An alternative approach to the synthesis of anticancer molecule spirobrassinin and its 2′-amino analogues

Article abstract of DOI:10.1007/s00706-019-02528-x

Abstract: A convenient synthesis of the cruciferous phytoalexin (S)-(?)-spirobrassinin and its (±)-2′-amino analogues have been achieved. Our synthetic route towards (S)-(?)-spirobrassinin is based on the CrO₃-mediated cyclization of chiral 1-(2′,3′,4′,6′-tetra-O-acetyl-?-D-glucopyranosyl)brassinin and subsequent removal of the chiral auxiliary. (±)-2′-Amino analogues of spirobrassinin and 1-methoxyspirobrassinin were obtained from thioureas in one step in an efficient manner with the aid of CrO₃. New synthesized compounds were screened in vitro for antiproliferative/cytotoxic activity against six human cancer cell lines by MTT assay. Amino analogues with CF₃ functionality displayed good antiproliferative effect. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-019-02528-x

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-019-02528-x
ORIGINAL PAPER
An alternative approach to the synthesis of anticancer molecule
spirobrassinin and its 2′‑amino analogues
Mariana Budovská1 · Viera Tischlerová · Ján Mojžiš · Oleksandr Kozlov · Taťána Gondová
2
2
3
3
Received: 25 June 2019 / Accepted: 2 December 2019
©
Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
A convenient synthesis of the cruciferous phytoalexin (S)-(−)-spirobrassinin and its (±)-ꢀ′-amino analogues have
been achieved. Our synthetic route towards (S)-(−)-spirobrassinin is based on the CrO -mediated cyclization of chiral
3
1
-(ꢀ′,3′,4′,6′-tetra-O-acetyl-ß-D-glucopyranosyl)brassinin and subsequent removal of the chiral auxiliary. (±)-ꢀ′-Amino
analogues of spirobrassinin and 1-methoxyspirobrassinin were obtained from thioureas in one step in an eꢁcient manner
with the aid of CrO . New synthesized compounds were screened in vitro for antiproliferative/cytotoxic activity against six
3
human cancer cell lines by MTT assay. Amino analogues with CF functionality displayed good antiproliferative eꢂect.
3
Graphic abstract
Keywords Spirobrassinin · Phytoalexins · Oxidative spirocyclization · Antiproliferative activity
Introduction
several classes including terpenoids, glycosteroids, and alka-
loids; however, researchers often ꢃnd it convenient to extend
the deꢃnition to include all phytochemicals that are part of
the plant’s defensive arsenal. Phytoalexins show biological
activity towards a variety of pathogens and are considered
as molecular markers of disease resistance. Brassica crop
species are proliꢃc producers of indole phytoalexins that
are an important part of the plant defence repertoire. These
indole phytoalexins are a peculiarity of the family Brassi-
caceae (syn. Cruciferae) with diꢂerent subsets produced by
diꢂerent edible brassicas (e.g. cabbage, radish, cauliꢄower,
broccoli, turnip, oilseed rape). Common structural moiety is
indole, oxindole, or indoline nucleus with a linear chain or
annexed heterocycle (Fig. 1). The 47 cruciferous phytoalex-
ins have been divided into six subsets according to simple
structural features. A special subset comprises spiroindoline
phytoalexins with spiro attached thiazoline ring ((S)-(−)-
spirobrassinin ((−)-3), (R)-(+)-1-methoxyspirobrassinin
The phytoalexins are antimicrobial and often antioxidative
products of the metabolism of higher plants absent in healthy
tissues or present only in insigniꢃcant amounts that accu-
mulate rapidly in response to biotic (pathogen infection) or
abiotic elicitor (heavy metals, UV radiation). Phytoalexins
are chemically diverse with diꢂerent types of characteris-
tic of particular plant species. Phytoalexins tend to fall into
*
Mariana Budovská
mariana.budovska@upjs.sk
1
Department of Organic Chemistry, Institute of Chemical
Sciences, Faculty of Science, P.J. Šafárik University,
Moyzesova 11, 040 01 Kosice, Slovak Republic
ꢀ
3
Department of Pharmacology, Faculty of Medicine,
P.J. Šafárik University, SNP 1, 040 11 Kosice,
Slovak Republic
Department of Analytical Chemistry, Institute of Chemical
Sciences, Faculty of Science, P. J. Šafárik University,
Moyzesova 11, 040 01 Kosice, Slovak Republic
(
(+)-4), erucalexin (5), 1-methoxyspirobrassinol (6),
Vol.:(0
1
123456789)
3

M. Budovská et al.
Fig. 1 Selected indole phyto-
alexins
trans-(ꢀR,3R)-(−)-1-methoxyspirobrassinol methyl ether
cyclization of brassinin derivatives have been developed
[ꢀ8]. A remarkable synthetic method of spirooxindole phyto-
alexins (±)-spirobrassinin ((±)-3) and (±)-1-methoxyspiro-
(
(ꢀR,3R)-(−)-7); Fig. 1) [1, ꢀ].
Indole phytoalexins show a wide range of biological
activities. Indole phytoalexins provide a broad spectrum of
antifungal activities and moderate antibacterial eꢂect [3–6].
Indole phytoalexins exhibit antitrypanosomal potencies [7].
The anti-aggregation eꢂect of spirobrassinin ((±)-3) was
revealed in the cerebrospinal ꢄuid of patients with multi-
ple sclerosis [8]. Indole phytoalexins possess also cancer
chemopreventive properties and an antiproliferative eꢂect
against human cancer cell lines [9–17]. Synthetic structural
modiꢃcations of indole phytoalexins have been shown to
display higher antiproliferative activities than the natural
lead compounds [18–ꢀ3].
brassinin ((±)-4) through the unusual CrO or pyridinium
3
chlorochromate-mediated cyclization of brassinin (1) and
1-methoxybrassinin (2) was described [ꢀ9]. ꢀ′-Phenylamino
analogues of spirobrassinin and 1-methoxyspirobrassinin
10, 11 were prepared by replacement of the methylsulfanyl
moiety of corresponding phytoalexins using aniline in the
absence of solvent at 1ꢀ0 or 140 °C [30].
Eꢁcient and practical protocols using transition metals
for the construction of spiro[indoline-3,5′-[4′,5′]dihydrothia-
zole] skeleton are rare in the literature [ꢀ8, ꢀ9].
As a continuation of our work, we set out to extend oxida-
With respect to the structural and biological ꢃnding relat-
ing to the spiroindoline phytoalexins, there have been devel-
oped several synthetic approaches for their construction to
date (Fig. ꢀ).
tive cyclization initiated with chromium oxidants CrO or
3
pyridinium chlorochromate to diꢂerent derivatives and to
investigate the scope and limitation of the reaction. In this
paper we report a stereoselective approach to the spiroox-
indole (S)-(−)-spirobrassinin ((−)-3) using direct oxidative
cyclization of chiral derivatives of brassinin (15, 16; Fig. 3).
Our further aim was to devise an eꢁcient route for the syn-
thesis of ꢀ′-phenylamino analogues of spirooxindole phy-
toalexins 13, 14 with potential antiproliferative eꢂect. For
the construction of the ꢀ′-phenylamino analogues 13, 14 we
applied the oxidative cyclizations of thioureas (phenylamino
analogues of brassinin 17, 18; Fig. 3).
First synthesis of spirobrassinin ((±)-3) was achieved
by thionyl chloride or methanesulfonyl chloride-mediated
cyclization of (±)-dioxibrassinin (10) [ꢀ4]. A stereoselec-
tive approach toward the (S)-(−)-spirobrassinin ((−)-3)
involves bromine-mediated cyclization of (−)-1-(8-phenyl-
menthoxycarbonyl)brassinin (11) in the presence of water
with the formation of chiral derivatives of spirobrassinol,
followed by oxidation and removal of the chiral auxiliary
[ꢀ5]. Oxidative rearrangement of 9-substituted chiral deriva-
tives of cyclobrassinin 12 using oxone and sodium chlo-
ride aꢂorded corresponding 1-substituted spirobrassinin
derivatives [ꢀ5]. Spirocyclization protocol using bromine
and various nucleophile was applied to the synthesis of
Results and discussion
For the study of oxidative cyclizations in the synthesis of
(S)-(−)-spirobrassinin ((−)-3), the 1-[(1R,ꢀS,5R)-menthoxy-
carbonyl]brassinin (15) and 1-(ꢀ′,3′,4′,6′-tetra-O-acetyl-ß-D-
glucopyranosyl)brassinin (16) were chosen as chiral starting
compounds considering easily removable chiral auxiliary.
The key starting intermediate, 1-[(1R,ꢀS,5R)-menthoxy-
carbonyl]brassinin (15), was previously prepared by our
research group [ꢀ5]. Our aim was to prepare 1-substituted
spirobrassinin derivatives 19a and 19b in one step direct
1
-methoxyspiroindoline phytoalexin 4,6,7 and its derivatives
[
19, ꢀ0, ꢀ3, ꢀ6]. The stereoselective syntheses of (R)-(+)-
1
-methoxyspirobrassinin ((+)-4) and trans-(ꢀR,3R)-(−)-1-
methoxyspirobrassinol methyl ether ((ꢀR,3R)-(−)-7) were
achieved using spirocylization methodology of 1-methoxy-
brassinin (2) with chiral reagent (+)-menthol as nucleophile
[ꢀ7]. In ꢀ014, a novel synthetic strategy toward spiroindoline
phytoalexins through the uncommon palladium-catalysed
1
3

An alternative approach to the synthesis of anticancer molecule spirobrassinin and its 2′-amino…
Fig. 2 Reported approaches to spirobrassinin (3) and 1-methoxyspirobrassinin (4)
Fig. 3 Retrosynthetic route towards spiroindoline phytoalexins spirobrassinin (3) and its ꢀ′-phenylamino analogues 13 and 14
from brassinin 15. Oxidative cyclization of 1-[(1R,ꢀS,5R)-
menthoxycarbonyl]brassinin (15) using oxidizing agents
group with the formation S-oxide (20; Table 1). We sup-
posed that the problem is in the bulkiness of menthoxycar-
bonyl group, but same situation was observed using Boc
group at the position 1 of indole ring.
PCC or CrO (adopting the known conditions reported by
3
Pedras [ꢀ9]) did not aꢂord expected 1-[(1R,ꢀS,5R)-menth-
oxycarbonyl]spirobrassinin (19). Exposure of 15 to PCC or
We also investigated the efficiency and limita-
tion of oxidative cyclization by employing of chiral
1-(ꢀ′,3′,4′,6′-tetra-O-acetyl-β-D-glucopyranosyl)brassinin
(16). 1-(ꢀ′,3′,4′,6′-Tetra-O-acetyl-β-D-glucopyranosyl)
CrO resulted in the formation of undesired S-oxide (20;
3
Scheme 1, Table 1). The use of several other oxidants has
also resulted in the oxidation of sulphur of the thiocarbonyl
1
3

