reported
3
1
procedure. Commercially available indole-3-carboxaldehyde (13) was used as a starting
compound for the preparation 1-acetyl (23), 1-benzoyl (24) and 1-(methoxycarbonyl)brassinin
(
25, Scheme 4). N-Acyl aldehydes 14-16 were synthesized by various methods, using acetic
anhydride/DMAP in THF (14, 91%), benzoyl chloride/Et N in THF (15, 98%) or methyl
3
chloroformate/NaH in acetonitrile (16, 85%). Treatment of aldehydes 14 and 15 with
hydroxylamine hydrochloride in THF in the presence of sodium acetate as the base provided
oximes 17 and 18 as mixtures of Z- and E-isomers. Oxime 19 was obtained from aldehyde 16
using NH
7 using sodium borohydride afforded the unstable amine 20 which was employed as a crude
product immediately after isolation. Subsequent reaction of amine 20 with CS and CH I in
2
OH.HCl, Na
2
CO
3
, EtOH, H O in 80% yield. Nickel boride-catalyzed reduction of
2
1
2
3
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methanol in the presence of Et
3
N resulted in the formation of 1-acetylbrassinin (23) in 22%
yield. A better result was obtained, when dichloromethane was used for the extraction of amine
0 and also as a solvent in the reaction with CS and CH I. Under these conditions 1-
acetylbrassinin (23) was isolated in 78% yield. The reduction of oxime 18 by sodium
borohydride and subsequent treatment of amine 21 with CS and CH I in methanol afforded 1-
2
2
3
2
3
benzoylbrassinin (24) in 20% yield. Replacement of the methanol as solvent by
dichloromethane again improved the yield to 36% (Scheme 4). If we performed the reduction
of 18 with sodium cyanoborohydride, the yield of 1-benzoylbrassinin (24) was 32%.
1
-(Methoxycarbonyl)brassinin
-(methoxycarbonyl)indole-3-ylmethyl amine (22, obtained by nickel boride-catalyzed
reduction of 19 with sodium borohydride) by the reaction with CS and CH I in methanol in
8% yield after two reaction steps .
(25)
was
prepared
from
the
unstable
1
2
3
5
Scheme 4. Synthesis of 1-acyl derivatives 12, 23-25 of brassinin.
Derivatives of brassinin 12, 23-25 were dissolved in a mixture dichloromethane/water or
methanol (v/v 9:1) and then 1.1 equivalents of bromine were added (Scheme 5). After 15
minutes of stirring triethylamine was added for the neutralization of hydrogen bromide
liberated by the spirocyclization. In such a way were prepared 1-acetyl- (26), 1-benzoyl- (27),
1
-methoxycarbonyl- (28) and 1-Boc-spirobrassinol (29) as well as 1-acetyl- (30), 1-benzoyl-
(
31), 1-methoxycarbonyl- (32) and 1-Boc-spirobrassinol methyl ether (33). The ratios of
diastereoisomers (±)-26a-(±)-33b and yields are summarized in Table 5. In all cases the trans-
diastereoisomer trans-(±)-26a-(±)-33a was obtained in preference. trans-Diastereoisomer
trans-(±)-33a was also the major product using 1,4-dioxane as the solvent. Cooling the reaction
mixture did not change the stereoselectivity. The cyclization reaction of 12 accomplished at –
6
0 °C also led predominantly to the trans-diastereoisomer trans-(±)-33a (Table 5). For
comparison, Table 5 includes the ratios of diastereoisomers of 1-methoxyspirobrassinol [(±)-
3
a-(±)-3b] and 1-methoxyspirobrassinol methyl ether [(±)-4a-(±)-4b].
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Scheme 5. Bromospirocyclization of 1-acyl derivatives 12, 23-25
Table 5. Ratios and yields of diastereoisomers of 1-acyl derivatives of 1-
methoxyspirobrassinol methyl ether (±)-26a-(±)-33b
Compounds
R1
R2
Conditions
Ratioa
trans-(±)- : cis-(±)-
71:29
Yieldb
(%)
79
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
)-26a, ()-26b
)-27a, ()-27b
)-28a, ()-28b
)-29a, ()-29b
)-29a, ()-29b
)-3a, ()-3b
COCH
3
H
CH
CH
CH
CH
2
2
2
2
Cl
Cl
Cl
Cl
2
, rt
, rt
, rt
, rt
COC
6
H
5
2
64:36
77
COOCH
3
2
83:17
38
COOC(CH
3
3
)
3
3
2
77:23
80:2012
53
COOC(CH
)
1,4-dioxane, rt
CH Cl , rt
1,4-dioxane, rt
47
OCH
OCH
3
3
2
2
21:79
90
)-3a, ()-3b
20:8012
67:33
90
)-30a, ()-30b
)-31a, ()-31b
)-32a, ()-32b
)-33a, ()-33b
)-33a, ()-33b
)-33a, ()-33b
)-4a, ()-4b
COCH
COC
COOCH
3
OCH
3
CH
CH
CH
CH
CH
2
2
2
2
2
Cl
Cl
Cl
Cl
Cl
2
, rt
67
6
H
5
2
, rt
74:26
66
3
2
, rt
71:29
49
COOC(CH
COOC(CH
COOC(CH
3
3
3
)
3
)
3
)
3
2
, rt
71:29
65
2
, -60 °C
78:22
65
1,4-dioxane, rt
71:29
51
OCH
OCH
OCH
3
3
3
CH
CH
2
Cl
2
, rt
54:46
65
)-4a, ()-4b
)-4a, ()-4b
2
Cl
2
, -60 °C
74:26
36:6412
70
1,4-dioxane, rt
60
a
The ratios of diastereoisomers (±)-26a-(±)-33b were determined by integration of separate
1
signals corresponding to H-2, H
a
and H
b
protons in the H NMR spectrum of the crude product
mixture.
b
Crude product.
Study of the bromine-mediated spirocyclization reaction of 1-methoxybrassinin (5a) and 1-
acyl derivatives 12, 23-25 in the presence of water or methanol revealed different
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diastereoselectivity. Under the same conditions, trans-diastereoisomers predominated from 1-
acyl derivatives, whereas with 1-methoxybrassinin (5a) the cis- and trans-isomers were
obtained in a near 1: 1 ratio (Tables 1, 4 and 5). The diastereoselectivity can be explained by a
different mechanism. In both cases reactions probably start at the thiocarbamoyl group creating
a sulfenyl bromide 6, 35. In the case of the methoxy derivative, the sulfenyl bromide 6
undergoes electrophilic attack on the sulfur with the formation of 1-methoxyspiroindoleninium
intermediate A. Subsequent nucleophilic addition of methanol gives spiroindoline structures
trans-(±)-4a, cis-(±)-4b (Scheme 6). In the case of the 1-acyl derivatives, delocalization of the
lone electron pair on nitrogen to the carbonyl group stabilizes sulfonium intermediate 36 and
the nucleophile approaches from the side opposite to sulfur with formation predominantly of
trans-diastereoisomers, trans-(±)-29a, side trans-(±)-33a (Scheme 6).
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Scheme 6. Different mechanisms of the bromine-mediated spirocyclization reactions of
-methoxybrassinin (5a) and 1-acyl derivatives.
1
1
The ratios of diastereoisomers (±)-26a-(±)-33b were determined by the H NMR spectra of
the crude products after dilution with dichloromethane, washing with brine, drying and
evaporation of the solvent. The ratios of diastereoisomers (±)-26a-(±)-33b were determined by
integration of well separated signals corresponding to the H-2, H
Chromatographic separation of the mixture of diastereoisomers
-methoxycarbonylspirobrassinol afforded pure trans-(±)-28a and pure cis-diastereoisomer
a
and H
b
protons.
of
1
cis-(±)-28b as crystalline substances. trans- and cis-Diastereoisomers 30a,30b-33a,33b were
separated by column chromatography. In the case of 1-acetylspirobrassinol (26) and 1-
benzoylspirobrassinol (27), the trans- and cis-diastereoisomers were not separable owing to
isomerization during the attempted separation on silica gel. This fact was confirmed by a simple
experiment. Prepared products 26 or 27 were applied to a TLC plate and the plate was
developed. After waiting for one hour, the plate was turned by 90° and developed again.
Detection using UV showed that from each original spot there were now two spots for the two
diastereoisomers (Figure 2). The products 26 and 27 were isolated as a mixture of trans- and
cis-diastereoisomers by column chromatography. It is supposed that diastereoisomers (±)-26a-
(
±)-26b and (±)-27a-(±)-27b isomerize at C-2 atom like the diastereoisomers of 1-
methoxyspirobrassinol [trans-(±)-3a, cis-(±)-3b]. In the case of 1-methoxyspirobrassinol (3),
7
isomerization was explained by facile interconversion of hemi-aminal and aminoaldehyde.
Figure 2. Evidence of isomerization of trans- and cis-diastereoisomers of 1-acetyl-(±)-26a-
(
±)-26b and 1-benzoylspirobrassinol (±)-27a-(±)-27b.
The structures of individual diastereoisomers were confirmed by NMR studies, including
COSY, HSQC, HMBC and NOESY experiments. The cis-diastereoisomers 7b-11b and 26b-
3b exhibited in their NOESY spectra a cross peak between H-2 and H protons confirming
their cis-configuration. The NOESY specta of structures 7a-11a and 26a-33a did not show the
interactions between H and OH or alkoxy group, which would have confirmed their trans-
diastereoisomeric structure. However, interactions between H-2 and H were also not observed
3
b
b
b
thus the structures of trans-diastereoisomer was assigned to these products.
1
Inspection of the H NMR spectra of 7-11 and 26-33 revealed a significant difference in the
chemical shifts between the H-2 protons of the trans- and cis-diastereoisomers. In all cases the
δ(H-2)trans appeared at lower field compared to δ(H-2)cis (Table 6). The higher shielding of H-
2
in the cis-diastereoisomers is probably caused by anisotropic shielding by the C=N double
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bond of the thiazoline ring. This correlation is valid for trans- and cis-diastereoisomers of 1-
7
7,11
methoxyspirobrassinol (3), 1-methoxyspirobrassinol methyl ether (4)
and 1-Boc-
spirobrassinol (29), in which the diastereoisomeric structures were confirmed by NOE
experiments. This consistent chemical shift difference was observed in CDCl or DMSO-d
1
2
3
6
.
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Table 6. Chemical shifts of H-2 proton in trans- and cis-diastereoisomers 7a-11b and 26a-33b
Ha
N
Ha
N
SCH3
SCH3
Hb
Hb
S
S
H-2
OR
OR2
H-2
2
N
N
R1
R1
trans-
NOESY
1H NMR
cis-
Compound
R1
R2
Isomer
δ(H-2) ppm
5.307
4.807
4.947
4.6211
5.02
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
)-3a
)-3b
)-4a
)-4b
)-7a
)-7b
)-8a
)-8b
transa
cisa
transa
cisa
transa
cisa
transa
cisa
transa
cisa
transa
cisa
transa
cisa
transa
cisa
transa
cisa
transa
cisa
transa
cisa
transa
cisa
transa
cisa
transb
cisb
transb
cisb
H
CH
CH
3
2
CH
3
3
4.70
5.07
OCH
3
CH(CH
)
2
4.75
)-9a
)-9b
5.26
C(CH
Ph
)
3 3
4.93
)-10a
)-10b
)-11a
)-11b
)-26a
)-26b
)-30a
)-30b
)-27a
)-27b
)-31a
)-31a
)-28a
)-28b
)-32a
)-32b
)-29a
)-29b
)-33a
)-33b
5.7823
5.4923
5.96
2
-naphthyl
5.67
5.73
H
5.42
COCH
3
5.41
CH
H
3
3
3
3
5.20
5.95
5.49
COC
6
H
5
5.52
CH
H
5.22
5.95
5.64
COOCH
3
5.56
CH
H
5.29
5.6312
5.4912
5.4231
5.3131
COOC(CH
)
3 3
CH
aCDCl
.bDMSO-d
.
3
6
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Huggershoff´s oxidative bromocyclization of brassinin (5b) and 1-methylbrassinin (37)
provided cyclobrassinin (45) or 9-methylcyclobrassinin (46). The formation of cyclobrassinin
(
45) or 9-methylcyclobrassinin (46) was achieved using various brominating agents (pyridinum
3
,32
19
33
12
12
tribromide 45 34%, NBS 45 35%, 46 61%, 1,4-dioxane dibromide 45 45%, 46 40%,
phenyltrimethylammonium tribromide 45 59% ). No comment was made on whether or not
these cyclizations afforded spirocyclic structures as minor products.
3
4
Therefore we decided to examine the cyclization of brassinin (5b) and 1-methylbrassinin
(
37) using several cyclization agents (Br
2
, DDB, I
2
, NBS, NCS, Me
3
PhNBr
3
) and solvents
(dichloromethane, 1,4-dioxane, methanol). Bromocyclizations of brassinin (5b) and 1-
methylbrassinin (37) in dichloromethane and 1,4-dioxane with water as a nucleophile did not
provide the desired spiroindoline[3,5']thiazoline derivatives (±)-38a-(±)-38b and (±)-41a-(±)-
4
1b but only unidentified products (Table 7, entries 1,2).
Bromine-mediated cyclization of brassinin (5b) and 1-methylbrassinin (37) in the presence
of methanol as a nucleophile led to the the formation of spirobrassinin [(±)-1] and 1-
methylspirobrassinin [(±)-44], respectively (Table 7, entry 11). It is postulated that the initially
formed unstable and nonisolable spirobrassinol methyl ether [(±)-39a,(±)-39b] and 1-
methylspirobrassinol methyl ether [(±)-42a,(±)-42b] undergo oxidation with bromine to
provide spirobrassinin [(±)-1] and 1-methylspirobrassinin [(±)-44] (Scheme 7). Transformation
of brassinin (5b) into spirobrassinin [(±)-1] was studied with an excess of bromine. The use of
2
.2 equivalents of bromine afforded 5-bromospirobrassinin [(±)-43] in 18% yield (Table 7,
entry 15). On the basis of the low yield it is assumed that firstly, bromation takes place on the
indole core of compounds (±)-39a-(±)-39b at C-5 and subsequently oxidation resulted in the
formation of 5-bromospirobrassinin [(±)-43]. Application of four equivalents of bromine led
to an increased yield (Table 7, entry 16). To prevent competitive bromination of the aromatic
core, 5-bromobrassin (5c) was used in a cyclization with four equivalents of bromine. 5-
Bromospirobrassinin [(±)-43] was obtained in 64% yield (Table 7, entry 19). The proposed
mechanism of oxidation of spirobrassinol methyl ether [(±)-39a,(±)-39b] is depicted in Scheme
8
.
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S
N
R2
N
R2
SCH3
SCH3
SCH3
S
S
NH
R2
OR3
OR3
N
R1
N
+
R1
N
1
2
3
R1
trans-(±)-38a
trans-(±)-39a
cis-(±)-38b R = H, R = H, R = H
1
2
3
cis-(±)-39b R = H, R = H, R = CH
3
1
2
5
5
3
b R = H, R = H
1
2
3
trans-(±)-40a
trans-(±)-41a
trans-(±)-42a
cis-(±)-40b R = H, R = Br R = CH
,
3
1
2
c R = H, R = Br
1
2
3
cis-(±)-41b R = Me, R = H, R = H
1
2
7 R = Me, R = H
1
2
3
cis-(±)-42b R = Me, R = H, R = CH3
R2
N
SCH3
N
S
SCH3
S
N
R1
N
O
+
R1
1
2
1
1
R = H, R = H
45 R = H
1
2
1
4
3 R = H, R = Br
46 R = Me
1
2
4
4 R = Me, R = H
Scheme 7. Bromine-mediated cyclization of brassinin (5b) and 1-methylbrassinin (37).
Table 7. Spirocyclization of brassinin (5b) and 1-methylbrassinin (37): reaction conditions and
yields
1
1
R = H
R = CH
3
Entry
Conditions
Yield (%)
Yield (%)
1
45
44
46
1
1.1eq. Br
2
, CH
2
Cl
2
/H
2
3
O, Et N, rt
decomposition
-
decomposition
-
1
.1eq. DDB, 1,4-dioxane/H
2
O, Et
3
N,
2
decomposition
-
decomposition
-
rt
3
4
5
6
1.1eq. Br
2
, MeOH, Et
N, rt
, MeOH, Et
Cl /MeOH, Et
, 1,4-dioxane/MeOH,
3
N, rt
24
-
-
-
-
27
30
13
33
-
-
-
-
1 eq. I
1 eq. Me
1.1eq. Br
.1eq. SOCl
Et N, rt
2
, MeOH, Et
PhNBr
, CH
3
decomposition
3
3
3
N, rt
18
32
2
2
2
3
N
1
2
7
16
-
25
-
3
8
9
1.1eq. NBS, CH
1.1eq. NCS, CH
2
Cl
2
2
/MeOH, Et
/MeOH, Et
3
3
N, rt
N, rt
21
40
-
-
33
40
-
-
2
Cl
1
.1eq. NBS, 1,4-dioxane/MeOH,
Et N, rt
.1eq. NCS, 1,4-dioxane/MeOH,
Et N, rt
.1eq. DDB, 1,4-dioxane/MeOH,
Et N, rt
3
2
-
-
-
45
35
55
-
-
-
3
1
1
1
0
1
42
47
3
1
3
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Table 7 (continued)
1
.1eq. DDB, 1,4-dioxane/EtOH,
1
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
39
-
11
42
-
68
65
60
-
-
8
13
-
Et
3
N, rt
1
.1eq. DDB, 1,4-dioxane/i-PrOH,
Et N, rt
.1eq. DDB, 1,4-dioxane/t-BuOH,
Et N, rt
.2 eq. DDB, 1,4-dioxane/MeOH,
Et N, rt
eq. DDB, 1,4-dioxane/MeOH,
Et N, rt
c, 1.1eq. DDB, 1,4-dioxane/MeOH,
Et N, rt
c, 2.2eq. DDB, 1,4-dioxane/MeOH,
Et N, rt
c, 4 eq. DDB, 1,4-dioxane/MeOH,
Et N, rt
31
3
1
-
3
2
18 (43)
49 (43)
48 (43)
64 (43)
64 (43)
3
4
-
-
-
3
5
-
-
-
3
5
-
-
-
3
5
-
-
-
3
Scheme 8. A plausible reaction mechanism.
The antiproliferative effect (using the colorimetric MTT assay) of the newly synthesized
substances was evaluated on six human cancer cell lines; Jurkat (acute T-lymphoblastic
leukemia), MCF-7 and MDA-MB-231 (mammary gland adenocarcinomas), HeLa (cervical
adenocarcinoma), CEM (acute T-lymphoblastic leukemia) and A-549 (non-small cell lung
cancer). IC50 values for the synthesized compounds are presented in Tables 8 and 9. For
comparison, Table 8 also includes IC50 values for conventional chemotherapeutic agents
(
cisplatin and etoposide) and 1-methoxybrassinin (5a), brassinin (5b), 1-Boc-brassinin (12)
synthesized previously.
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1
-(Methoxycarbonyl)brassinin (25) displayed the highest antiproliferative activity with IC50
-1
from 10 to 32.5 µmol × L with the greatest activity in CEM cells (Table 8). 1-
Benzoylbrassinin (24) reduced the proliferation capacity of CEM cells with IC50 25.8 µmol ×
L . 1-Acetylbrassinin (23) did not demonstrate any activity in all the cancer cell lines
examined. 1-(Methoxycarbonyl)brassinin (25) and 1-benzoylbrassinin (24) exhibited more
significant inhibitory effects than natural phytoalexins 1-methoxybrassinin (5a) and brassinin
-
1
(
5b) against all of the tested cancer lines.
-Alkoxy analogues of 1-methoxyspirobrassinol methyl ether 7a-11b possess relatively
2
-1
weak antiproliferative activity with IC50 values ranging from 50 to >100 µmol × L (Table 9).
Similar results were obtained with the 1-acyl analogues of 1-methoxyspirobrassinol methyl
ether 26a-33b. The highest antiproliferative effects were noted with 1-Boc-spirobrassinol [(±)-
2
9a,(±)-29b] and 1-Boc-spirobrassinol methyl ether [(±)-33a,(±)-33b], where measured IC50
-1
values 29.8–43.4 µmol × L were obtained with leukemic cells (Jurkat and CEM).
Table 8. Antiproliferative activity of 1-methoxybrassinin (5a) and its derivatives
-1
Cancer Cell line, IC50 (µmol × L )
Compound R
Jurkat MCF-7 MDA HeLa CEM A-549
5
5
2
2
2
1
b24
a24
3
H
>100
37.5
>100
32.4
32.5
17.8
12
>100
100
>100 >100 90.2 >100
100 100 63.5 100
>100 >100 >100 >100
OCH
3
COCH
3
>100
56.1
32.5
23.0
11.4
10.9
4
COC
6
H
5
35.2 29.0 25.8
32 28.5 10
21.4 16.9 19.6
55.2
31.8
21.4
12.2
14.3
5
224
COOCH
3
Boc
Cisplatin
Etoposide
14.7
21.2
7.7
3.9
4.4
1.1
1.2
The potency of 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 amount of viable cells to 50% relative to untreated control cells).