M. Budovská et al.
Scheme 1
Table 1 Oxidation of 1-[(1R,ꢀS,5R)-menthoxycarbonyl]brassinin (15)
Scheme 2
Reaction conditions
Yield of 20/%
PCC, CH Clꢀ
60
49
65
70
65
58
60
ꢀ
CrO , AcOH, H O
3
ꢀ
H O , CH COOH, THF
ꢀ
ꢀ
3
H O , Na CO , CH Cl
ꢀ
ꢀ
ꢀ
ꢀ
3
ꢀ
TBHP, CH Clꢀ
ꢀ
Oxone, acetone, H O, NaHCO
m-CPBA, CH Clꢀ
ꢀ
3
ꢀ
brassinin (16) was prepared according to published proce-
dures [31]. The oxidation of 1-(ꢀ′,3′,4′,6′-tetra-O-acetyl-β-
D-glucopyranosyl)brassinin (16) using PCC was found to
proceed without any problems and furnished the correspond-
ing protected derivatives of spirobrassinin 21a and 21b in
good yield (47% and 16%, Scheme ꢀ) after 45 min as a sep-
arable mixture of diastereoisomers. Column chromatogra-
phy on silica gel (eluent n-hexane/acetone ꢀ:1) allowed the
straightforward isolation of both diastereoisomers 21a and
2
1b. The ratio of diastereoisomers 21a and 21b (75:ꢀ5) was
determined by the integration of non-overlapping singlets
using the eluent n-hexane/ꢀ-propanol/triꢄuoroacetic acid
1
3
of SCH protons in H NMR spectra of crude product mix-
(80:ꢀ0:0.1, v/v) at a ꢄow rate of 0.8 cm /min). The optical
3
ꢀ
0
ture. On the other hand, reaction of 16 with CrO aꢂorded
rotation of prepared product (S)-3 ([α] =−36.3 (c=0.08,
3
D
oxidation products 21a and 21b in a ratio 8ꢀ:18 isolated
CHCl )) had a comparable magnitude and the same sign
3
ꢀ
0
in 47% and 13% yields after chromatography on silica gel
as (S)-spirobrassinin ((S)-(−)-3) ([α] =−143.6 (c=0.ꢀ5,
D
(
Scheme ꢀ). To continue a synthesis towards indole phyto-
CH Cl )) [ꢀ4].
ꢀ
ꢀ
alexins spirobrassinin (3), treatment of spirocyclic product
1a with NaOH in MeOH under reꢄux removed the tetra-
To expand the scope of oxidative cyclization we have
also studied reactions of derivatives of brassinin possess-
ing side-chain chirality 23 or 24. (R)-ꢀ-(1H-Indol-3-yl)-
ꢀ-[N-(methylsulfanylthiocarbonyl)amino]acetic acid (23)
was prepared by treatment of (R)-ꢀ-(1H-indol-3-yl)gly-
cine (22) with carbon disulꢃde and iodomethane in 86%
2
O-acetyl-ß-D-glucopyranosyl group and gave a (S)-enan-
tiomer of spirobrassinin ((S)-(−)-3). The conꢃguration of
the product was assigned to be (3S) by direct comparison
of the ECD spectrum with the published spectrum of the
spirobrassinin ((S)-(−)-3) [ꢀ4]. The enantiomeric excess of
the obtained (S)-(−)-spirobrassinin ((−)-3) was 93% (deter-
mined by analytical chiral HPLC, on the column LAR-
IHC CF6–RN (AZYP LLC, ꢀ5 cm × 0.46 cm i.d.; 5 μm),
yield. The PCC or CrO -mediated oxidation of acid 23
3
did not aꢂord desired oxidation products 26a and 26b and
led to decomposition. Therefore, acid 23 was converted
to the corresponding methyl ester 24. The conversion was
1
3

An alternative approach to the synthesis of anticancer molecule spirobrassinin and its 2′-amino…
achieved using thionyl chloride and methanol (Scheme 3).
With the substance 24 in the hand, we focused on the sub-
sequent oxidation reaction, which was carried out using
PCC in CH Cl at rt and provided the required spiro com-
H-4 of indole and OCH protons) revealed that the newly
3
formed stereogenic centre possesses the relative (3S)-
conꢃguration. The relative (3R)-conꢃguration of 25b was
conꢃrmed by interaction between H-4 of indole and H-4′
of thiazoline ring in NOESY spectra (Fig. 4). Because of
the unsatisfactory dr of used oxidation reaction of 24 we
did not continue with our elaborated strategy.
ꢀ
ꢀ
pounds 25a and 25b (38:6ꢀ) in good combined yield (70%)
after 1 h. The CrO -initiated oxidation of 24 aꢂorded oxi-
3
dation products 25a and 25b in a 41:59 ratio in low yield
(
9% and 16%). Diastereoisomers 25a and 25b were easily
In order to check the eꢁciency of our synthetic approach,
we explored oxidation spirocyclization of amino analogues
of brassinin and 1-methoxybrassinin 28–40. Thioureas
28–35 (amino analogues of brassinin) were prepared by
treatment of amine 27 with phenyl isothiocyanate or sub-
stituted phenyl isothiocyanate in dioxane/chloroform (ꢀ:1)
in the presence of triethylamine in 55–67% yield (Table ꢀ,
Scheme 4). Thioureas 36–40 (amino analogues of 1-meth-
oxybrassinin) were synthesized according to previously
published procedures [ꢀ3, ꢀ8]. With the requisite thioureas
in the hand, we directed our eꢂorts towards construction
of the required ꢀ′-phenylamino analogues of spirobrassinin
or 1-methoxyspirobrassinin 41–53. Oxidation of thioureas
separated by column chromatography (eluent n-hexane/
ethyl acetate ꢀ:1). The ratio of product 25a and 25b was
determined by integration of the non-overlapping signals
of the H-4′ proton of thiazoline ring and OCH protons
3
1
in the H NMR spectrum of the crude product mixture.
A realized NOESY study of 25a (interaction between
Scheme 3
2
8–40 using PCC led to decompositon. The reaction condi-
tions CrO /AcOH/H O/dioxane were successfully applied to
3
ꢀ
the cyclization of thioureas 28–40 (Scheme 5). The reactions
proceeded cleanly and provided a series of ꢀ′-phenylamino
analogues of spirobrassinin and 1-methoxyspirobrassinin
4
1–53 in 39–70% yields (Table 3).
Table 2 Yields of amino analogues of brassinin 28–35 prepared via
Scheme 4
R
Compound
Yield/%
4
4
4
4
4
4
4
3
-H
-F
-Cl
-Br
28
29
30
31
32
33
34
35
55
67
64
67
59
65
6ꢀ
63
-Me
-MeO
-CF3
,5-bis-CF3
Fig. 4 NOESY correlations for
diastereoisomers 25a and 25b
1
3

M. Budovská et al.
Scheme 4
gland adenocarcinomas), HeLa (cervical adenocarci-
noma), A-549 (non-small cell lung cancer) using MTT
(
thiazolyl blue tetrazolium bromide) assay [3ꢀ]. All assays
were carried out in triplicate. For comparisons, Tables 4
and 5 also include IC values for natural brassinin (1) and
5
0
spirobrassinin (3) and conventional anticancer substance
cisplatin.
Amino analogues of brassinin 28–35 displayed good
antiproliferative activities against MCF-7 and CEM cancer
cell lines. The obtained IC values showed (Table 4) that
5
0
aminoanalogue 35 is the most active with IC from < 10
5
0
3
to ꢀ4.5 µmol/dm . These values are comparable to that of
cisplatin.
ꢀ
′-Amino analogues of spirobrassinin (±)-41–48 exhib-
Scheme 5
ited higher potencies on leukaemia cell lines (Jurkat and
CEM, Table 5). Compounds (±)-41–46 were inactive
against HeLa, MCF-7, MDA-MB-ꢀ31, and A-549 cells.
Among the eight tested spiro compounds, compounds
with CF moiety (±)-47 and (±)-48 displayed signiꢃ-
3
cant potency on our panel (Table 5). Compound (±)-47
exhibited the most potent activity with IC from < ꢀ5.8
5
0
3
to 35.5 µmol/dm .
Cytotoxic eꢂect of most active compounds (±)-47 and
±)-48 on human nonmalignant endothelial cells HUVEC
human umbilical vein endothelial cells Table 5) was also
(
(
investigated. Compounds (±)-47 and (±)-48 were inac-
3
tive against HUVEC cells (IC values > 100 µmol/dm ).
5
0
The results showed that derivatives (±)-47 and (±)-48 had
great lower toxicity than cisplatin on HUVEC cells.
As seen, compounds containing CF group 34, 35, (±)-
3
Table 3 Yields of ꢀ′-amino analogues of spirobrassinin and 1-meth-
oxyspirobrassinin 41–53 prepared via Scheme 5
47, and (±)-48 demonstrated signiꢃcant antiproliferative
activity and this indicates the importance of CF moiety
3
for the biological activity. Prepared amino analogues are
more eꢂective than parental compounds brassinin (1) and
spirobrassinin (3). This makes compounds 34, 35, (±)-47,
and (±)-48 very promising candidates for further screen-
ing and evaluation.
Compound
Rꢀ
Yield/%
1
1
R =H
R =OCH3
4
4
4
4
4
4
4
3
-H
-F
-Cl
-Br
-Me
-MeO
-CF3
,5-bis-CF3
(±)-41
(±)-42
(±)-43
(±)-44
(±)-45
(±)-46
(±)-47
(±)-48
66
69
68
6ꢀ
70
67
68
65
(±)-49
–
(±)-50
(±)-51
(±)-52
–
39
–
4ꢀ
43
46
–
Conclusion
(±)-53
46
–
–
In summary, we have developed a convenient stereose-
lective synthetic method for the preparation of (S)-spiro-
brassinin from functionalized brassinin derivative via
oxidative cyclization using PCC or CrO3 as an oxidizing
agent. An eꢁcient one-pot synthesis of (±)-ꢀ′-amino ana-
logues of spirobrassinin and 1-methoxyspirobrassinin was
described through CrO3-mediated spirocyclization of cor-
responding thioureas. The antiproliferative activity results
revealed that ꢃnal synthesized amino analogues containing
CF3 moiety exhibited signiꢃcant potencies on our panel.
Antiproliferative activity
Our newly prepared compounds (amino analogues of
brassinin and ꢀ′-amino analogues of spirobrassinin) were
evaluated for in vitro antiproliferative eꢂects against six
human cancer cell lines Jurkat and CEM (acute T-lympho-
blastic leukaemia), MCF-7 and MDA-MB-ꢀ31 (mammary
1
3

An alternative approach to the synthesis of anticancer molecule spirobrassinin and its 2′-amino…
Table 4 Antiproliferative activity of amino analogues of brassinin 28–35
−3
Comp.
R
Cell lines, IC /μmol dm
50
Jurkat
MCF-7
MDA
HeLa
CEM
A-549
1
2
2
3
3
3
3
3
3
[ꢀ0]
8
SCH3
PhNH
4-FPhNH
4-ClPhNH
4-BrPhNH
4-MePhNH
4-MeOPhNH
>100
73.6
80.3
74.5
35
46.3
71.7
31.5
<10
1ꢀ
>100
38.5
48
31.4
40.6
ꢀ5.7
ꢀ6.6
30.3
<10
11.4
>100
>100
>100
7ꢀ.3
71.7
84
>100
5ꢀ.3
18.9
14.7
>100
>100
>100
74.ꢀ
69.3
87
>100
61.7
ꢀ4.5
7.7
90.ꢀ
38
47.1
37.3
30.5
ꢀꢀ.4
44
ꢀ7.1
<10
4.4
>100
>100
>100
76.5
65.4
77
>100
35.ꢀ
<10
1ꢀ.ꢀ
9
0
1
2
3
4
5
4-CF PhNH
3
3,5-bis-CF PhNH
−
3
Cisplatin [ꢀ0]
The potency of compounds was estimated using the MTT (thiazolyl blue tetrazolium bromide) assay after 7ꢀ h incubation of cells and presented
as IC (concentration of a given compound that decreased amount of viable cells to 50% relative to untreated control cells)
50
Table 5 Antiproliferative activity of ꢀ′-amino analogues of spirobrassinin 41–48 on six human cancer cell lines (Jurkat and CEM, MCF-7,
MDA-MB-ꢀ31, HeLa, A-549)
−3
Comp.
R
Cell lines, IC /μmol dm
50
Jurkat
MCF-7
MDA
HeLa
CEM
A-549
HUVEC
(
(
(
(
(
(
(
(
(
±)-3 [30]
±)-41
±)-42
±)-43
±)-44
±)-45
±)-46
±)-47
±)-48
SCH3
PhNH
4-FPhNH
4-ClPhNH
4-BrPhNH
4-MePhNH
4-MeOPhNH
63.4
38
35
48
ꢀ7
8ꢀ
54
ꢀ6.1
3ꢀ
1ꢀ
>100
>100
>100
>100
>100
>100
>100
33.6
>100
>100
>100
>100
>100
>100
>100
35.5
>100
>100
>100
>100
>100
>100
>100
33.6
>100
67
4ꢀ
56
34
76
63
ꢀ5.8
36
4.4
>100
>100
>100
>100
>100
>100
>100
3ꢀ.ꢀ
NT
NT
NT
NT
NT
NT
NT
>100
>100
5.06
4-CF PhNH
3
3,5-bis-CF PhNH
−
41
11.4
39
14.7
48
7.7
39.9
1ꢀ.ꢀ
3
Cisplatin [ꢀ0]
NT not tested
The potency of compounds was estimated using the MTT (thiazolyl blue tetrazolium bromide) assay after 7ꢀ h incubation of cells and presented
as IC (concentration of a given compound that decreased amount of viable cells to 50% relative to untreated control cells)
50
1
3
Experimental
and C NMR (100 MHz) spectra were measured on a
Varian Mercury Plus spectrometer. Chemical shifts (δ) are
reported in ppm downꢃeld from TMS as internal standard
and coupling constants (J) are given in hertz. Microanal-
yses were performed with a Perkin-Elmer, Model ꢀ400
Melting points were determined on a Koꢄer micro melting
point apparatus. IR spectra were recorded on an Avatar
1
FT-IR 6700 (Thermo Scientiꢃc, IK). H NMR (400 MHz)
1
3