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Table 9. Antiproliferative activity of 1-methoxyspirobrassinol methyl ether (4) and its
analogues
-
1
Cancer Cell line, IC50 (µmol × L )
Compound
R1
R2
Jurkat
30.2
57.4
70.4
>100
73.8
>100
50.0
59.6
100
MCF-7 MDA HeLa CEM A-549
trans-(±)-4a24
cis-(±)-4b24
trans-(±)-7a
cis-(±)-7b
CH
100
100
100
100
48.9
53.2
100
100
100
100
3
2
CH
CH
3
3
85.6
>100
NT
>100 >100 83.7
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 84.8 >100
>100 >100 >100
85.4
>100
>100
>100
72.8
68.8
100
trans-(±)-8a
cis-(±)-8b
CH(CH
)
2
NT
OCH
3
trans-(±)-9a
cis-(±)-9b
C(CH
Ph
3
)
3
NT
NT
trans-(±)-10a23
cis-(±)-10b23
trans-(±)-11a
cis-(±)-11b
100
100
100
100
100
100
100
100
100
100
2-naphthyl
H
>100
>100
>100
49.4
>100
50.0
42.0
53.0
>100
>100
>100
>100
34.0
29.8
37.3
43.4
>100
>100
>100
>100
>100
>100
>100
78.0
>100
>100
>100
>100
100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100
>100
>100
>100
>100
>100
>100
68.0
>100
>100
>100
85.0
100
(±)-26a,b
trans-(±)-30a
cis-(±)-30b
COCH
3
CH
3
(±)-27a,b
H
trans-(±)-31a
cis-(±)-31b
COC
6
H
5
CH
3
>100 74.0
>100 67.0
38.0
31.0
trans-(±)-28a
cis-(±)-28b
H
>100 >100 >100
>100 >100 >100
>100 96.0 >100
>100 >100 >100
COOCH
3
trans-(±)-32a
cis-(±)-32b
CH
H
3
3
trans-(±)-29a
cis-(±)-29b
trans-(±)-33a24
cis-(±)-33b24
82.8
95.0
87.0
97.7
78.0
93.6
74.3
77.6
30.6
27.1
37.9
41.9
100
100
Boc
CH
70.2
100
70.5
96.3
The potency of 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 amount of viable cells to 50% relative to untreated control cells).
NT not tested
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ARKIVOC 2016 (vi) 198-234
Conclusions
The effect of the solvent and temperature was investigated with the aim of influencing the
diastereoselectivity of the bromine-initiated spirocyclization of 1-methoxybrassinin (5a) with
methanol. It was found that the use of ether solvents gives rise to a preference for the cis-
diastereoisomer cis-(±)-4b, whereas at low temperature the trans-diastereoisomer trans-(±)-4a
is preferred. The bromospirocyclization of brassinin bearing an acyl group (acetyl, benzoyl,
methoxycarbonyl and tert-butoxycarbonyl) 12, 23-25 on the indole nitrogen afforded
predominantly the trans-diastereoisomer. Bromine-induced spirocyclization reactions of
brassinin (5b) and 1-methylbrassinin (37) in the presence of methanol produced spirobrassinin
[
(±)-1] and 1-methylspirobrassinin [(±)-44]. The antiproliferative activity of the newly
synthesized compounds against selected human cancer cell lines was examined. Substances 25,
±)-29a, (±)-29b, (±)-33a, (±)-33b exhibited the highest inhibitory effects on the growth of
(
CEM cells.
Experimental Section
General. Melting points were determined on a Koffler hot-stage apparatus and are uncorrected.
1
13
H- and C-NMR spectra were measured on a Varian Mercury Plus spectrometer (400 MHz
1
13
for H and 100 MHz for C). Chemical shifts () are reported in ppm downfield from TMS as
the internal standard and the coupling constants (J) are given in Hertz. Microanalyses were
performed with a Perkin-Elmer, Model 2400 analyzer. The EI mass spectra were recorded on
a GS-MS Trio 1000 (Fisons Instruments) spectrometer at an ionization energy of 70 eV. IR
spectra were recorded on an IR-75 spectrometer (Zeiss Jena). Flash column chromatography
was performed on the Kieselgel Merck Type 9385 at 230-400 mesh. The progress of chemical
reactions was monitored by thin layer chromatography (TLC), using Macherey–Nagel plates
Alugram Sil G/UV254. Preparative column chromatography was performed on Kieselgel 60
Merck Type 9385 (0.040–0.063).
Spirocyclization of 1-methoxybrassinin (5a) in the presence of methanol
trans-(±)- and cis-(±)-1-Methoxyspirobrassinol methyl ether [trans-(±)-4a and cis-(±)-4b].
Method A (Table 1 and Table 2): To a stirred solution of 1-methoxybrassinin (5a; 0.027 g, 0.1
mmol) in a mixture of anhydrous solvent/methanol (0.9 mL/0.1 mL) at rt (or 0 °C, -20 °C, -60
°
C) was added a freshly prepared solution of Br
was obtained by dissolving bromine (0.04 mL) in 1.76 mL of the used solvent. The reaction
mixture was stirred for 15 min, then Et N (0.022 g, 0.031 mL, 0.22 mmol) was added. Stirring
was continued for 5 min and the reaction mixture was diluted with CH Cl (5 mL) and washed
SO and the crude
2
(0.25 mL, 0.11 mmol). The stock solution
3
2
2
with brine (2 × 5 mL). The organic layer was dried over anhydrous Na
product, obtained after evaporation of the solvent, was subjected to H NMR spectroscopy to
determine the ratio of diastereoisomers trans-(±)-4a and cis-(±)-4b.
2
4
1
Method B (Table 2, entries 28 and 29): To a stirred solution of 1-methoxybrassinin (5a; 0.027
g, 0.1 mmol) in dichloromethane (0.9 mL) at rt (or -75 °C) was added a freshly prepared
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General Papers
ARKIVOC 2016 (vi) 198-234
solution of Br
2
(0.25 mL, 0.11 mmol). The stock solution was obtained by dissolving bromine
(
0.04 mL) in anhydrous CH
methanol (0.004 g, 0.005 mL, 0.11 mmol) and Et
added. Stirring was continued for 15 min and the reaction mixture was diluted with CH
2
Cl
2
(1.76 mL). The reaction mixture was stirred for 1 min, then
N (0.101 g, 0.139 mL, 1.00 mmol) were
Cl (5
SO
3
2
2
mL) and washed with brine (2 × 5 mL). The organic layer was dried over anhydrous Na
2
4
1
and the crude product, obtained after evaporation of the solvent, was subjected to H NMR
spectroscopy to determine the ratio of diastereoisomers trans-(±)-4a and cis-(±)-4b.
Method C (Table 3, entry 3): To a stirred mixture of 1-methoxybrassinin (5a; 0.210 g, 0.79
mmol) and powdered molecular sieves (3 Å) in anhydrous CH
powdered anhydrous K CO (0.220 g, 1.6 mmol) and a freshly prepared solution of bromine
2.1 mL, 0.9 mmol; the stock solution was obtained by dissolving bromine (0.05 mL) in
anhydrous CH Cl (2.25 mL)]. After stirring for 1 min, a freshly prepared solution of complex
CH ONa-15-crown-5-ether in anhydrous CH Cl (1.9 mL, 0.90 mmol) was added. The stock
solution was prepared by dissolving of CH ONa (0.054 g 1.0 mmol) in anhydrous MeOH (2
mL) with a subsequent addition of 15-crown-5-ether (0.220 g, 0.20 mL, 1 mmol). MeOH was
thoroughly evaporated and the residue was dissolved in anhydrous CH Cl (2 mL). Stirring
was continued for 10 min, and the reaction mixture was diluted with CH Cl (10 mL) and
washed with brine (2 × 10 mL). The organic layer was dried over anhydrous Na SO . The
residue obtained after evaporation of the solvent was subjected to chromatography on 25 g
silica gel (n-hexane/Et O 3:1), affording natural diastereoisomer trans-(±)-4a (0.086 g, 37%)
and unnatural cis-(±)-4b (0.026 g, 19%).
The spectral data were identical with those of the natural product trans-(±)-4a and unnatural
2
Cl (4.2 mL) were added
2
2
3
[
2
2
3
2
2
3
2
2
2
2
2
4
2
7
1
1
product cis-(±)-4b.
Spirocyclization of 1-methoxybrassinin (5a) in the presence of ethanol
trans-(±)- and cis-(±)-1-Methoxyspirobrassinol ethyl ether [trans-(±)-7a and cis-(±)-7b].
To a stirred solution of 1-methoxybrassinin (5a; 0.081 g, 0.3 mmol) in a mixture of anhydrous
CH
.33 mmol). The stock solution was obtained by dissolving of bromine (0.04 mL) in 1.76 mL
of anhydrous CH Cl . The reaction mixture was stirred for 15 min, then Et N (0.067 g, 0.09
mL, 0.66 mmol) was added. Stirring was continued for 5 min and the reaction mixture was
2
Cl
2
/EtOH (2.7 mL/0.3 mL) at rt was added a freshly prepared solution of Br
2
(0.77 mL,
0
2
2
3
diluted with CH
over anhydrous Na
2
Cl
2
(15 mL) and washed with brine (2 × 15 mL). The organic layer was dried
SO . The residue obtained after evaporation of the solvent was subjected
CO 5:1) and diastereoisomers trans-(±)-
2
4
to chromatography on silica gel (10 g, n-hexane/Me
a, cis-(±)-7b were separated.
trans-(±)-1-Methoxyspirobrassinol ethyl ether [trans-(±)-7a]. Yield: 0.036 g (39%), bright
2
7
yellow oil, R
f
0.62 (n-hexane/Me
2
CO 5:1). Anal. Calcd for C14
H
18
N
2
O
2
S
2
requires: C, 54.17;
+
H, 5.84; N, 9.02. Found: C, 54.39; H, 6.01; N, 9.23. MS (EI), m/z (%): 310 [M] (8), 279 (83),
-1 1
2
(
(
CH
2
51 (30), 117 (100). IR (CHCl
400 MHz, CDCl )  7.29-7.22 (m, 2H, H-4, H-6), 7.01 (ddd, J 7.5, J 7.5, J 1.0, 1H, H-5), 6.93
d, J 7.8, 1H, H-7), 5.02 (s, 1H, H-2), 4.97 (d, J 15.3, 1H, H ), 3.98 (dq, J 9.7, J 7.0, 1H,
CH ), 3.95 (s, 3H, N-OCH ), 3.89 (d, J 15.3, 1H, H ), 3.82 (dq, J 9.7, J 7.0, 1H, CH CH ),
.57 (s, 3H, SCH ), 1.30 (t, J 7.0, 3H, CH ). C NMR (100 MHz, CDCl )  163.4 (C=N),
3
) max: 3007, 1567 (C=N), 1460, 1380, 1193 cm . H NMR
3
b
2
3
3
a
2
3
1
3
3
2
CH
3
3
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ARKIVOC 2016 (vi) 198-234
1
7
48.3 (C-7a), 129.8 (C-6), 127.9 (C-3a), 124.1 (C-4), 123.9 (C-5), 113.1 (C-7), 107.8 (C-2),
0.1 (CH ), 69.1 (C-3), 67.9 (CH CH ), 64.1 (N-OCH ), 15.8 (CH CH ), 15.2 (SCH ). NOESY
): H /H , H /H-4, H-6/H-7, H-4/H-5.
cis-(±)-1-Methoxyspirobrassinol ethyl ether [cis-(±)-7b]. Yield: 0.027 g (29%), bright
yellow oil, R 0.43 (n-hexane/Me CO 5:1). Anal. Calcd for C14 requires: C, 54.17;
H, 5.84; N, 9.02. Found: C, 53.86; H, 5.67; N, 9.18. MS of compound cis-(±)-7b was fully
identical with MS of trans-(±)-7a diastereoisomer. IR (CHCl ) max: 3013, 1560 (C=N), 1447,
)  7.28-7.24 (m, 2H, H-6, H-4), 7.01 (ddd, J 7.5,
J 7.5, J 0.7, 1H, H-5), 6.93 (d, J 7.7, 1H, H-7), 4.70 (s, 1H, H-2), 4.49 (d, J 15.2, 1H, H ), 4.31
d, J 15.2, 1H, H ), 3.98 (dq, J 7.1, J 9.5, 1H, CH CH ), 3.95 (s, 3H, N-OCH ), 3.81 (dq, J 7.1,
J 9.5, 1H, CH CH ), 2.56 (s, 3H, SCH ), 1.32 (t, J 7.1, 3H, CH ). C NMR (100 MHz,
CDCl )  166.9 (C=N), 147.9 (C-7a), 130.1 (C-6), 128.6 (C-3a), 124.1 (C-5), 123.3 (C-4),
12.9 (C-7), 104.5 (C-2), 73.1 (CH ), 70.4 (C-3), 67.9 (CH CH ), 64.1 (N-OCH ), 15.8
CH CH ), 15.3 (SCH ). NOESY correlations (400 MHz, CDCl ): H /H , H-2/H , H-6/H-7, H-
/H-5.
2
2
3
3
2
3
3
correlations (400 MHz, CDCl
3
a
b
a
f
2
H
18
N
2
O
2 2
S
3
-
1 1
1
380, 1193 cm . H NMR (400 MHz, CDCl
3
a
(
b
2
3
3
1
3
2
3
3
2
CH
3
3
1
(
4
2
2
3
3
2
3
3
3
a
b
b
Spirocyclization of 1-methoxybrassinin (5a) in the presence of isopropyl alcohol
trans-(±)- and cis-(±)-1-Methoxyspirobrassinol isopropyl ether [trans-(±)-8a and cis-(±)-
8
b]. To a stirred solution of 1-methoxybrassinin (5a; 0.081 g, 0.3 mmol) in a mixture of
anhydrous CH Cl /i-PrOH (2.7 mL/0.3 mL) at rt was added a freshly prepared solution of Br
0.77 mL, 0.33 mmol). The stock solution was obtained by dissolving of bromine (0.04 mL) in
2
2
2
(
1
.76 mL of anhydrous CH
2
Cl
2
. The reaction mixture was stirred for 15 min, then Et N (0.067
3
g, 0.09 mL, 0.66 mmol) was added. Stirring was continued for 5 min and the reaction mixture
was diluted with CH Cl (15 mL) and washed with brine (2 × 15 mL). The organic layer was
dried over anhydrous Na SO . The residue obtained after evaporation of the solvent was
2
2
2
4
subjected to chromatography on silica gel (10 g, n-hexane/Me
trans-(±)-8a, cis-(±)-8b were separated.
2
CO 5:1) and diastereoisomers
trans-(±)-1-Methoxyspirobrassinol isopropyl ether [trans-(±)-8a]. Yield: 0.046 g (47%),
bright yellow oil, R
f
0.59 (n-hexane/Me
2
CO 5:1). Anal. Calcd for C15
H
20
N
2
O
2
S
2
requires: C,
+
5
2
5.53; H, 6.21; N, 8.63. Found: C, 55.81; H, 6.47; N, 8.35. MS (EI), m/z (%): 324 [M] (7),
-1 1
93 (30), 251 (93), 117 (60), 43 (100). IR (CHCl
)  7.29 (d, J 7.7, 1H, H-4), 7.23 (ddd, J 7.7, J 7.7, J =1.2, 1H, H-6), 7.00
ddd, J 7.7, J 7.7, J =1.0, 1H, H-5), 6.92 (d, J 7.7, 1H, H-7), 5.07 (s, 1H, H-2), 4.99 (d, J 15.2,
H, H ), 4.06 [sep, J 6.1, 1H, CH(CH ], 3.95 (s, 3H, N-OCH ), 3.85 (d, J 15.2, 1H, H ), 2.57
s, 3H, SCH ), 1.31 [d, J 6.1, 3H, CH(CH ], 1.25 [d, J 6.1, 3H, CH(CH ]. C NMR (100
MHz, CDCl )  163.3 (C=N), 148.6 (C-7a), 129.6 (C-6), 127.9 (C-3a), 124.3 (C-4), 123.9 (C-
), 112.5 (C-7), 106.3 (C-2), 76.3 [CH(CH ], 70.3 (CH ), 69.7 (C-3), 64.4 (N-OCH ), 24.1
and 24.0 [CH(CH ], 15.2 (SCH ). NOESY correlations (400 MHz, CDCl ): H /H , H /H-4,
OCH /CH(CH , H-6/H-7, H-4/H-5.
cis-(±)-1-Methoxyspirobrassinol isopropyl ether [cis-(±)-8b].Yield: 0.029 g (30%), bright
yellow oil, R 0.40 (n-hexane/Me CO 5:1). Anal. Calcd for C15 requires: C, 55.53;
H, 6.21; N, 8.63. Found: C, 55.72; H, 5.96; N, 8.85. MS of compound cis-(±)-8b was fully
3
) max: 2980, 1547, 1373, 1187 cm . H NMR
(400 MHz, CDCl
3
(
1
b
3
)
2
3
a
1
3
(
3
3
)
2
)
3 2
3
5
3
)
2
2
3
3
)
2
3
3
a
b
a
3
)
3 2
f
2
H N
20 2
O
S
2 2
identical with MS of trans-(±)-8a diastereoisomer. IR (CHCl
3
) max: 2973, 1563, 1367, 1187,
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ARKIVOC 2016 (vi) 198-234
-1 1
1
106 cm . H NMR (400 MHz, CDCl
3
)  7.28-7.24 (m, 2H, H-6, H-4), 7.00 (ddd, J 7.6, J 7.6,
J 1.1, 1H, H-5), 6.93 (dd, J 8.2, J 1.0, 1H, H-7), 4.75 (s, 1H, H-2), 4.48 (d, J 15.2, 1H, H
.32 (d, J 15.2, 1H, H ), 3.98 [sep, J 6.1, 1H, CH(CH ], 3.94 (s, 3H, N-OCH ), 2.55 (s, 3H,
SCH ), 1.34 [d, J 6.1, 3H, CH(CH ], 1.26 [d, J 6.1, 3H, CH(CH ]. C NMR (100 MHz,
CDCl )  167.0 (C=N), 148.2 (C-7a), 130.6 (C-6), 127.7 (C-3a), 123.8 (C-5), 123.4 (C-4),
12.7 (C-7), 103.2 (C-2), 74.1 [CH(CH ], 72.8 (CH ), 70.5 (C-3), 64.2 (N-OCH ), 22.8 and
2.7 [CH(CH ], 15.3 (SCH ). NOESY correlations (400 MHz, CDCl ): H /Hb, H-2/H
/CH(CH , H-6/H-7, H-4/H-5.
a
),
4
b
3
)
2
3
1
3
3
3
)
2
)
3 2
3
1
2
3
)
2
2
3
3
)
2
3
3
a
b
,
CH(CH
3
)
2
)
3 2
Spirocyclization of 1-methoxybrassinin (5a) in the presence of tert-butanol
trans-(±)- and cis-(±)-1-Methoxyspirobrassinol tert-butyl ether [trans-(±)-9a and cis-(±)-
b]. To a stirred solution of 1-methoxybrassinin (5a; 0.081 g, 0.3 mmol) in a mixture of
anhydrous CH Cl /t-BuOH (2.7 mL/0.3 mL) at rt was added a freshly prepared solution of Br
0.77 mL, 0.33 mmol). The stock solution was obtained by dissolving of bromine (0.04 mL) in
9
2
2
2
(
1
.76 mL of anhydrous CH
2
Cl
2
. The reaction mixture was stirred for 15 min, then Et N (0.067
3
g, 0.09 mL, 0.66 mmol) was added. Stirring was continued for 5 min and the reaction mixture
was diluted with CH Cl (15 mL) and washed with brine (2 × 15 mL). The organic layer was
dried over anhydrous Na SO . The residue obtained after evaporation of the solvent was
2
2
2
4
subjected to chromatography on silica gel (10 g, n-hexane/Me
trans-(±)-9a, cis-(±)-9b were separated.
2
CO 5:1) and diastereoisomers
trans-(±)-1-Methoxyspirobrassinol tert-butyl ether [trans-(±)-9a]. Yield: 0.042 g (41%),
bright yellow oil, R
f
0.64 (n-hexane/Me
2
CO 5:1). Anal. Calcd for C16
H
22
N
2
O
2
S
2
requires: C,
+
5
2
6.77; H, 6.55; N, 8.28. Found: C, 56.52; H, 6.74; N, 8.06. MS (EI), m/z (%): 338 [M] (2),
-1 1
51 (100), 57 (77). IR (CHCl
)  7.30-7.28 (m, 1H, H-4), 7.22 (ddd, J 7.5, J 7.5, J 1.2, 1H, H-6), 6.99 (ddd, J 7.5, J
.5, J 1.0, 1H, H-5), 6.98 (m, 1H, H-7), 5.26 (s, 1H, H-2), 5.07 (d, J 15.2, 1H, H ), 3.92 (s, 3H,
3
) max: 3000, 1560, 1387, 1186, 1120 cm . H NMR (400 MHz,
CDCl
7
3
b
1
3
N-OCH
MHz, CDCl
), 112.5 (C-7), 101.0 (C-2), 76.3 [C(CH
C(CH ], 15.2 (SCH ). NOESY correlations (400 MHz, CDCl
, H-6/H-7, H-4/H-5.