M. Budovská et al.
analyzer. The EI mass spectra were recorded on a GS-MS
Trio 1000 (Fisons Instruments) spectrometer at an ioniza-
tion energy of 70 eV. The samples were ionized with a
chromathography on silica gel (4 g, n-hexane/ethyl acetate
4:1).
ꢀ
0
Yield: 0.0ꢀ1 g (49%); colourless oil; [α] = − 19.5
D
1
N -laser (λ = 337 nm). Optical rotations were measured at
(c=0.ꢀ3, CHCl ); R =0.50 (n-hexane/ethyl acetate 4:1); H
ꢀ
3
f
room temperature in a 10-cm cell on a P-ꢀ000 Jasco pola-
rimeter at the sodium D-line. CD spectra were obtained
in a 10-mm quartz cell on a JASCO J-810 spectrometer.
Enantiomeric separations were performed on Agilent 1ꢀ60
Inꢃnity HPLC system (Agilent Technologies, Waldbronn,
Germany) consisting of a quaternary pump, an injection
NMR (400 MHz, CDCl ): δ=8.16 (d, J=7.5 Hz, 1H, H-7),
3
7.59–7.58 (m, 1H, H-4), 7.56 (s, 1H, H-ꢀ), 7.38–7.34 (m,
1H, H-6), 7.ꢀ9–7.ꢀ6 (m, 1H, H-5), 5.58 (bs, 1H, NH), 4.94
(dt, J=10.9 Hz, J=4.4 Hz, 1H, H-1′), 4.63 (d, J=4.9 Hz,
ꢀH, CH ), ꢀ.39 (s, 3H, SCH ), ꢀ.ꢀꢀ–ꢀ.16 (m, 1H, H-8′),
ꢀ
3
ꢀ.04–1.96 (m, 1H, H-6′), 1.80–1.73 (m, ꢀH, H-3′, H-4′),
1.67–1.51 (m, ꢀH, H-ꢀ′, H-5′), 1.ꢀ8–0.94 (m, 3H, H-3′, H-4′,
H-6′), 0.95 (d, J=7.0 Hz, 3H) and 0.94 (d, J=7.0 Hz, 3H)
3
valve Rheodyne model 71ꢀ5 with a ꢀ0-mm loop, a column
oven, and an UV/Vis detector with variable wavelength.
The collection and evaluation of data was performed using
Agilent ChemStation software. The enantiomeric excess
of synthesized compound was determined on a LARIHC
CF6–RN (AZYP LLC, ꢀ5 cm×0.46 cm i.d.; 5 μm) column
with a mobile phase consisting of n-hexane/ꢀ-propanol/tri-
1
3
[H-9′, H-10′], 0.8ꢀ (d, J=6.9 Hz, 3H, H-7′) ppm; C NMR
(100 MHz, CDCl ): δ = 168.0 (C=S=O), 150.7 (C=O),
3
135.9 (C-7a), 1ꢀ9.3 (C-3a), 1ꢀ5.3 (C-6), 1ꢀ4.ꢀ (C-ꢀ), 1ꢀ3.3
(C-5), 119.4 (C-4), 117.7 (C-3), 115.7 (C-7), 78.4 (C-1′),
47.5 (C-ꢀ′), 41.ꢀ (C-6′), 36.7 (CH ), 34.3 (C-4′), 31.7 (C-5′),
ꢀ
3
ꢄuoroacetic acid (80:ꢀ0:0.1, v/v) at a ꢄow rate of 0.8 cm /
ꢀ6.7 (C-8′), ꢀ3.7 (C-3′), ꢀꢀ.ꢀ, ꢀ1.0 (C-9′, C-10′), 16.4
min and UV detection at ꢀꢀ0 nm. The progress of chemical
reactions was monitored by thin layer chromatography,
(C-7′) 1ꢀ.6 (SCH ) ppm; IR: ̄ꢀ = 3360 (NH), ꢀ958, ꢀ877,
3
−
1
1736 (C=O), 1657, 1456, 1399, 1ꢀ59 cm ; MS (EI): m/z
®
+
using Macherey–Nagel plates Alugram Sil G/UVꢀ54.
(%)=434 (M , ꢀ), 40ꢀ (46), ꢀ64 (51), 130 (53), 83 (100).
Preparative column chromatography was performed on
Kieselgel 60 Merck Type 9385 (0.040–0.063). (R)-ꢀ-(1H-
indol-3-yl)glycine (ee> 99.0%) was synthesized in labora-
tory of Institute of Organic Chemistry, Catalysis and Pet-
rochemistry, Faculty of Chemical and Food Technology,
Slovak University of Technology, Slovakia.
1‑(2′,3′,4′,6′‑Tetra‑O‑acetyl‑ß‑D‑glucopyranosyl)‑
spirobrassinin (21, C H N O S )
2
5 28 2 10 2
Procedure A A solution of 0.071 g brassinin derivative 16
3
(0.1ꢀ5 mmol) in 1.0 cm dichloromethane was added to a
vigorously stirred slurry of 0.193 g pyridinium chlorochro-
3
mate (0.875 mmol) in 1.0 cm dichloromethane. The reac-
1
‑[(1R,2S,5R)‑Menthoxycarbonyl]brassinin‑S‑oxide
tion mixture was stirred for 45 min at room temperature and
3
(
20, C H N O S )
then diluted with 8 cm dichloromethane. After adding a
22 30 2 3 2
small amount of silica gel, the solvent was evaporated and
residue preabsorbed on silica was subjected to silica gel col-
umn chromatography (ꢀ0 g, n-hexane/acetone ꢀ:1) aꢂording
diastereoisomers 21a and 21b. The obtained products were
further crystallized from dichloromethane/n-hexane to aꢂord
pure diastereoisomers 21a (0.034 g, 47%) and 21b (0.01ꢀ g,
16%).
Procedure A A solution of 0.04ꢀ g 1-[(1R,ꢀS,5R)-men-
3
thoxycarbonyl]brassinin (15, 0.10 mmol) in 1.ꢀ cm
dichloromethane was added to a vigorously stirred slurry
of 0.151 g pyridinium chlorochromate (0.70 mmol) in
3
1
.0 cm dichloromethane. The reaction mixture was stirred
3
for ꢀ.5 h at room temperature and then diluted with 5 cm
dichloromethane. After adding a small amount of silica
gel, the solvent was evaporated and residue preabsorbed on
silica was subjected to silica gel column chromatography
Procedure B To a solution of 0.057 g brassinin derivative
3
16 (0.10 mmol) in 0.9 cm AcOH, a solution of 0.050 g CrO
3
3
(0.50 mmol) in 0.3 cm water was added in one portion at
(
4 g, n-hexane/ethyl acetate 4:1). Yield: 0.0ꢀ6 g (60%);
rt. The reaction mixture was stirred for ꢀ h at rt. The reac-
3
colourless oil.
tion mixture was diluted with 15 cm brine and extracted
3
Procedure B To a solution of 0.040 g brassinin deriva-
with EtOAc (ꢀ×15 cm ). The combined organic extract was
3
3
tive 15 (0.10 mmol) in 0.6 cm AcOH, a solution of
washed with 10% K CO solution (ꢀ × 15 cm ) and dried
ꢀ
3
3
0
.050 g CrO (0.50 mmol) in 0.3 cm water was added
over Na SO . The residue obtained after evaporation of
3
ꢀ 4
in one portion at rt. The reaction mixture was stirred for
the solvent was subjected to chromathography on silica gel
(10 g, n-hexane/acetone ꢀ:1) aꢂording only diastereoisomers
21a (0.0ꢀ7 g, 47%) and 21b (0.008 g, 13%).
3
1
h at rt. The reaction mixture was diluted with 15 cm
3
brine and extracted with EtOAc (ꢀ × 15 cm ). The com-
bined organic extract was washed with 10% K CO solu-
ꢀ
3
3
tion (ꢀ × 15 cm ) and dried over Na SO . The residue
(3S)‑1‑(2′,3′,4′,6′‑Tetra‑O‑acetyl‑ß‑D‑glucopyranosyl)spiro‑
brassinin (21a, C H N O S ) Yellow crystals; m.p.:
ꢀ
4
obtained after evaporation of the solvent was subjected to
2
5 28 2 10 2
ꢀ
0
8
9–91 °C (dichloromethane/n-hexane); [α] = − ꢀ9.ꢀ
D
1
3