3
), 3.86 (d, J 15.2, 1H, H
)  163.3 (C=N), 148.6 (C-7a), 129.6 (C-6), 127.9 (C-3a), 124.3 (C-4), 123.6 (C-
], 70.3 (CH ), 69.7 (C-3), 64.4 (N-OCH ), 29.3
): H /H , H /H-4, H-
a
), 2.53 (s, 3H, SCH
3
), 1.35 [s, 9H, C(CH
3
)
3
]. C NMR (100
3
5
[
3
)
3
2
3
3
)
3
3
3
a
b
a
2
3 3
/C(CH )
cis-(±)-1-Methoxyspirobrassinol tert-butyl ether [cis-(±)-9b]. Yield: 0.031 g (30%), bright
yellow oil, R 0.51 (n-hexane/Me CO 5:1). Anal. Calcd for C16 requires: C, 56.77;
H, 6.55; N, 8.28. Found: C, 56.94; H, 6.37; N, 8.12. MS of compound cis-(±)-9b was fully
identical with MS of trans-(±)-9a diastereoisomer. IR (CHCl ) max: 3020, 1500, 1400, 1200,
)  7.28-7.23 (m, 2H, H-6, H-4), 7.01-6.97 (m,
H, H-5), 6.93-6.91 (m, 1H, H-7), 4.93 (s, 1H, H-2), 4.43 (d, J 15.4, 1H, H ), 4.37 (d, J 15.4,
H, H ), 3.91 (s, 3H, N-OCH ), 2.54 (s, 3H, SCH ), 1.35 [s, 9H, C(CH
)  166.8 (C=N), 148.8 (C-7a), 129.9 (C-6), 126.9 (C-3a), 123.5 (C-5, C-4), 112.5
C-7), 98.7 (C-2), 76.3 [C(CH ], 72.4 (CH ), 71.3 (C-3), 64.3 (N-OCH ), 28.9 [C(CH ],
5.3 (SCH ). NOESY correlations (400 MHz, CDCl ): H /Hb, H-2/H , H-2/C(CH , H-6/H-7,
H-4/H-5.
f
2
H N
22 2
O S
2 2
3
-1 1
9
1
1
13, 720, 660 cm . H NMR (400 MHz, CDCl
3
a
1
3
b
3
3
3
) ]. C NMR (100
3
MHz, CDCl
3
(
3
)
3
2
3
)
3 3
1
3
3
a
b
)
3 3
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Spirocyclization of 1-methoxybrassinin (5a) in the presence of naphth-2-ol
trans-(±)- and cis-(±)-1-Methoxyspirobrassinol naphth-2-yl ether [trans-(±)-11a and cis-
(
±)-11b]. To a stirred solution of 1-methoxybrassinin (5a; 0.054 g, 0.2 mmol) in anhydrous
CH Cl (3 mL) at rt was added a freshly prepared solution of Br (0.52 mL, 0.22 mmol). The
stock solution was obtained by dissolving of bromine (0.04 mL) in 1.76 mL of anhydrous
CH Cl . After stirring for 1 min, the solution of naphth-2-ol (0.032 g, 0.22 mmol) and
triethylamine (0.202 g, 0.279 mL, 2.0 mmol) in anhydrous CH Cl (3 mL) was added. Stirring
was continued for 15 min, then the reaction mixture was diluted with CH Cl (10 mL), washed
with 1M HCl (5 mL) and brine (2 × 10 mL). The organic layer was dried over anhydrous
Na SO . The residue obtained after evaporation of the solvent was subjected to
2
2
2
2
2
2
2
2
2
2
4
chromatography on silica gel (10 g, n-hexane/EtOAc 3:1) and diastereoisomers trans-(±)-11a,
cis-(±)-11b were separated. trans-Diastereoisomer trans-(±)-11a contained small amount of
naphth-2-ol as an impurity which was removed by repeated chromatography on silica gel (20
g, n-hexane/Me
trans-(±)-1-Methoxyspirobrassinol naphth-2-yl ether [trans-(±)-11a]. Yield: 0.016 g (20%),
bright yellow oil, R 0.68 (n-hexane/EtOAc 3:1). Anal. Calcd for C22 requires: C,
4.68; H, 4.93; N, 6.86. Found: C, 64.49; H, 4.61; N, 6.61. IR (CHCl ) max: 3054, 2929, 2847,
)  7.82-7.75 (m, 3H, H-arom),
.65-7.58 (m, 1H, H-arom), 7.48 -7.29 (m, 5H, H-arom), 7.14-7.02 (m, 2H, H-arom), 5.96 (s,
H, H-2), 5.25 (d, J 15.4, 1H, H ), 4.06 (d, J 15.4, 1H, H ), 3.91 (s, 3H, N-OCH ), 2.50 (s, 3H,
). C NMR (100 MHz, CDCl )  163.8 (C=N), 155.9 (C-arom), 147.9 (C-arom), 134.3
C-arom), 129.9 (CH-arom), 129.6 (CH-arom), 127.8 (C-arom), 127.6 (CH-arom), 127.2 (CH-
arom), 126.9 (C-arom), 126.4 (CH-arom), 124.5 (CH-arom), 124.0 (CH-arom), 123.9 (CH-
arom), 119.5 (CH-arom), 113.0 (CH-arom), 112.4 (CH-arom), 107.4 (C-2), 70.3 (CH ), 69.4
C-3), 64.1 (N-OCH ), 15.0 (SCH ). NOESY correlations (400 MHz, CDCl ): H /H
cis-(±)-1-Methoxyspirobrassinol naphth-2-yl ether [cis-(±)-11b]. Yield: 0.037 g (45%),
bright yellow oil, R 0.55 (n-hexane/EtOAc 3:1). Anal. Calcd for C22 requires: C,
4.68; H, 4.93; N, 6.86. Found: C, 64.45; H, 4.72; N, 6.58. IR (CHCl ) max: 3054, 2929, 2847,
)  7.81-7.68 (m, 3H, H-arom),
.63-7.56 (m, 1H, H-arom), 7.48 -7.24 (m, 5H, H-arom), 7.11-7.01 (m, 2H, H-arom), 5.67 (s,
H, H-2), 4.46 (d, J 15.3, 1H, H ), 4.35 (d, J 15.3, 1H, H ), 3.88 (s, 3H, N-OCH ), 2.53 (s, 3H,
). C NMR (100 MHz, CDCl )  166.9 (C=N), 155.9 (C-arom), 147.3 (C-arom), 134.2
C-arom), 130.1 (CH-arom), 130.0 (CH-arom), 128.8 (C-arom), 127.7 (CH-arom), 127.2 (CH-
arom), 126.5 (CH-arom), 126.3 (C-arom), 124.6 (CH-arom), 124.0 (CH-arom), 123.3 (CH-
arom), 119.6 (CH-arom), 112.8 (CH-arom), 112.4 (CH-arom), 103.1 (C-2), 72.6 (CH ), 70.7
C-3), 63.9 (N-OCH ), 15.1 (SCH ). NOESY correlations (400 MHz, CDCl ): H /H , H-2/H
2
CO 1:1).
f
H
20
N
2
O S
2 2
6
1
7
1
3
-
1 1
585, 1462, 1212, 941, 746 cm . H NMR (400 MHz, CDCl
3
b
a
3
1
3
SCH
3
3
(
2
(
3
3
3
a
b
.
f
H
20
N
2
O S
2 2
6
1
7
1
3
-
1 1
585, 1462, 1212, 941, 746 cm . H NMR (400 MHz, CDCl
3
a
b
3
1
3
SCH
3
3
(
2
(
3
3
3
a
b
b
,
1
-Acetylindole-3-carboxaldehyde (14). To a solution of indole-3-carboxaldehyde (13; 2.90
g, 20.0 mmol) in THF (66 mL) at 0 ºC was added Ac O (6.12 g, 5.6 mL, 60.0 mmol) and
catalytic amount of DMAP. The reaction mixture was stirred for 1 h at rt. After the reaction
was finished, THF was evaporated. The residue was dissolved in CH Cl (120 ml) and the
solution washed with 5% solution of KOH (100 mL), 1M HCl (100 ml) and H O (80 ml). After
drying over anhydrous Na SO and evaporation of solvent, aldehyde 14 was obtained by
2
2
2
2
2
4
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crystallization from the hot EtOH. Yield: 3.42 g (91%), bright yellow crystals, R
f
0.47 (n-
3
5
hexane/Me
Spectral and analytical data are consistent with literature values.
-Benzoylindole-3-carboxaldehyde (15). To a solution of indole-3-carboxaldehyde (13; 3.0
g, 20.0 mmol) in THF (70 mL) at 0 ºC was added Et N (10.12 g, 14.0 mL, 100 mmol). The
2
CO 2:1), m.p. 165-166 ºC (hot ethanol), lit. 167-169 ºC (n-hexane/EtOAc).
3
5
1
3
reaction mixture was stirred at 0 ºC for 10 min. After that, PhCOCl (3.93 g, 3.25 mL, 28.0
mmol) was added and the reaction mixture was stirred at 0 ºC for 45 min. After the reaction
was finished, THF was evaporated. The residue obtained after evaporation of the solvent was
subjected to column chromatography (30 g silica gel, n-hexane/EtOAc 4:1). The obtained
compound was further crystallized from CH
98%), white crystals, R 0.56 (n-hexane/Me
Calcd for C16 requires: C, 77.10; H, 4.45; N, 5.62. Found: C, 76.77; H, 4.69; N, 5.41.
MS (EI), m/z (%): 249 [M] (43), 105 [C
026, 1686 (C=O), 1673 (C=O), 1440, 706 cm . H NMR (400 MHz, CDCl
2
Cl
2
/n-hexane to afford aldehyde 15. Yield: 4.88 g
(
f
2
CO 2:1), m.p. 68-71 ºC (CH
2
Cl /n-hexane). Anal.
2
H11NO
2
+
+
+
6
H
5
C=O] (100), 77 [C
6
H
5
] (79). IR (CHCl
3
) max:
-1 1
3
3
)  10.05 (s, 1H,
CHO), 8.32-8.30 (m, 1H, H-7), 8.12-8.10 (m, 1H, H-4), 7.94 (s, 1H, H-2) , 7.78-7.76 (m, 2H,
H-2´, H-6´), 7.70-7.66 (m, 1H, H-4´), 7.62-7.56 (m, 2H, H-3´, H-5´), 7.49-7.42 (m, 2H, H-5,
1
3
H-6). C NMR (100 MHz, CDCl
3
)  185.8 (CHO), 168.5 (C=O), 137.6 (C-2), 136.8 (C-1´),
1
1
1
33.0 (C-4´), 129.4 (C-2´, C-6´), 129.3 (C-7a), 129.0 (C-3´, C-5´), 126.6 (C-6), 126.2 (C-3a),
25.6 (C-5), 122.2 (C-3), 122.0 (C-4), 116.1 (C-7).
-Methoxycarbonylindole-3-carboxaldehyde (16). To a suspension of NaH (2.4 g, 60.0
mmol, 60% suspension in mineral oil) in anhydrous MeCN (60 mL) was added indole-3-
carboxaldehyde (13; 2.17 g, 15.0 mmol). After stirring for 5 min at rt, methyl chloroformate
(
2.83 g, 2.3 mL, 30.0 mmol) was added. The reaction mixture was stirred for 10 min, then
poured into cold water (200 mL) and the product was extracted with EtOAc (1 × 150 mL and
× 100 mL). The extract was dried over Na SO . The residue obtained after evaporation of the
solvent was crystallized from CH Cl /n-hexane to afford aldehyde 16. Yield: 2.59 g (85%),
bright yellow crystals, R 0.54 (n-hexane/Me CO 2:1), m.p. 94-96 ºC (CH Cl /n-hexane). Anal.
Calcd for C11 NO requires: C, 65.02; H, 4.46; N, 6.89. Found: C, 64.79; H, 4.61; N, 6.73.
MS (EI), m/z (%): 203 [M] (100), 158 (78), 130 (47), 116 (81), 89 (35), 59 [CH
1
2
4
2
2
f
2
2
2
H
9
3
+
+
3
OCO] (37).
-1 1
IR (CHCl
MHz, CDCl
d, J 7.3, 1H, H-7), 7.43 (ddd, J 7.3, J 7.3, J 1.4, 1H, H-6), 7.38 (ddd, J 7.3, J 7.3, J 1.1, 1H,
3
) max: 3016, 1755 (C=O), 1673 (C=O), 1440, 1345, 1226, 1096 cm . H NMR (400
3
)  10.08 (s, 1H, CHO), 8.28 (dd, J 7.3, J 1.4, 1H, H-4), 8.22 (s, 1H, H-2), 8.16
(
1
3
H-5), 4.11 (s, 3H, OCH
(
(
3
). C NMR (100 MHz, CDCl
C-2), 131.1 (C-7a), 121.5 (C-6), 121.1 (C-3a), 120.1 (C-5), 117.5 (C-3), 117.4 (C-4), 110.3
C-7), 49.8 (CH ).
3
)  180.9 (CHO), 145.9 (C=O), 131.2
3
1
1
2
4
-Acetylindole-3-carboxaldehyde oxime (17). To a stirred solution of aldehyde (14; 3.42 g,
8.3 mmol) in THF (80 mL) was added a solution of hydroxylammonium chloride (1.98 g,
8.5 mmol) and NaOAc (1.72 g, 12.6 mmol) in water (14 mL) and the mixture was stirred for
h at rt. After evaporation of THF and addition of water (80 mL), the oxime 17 was extracted
with EtOAc (1 × 350 mL, 1 × 250 mL). The extract was dried over Na
2
SO
4
and the residue
obtained after evaporation of the solvent was further crystallized from Me
2
CO/n-hexane to
afford oxime 17 as a mixture of E- and Z-isomer.
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General Papers
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Yield: 3.26 g (88%), white crystals, R
f
0.44 (n-hexane/Me
2
CO 2:1), m.p. 145-148 ºC
(
Me
2
CO/n-hexane). Anal. Calcd for C11
H
10
N
2
O requires: C, 65.34; H, 4.98; N, 13.85. Found:
2
+
+
C, 65.27; H, 4.80; N, 13.51. MS (EI), m/z (%): 203 [M+H] (7), 202 [M] (66), 160 (100), 43
[
1
0
8
+
CH
3
CO] (78). IR (KBr) max: 3229 (OH); 1706 (C=O); 1620 (C=N); 1539; 1433; 1365; 1200;
-1 1
119; 932; 745 cm . H NMR (400 MHz, DMSO-d )  11.64 (bs, 0.3H, OH min.), 10.73 (bs,
.7H, OH maj.), 8.60 (s, 0.3H, CH= min.), 8.40 (d, J 8.2, 1H, H-7), 8.26 (s, 0.7H, CH= maj.),
.14 (d, J 7.6, 1H, H-4), 7.72 (s, 0.3H, H-2 min.), 7.63 (s, 0.7H, H-2 maj.), 7.39-7.34 (m, 1H,
6
1
3
H-6), 7.31-7.28 (m, 1H, H-5), 2.67 (s, 0.9H, CH
3
min.), 2.64 (s, 2.1H, CH maj.). C NMR
3
(
100 MHz, DMSO-d )  169.3 (C=O min.), 168.7 (C=O maj.), 143.5 (CH= maj.), 137.3 (C-7a
6
min.), 136.4 (C-7a maj.), 134.8 (CH= min.), 130.3 (C-3a min.), 129.0 (C-2 min.), 127.4 (C-3a
maj.), 126.8 (C-2 maj.), 126.1 (C-6 maj.), 125.6 (C-6 min.), 124.3 (C-5), 122.6 (C-4), 118.3
(
1
4
C-7 min.), 116.7 (C-3 maj.), 116.5 (C-7 maj.), 111.7 (C-3 min.), 24.1 (CH
3
).
-Benzoylindole-3-carboxaldehyde oxime (18). To a stirred solution of aldehyde (15; 1.0 g,
.0 mmol) in THF (26 mL) was added a solution of hydroxylammonium chloride (0.43 g, 6.3
mmol) and NaOAc (0.38 g, 2.8 mmol) in water (5 mL) and the mixture was stirred for 4 h at
rt. After evaporation of THF and addition of water (26 mL), the oxime 18 was extracted with
EtOAc (2 × 80 mL). The extract was dried over Na
evaporation of the solvent was further crystallized from EtOAc/n-hexane to afford oxime 18
as a mixture of E- and Z-isomer. Yield: 0.93 g (88%), bright yellow crystals, R 0.46 (n-
hexane/Me CO 2:1), m.p. 115-117 ºC (EtOAc/n-hexane). Anal. Calcd for C16
requires: C, 72.72; H, 4.58; N, 10.60. Found: C, 72.48; H, 4.90; N, 10.77. MS (EI), m/z (%):
2
SO
4
and the residue obtained after
f
2
H
12
N
2
O
2
+
+
+
2
1
64 [M] (27), 105 [C
6
H
5
C=O] (100), 77 [C
6
H
5
] (73). IR (CHCl
3
) max: 3579 (OH), 3020,
)  11.35 (bs, 0.3H, OH
-1 1
680 (C=O), 1446, 1339, 1165, 1125 cm . H NMR (400 MHz, CDCl
3
min), 10.34 (bs, 0.7H, OH maj.), 8.46 (s, 0.3H, CH=N min.), 8.42-8.40 (m, 0.3H, H-7 min),
8
7
.37-8.35 (m, 0.7H, H-7 maj.), 8.21 (s, 0.7H, CH=N maj.), 8.20-8.18 (m, 0.7H, H-4 maj.), 7.78-
.76 (m, 0.3H, H-4 min.), 7.74-7.72 (m, 2H, H-2´, H-6´), 7.65-7.61 (m, 1H, H-4´), 7.56-7.51
1
3
(
m, 2H, H-3´, H-5´), 7.44 (s, 1H, H-2), 7.44-7.39 (m, 1H, H-6), 7.37-7.33 (m, 1H, H-5). C
NMR (100 MHz, CDCl )  168.9 (C=O min.), 168.3 (C=O maj.), 143.4 (CH=N maj.), 137.3
C-4´ min.), 136.4 (C-7a maj.), 135.1 (C-7a min.), 134.0 (CH=N min.), 133.8 (C-1´ min.),
3
(
1
32.3 (C-1´ maj.), 132.2 (C-4´ maj.), 129.3 (C-2´, C-6´ min.), 129.1 (C-2´, C-6´ maj.), 128.7
(
(
C-3´, C-5´ maj.), 128.6 (C-3´, C-5´ min.), 128.3 (C-2), 127.6 (C-3a), 125.7 (C-6 maj.), 125.3
C-6 min.), 124.4 (C-5 maj.), 124.2 (C-5 min.), 122.5 (C-4 maj.), 118.2 (C-4 min.), 116.3 (C-
3
maj.), 116.2 (C-7 min.), 116.1 (C-7 maj.), 111.0 (C-3 min.).
1
-Methoxycarbonylindole-3-carboxaldehyde oxime (19). To a stirred solution of aldehyde
(
(
16; 2.03 g, 10.0 mmol) in EtOH (40 mL) was added a solution of hydroxylammonium chloride
1.04 g, 15.0 mmol) and Na CO (0.73 g, 7.0 mmol) in water (5 mL) and the mixture was
2
3
stirred for 10 min at rt. After evaporation of EtOH and addition of water (10 mL), the oxime
9 was extracted with EtOAc (1 × 80 mL and 1 × 50 mL). The extract was dried over Na SO
and the residue obtained after evaporation of the solvent was further crystallized from
CH Cl /n-hexane to afford oxime 19 as a mixture of E- and Z-isomer. Yield: 1.74 g (80%),
white crystals, R 0.47 (n-hexane/Me2CO 2:1), m.p. 122-124 ºC (CH Cl /n-hexane). Anal.
Calcd for C11 requires: C, 60.55; H, 4.62; N, 12.84. Found: C, 60.83; H, 4.37; N, 12.58.
MS (EI), m/z (%): 219 [M+H] (9), 218 [M] (90), 175 (47), 159 (56), 142 (76), 132 (57), 131
1
2
4
2
2
f
2
2
H
10
N
2
O
3
+
+
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General Papers
ARKIVOC 2016 (vi) 198-234
+
(
1
79), 130 (87), 115 (76), 114 (76), 77 (52), 59 [CH
3
OCO] (100). IR (CHCl
3
) max: 3578 (OH),
3
-1 1
732 (C=O), 1433, 1345, 1246, 1082, 932 cm . H NMR (400 MHz, CDCl )  8.67 (s, 0.3H,
CH= min.), 8.29 (s, 0.7H, CH= maj.), 8.24 (d, J 8.1, 0.3H, H-7 min.), 8.19 (d, J 8.0, 0.7H, H-
maj.), 8.11 (d, J 7.8, 0.7H, H-4 maj.), 7.79 (s, 0.7 H, H-2 maj.), 7.77 (s, 0.3H, H-2 min.), 7.72
d, J 7.8, 0.3H, H-4 min.), 7.42-7.31 (m, 2H, H-6, H-5), 4.08 (s, 0.9H, OCH ), 4.06 (s, 2.1H,
)  151.0 (C=O), 144.9 (CH=N maj.), 138.8 (C-2 min.),
35.9 (C-7a maj.), 134.4 (C-7a min.), 130.9 (CH=N min.), 128.6 (C-3a min.), 127.5 (C-2 maj.),
7
(
3
1
3
OCH
3
). C NMR (100 MHz, CDCl
3
1
1
1
26.9 (C-3a maj.), 125.7 (C-6 maj.), 125.2 (C-6 min.), 123.9 (C-5 maj.), 123.6 (C-5 min.),
22.5 (C-4 maj.), 118.3 (C-4 min.), 115.3 (C-7 min.), 115.0 (C-7 maj., C-3 maj.), 109.9 (C-3
min.), 55.2 (CH
3
O min.), 55.1 (CH O maj.).
3
General procedure for the preparation of 1-acyl derivatives of indole-3-ylmethyl amine
0-22.