An alternative approach to the synthesis of anticancer molecule spirobrassinin and its 2′-amino…
1
(
c = 0.17, CHCl ); R = 0.50 (n-hexane/acetone ꢀ:1); H
ee; ECD (CH OH, c~0.1 mM): λ (Δε)=ꢀ00 (−15.39),
3 ext
3
f
NMR (400 MHz, CDCl ): δ = 7.40–7.36 (m, 1H, H-4),
ꢀ06 (− ꢀ0.86), ꢀ10 (− 5.06), ꢀ18 (+17.96), ꢀꢀ5 (+ꢀ5.11),
ꢀ37 (+8.51), ꢀ4ꢀ (−0.9ꢀ), ꢀ60 (−3.ꢀ6), ꢀ68 (−6.3ꢀ), ꢀ78
(− ꢀ.19), ꢀ88 (− 1.71), 305 (− 5.65), 309 (− 5.70), 340
(−0.66) nm. Spectral data of (S)-(−)-3 were fully identical
with those of natural product [3, ꢀ4].
3
7
.33–7.31 (m, 1H, H-6), 7.ꢀꢀ (d, J=7.9 Hz, 1H, H-7), 7.16
dd, J=7.6 Hz, J=7.6 Hz, 1H, H-5), 5.70 (d, J=9.3 Hz, 1H,
H-1′), 5.53 (dt, J=9.3 Hz, J=ꢀ.3 Hz, 1H, H-ꢀ′), 5.45–5.39
m, 1H, H-3′), 5.ꢀ8–5.ꢀ3 (m, 1H, H-4′), 4.67 (d, J=15.3 Hz,
H, H ), 4.48 (d, J=15.3 Hz, 1H, H ), 4.ꢀ8–4.14 (m, ꢀH,
(
(
1
b
a
H-6′ , H-6′ ), 3.96–3.9ꢀ (m, 1H, H-5′), ꢀ.61 (s, 3H, SCH ),
(R)‑2‑(1H‑Indol‑3‑yl)‑2‑[N‑(methylsulfanylthiocarbonyl)‑
amino]acetic acid (23, C H N O S ) (R)-ꢀ-(1H-Indol-
b
a
3
ꢀ
.11 (s, 3H, CH CO), ꢀ.09 (s, 3H, CH CO), ꢀ.0ꢀ (s, 3H,
3 3
12 12 2 2 2
1
3
CH CO), 1.93 (s, 3H, CH CO) ppm; C NMR (100 MHz,
3-yl)glycine (22, 0.095 g, 0.5 mmol) was dissolved in
3
3
3
3
CDCl ): δ = 176.1 (CO), 170.5 (CO), 169.9 (CO), 169.5
1.ꢀ5 cm methanol and 0.75 cm water. Triethylamine
3
3
(
CO), 169.3 (CO), 163.4 (C=N), 138.4 (C-7a), 1ꢀ9.9 (C-3a),
(0.11ꢀ g, 0.154 cm , 1.1 mmol) and 0.04ꢀ g carbon
3
1
ꢀ9.7 (C-6), 1ꢀ4.4 (C-5), 1ꢀ4.3 (C-4), 111.9 (C-7), 80.ꢀ
disulꢃde (0.033 cm , 0.55 mmol) were added and mixture
(
(
(
(
(
C-1′), 74.6 (CH ), 74.5 (C-5′), 7ꢀ.8 (C-3′), 68.0 (C-ꢀ′), 67.6
was stirred for 5 min at room temperature. Then 0.078 g
ꢀ
3
C-4′), 64.1 (C-3), 61.6 (C-6′), ꢀ0.7 (CH ), ꢀ0.6 (CH ), ꢀ0.4
iodomethane (0.034 cm , 0.55 mmol) was added and the
3
3
CH ), ꢀ0.3 (CH ), 15.7 (SCH ) ppm; IR: ̄ꢀ = 3061, 1740
stirring was continued for 3 h. The reaction mixture was
3
3
3
−
1
3
C=O), 1654, 1465, 1367, 1ꢀꢀ0, 1034 cm ; MS (EI): m/z
diluted with 15 cm brine and extracted with dichlorometh-
+
3
%)=580 (M , 7), 169 (44), 109 (33), 43 (73).
ane (ꢀ×15 cm ). The combined organic extract was dried
over Na SO . The solvent was evaporated and the residue
ꢀ
4
(
3R)‑1‑(2′,3′,4′,6′‑Tetra‑O‑acetyl‑ß‑D‑glucopyranosyl)‑
was puriꢃed by silica gel column ꢄash chromatography
spirobrassinin (21b, C H N O S ) White crystals; m.p.:
(ꢀ0 g, dichloromethane/methanol/ammonia 80:ꢀ0:1). Yield:
2
5 28 2 10 2
ꢀ
0
ꢀ0
1
83–185 °C (dichloromethane/n-hexane); [α] = + 13.6
0.1ꢀ0 g (86%); yellow oil; [α] =−130.0 (c=0.55, CHCl );
f
D
D
3
1
1
(
c = 0.14, CHCl ); R = 0.44 (n-hexane/acetone ꢀ:1); H
R =0.59 (dichloromethane/methanol/ammonia 80:ꢀ0:1); H
3
f
NMR (400 MHz, CDCl ): δ=7.60 (d, J=7.8 Hz, 1H, H-4),
NMR (400 MHz, CDCl ): δ=10.14 (bs, 1H, NH), 8.68 (d,
3
3
7
7
5
ꢀ
1
4
.40 (d, J=8.3 Hz, 1H, H-7), 7.31–7.ꢀ9 (m, 1H, H-6), 7.19–
.17 (m, 1H, H-5), 5.60 (d, J = 8.8 Hz, 1H, H-1′), 5.5ꢀ–
.4ꢀ (m, ꢀH, H-ꢀ′, H-3′), 5.ꢀ9–5.ꢀ5 (m, 1H, H-4′), 4.63 (d,
H, J=4.6 Hz, H , H ), 4.30 (dd, J=1ꢀ.5 Hz, J=4.9 Hz,
J = 6.6 Hz, 1H, NH), 7.70 (d, J = 7.8 Hz, 1H, H-4), 7.39
(dd, J = 7.ꢀ Hz, J = 0.5 Hz, 1H, H-7), 7.34 (d, J = ꢀ.5 Hz,
1H, H-ꢀ), 7.16 (dd, J=7.ꢀ Hz, J=7.ꢀ Hz, 1H, H-6), 7.08
(dd, J = 7.8 Hz, J = 7.ꢀ Hz, 1H, H-5), 6.38 (d, J = 6.6 Hz,
a
b
1
3
H, H-6′ ), 4.14 (dd, J = 1ꢀ.5 Hz, J = ꢀ.1 Hz, 1H, H-6′ ),
1H, CH), ꢀ.60 (s, 3H, SCH ) ppm; C NMR (100 MHz,
b
a
3
.01–3.97 (m, 1H, H-5′), ꢀ.39 (s, 3H, SCH ), ꢀ.08 (s, 3H,
CDCl ): δ = 198.5 (C=S), 171.7 (COOH), 136.4 (C-7a),
3
3
CH CO), ꢀ.0ꢀ (s, 6H, ꢀ × CH CO), 1.67 (s, 3H, CH CO)
1ꢀ5.7 (C-3a), 1ꢀ4.7 (C-ꢀ), 1ꢀ1.9 (C-6), 119.6 (C-5), 118.9
3
3
3
1
3
ppm; C NMR (100 MHz, CDCl ): δ=179.1 (CO), 170.6
(C-4), 111.7 (C-7), 109.ꢀ (C-3), 56.4 (CH), 17.9 (SCH )
3
3
(
CO), 170.1 (CO), 169.4 (CO), 168.8 (CO), 167.6 (CN),
ppm; IR: ̄ꢀ = 3178 (NH), ꢀ978, ꢀ914, 1704 (C=O), 159ꢀ,
−
1
1
36.7 (C-7a), 1ꢀ7.5 (C-3a), 1ꢀ3.1 (C-6), 1ꢀ1.0 (C-5), 119.5
1455, 1353, 1094 cm .
(
(
(
C-4), 109.7 (C-7), 8ꢀ.9 (C-1′), 74.6 (CH , C-5′), 73.ꢀ
ꢀ
C-3′), 70.4 (C-ꢀ′), 67.9 (C-4′), 65.7 (C-3), 61.8 (C-6′), ꢀ0.8
(R)‑2‑(1H‑Indol‑3‑yl)‑2‑[N‑(methylsulfanylthiocarbonyl)‑
amino]acetic acid methyl ester (24, C H N O S ) SOCl
CH ), ꢀ0.7 (CH ), ꢀ0.6 (CH ), ꢀ0.1 (CH ), 1ꢀ.4 (SCH )
3
3
3
3
3
13 14
2
2
2
ꢀ
3
ppm; IR: ̄ꢀ = 3061, 1740 (C=O), 1654, 1465, 1367, 1ꢀꢀ0,
(0.041 g, 0.0ꢀ5 cm , 0.347 mmol) was slowly added to a
−
1
+
1
1
034 cm ; MS (EI): m/z (%) = 580 (M , 0.ꢀ), 169 (74),
cooled (0 °C) solution of 0.075 g brassinin derivative 23
3
09 (5ꢀ), 43 (50).
(0.ꢀ67 mmol) in 0.8 cm dry MeOH. The reaction mixture
was stirred for 30 min at room temperature. The residue
obtained after evaporation of the solvent was dissolved in
ethyl acetate, small amount of silica gel added, ethyl acetate
evaporated and product preabsorbed on silica gel was chro-
(
S)‑(−)‑Spirobrassinin ((S)‑(−)‑3, C H N OS ) from 21a To
1
1
10
2
2
3
a stirred solution of 0.0ꢀꢀ g 21a (0.040 mmol) in 4.0 cm
3
dry methanol was added 1.0 cm solution of NaOH (1 M,
1
.0 mmol). The mixture was stirred for ꢀ1 h under reꢄux;
matographed on silica gel (5 g, n-hexane/ethyl acetate ꢀ:1).
3
ꢀ0
then the reaction mixture was poured into 10 cm of 1 M
Yield: 0.063 g (80%); yellow oil; [α] =−189.0 (c=0.31,
D
3
1
solution of HCl, diluted with ꢀ0 cm water, and extracted
CHCl ); R = 0.47 (n-hexane/ethyl acetate ꢀ:1); H NMR
3
f
3
with EtOAc (ꢀ×ꢀ0 cm ). The combined extract was dried
(400 MHz, CDCl ): δ=8.33 (s, 1H, NH), 7.70 (d, J=7.1 Hz,
3
over over Na SO . The solvent was evaporated and the
1H, H-4), 7.39 (dd, J=7.1 Hz, J=1.1 Hz, 1H, H-7), 7.ꢀ8
(d, J=ꢀ.3 Hz, 1H, H-ꢀ), 7.ꢀ5 (ddd, J=7.1 Hz, J=7.1 Hz,
J = 1.1 Hz, 1H, H-6), 7.17 (ddd, J = 7.1 Hz, J = 7.1 Hz,
J=1.1 Hz, 1H, H-5), 6.39 (d, J=6.3 Hz, 1H, CH), 3.75 (s,
ꢀ
4
residue was puriꢃed by silica gel column ꢄash chromatog-
raphy (ꢀ g, n-hexane/ethyl acetate ꢀ:1) aꢂording (S)-(−)-
3
(0.006 g, 60%). Colourless needles; m.p.: 153–154 °C
ꢀ
0
13
(
n-hexane/acetone); [α] =−36.3 (c=0.08, CHCl ); 93%
3H, OCH ), ꢀ.6ꢀ (s, 3H, SCH ) ppm; C NMR (100 MHz,
D
3
3
3
1
3