To a solution of NiCl
.0 mmol) in MeOH (30 mL) followed by NaBH
stirring and cooling with flowing cold water. After 5 min, MeOH in the mixture was evaporated
to ¼ of its original volume and mixture was poured into a saturated solution of NH Cl (250
mL). After extraction with CH Cl for compounds 20 and 21 or EtOAc for compound 22 (1 ×
50 mL, 1 × 100 mL, 2 × 50 mL), drying the extract over Na SO and evaporation of the solvent,
2
2
6H
2
O (1.05 g, 4.4 mmol) in MeOH (40 mL) was added oxime (17-19;
(1.51 g, 40.0 mmol) in one portion with
4
4
4
2
2
1
2
4
the crude amine 20-22 was obtained. The crude amine 20-22 was employed in the next reaction
without purification.
1
-(Acetyl)indole-3-ylmethyl amine (20). Following the general procedure, amine 20 was
obtained using oxime (17; 0.6 g, 3.0 mmol).
-(Benzoyl)indole-3-ylmethyl amine (21). Following the general procedure, amine 21 was
obtained using oxime (18; 1.06 g, 4.0 mmol).
1
1
2
-(Methoxycarbonyl)indole-3-ylmethyl amine (22). Following the general procedure, amine
2 was obtained using oxime (19; 0.87 g, 4.0 mmol).
General procedure for the preparation of 1-acetylbrassinin (23) and 1-benzoylbrassinin
24).
To a stirred solution of crude freshly prepared amine (20; 0.565 g, 3.0 mmol or 21; 1.00 g, 4.0
mmol) in CH Cl (25 mL or 40 mL) was added Et N (0.91 g, 1.25 mL, 9.0 mmol or 1.21 g,
.67 mL, 12.0 mmol) and CS (0.685 g, 0.54 mL, 9.0 mmol or 0.91 g, 0.72 mL, 12.0 mmol).
(
2
2
3
1
2
After stirring for 5 min at rt, MeI (1.28 g, 0.57 mL, 9.0 mmol or 1.70 g, 0.75 mL, 12.0 mmol)
was added and stirring was continued for 1 h or 30 min. The solvent was evaporated and the
residue obtained after evaporation of the solvent was subjected to chromatography on silica gel
1
-Acetylbrassinin (23). Following the general procedure, product 23 was obtained using of
amine (20; 0.565 g, 3.0 mmol) and isolated on silica gel (25 g, n-hexane/Me CO 2:1). The
obtained compound was crystallized from Me CO/n-hexane to afford 1-acetylbrassinin (23).
Yield: 0.651 g (78%), bright yellow crystals, R 0.59 (n-hexane/Me CO 2:1), m.p. 155-156 ºC
Me CO/n-hexane). Anal. Calcd for C13 OS requires: C, 56.09; H, 5.07; N, 10.06. Found:
C, 55.72; H, 4.89; N, 10.30. MS (EI), m/z (%): 279 [M+H] (2), 278 [M] (10), 130 (100), 43
2
2
f
2
(
2
H
14
N
2
2
+
+
+
-1 1
[
CH
3
CO] (27). IR (CHCl ) max: 3366 (NH), 1687 (C=O), 1440, 1373, 1120, 1080 cm . H
3
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General Papers
ARKIVOC 2016 (vi) 198-234
NMR (400 MHz, DMSO-d
6
)  9.83 (bs, 1H, NH), 8.38 (d, J 8.0, 1H, H-7), 7.65 (d, J 7.7, 1H,
H-4), 7.58 (s, 1H, H-2), 7.36-7.26 (m, 2H, H-6, H-5), 5.01 (d, J 5.0, 1.8H, CH
2
), 4.74 (d, J 5.4,
). C NMR (100 MHz, DMSO-d )  198.9
C=S), 168.8 (C=O), 135.9 (C-7a), 129.8 (C-3a), 125.5 (C-2), 125.0 (C-6), 123.8 (C-5), 119.6
C-4), 118.0 (C-3), 116.7 (C-7), 42.1 (CH ), 24.2 (CH ), 18.1 (SCH ).
-Benzoylbrassinin (24). Following the general procedure, product 24 was obtained using of
1
3
0
(
(
.2H, CH
2
), 2.62 (s, 3H, CH
3
), 2.61 (s, 3H, SCH
3
6
2
3
3
1
amine (21; 1.00 g, 4.0 mmol) and isolated on silica gel (60 g, n-hexane/EtOAc 2:1). The
obtained compound was further crystallized from dichloromethane/n-hexane to afford 1-
benzoylbrassinin (24). Yield: 0.490 g (36%), bright yellow crystals, R 0.66 (n-hexane/EtOAc
f
2
4
1
1
2
1
0
1
2
:1), m.p. 109-111 ºC (CH
2
Cl
2
/n-hexane). Anal. Calcd for C18
H
16
N
2
OS
2
requires: C, 63.50; H,
+
.74; N, 8.23. Found: C, 63.21; H, 4.99; N, 8.01. MS (EI), m/z (%): 340 [M] (35), 234 (80),
+
+
05 [C
352, 1172, 1086 cm . H NMR (400 MHz, CDCl
H, H-2´, H-6´), 7.64-7.59 (m, 2H, H-4, H-4´), 7.55-7.51 (m, 2H, H-3´, H-5´), 7.43-7.39 (m,
H, H-6), 7.36-7.32 (m, 2H, H-5, H-2), 7.11 (bs, 1H, NH), 5.01 (d, J 4.3, 1.5H, CH ), 4.71 (s,
). C NMR (100 MHz, CDCl ) 
6
H
5
C=O] (100), 77 [C
6
H
5
] (81). IR (CHCl
3
) max: 3365 (NH), 1679 (C=O), 1446,
-1 1
3
)  8.36 (d, J 8.2, 1H, H-7), 7.71-7.69 (m,
2
1
3
.5H, CH
2
), 2.71 (s, 0.75H, SCH
3
), 2.63 (s, 2.25H, SCH
3
3
99.1 (C=S), 168.4 (C=O), 136.4 (C-7a), 134.1 (C-1´), 132.1 (C-4´), 129.3 (C-3a), 129.1 (C-
´, C-6´), 128.7 (C-3´, C-5´), 126.7 (C-2), 125.6 (C-6), 124.2 (C-5), 119.0 (C-4), 116.6 (C-3,
C-7), 42.3 (CH
-(Methoxycarbonyl)brassinin (25). To a stirred solution of crude freshly prepared amine
22; 0.817 g, 4.0 mmol) in MeOH (25 mL) was added Et N (1.21 g, 1.67 mL, 12.0 mmol) and
CS (0.91 g, 0.72 mL, 12.0 mmol). After stirring for 5 min at rt, MeI (1.70 g, 0.75 mL, 12.0
2
), 18.2 (SCH
3
).
1
(
3
2
mmol) was added and stirring was continued for 15 min. The solvent was evaporated and the
residue obtained after evaporation of the solvent was subjected to chromatography on silica gel
(
25 g, n-hexane/EtOAc 2:1). The obtained compound was further crystallized from CH
hexane to afford 1-(methoxycarbonyl)brassinin (25). Yield: 0.683 g (58%), bright yellow
crystals, R 0.46 (n-hexane/EtOAc 2:1), m.p. 128-131 ºC (CH Cl /n-hexane). Anal. Calcd for
requires: C, 53.04; H, 4.79; N, 9.52. Found: C, 52.81; H, 5.08; N, 9.74. MS (EI),
2
Cl /n-
2
f
2
2
C
13
H
14
N
2
O
S
2 2
+
+
m/z (%): 294 [M] (13), 188 (78), 59 [CH
3
1 1
OCO] (100). IR (CHCl
3
) max: 3367 (NH), 3020,
725 (C=O), 1439, 1371, 1276, 732 cm . H NMR (400 MHz, CDCl )  8.15 (d, J 7.5, 1H, H-
), 7.59 (s, 1H, H-2), 7.56 (d, J 7.7, 1H, H-4), 7.39-7.35 (m, 1H, H-6), 7.30-7.27 (m, 1H, H-5),
.12 (s, 1H, NH), 5.03 (d, J 4.7, 1.7H, CH ), 4.74 (s, 0,3H, CH ), 4.01 (s, 3H, OCH ), 2.73 (s,
). C NMR (100 MHz, CDCl )  199.1 (C=S), 151.1
-
1
7
7
0
3
2
2
3
1
3
.45H, SCH
3
), 2.66 (s, 2.55H, SCH
3
3
(C=O), 135.5 (C-7a), 128.9 (C-3a), 125.2 (C-6), 124.5 (C-2), 123.3 (C-5), 119.0 (C-4), 116.3
(
C-3), 115.3 (C-7), 53.9 (OCH ), 42.3 (CH ), 18.2 (SCH ).
3
2
3
General procedure for the spirocyclization of 1-acyl derivatives of brassinin 23-25 with
bromine in the presence of water. To a stirred solution of 1-acyl derivatives of brassinin 23-
2
5 (0.5 mmol) in a mixture of CH
solution of Br (1.26 mL, 0.55 mmol). The stock solution was obtained by dissolving of 0.04
mL of bromine in 1.76 mL of anhydrous CH Cl . The reaction mixture was stirred for 15 min,
then Et N (0.111 g, 0.15 mL, 1.1 mmol) was added. Stirring was continued for 5 min and the
reaction mixture was diluted with CH Cl (25 mL) and washed with brine (2 × 25 mL). The
2
Cl /water (3.6 mL/0.4 mL) at rt was added freshly prepared
2
2
2
2
3
2
2
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General Papers
ARKIVOC 2016 (vi) 198-234
organic layer was dried over anhydrous Na
the solvent subjected to chromatography.