M. Budovská et al.
CDCl ): δ=199.ꢀ (C=S), 170.6 (C=O), 136.ꢀ (C-7a), 1ꢀ5.7
H-6),), 7.03 (dd, J=7.6 Hz, J=7.6 Hz, 1H, H-5), 6.91 (d,
3
(
C-3a), 1ꢀ4.5 (C-ꢀ), 1ꢀꢀ.9 (C-6), 1ꢀ0.6 (C-5), 118.9 (C-4),
J=7.6 Hz, 1H, H-7), 5.54 (s, 1H, CH), 3.3ꢀ (s, 3H, OCH ),
3
1
3
1
11.6 (C-7), 109.6 (C-3), 56.ꢀ (CH), 5ꢀ.9 (OCH ), 18.3
ꢀ.68 (s, 3H, SCH ) ppm; C NMR (100 MHz, CDCl ):
3
3
3
(
SCH ) ppm; IR: ̄ꢀ = 3390 (NH), 3301 (NH), ꢀ997, ꢀ949,
δ=176.3 (C=O), 167.8 (C=O), 167.4 (C=N), 139.3 (C-7a),
3
−
1
1
7ꢀ4 (C=O), 1601, 1456, 1337, 1ꢀꢀ9, 1100 cm .
130.ꢀ (C-6), 1ꢀ8.7 (C-3a), 1ꢀ4.9 (C-4), 1ꢀ3.4 (C-5), 110.3
(
C-7), 85.7 (CH), 66.3 (C-3), 5ꢀ.1 (OCH ), 16.0 (SCH )
3 3
2
′‑(Methylsulfanyl)‑4′‑(methoxycarbonyl)‑
ppm; IR: ̄ꢀ = 3ꢀ55 (NH), ꢀ948, ꢀ9ꢀ6, ꢀ849, 17ꢀ0 (C=O),
−
1
spiro[indoline‑3,5′‑[4′,5′]dihydrotiazole]‑2‑one (25,
1615, 1566, 1470, 1197, 117ꢀ cm .
C H N O S )
13 12 2 2 2
General procedure for the synthesis of phenylamino
analogues of brassinin 28–35
Procedure A A solution of 0.05ꢀ g 24 (0.176 mmol) in
3
3
.0 cm dichloromethane was added to a vigorously stirred
slurry of 0.ꢀ64 g pyridinium chlorochromate (1.ꢀ3ꢀ mmol)
Crude product of (indol-3-yl)methylamine (27), freshly pre-
3
in 1.0 cm dichloromethane. The reaction mixture was
pared by the reduction of 0.481 g indole-3-carboxaldehyde
stirred for 1 h at room temperature and then diluted with
oxime (3 mmol) with 1.13 g NaBH (30 mmol) in the pres-
4
3
1
0 cm dichloromethane. After adding a small amount of sil-
ence of 0.713 g NiCl ·6H O [33] (3 mmol), was dissolved in
ꢀ
ꢀ
3
3
ica gel, the solvent was evaporated and residue preabsorbed
on silica was subjected to silica gel column chromatography
dioxane/chloroform (ꢀ0 cm /10 cm ). Triethylamine (1.5ꢀ g,
3
ꢀ.10 cm , 15 mmol) and corresponding phenyl isothiocy-
(
ꢀ4 g, n-hexane/ethyl acetate ꢀ:1) aꢂording diastereoisomers
anate or 4-substituted phenyl isothiocyanate (3 mmol) were
added and mixture was stirred for 1ꢀ h at room temperature.
The residue obtained after evaporation of the solvent was
dissolved in acetone, small amount of silica gel was added,
acetone evaporated, and product preabsorbed on silica gel
was chromatographed on silica gel. The obtained com-
pounds were further crystallized from acetone/n-hexane to
aꢂord pure products 28–35.
2
5a (0.016 g, ꢀ9%) and 25b (0.0ꢀꢀ g, 41%).
Procedure B To a solution of 0.06ꢀ g brassinin derivative
3
3
2
4 (0.ꢀ10 mmol) in ꢀ.0 cm AcOH and 0.90 cm dioxane,
3
a solution of 0.150 g CrO (1.50 mmol) in ꢀ.7 cm water
3
was added in one portion at rt. The reaction mixture was
stirred for 30 min at rt. The reaction mixture was diluted
3
3
with 60 cm brine and extracted with EtOAc (ꢀ×60 cm ).
The combined organic extract was washed with 10% K CO
ꢀ
3
3
solution (ꢀ × 30 cm ) and dried over Na SO . The residue
N‑[(I ndol‑3‑yl)methyl]‑N′‑phenylthiourea (28,
C H N S) Following the general procedure, product 28
ꢀ
4
obtained after evaporation of the solvent was subjected to
1
6 15 3
3
chromathography on silica gel (16 g, n-hexane/ethyl acetate
was obtained using 0.406 g phenyl isothiocyanate (0.36 cm ,
3 mmol) and isolated on silica gel (30 g, n-hexane/ethyl
acetate ꢀ:1). Yield: 0.468 g (55%); white crystals; m.p.:
ꢀ
:1) aꢂording diastereoisomers 25a (0.006 g, 9%)and 25b
(
0.010 g, 16%).
1
56–157 °C (acetone/n-hexane); R =0.ꢀꢀ (n-hexane/ethyl
f
1
(
3S),(4′R)‑2′‑(Methylsulfanyl)‑4′‑(methoxycarbonyl)‑
acetate ꢀ:1); H NMR (400 MHz, acetone-d ): δ=10.16 (s,
6
spiro[indoline‑3,5′‑[4′,5′]dihydrotiazole]‑2‑one (25a,
1H, NH), 8.84 (s, 1H, NH), 7.77–7.70 (m, 1H, H-4), 7.44–
7.38 (m, 4H, H-ꢀ′, H-6′, H-7, H-ꢀ), 7.31–7.ꢀ7 (m, ꢀH, H-3′,
H-5′), 7.18 (s, 1H, NH), 7.15–7.03 (m, 3H, H-4′, H-6, H-5),
ꢀ
0
C H N O S ) Yellow oil; [α] =−44.3 (c=0.14, CHCl );
f
1
3
12
2
2
2
D
3
1
R =0.38 (n-hexane/ethyl acetate ꢀ:1); H NMR (400 MHz,
1
3
CDCl ): δ = 7.77 (s, 1H, NH), 7.40 (d, J = 7.6 Hz, 1H,
5.01 (d, J = 4.9 Hz, ꢀH, CH ) ppm; C NMR (100 MHz,
3
ꢀ
H-4), 7.ꢀ7 (dt, J = 7.6 Hz, J = 1.ꢀ Hz, 1H, H-6),), 7.10
acetone-d ): δ = 180.9 (C=S), 138.9 (C-1′), 136.8 (C-7a),
6
(
dt, J = 7.6 Hz, J = 1.0 Hz, 1H, H-5), 6.87 (d, J = 7.6 Hz,
1ꢀ8.9 (C-3′, C-5′), 1ꢀ6.9 (C-3a), 1ꢀ4.8 (C-4′), 1ꢀ4.ꢀ (C-ꢀ),
1
3
H, H-7), 5.43 (s, 1H, CH), 3.73 (s, 3H, OCH ), ꢀ.66 (s,
1ꢀ3.7 (C-ꢀ′, C-6′), 1ꢀ1.6 (C-6), 119.0 (C-5), 118.9 (C-4),
3
1
3
H, SCH ) ppm; C NMR (100 MHz, CDCl ): δ = 169.3
111.9 (C-3), 111.4 (C-7), 40.3 (CH ) ppm; IR: ̄ꢀ = 3351
3
3
ꢀ
−
1
(
(
C=O), 168.5 (C=O), 167.ꢀ (C=N), 140.5 (C-7a), 1ꢀ9.9
(NH), 3ꢀ66 (NH), 3118, ꢀ97ꢀ, ꢀ914, 1540, 1ꢀ94, 748 cm .
C-6), 1ꢀ8.3 (C-3a), 1ꢀ4.3 (C-4), 1ꢀ3.4 (C-5), 110.ꢀ (C-7),
8
5.5 (CH), 66.6 (C-3), 5ꢀ.8 (OCH ), 15.8 (SCH ) ppm; IR:
N‑[(Indol‑3‑yl)methyl]‑N′‑(4‑fluorophenyl)thiourea (29,
C H FN S) Following the general procedure, product 29
3
3
̄
ꢀ=3ꢀ55 (NH), ꢀ948, ꢀ9ꢀ6, ꢀ849, 17ꢀ0 (C=O), 1615, 1566,
1
6
14
3
−
1
1
470, 1197, 117ꢀ cm .
was obtained using 0.459 g 4-ꢄuorophenyl isothiocyanate
(
3 mmol) and isolated on silica gel (30 g, n-hexane/ethyl
(
3R),(4′R)‑2′‑(Methylsulfanyl)‑4′‑(methoxycarbonyl)‑
acetate 1:1). Yield: 0.60ꢀ g (67%); white crystals; m.p.:
spiro[indoline‑3,5′‑[4′,5′]dihydrotiazole]‑2‑one (25b,
180–18ꢀ °C (acetone/n-hexane); R =0.4ꢀ (n-hexane/ethyl
f
ꢀ
0
1
C H N O S ) Yellow oil; [α] =+58.0 (c=0.ꢀ0, CHCl );
f
acetate 1:1); H NMR (400 MHz, acetone-d ): δ=10.14 (s,
1
3
12
2
2
2
D
3
6
1
R =0.47 (n-hexane/ethyl acetate ꢀ:1); H NMR (400 MHz,
1H, NH), 8.78 (s, 1H, NH), 7.71 (d, J =7.9 Hz, 1H, H-4),
CDCl ): δ=8.88 (s, 1H, NH), 7.ꢀ4 (d, J=7.6 Hz, ꢀH, H-4,
7.48–7.38 (m, 4H, H-ꢀ′, H-6′, H-7, H-ꢀ), 7.ꢀ0 (s, 1H, NH),
3
1
3

An alternative approach to the synthesis of anticancer molecule spirobrassinin and its 2′-amino…
7
.14–7.10 (m, 1H, H-6), 7.09–7.0ꢀ (m, 3H, H-3′, H-5′, H-5),
NH), 8.75 (s, 1H, NH), 7.74 (d, J=8.3 Hz, 1H, H-4), 7.4ꢀ–
7.40 (m, 1H, H-7), 7.38 (s, 1H, H-ꢀ), 7.ꢀ8–7.ꢀ5 (m, ꢀH,
H-ꢀ′, H-6′), 7.16–7.10 (m, 3H, H-3′, H-5′, H-6), 7.07–7.04
1
3
4
.99 (d, J = 4.6 Hz, ꢀH, CH ) ppm; C NMR (100 MHz,
ꢀ
acetone-d ): δ = 180.0 (C=S), 158.5 (d, J = ꢀ46.8 Hz,
6
C-4′), 135.4 (C-7a), 133.9 (C-1′), 1ꢀ5.5 (C-3a), 1ꢀ4.9 (d,
(m, ꢀH, H-5, NH), 5.01 (d, J= 4.9 Hz, ꢀH, CH ), ꢀ.ꢀ6 (s,
ꢀ
1
3
J = 8.ꢀ Hz, C-ꢀ′, C-6′), 1ꢀꢀ.7 (C-ꢀ), 1ꢀ0.ꢀ (C-6), 117.6
3H, CH ) ppm; C NMR (100 MHz, acetone-d ): δ=179.7
3
6
(
C-5), 117.4 (C-4), 113.8 (d, J=ꢀꢀ.7 Hz, C-3′, C-5′), 110.4
(C=S), 135.5 (C-7a), 134.8 (C-1′), 133.5 (C-4′), 1ꢀ8.ꢀ (C-3′,
C-5′), 1ꢀ5.7 (C-3a), 1ꢀꢀ.9 (C-ꢀ′, C-6′), 1ꢀꢀ.8 (C-ꢀ), 1ꢀ0.3
(C-6), 117.7 (C-5), 117.6 (C-4), 110.6 (C-7), 110.1 (C-3),
(
C-3), 110.0 (C-7), 38.9 (CH ) ppm; IR: ̄ꢀ = 3395 (NH),
ꢀ
−
1
3
ꢀ31 (NH), 3074, 3053, 3031, 1537, 1ꢀ16, 739 cm .
3
9.0 (CH ), 18.6 (CH ) ppm; IR: ̄ꢀ=3ꢀ74 (NH), 3147 (NH),
ꢀ 3
3053, ꢀ970, ꢀ914, 1540, 1ꢀ54, 746 cm .
−
1
N‑[(Indol‑3‑yl)methyl]‑N′‑(4‑chlorophenyl)thiourea (30,
C H ClN S) Following the general procedure, product 30
1
6
14
3
was obtained using 0.509 g 4-chlorophenyl isothiocyanate
N‑[(Indol‑3‑yl)methyl]‑N′‑(4‑methoxyphenyl)thiourea (33,
C H N OS) Following the general procedure, product 33
(
3 mmol) and isolated on silica gel (30 g, n-hexane/ethyl
1
7 17 3
acetate 1:1). Yield: 0.606 g (64%); white crystals; m.p.:
was obtained using 0.496 g 4-methoxyphenyl isothiocy-
3
1
67–168 °C (acetone/n-hexane); R =0.47 (n-hexane/ethyl
anate (0.4ꢀ cm , 3 mmol) and isolated on silica gel (30 g,
f
1
acetate 1:1); H NMR (400 MHz, acetone-d ): δ=10.16 (s,
n-hexane/ethyl acetate 1:1). Yield: 0.607 g (65%); bright
6
1
7
7
H, NH), 8.86 (s, 1H, NH), 7.71 (d, J =7.9 Hz, 1H, H-4),
.58–7.50 (m, ꢀH, H-ꢀ′, H-6′), 7.4ꢀ–7.39 (m, ꢀH, H-7, H-ꢀ),
.3ꢀ–7.ꢀ7 (m, 3H, NH, H-3′, H-5′), 7.13 (ddd, J=8.ꢀ Hz,
yellow crystals; m.p.: 159–161 °C (acetone/n-hexane);
1
R =0.34 (n-hexane/ethyl acetate 1:1); H NMR (400 MHz,
f
acetone-d ): δ=10.13 (s, 1H, NH), 8.64 (s, 1H, NH), 7.73
6
J = 7.1 Hz, J = 1.1 Hz, 1H, H-6), 7.05 (ddd, J = 8.0 Hz,
(d, J=7.8 Hz, 1H, H-4), 7.40 (d, J=7.5 Hz, 1H, H-7), 7.36
(s, 1H, H-ꢀ), 7.36–7.ꢀꢀ (m, ꢀH, H-ꢀ′, H-6′), 7.14–7.10 (m,
1H, H-6), 7.06–7.03 (m, 1H, H-5), 6.9ꢀ (s, 1H, NH), 6.86–
J = 7.1 Hz, J = 1.0 Hz, 1H, H-5), 4.98 (d, J = 4.4 Hz, ꢀH,
1
3
CH ) ppm; C NMR (100 MHz, acetone-d ): δ = 179.8
ꢀ
6
(
C=S), 137.1 (C-4′), 135.6 (C-7a), 1ꢀ7.7 (C-1′), 1ꢀ7.3 (C-3′,
6.84 (m, ꢀH, H-3′, H-5′), 4.99 (d, J = 4.7 Hz, ꢀH, CH ),
ꢀ
1
3
C-5′), 1ꢀ5.7 (C-3a), 1ꢀ3.7 (C-ꢀ′, C-6′), 1ꢀꢀ.9 (C-ꢀ), 1ꢀ0.4
3.74 (s, 3H, OCH ) ppm; C NMR (100 MHz, acetone-d ):
3
6
(
C-6), 117.8 (C-5), 117.6 (C-4), 110.5 (C-3), 110.ꢀ (C-7),
δ=181.4 (C=S), 157.7 (C-4′), 136.6 (C-7a), 131.1 (C-1′),
1ꢀ6.9 (C-3a), 1ꢀ6.6 (C-ꢀ′, C-6′), 1ꢀ4.0 (C-ꢀ), 1ꢀ1.5 (C-6),
118.9 (C-5), 118.8 (C-4), 114.ꢀ (C-3′, C-5′), 11ꢀ.1 (C-3),
3
9.9 (CH ) ppm; IR: ̄ꢀ=3383 (NH), 3310 (NH), 3090, 3053,
ꢀ
−
1
1
538, 1ꢀ93, 739 cm .
1
11.4 (C-7), 54.8 (OCH ), 40.4 (CH ) ppm; IR: ̄ꢀ = 3393
3 ꢀ
−1
N‑[(Indol‑3‑yl)methyl]‑N′‑(4‑bromophenyl)thiourea (31,
C H BrN S) Following the general procedure, product 31
(NH), 3ꢀ40 (NH), 3040, ꢀ956, ꢀ837, 1536, 1ꢀꢀ5, 739 cm .
1
6
14
3
was obtained using 0.64ꢀ g 4-bromophenyl isothiocyanate
N‑[(Indol‑3‑yl)methyl]‑N′‑[4‑(triꢀuoromethyl)phenyl]thio‑
urea (34, C H F N S) Following the general procedure,
(
3 mmol) and isolated on silica gel (30 g, n-hexane/ethyl
1
7 14 3 3
acetate 1:1). Yield: 0.7ꢀ4 g (67%); white crystals; m.p.:
product 34 was obtained using 0.610 g 4-(triꢄuoromethyl)
phenyl isothiocyanate (3 mmol) and isolated on silica gel
(30 g, n-hexane/ethyl acetate 1:1). Yield: 0.651 g (6ꢀ%);
1
61–163 °C (acetone/n-hexane); R =0.47 (n-hexane/ethyl
f
1
acetate 1:1); H NMR (400 MHz, acetone-d ): δ=10.17 (s,
6
1
7
H, NH), 8.87 (s, 1H, NH), 7.71 (d, J =7.9 Hz, 1H, H-4),
.51–7.35 (m, 6H, H-ꢀ′, H-6′, H-7, H-ꢀ, H-3′, H-5′), 7.35
bright yellow crystals; m.p.: 166–167 °C (acetone/n-
1
hexane); R = 0.5 (n-hexane/ethyl acetate 1:1); H NMR
f
(
s, 1H, NH), 7.13 (ddd, J=7.9 Hz, J=7.5 Hz, J=0.8 Hz,
(400 MHz, acetone-d ): δ=10.ꢀ0 (s, 1H, NH), 9.1ꢀ (s, 1H,
6
1
H, H-6), 7.06–7.03 (m, 1H, H-5), 4.98 (d, J=3.9 Hz, ꢀH,
NH), 7.85–7.83 (m, ꢀH, H-ꢀ′, H-6′), 7.70 (d, J = 7.9 Hz,
1H, H-4), 7.6ꢀ–7.55 (m, ꢀH, H-3′, H-5′), 7.55 (s, 1H, NH),
7.43–7.41 (m, ꢀH, H-7, H-ꢀ), 7.16–7.1ꢀ (m, 1H, H-6), 7.08–
1
3
CH ) ppm; C NMR (100 MHz, acetone-d ): δ = 180.0
ꢀ
6
(
(
(
C=S), 138.ꢀ (C-4′), 136.1 (C-7a), 130.8 (C-3′, C-5′), 1ꢀ6.3
C-3a), 1ꢀ4.5 (C-ꢀ′, C-6′), 1ꢀ3.5 (C-ꢀ), 1ꢀ0.9 (C-6), 118.3
C-5), 118.1 (C-4), 115.9 (C-1′), 110.9 (C-3), 110.6 (C-7),
1
3
7.04 (m, 1H, H-5), 5.00 (d, J=4.5 Hz, ꢀH, CH ) ppm;
C
ꢀ
NMR (100 MHz, acetone-d ): δ=180.0 (C=S), 14ꢀ.8 (C-1′),
6
3
9.4 (CH ) ppm; IR: ̄ꢀ=3384 (NH), 3ꢀ37 (NH), 3070, 3047,
136.ꢀ (C-7a), 1ꢀ6.3 (C-3a), 1ꢀ4.9 (C-3′, C-5′), 1ꢀ3.9 (q,
ꢀ
−
1
1
535, 1ꢀꢀ0, 746 cm .
J=3ꢀ.1 Hz, C-4′), 1ꢀ3.6 (C-ꢀ), 1ꢀꢀ.6 (q, J=ꢀ70.9 Hz, CF ),
3
1
ꢀ1.5 (C-ꢀ′, C-6′), 1ꢀ0.9 (C-6), 118.4 (C-5), 118.1 (C-4),
N‑[(Indol‑3‑yl)methyl]‑N′‑(4‑methylphenyl)thiourea (32,
C H N S) Following the general procedure, product 32
110.7 (C-7), 110.6 (C-3), 39.4 (CH ) ppm; IR: ̄ꢀ = 3375
ꢀ
−
1
(NH), 3ꢀ68 (NH), 3094, 3070, 1557, 133ꢀ, 746 cm .
1
7 17 3
was obtained using 0.448 g 4-methylphenyl isothiocyanate
(
3 mmol) and isolated on silica gel (30 g, n-hexane/ethyl
N‑[(Indol‑3‑yl)methyl]‑N′‑[3,5‑bis(trifluoromethyl)phe‑
nyl]thiourea (35, C H F N S) Following the gen-
acetate ꢀ:1). Yield: 0.5ꢀ5 g (59%); white crystals; m.p.: 154–
1
8
13
6
3
1
ꢀ
55 °C (acetone/n-hexane); R =0.ꢀ (n-hexane/ethyl acetate
eral procedure, product 35 was obtained using 0.813 g
f
1
3
:1); H NMR (400 MHz, acetone-d ): δ = 10.15 (s, 1H,
3,5-bis(triꢄuoromethyl)phenyl isothiocyanate (0.548 cm ,
6
1
3