2
SO
4
and the residue obtained after evaporation of
trans-(±)- and cis-(±)-1-Acetylspirobrassinol [trans-(±)-26a and cis-(±)-26b]. Following
the general procedure, products trans-(±)-26a and cis-(±)-26b were obtained using 0.139 g (0.5
mmol) of 1-acetylbrassinin (23) and isolated on silica gel (15 g, n-hexane/EtOAc 1:3) as
mixture of products trans-(±)-26a : cis-(±-)-26b in a 71:29 ratio. Yield: 0.116 g (79%), sallow
oil, R
f
(trans) 0.52 (n-hexane/EtOAc 1:3), R
f
(cis) 0.37 (n-hexane/EtOAc 1:3). Anal. Calcd for
C
13
H
14
N
2
O
2 2
S
requires: C, 53.04; H, 4.79; N, 9.52. Found: C, 53.31; H, 4.50; N, 9.83. MS (EI),
+
+
+
m/z (%): 295 [M+H] (9), 294 [M] (47), 251 (50), 43 [CH
3
CO] (100). IR (CHCl ) max: 3279
3
-1 1
(OH), 3013, 1649 (C=O), 1547 (C=N), 1466, 1378, 1099 cm . H NMR (400 MHz, CDCl
3
) 
8
.06 (d, J 7.6, 0.7H, H-7 trans), 7.82 (d, J 7.6, 0.3H, H-7 cis), 7.43 (dd, J 7.5, J 0.6, 0.7H, H-4
trans), 7.36 (d, J 7.4, 0.3H, H-4 cis), 7.32-7.27 (m, 1H H-6), 7.11 (ddd, J 0.9, J 7.5, J 7.5, 1H,
H-5), 6.11 (s, 0.3H, OH cis), 5.73 (s, 0.7H, H-2 trans), 5.42 (s, 0.3H, H-2 cis), 5.17 (s, 0.7H,
OH trans), 4.95 (d, J 15.6, 0.7H, H
b
trans), 4.35 (d, J 15.3, 0.3H, H
cis), 2.57 (s, 2.1H, SCH trans), 2.54 (s, 0.9H, SCH
cis, trans). C NMR (100 MHz, CDCl )  170.1 (C=O), 166.2 (C=N), 141.4
C-7a), 130.0 (C-6), 128.5 (C-3a), 124.7 (C-5), 123.9 (C-4), 117.2 (C-7 trans), 114.1 (C-7 cis),
3.0 (C-2 cis), 88.5 (C-2 trans), 75.1 (CH cis), 71.5 (C-3 trans), 67.6 (C-3 cis), 66.3 (CH
trans), 23.3 (CH ), 15.2 (SCH ). NOESY correlations (400 MHz, CDCl ): H /H-4 (trans), H-
/H (cis), H-5/H-4, H-6/H-7.
b
cis), 4.31 (d, J 15.6, 0.7H,
H
a
trans), 3.93 (d, J 15.3, 0.3H, H
a
3
3
cis),
1
3
2
.39 (s, 3H, CH
3
3
(
9
2
2
3
3
3
a
2
b
trans-(±)- and cis-(±)-1-Benzoylspirobrassinol [trans-(±)-27a and cis-(±)-27b]. Following
the general procedure, products trans-(±)-27a and cis-(±)-27b were obtained using 0.170 g (0.5
mmol) of 1-benzoylbrassinin (24) and isolated on silica gel (15 g, CH
mixture of products trans-(±)-27 : cis-(±-)-27b in a 64:36 ratio. Yield: 0.137 g (77%), sallow
oil, R (trans) 0.67 (CH Cl /Me CO 8:1), R (cis) 0.43 (CH Cl /Me CO 8:1). Anal. Calcd for
requires: C, 60.65; H, 4.52; N, 7.86. Found: C, 60.31; H, 4.79; N, 7.53. MS (EI),
2
Cl
2
/Me
2
CO 8:1) as a
f
2
2
2
f
2
2
2
C
18
H
16
N
2
O
2 2
S
+
+
+
+
m/z (%): 357 [M+H] (11), 356 [M] (35), 251 (71), 105 [C
IR (CHCl ) max: 3365 (OH), 3006, 1666 (C=O), 1560 (C=N), 1466, 1365, 1086, 939 cm . H
NMR (400 MHz, CDCl )  7.70 (d, J 7.1, 0.6 H, H-2´, H-6´ cis), 7.62 (d, J 7.5, 1.4 H, H-2´,
H-6´ trans), 7.56-7.53 (m, 1H, H-4´), 7.48-7.38 (m, 4H, H-7, H-4, H-3´, H-5´), 7.22-7.05 (m,
H, H-6, H-5), 5.95 (s, 0.7H, H-2 trans), 5.49 (s, 0.3H, H-2 cis), 4.97 (d, J 15.6, 0.7H, H
trans), 4.63 (s, 1H, OH cis, trans), 4.37 (d, J 15.2, 0.3H, H
6
H
5
C=O] (100), 77 [C
6
H
5
] (92).
-1 1
3
3
2
b
b
cis), 4.34 (d, J 15.6, 0.7H, H
). C NMR (100 MHz, CDCl
a
1
3
trans), 3.98 (d, J 15.2, 0.3H, H
a
cis), 2.55 (s, 3H, SCH
3
3
)  170.2
(
(
C=O trans), 170.1 (C=O cis), 165.9 (C=N cis), 164.7 (C=N trans), 140.5 (C-7a trans), 140.0
C-7a cis), 135.1 (C-1´ cis), 134.8 (C-1´ trans), 131.6 (C-4´ trans), 131.4 (C-4´ cis), 131.2 (C-
3
(
1
(
a), 129.5 (C-6 trans), 129.4 (C-6 cis), 128.7 (C-3´, C-5´ trans), 128.6 (C-3´, C-5´ cis), 127.9
C-2´, C-6´ trans), 127.8 (C-2´, C-6´ cis), 125.0 (C-5 cis), 124.4 (C-5 trans), 124.3 (C-4 cis),
24.1 (C-4 trans), 117.0 (C-7 cis), 116.1 (C-7 trans), 92.7 (C-2 trans), 89.2 (C-2 cis), 74.1
CH cis), 70.4 (C-3 trans), 66.7 (CH trans), 64.3 (C-3 cis), 15.2 (SCH trans), 15.1 (SCH
cis). NOESY correlations (400 MHz, CDCl ): H /H-4 (trans), H /H (trans), H-2/H (cis),
/H (cis).
2
2
3
3
3
a
a
b
b
H
a
b
trans-(±)- and cis-(±)-1-Methoxycarbonylspirobrassinol [trans-(±)-28a and cis-(±)-28b].
Following the general procedure, products trans-(±)-28a and cis-(±)-28b were obtained using
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General Papers
ARKIVOC 2016 (vi) 198-234
0
.147 g (0.5 mmol) of 1-(methoxycarbonyl)brassinin (25) and separated on silica gel (30 g,
CH Cl /Me CO 9:1). Both diastereoisomers trans-(±)-28a and cis-(±)-28b were crystallized
from CH Cl /n-hexane.
2
2
2
2
2
trans-(±)-1-Methoxycarbonylspirobrassinol [trans-(±)-28a]. Yield: 0.048 g (31%), white
o
crystals, R
f
0.48 (CH
2
Cl
2
/Me
2
CO 9:1), mp 135-138 C (CH
2
Cl /n-hexane). Anal. Calcd for
2
C
13
H
14
N
2
O
3
S requires: C, 50.30; H, 4.55; N, 9.03. Found: C, 50.49; H, 4.37; N, 8.85. MS (EI),
2
+
+
m/z (%): 311 [M+H] (16), 310 [M] (65), 203 (100), 159 (87), 117 (47), 87 (87), 72 (63), 59
+
[
CH
3
OCO] (58). IR (CHCl
3
) max: 3567 (OH), 3099, 1699 (C=O), 1547 (C=N), 1433, 1073
)  7.80 (bs, 1H, H-7), 7.38 (d, J 7.5, 1H, H-4), 7.31-7.26 (m,
H, H-6), 7.10-7.06 (m, 1H, H-5), 5.95 (s, 1H, H-2), 5.04 (d, J 15.5, 1H, H ), 4.64 (bs, 1H,
). C NMR (100 MHz,
)  164.8 (C=N), 153.8 (C=O), 140.6 (C-7a), 129.9 (C-6), 129.8 (C-3a), 123.9 (C-4, C-
), 114.9 (C-7), 91.6 (C-2), 70.6 (C-3), 67.2 (CH ), 53.2 (OCH ), 15.2 (SCH ). NOESY
correlations (400 MHz, CDCl ): H /H , H /H-4, H-4/H-5, H-5/H-6, H-6/H-7.
-
1 1
cm . H NMR (400 MHz, CDCl
1
OH), 4.31 (d, J 15.5, 1H, H
CDCl
3
b
1
3
a
), 3.91 (s, 3H, OCH
3
), 2.55 (s, 3H, SCH
3
3
5
2
3
3
3
a
b
a
cis-(±)-1-Methoxycarbonylspirobrassinol [cis-(±)-28b]. Yield: 0.011 g (7%), white crystals,
o
R
f
0.57 (CH
2
Cl
2
/Me
2
CO 9:1), mp 129-132 C (CH
2
Cl /n-hexane). Anal. Calcd for
2
C
13
H
14
N O
2 3
S
2
requires: C, 50.30; H, 4.55; N, 9.03. Found: C, 50.58; H, 4.39; N, 9.31. MS of
compound cis-(±)-28b was fully identical with MS of trans-(±)-28a diastereoisomer. IR
-1 1
(CHCl
3
) max: 3526 (OH), 3132, 1706 (C=O), 1567 (C=N), 1476, 1378, 1083 cm . H NMR
(
7
1
400 MHz, CDCl
3
)  7.68 (bs, 1H, H-7), 7.39 (d, J 7.5, 1H, H-4), 7.31-7.26 (m, 1H, H-6), 7.10-
); 3.99 (d, J
). C NMR (100 MHz, CDCl )  166.3
.06 (m, 1H, H-5), 6.11 (bs, 1H, OH), 5.64 (s, 1H, H-2), 4.37 (d, J 15.1, 1H, H
b
1
3
5.1, 1H, H
a
), 3.91 (s, 3H, OCH
3
), 2.58 (s, 3H, SCH
3
3
(
(
C=N), 153.7 (C=O), 139.1 (C-7a), 130.1 (C-3a), 129.8 (C-6), 124.1 (C-4), 124.0 (C-5), 114.9
C-7), 88.0 (C-2), 75.2 (CH ), 73.4 (C-3), 53.2 (OCH ), 15.1 (SCH ). NOESY correlations (400
): H /H , H /H-2, H-4/H-5, H-5/H-6.
2
3
3
MHz, CDCl
3
a
b
b
trans-(±)- and cis-(±)-1-Boc-spirobrassinol [trans-(±)-29a and cis-(±)-29b]. To a stirred
solution of 1-Boc-brassinin (12; 0.027 g, 0.08 mmol) in a mixture of CH Cl /water (0.9 mL/0.1
mL) at rt was added freshly prepared solution of Br (0.20 mL, 0.088 mmol). The stock solution
was obtained by dissolving of 0.04 mL of bromine in 1.76 mL of anhydrous CH Cl . The
reaction mixture was stirred for 15 min, then Et N (0.017 g, 0.024 mL, 0.18 mmol) was added.
Stirring was continued for 5 min and the reaction mixture was diluted with CH Cl (5 mL) and
washed with brine (2 × 5 mL). The organic layer was dried over anhydrous Na SO . The residue
obtained after evaporation of the solvent was subjected to chromatography on 8 g silica gel (n-
hexane/Me CO 3:1) and diastereoisomers trans-(±)-29a and cis-(±)-29b were separated.
2
2
2
2
2
3
2
2
2
4
2
trans-(±)-1-Boc-spirobrassinol [trans-(±)-29a]. Yield: 0.012 g (42%), white solid, R
f
0.35 (n-
o
hexane/Me
2
CO 3:1), mp 73-75 C (CHCl
3
/light petroleum). The spectral data were fully
1
2
identical with those of previously described product trans-(±)-29a.
cis-(±)-1-Boc-spirobrassinol [cis-(±)-29b]. Yield: 0.003 g (11%), colourless plates, R
f
0.67
o
(
n-hexane/Me
2
CO 3:1), mp 126-128 C (CH
2
Cl /light petroleum). The spectral data were fully
2
1
2
identical with those of previously described product cis-(±)-29b.
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