M. Budovská et al.
3
mmol) and isolated on silica gel (30 g, n-hexane/ethyl
(C-6), 1ꢀ3.9 (C-4), 1ꢀꢀ.4 (C-5), 119.3 (C-ꢀ′, C-6′), 115.1
acetate ꢀ:1). Yield: 0.79ꢀ g (63%), white crystals; m.p.:
(d, J=ꢀꢀ.ꢀ Hz, C-3′, C-5′), 109.8 (C-7), 71.3 (CH ), 61.6
ꢀ
1
39–141 °C (acetone/n-hexane); R = 0.3 (n-hexane/ethyl
(C-3) ppm; IR: ̄ꢀ = 313ꢀ, 3086, 3033, ꢀ833, 1716 (C=O),
f
1
−1
acetate ꢀ:1); H NMR (400 MHz, acetone-d ): δ=10.ꢀ3 (s,
1647, 1530 (C=N), 1ꢀ00, 745 cm .
6
1
7
7
H, NH), 9.31 (s, 1H, NH), 8.36 (s, ꢀH, H-ꢀ′, H-6′), 7.73–
.68 (m, 3H, H-4, H-4′, NH), 7.44–7.4ꢀ (m, ꢀH, H-7, H-ꢀ),
.17–7.13 (m, 1H, H-6), 7.08–7.04 (m, 1H, H-5), 5.00 (d,
(±)‑2′‑(4‑Chlorophenylamino)spiro[indoline‑3,5′‑[4′,5′]‑
dihydrothiazole]‑2‑one ((±)‑43, C H ClN OS) Following
1
6
12
3
1
3
J=4.5 Hz, ꢀH, CH ) ppm; C NMR (100 MHz, acetone-
the general procedure, product (±)-43 was obtained using
0.158 g thiourea 30 (0.5 mmol) and isolated on silica gel
(15 g, n-hexane/ethyl acetate 1:1). Yield: 0.11ꢀ g (68%);
bright yellow crystals; m.p.: ꢀꢀ3–ꢀꢀ5 °C (acetone/n-hex-
ꢀ
d ): δ=179.7 (C=S), 140.5 (C-1′), 135.ꢀ (C-7a), 1ꢀ9.3 (q,
6
J=33.3 Hz, C-3′, C-5′), 1ꢀ5.3 (C-3a), 1ꢀꢀ.7 (C-ꢀ), 1ꢀ1.8 (q,
J=ꢀ7ꢀ.4 Hz, ꢀ×CF ), 1ꢀ0.5 (C-ꢀ′, C-6′), 1ꢀ0.0 (C-6), 117.5
3
(
C-5), 116.9 (C-4), 114.6 (C-4′), 109.8 (C-7), 109.4 (C-3),
ane); R =0.ꢀ6 (n-hexane/ethyl acetate 1:1). All spectral data
f
38.4 (CH ) ppm; IR: ̄ꢀ=3490 (NH), 3ꢀꢀ6 (NH), 3080, 3037,
are fully identical with previously described product [30].
ꢀ
−
1
1
540, 1ꢀ85, 744 cm .
(
±)‑2′‑(4‑Bromophenylamino)spiro[indoline‑3,5′‑[4′,5′]‑
General procedure for synthesis of 2′‑amino
dihydrothiazole]‑2‑one ((±)‑44, C H BrN OS) Following
1
6
12
3
analogues of spirobrassinin ((±)‑41‑(±)‑48)
the general procedure, product (±)-44 was obtained using
0
.180 g thiourea 31 (0.5 mmol) and isolated on silica gel
To a solution of corresponding thiourea 28–35
(15 g, n-hexane/ethyl acetate 1:1). Yield: 0.116 g (6ꢀ%);
bright yellow crystals; m.p.: ꢀꢀ3–ꢀꢀ4 °C (acetone/n-hex-
3
3
(
0.50 mmol) in 4.ꢀ cm AcOH and ꢀ.ꢀ cm dioxane, a
3
solution of 0.ꢀ50 g CrO (ꢀ.50 mmol) in 1.70 cm water
ane); R =0.ꢀ6 (n-hexane/ethyl acetate 1:1). All spectral data
3
f
was added in one portion at rt. The reaction mixture was
are fully identical with previously described product [30].
stirred for 0.5 h at rt. The reaction mixture was diluted
3
3
with 80 cm brine and extracted with EtOAc (ꢀ × 80 cm ).
The combined organic extract was washed with 10%
(±)‑2′‑(4‑Methylphenylamino)spiro[indoline‑3,5′‑[4′,5′]‑
dihydrothiazole]‑2‑one ((±)‑45, C H N OS) Following
1
7 15 3
3
K CO solution (ꢀ × 80 cm ) and dried over Na SO . The
the general procedure, product (±)-45 was obtained using
0.148 g thiourea 32 (0.5 mmol) and isolated on silica gel
(15 g, n-hexane/ethyl acetate 1:1). Yield: 0.109 g (70%);
bright yellow crystals; m.p.: ꢀ31–ꢀ33 °C (acetone/n-hex-
ꢀ
3
ꢀ
4
residue obtained after evaporation of the solvent was sub-
jected to chromathography on silica gel.
(
±)‑2′‑(Phenylamino)spiro[indoline ‑3, 5′‑[4′, 5′]‑
ane); R =0.ꢀ0 (n-hexane/ethyl acetate 1:1). All spectral data
f
dihydrothiazole]‑2‑one ((±)‑41, C H N OS) Following
are fully identical with previously described product [30].
1
6 13 3
the general procedure, product (±)-41 was obtained using
0
.141 g thiourea 28 (0.5 mmol) and isolated on silica gel
(±)‑2′‑(4‑Methoxyphenylamino)spiro[indoline‑3,5′‑[4′,5′]‑
dihydrothiazole]‑2‑one ((±)‑46, C H N O S) Following
(
15 g, n-hexane/ethyl acetate 1:1). Yield: 0.097 g (66%);
1
7 15 3 2
bright yellow crystals; m.p.: ꢀ06–ꢀ08 °C (acetone/n-hex-
the general procedure, product (±)-46 was obtained using
0.156 g thiourea 33 (0.5 mmol) and isolated on silica gel
(15 g, n-hexane/ethyl acetate 1:ꢀ). Yield: 0.109 g (67%);
ane); R =0.ꢀꢀ (n-hexane/ethyl acetate 1:1). All spectral data
f
are fully identical with previously described product [30].
bright yellow crystals; m.p.: 1ꢀ9–131 °C (acetone/n-
1
(
±)‑2′‑(4‑fluorophenylamino)spiro[indoline‑3,5′‑[4′,5′]‑
hexane); R = 0.31 (n-hexane/ethyl acetate 1:ꢀ); H NMR
f
dihydrothiazole]‑2‑one ((±)‑42, C H FN OS) Following
(400 MHz, DMSO-d ): δ=10.65 (s, 1H, NH), 9.33 (s, 1H,
1
6
12
3
6
the general procedure, product (±)-42 was obtained using
NH), 7.50 (s, ꢀH, H-ꢀ′, H-6′), 7.36 (d, J=7.4 Hz, 1H, H-4),
7.ꢀ5 (ddd, J=7.7 Hz, J=7.7 Hz, J=1.1 Hz, 1H, H-6), 7.03
(dd, J = 7.7 Hz, J = 7.4 Hz, 1H, H-5), 6.89–6.85 (m, 3H,
0
.150 g thiourea 29 (0.5 mmol) and isolated on silica gel
(
15 g, n-hexane/ethyl acetate 1:1). Yield: 0.108 g (69%);
bright yellow crystals; m.p.: ꢀ48–ꢀ50 °C (acetone/n-hexane);
H-7, H-3′, H-5′), 4.30 (d, J = 13.8 Hz, 1H, H ), 4.ꢀ0 (d,
b
1
13
R =0.ꢀ6 (n-hexane/ethyl acetate 1:1); H NMR (400 MHz,
J = 13.8 Hz, 1H, H ), 3.7ꢀ (s, 3H, OCH ) ppm; C NMR
f
a
3
DMSO-d ): δ=10.68 (s, 1H, NH), 9.6ꢀ (s, 1H, NH), 7.61
(100 MHz, DMSO-d ): δ = 177.9 (C=O), 154.7 (C-4′),
6
6
(
s, ꢀH, H-ꢀ′, H-6′), 7.38 (d, J=7.5 Hz, 1H, H-4), 7.ꢀ6 (dd,
153.5 (C=N), 141.5 (C-7a), 135.4 (C-1′), 131.3 (C-3a),
J = 7.6 Hz, J = 7.6 Hz, 1H, H-6), 7.1ꢀ (t, J = 8.8 Hz, ꢀH,
1ꢀ9.7 (C-6), 1ꢀ4.4 (C-4), 1ꢀꢀ.9 (C-5), 119.8 (C-ꢀ′, C-6′),
H-3′, H-5′), 7.04 (dd, J=7.6 Hz, J=7.5 Hz, 1H, H-5), 6.89
114.3 (C-3′, C-5′), 110.3 (C-7), 7ꢀ.1 (CH ), 6ꢀ.0 (C-3), 55.6
ꢀ
(
(
d, J=7.6 Hz, 1H, H-7), 4.33 (d, J=14.3 Hz, 1H, H ), 4.ꢀ3
(OCH ) ppm; IR: ̄ꢀ=3188, 3080, ꢀ833, ꢀ671, 1693 (C=O),
b
3
1
3
−1
d, J=14.3 Hz, 1H, H ) ppm; C NMR (100 MHz, DMSO-
1615, 1504 (C=N), 1473, 1ꢀ43, 747 cm .
a
d ): δ=177.3 (C=O), 157.1 (d, J =ꢀ37.9 Hz, C-4′), 153.0
6
(
C=N), 141.0 (C-7a), 137.8 (C-1′), 130.6 (C-3a), 1ꢀ9.ꢀ
1
3

An alternative approach to the synthesis of anticancer molecule spirobrassinin and its 2′-amino…
(
± ) ‑ 2 ′ ‑ [ 4 ‑ ( T r i f l u o r o m e t h y l ) p h e n y l a m i n o ] ‑
( ± ) ‑ 1 ‑ M e t h o x y ‑ 2 ′ ‑ ( 4 ‑ c h l o r o p h e n y l a m i n o ) ‑
spiro[indoline‑3,5′‑[4′,5′]dihydrothiazole]‑2‑one ((±)‑50,
C H ClN O S) Following the general procedure, product
spiro[indoline‑3,5′‑[4′,5′]dihydrothiazole]‑2‑one ((±)‑47,
C H F N OS) Following the general procedure, product
1
7
12
3
3
17 14
3 2
(
±)-47 was obtained using 0.175 g thiourea 34 (0.5 mmol)
(±)-50 was obtained using 0.173 g thiourea 37 (0.5 mmol)
and isolated on silica gel (15 g, n-hexane/ethyl acetate 1:1).
and isolated on silica gel (ꢀ0 g, n-hexane/ethyl acetate ꢀ:1).
Yield: 0.1ꢀ4 g (68%); bright yellow crystals; m.p.: 87–89 °C
Yield: 0.075 g (4ꢀ%); yellow oil; R =0.61 (n-hexane/ethyl
f
(
acetone/n-hexane); R =0.37 (n-hexane/ethyl acetate 1:1).
acetate ꢀ:1). All spectral data are fully identical with previ-
f
All spectral data are fully identical with previously described
ously described product [30].
product [30].
(
± ) ‑ 1 ‑ M e t h o x y ‑ 2 ′ ‑ ( 4 ‑ b r o m o p h e n y l a m i n o ) ‑
(
± ) ‑ 2 ′ ‑ [ 3 , 5 ‑ B i s ( t r i f l u o r o m e t hy l ) p h e ny l a m i n o ] ‑
spiro[indoline‑3,5′‑[4′,5′]dihydrothiazole]‑2‑one ((±)‑51,
C H BrN O S) Following the general procedure, product
spiro[indoline‑3,5′‑[4′,5′]dihydrothiazole]‑2‑one ((±)‑48,
C H F N OS) Following the general procedure, product
1
7
14
3 2
(±)-51 was obtained using 0.195 g thiourea 38 (0.5 mmol)
1
8 11 6 3
(
±)-48 was obtained using 0.ꢀ16 g thiourea 35 (0.5 mmol)
and isolated on silica gel (ꢀ0 g, n-hexane/ethyl acetate 1:1).
and isolated on silica gel (15 g, n-hexane/ethyl acetate 1:1).
Yield: 0.086 g (43%); yellow oil; R =0.56 (n-hexane/ethyl
f
Yield: 0.140 g (65%); bright yellow crystals; m.p.: 109–
acetate 1:1). All spectral data are fully identical with previ-
1
1
1
11 °C (acetone/n-hexane); R =0.5 (n-hexane/ethyl acetate
ously described product [30].
f
1
:1); H NMR (400 MHz, DMSO-d ): δ=10.7ꢀ (s, 1H, NH),
6
0.40 (s, 1H, NH), 8.3ꢀ (s, ꢀH, H-ꢀ′, H-6′), 7.64 (s, 1H,
( ± ) ‑ 1 ‑ M e t h o x y ‑ 2 ′ ‑ ( 4 ‑ m e t h y l p h e n y l a m i n o ) ‑
spiro[indoline‑3,5′‑[4′,5′]dihydrothiazole]‑2‑one ((±)‑52,
C H N O S) Following the general procedure, product
H-4′), 7.4ꢀ (d, J= 7.5 Hz, 1H, H-4), 7.ꢀ7 (dd, J= 7.7 Hz,
J=7.7 Hz, 1H, H-6), 7.05 (dd, J=7.7 Hz, J=7.5 Hz, 1H,
H-5), 6.91 (d, J=7.7 Hz, 1H, H-7), 4.48 (d, J=14.0 Hz, 1H,
1
8 17 3 2
(±)-52 was obtained using 0.163 g thiourea 39 (0.5 mmol)
13
H ), 4.38 (d, J=14.0 Hz, 1H, H ) ppm; C NMR (100 MHz,
and isolated on silica gel (ꢀ0 g, n-hexane/ethyl acetate 1:1).
b
a
DMSO-d ): δ = 177.3 (C=O), 15ꢀ.8 (C=N), 14ꢀ.6 (C-1′),
Yield: 0.078 g (46%); yellow oil; R =0.61 (n-hexane/ethyl
6
f
1
41.ꢀ (C-7a), 130.8 (q, J = 3ꢀ.6 Hz, C-3′, C-5′), 130.1
C-3a), 1ꢀ9.5 (C-6), 1ꢀ4.1 (C-4), 1ꢀ3.ꢀ (q, J = ꢀ7ꢀ.7 Hz,
×CF ), 1ꢀꢀ.5 (C-5), 117.3 (C-ꢀ′, C-6′), 114.3 (C-4′), 110.0
acetate 1:1). All spectral data are fully identical with previ-
(
ously described product [30].
ꢀ
3
(
C-7), 71.5 (CH ), 6ꢀ.1 (C-3) ppm; IR: ̄ꢀ=3ꢀ46, 3106, 1707
(±)‑1‑Methoxy‑2′‑[4‑(trifluoromethyl)phenylamino]‑
spiro[indoline‑3,5′‑[4′,5′]dihydrothiazole]‑2‑one ((±)‑53,
C H F N O S) Following the general procedure, product
ꢀ
−
1
(
C=O), 1618, 147ꢀ (C=N), 1381, 1ꢀ76, 11ꢀ7, 749 cm .
1
8 14 3 3 2
General procedure for synthesis of 2′‑amino
(±)-53 was obtained using 0.190 g thiourea 40 (0.5 mmol)
analogues of 1‑methoxyspirobrassinin
and isolated on silica gel (ꢀ0 g, n-hexane/ethyl acetate 1:1).
(
(±)‑49‑(±)‑53)
Yield: 0.090 g (46%); yellow oil; R =0.58 (n-hexane/ethyl
f
acetate 1:1). All spectral data are fully identical with previ-
To a solution of corresponding thiourea 36–40 (0.50 mmol)
ously described product [30].
3
3
in 4.ꢀ cm AcOH and ꢀ.ꢀ cm dioxane, a solution of 0.ꢀ50 g
3
CrO (ꢀ.50 mmol) in 1.70 cm water was added in one por-
3
tion at rt. The reaction mixture was stirred for ꢀ.5 h at rt. The
Cancer cell lines
3
reaction mixture was diluted with 80 cm brine and extracted
3
with EtOAc (ꢀ×80 cm ). The combined organic extract was
Jurkat, CCRF-CEM (acute T-lymphoblastic leukaemia),
HeLa (cervical adenocarcinoma), MCF-7 (mammary gland
adenocarcinoma), MDA-MB-ꢀ31 (mammary gland adeno-
carcinoma), and A-549 (non-small cell lung cancer) cells
were maintained in RPMI 1640 medium supplemented with
Glutamax-I or in Dulbecco’s modiꢃed Eagle’s medium with
Glutamax-I and glucose. Both of these media were supple-
mented with 10% (v/v) foetal calf serum, penicillin (100 IU/
3
washed with 10% K CO solution (ꢀ × 80 cm ) and dried
ꢀ
3
over Na SO . The residue obtained after evaporation of the
ꢀ
4
solvent was subjected to chromathography on silica gel.
(
±)‑1‑Methoxy‑2′‑(phenylamino)spiro[indoline‑3,5′‑[4′,5′]‑
dihydrothiazole]‑2‑one ((±)‑49, C H N O S) Following
1
7 15 3 2
the general procedure, product (±)-49 was obtained using
.156 g thiourea 36 (0.5 mmol) and isolated on silica gel
3
3
0
cm ), and streptomycin (100 μg/cm ) (all from Invitrogen,
(
ꢀ0 g, n-hexane/ethyl acetate 1:1). Yield: 0.063 g (39%); yel-
UK), in humidiꢃed air with 5% CO at 37 °C. Before each
ꢀ
low oil; R =0.49 (n-hexane/ethyl acetate 1:1). All spectral
cytotoxicity assay, cell viability was determined by the
trypan blue (Invitrogen) exclusion method and found to be
greater than 95%.
f
data are fully identical with previously described product
[
30].
1
3

M. Budovská et al.
Human umbilical vein endothelial cells
(Sigma-Aldrich) were added into each well and incubated for
another 4 h. Then, the formazan crystals were dissolved with
3
Human umbilical vein endothelial cells (HUVEC) were iso-
lated, cultured, and characterized as previously described
100 mm of 10% SDS. The optical density (OD) was meas-
ured at 540 nm with an EL-31ꢀ microplate reader (Biotek
Instruments Inc., Winooski, VT USA) for wells with treated
cells and normalized to untreated control.
[
34, 35]. Cells were cultured on gelatin-coated dishes in
cM199 (= M199 medium supplemented with 10% heat-
inactivated human serum, 10% heat-inactivated newborn
3
Acknowledgments The present study was supported in part by
the Grant Agency of Ministry of the Education, Science, Research
and Sport of the Slovak Republic (VEGA 1/0753/17 and VEGA
1/0018/16), and the Slovak Research and Development Agency
under the Contract No. APVV-16-0446. Moreover, this publication
is the result of the project implementation: „Medicínsky univer-
zitný vedecký park v Košiciach (MediPark, Košice - Fáza II.)“, kód
ITMSꢀ014+313011D103 supported by the Operational Programme
Research & Innovation, funded by the ERDF“. The authors thank
Assoc. prof. D. Berkeš and Dr. J. Markus for synthesis and providing
of the (R)-ꢀ-(1H-indol-3-yl)glycine.
calf serum, 3.75 µg/cm crude endothelial cell growth fac-
3
3
tor (ECGF), 5 U/cm heparin, 100 IU/cm penicillin, and
3
1
00 µg/cm streptomycin) at 37 °C under 5% CO /95%
ꢀ
air atmosphere. Twenty-four hours prior to experiments
endothelial cell cultures were refreshed with medium with-
out crude endothelial cell growth factor and heparin.
Cytotoxicity assay
IC values (concentration of a given compound that
5
0
decreased amount of viable cells to 50% relative to untreated
control cells) were determined by 4 parameter logistic
non-linear regression model using normalized concentra-
tion–response data obtained by MTT (thiazolyl blue tetra-
zolium bromide) assay.
References
1
.
Pedras MSC, Yaya EE, Glawischnig E (ꢀ011) Nat Prod Rep
ꢀ
8:1381
ꢀ
3
.
.
Pedras MSC, Abdoli A (ꢀ017) RSC Adv 7:ꢀ3633
Takasugi M, Monde K, Katsui N, Shirata A (1987) Chem
Lett:1631
Cancer cells
4
.
Gross D, Porzel A, Schmidt J (1994) Z Naturforsch C Biosci
4
9:ꢀ81
The cytotoxic eꢂect of compounds was studied using the
5
.
Monde K, Takasugi M, Shirata A (1995) Phytochemistry 39:581
colorimetric microculture assay with the MTT endpoint
6. Storck M, Sacristan MD (1995) Z Naturforsch 50c:15
7. Mezencev R, Galizzi M, Kutschy P, Docampo R (ꢀ009) Exp
Parasitol 1ꢀꢀ:66
3
[
3ꢀ]. Brieꢄy, 5×10 cells were plated per well in 96-well
3
polystyrene microplates (Sarstedt, Germany) in 100 mm
of the culture medium containing tested chemicals at ꢃnal
8
.
Kristoꢃkova Z, Gazova Z, Siposova K, Bartos A, Ricny J,
Kotoucova J, Sirova J, Ripova D (ꢀ014) Neurochem Res
39:150ꢀ
−
6
−4
3
concentrations of 10 –10 mol/dm . After 7ꢀ h incubation,
3
3
9. Mehta RG, Liu J, Constantinou A, Hawthorne M, Pezzuto JM,
Moon RC, Moriarty RM (1994) Anticancer Res 14:1ꢀ09
1
0 mm of MTT (5 mg/cm , Sigma-Aldrich) was added into
each well. After an additional 4 h at 37 °C, during which
1
0. Mezencev R, Mojžiš J, Pilátová M, Kutschy P, Čurillová Z (ꢀ004)
Int J Canc Prev 1:105
3
insoluble formazan was produced, 100 mm of 10% (m/m)
sodium dodecylsulfate (SDS, Sigma-Aldrich) were added
into each well and another 1ꢀ h were allowed for the dis-
solution of formazan. The absorbance was measured at
11. Pilátová M, Šarišský M, Kutschy P, Miroššay A, Mezencev R,
Čurillová Z, Suchý M, Monde K, Miroššay L, Mojžiš J (ꢀ005)
Leukemia Res ꢀ9:415
1
ꢀ. Mezencev R, Mojžiš J, Pilátová M, Kutschy P, Čurillová Z (ꢀ007)
Eꢂects of phytoalexins on the growth of cancer cells. In: Colum-
bus F (ed) Trends in cancer prevention research. Nova Science
Publishers, New York, p 81
5
40 nm and 630 nm – reference wavelength by the auto-
mated uQuant™ Universal Microplate Spectrophotometer
(
Biotek Instruments Inc., Winooski, VT USA). The blank
1
3. Banerjee T, Duhadaway JB, Gaspari P, Sutanto-Ward E, Munn
DH, Mellor AL, Malachowski WP, Prendergast GC, Muller AJ
corrected absorbance of the control wells was taken as 100%
and the results were expressed as a percentage of the control.
(
ꢀ008) Oncogene ꢀ7:ꢀ851
1
1
1
4. Izutani Y, Yogosawa S, Sowa Y, Sakai T (ꢀ01ꢀ) Int J Oncol
4
0:816
5. Kim SM, Oh EY, Lee JH, Nam D, Lee SG, Lee J, Kim SH, Shim
BS, Ahn KS (ꢀ015) Phytother Res ꢀ9:18ꢀ8
Human umbilical vein endothelial cells
6. Kello M, Drutovic D, Chripková M, Pilatová M, Budovská M,
Kuliková L, Urdzík P, Mojžiš J (ꢀ014) Molecules 19:10877
The assay conditions were essentially the same as described
3
17. Chripkova M, Zigo F, Mojzis J (ꢀ016) Molecules ꢀ1:16ꢀ6
previously [36]. Brieꢄy, aliquots of 80 mm cell suspension
1
8. Mezencev R, Kutschy P, Salayova A, Curillova Z, Mojzis J, Pila-
tova M, McDonald J (ꢀ008) Chemotherapy 54:37ꢀ
(
8000 cells/well) were added to 96-well microculture plates.
3
Twenty-four hours after seeding, ꢀ0 mm aliquots of drug
1
9. Kutschy P, Salayová A, Čurillová Z, Kožár T, Mezencev R,
Mojžiš J, Pilátová M, Balentová E, Pazdera P, Sabol M, Zburová
M (ꢀ009) Bioorg Med Chem 17:3698
solutions were added to the HUVEC in triplicate wells for
3
4
8 h. After 48 h of culturing, 10 mm of the MTT solution
1
3

An alternative approach to the synthesis of anticancer molecule spirobrassinin and its 2′-amino…
ꢀ
ꢀ
0. Budovská M, Pilatová M, Varinská L, Mojžiš J, Mezencev R
30. Budovská M, Tischlerová V, Mojžiš J, Harvanová M, Kozlov O,
Gondová T, Tomášková N (ꢀ017) Tetrahedron 73:6356
31. Kutschy P, Sabol M, Marušková R, Čurillová Z, Dzurilla M, Géci
I, Alföldi J, Kováčik V (ꢀ004) Collect Czech Chem Commun
69:850
(
ꢀ013) Bioorg Med Chem ꢀ1:66ꢀ3
1. Chripkova M, Drutovic D, Pilatova M, Mikes J, Budovska M,
Vaskova J, Broggini M, Mirossay L, Mojzis J (ꢀ014) Toxicol
In Vitro ꢀ8:909
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ. Tischlerová V, Kello M, Budovská M, Mojžiš J (ꢀ017) World J
Gastroenterol ꢀ3:4341
3ꢀ. Mosmann T (1983) J Immunol Methods 65:55
33. Kutschy P, Dzurilla M, Takasugi M, Török M, Achbergerová I,
Homzová R, Rácová M (1998) Tetrahedron 54:3549
34. Deꢃlippi P, van Hinsbergh VWM, Bertolotto A, Rossino P, Sileng
L, Tarone G (1991) J Cell Biol 114:855
3. Budovská M, Baláž M, Mezencev R, Tischlerová V, Zigová M,
Mojžiš J (ꢀ018) J Fluorine Chem ꢀ16:ꢀ4
4. Suchý M, Kutschy P, Monde K, Goto H, Harada N, Takasugi M,
Dzurilla M, Balentová E (ꢀ001) J Org Chem 66:3940
5. Budovská M, Kutschy P, Kožár T, Gondová T, Petrovaj J (ꢀ013)
Tetrahedron 69:109ꢀ
35. van Hinsbergh VW, Sprengers ED, Kooistra T (1987) Thromb
Haemost 57:148
36. Kaspers GJ, Veerman AJ, Pieters R, Broekema GJ, Huijsmans
DR, Kazemier KM, Loonen AH, Rottier MA, van Zantwijk CH,
Hählen K (1994) Br J Cancer 70:1047
6. Kutschy P, Suchý M, Monde K, Harada N, Marušková R,
Čurillová Z, Dzurilla M, Miklošová M, Mezencev R, Mojžiš J
(
ꢀ00ꢀ) Tetrahedron Lett 43:9489
ꢀ
7. Čurillová Z, Kutschy P, Budovská M, Nakahashi A, Monde K
Publisher’s Note Springer Nature remains neutral with regard to
(
ꢀ007) Tetrahedron Lett 48:8ꢀ00
jurisdictional claims in published maps and institutional aꢁliations.
ꢀ
ꢀ
8. Budovská M (ꢀ014) RSC Adv 4:5575
9. Pedras MSC, Suchý M, Ahiahonu PWK (ꢀ006) Org Biomol Chem
4
:691
1
3