Spirocyclisation of phytoalexin 1-methoxybrassinin in the presence of Grignard reagents

Article abstract of DOI:10.2478/s11696-013-0335-7

The anti-cancer properties of naturally occurring (2R, 3R)-(-)-1- methoxyspirobrassinol methyl ether (I) and their synthetic amino or piperidyl analogues II inspired us to study the synthesis of new target compounds III with a C - C bond in the 2-position of indole rather than a C - N or C - O bond (I or II respectively). The goal was achieved via electrophilic-nucleophilic 3,2-difunctionalisation of 1-methoxybrassinin (IV) in the presence of bromine and the Grignard reagent leading to the formation of cis- and trans-C - C analogues of I. Finally, the anti-cancer activities of the new compounds were measured and compared with I and II in order to show the importance of a heteroatom in the 2-substituted indole on the anti-cancer activity of spirobrassinols.

Full text of DOI:10.2478/s11696-013-0335-7

Chemical Papers 67 (6) 631–642 (2013)
DOI: 10.2478/s11696-013-0335-7
ORIGINAL PAPER
Spirocyclisation of phytoalexin 1-methoxybrassinin
in the presence of Grignard reagents
a
a
b
c
aPeter Očenáš*, Lucia Tomášová, Peter Kutschy, Pavel Pazdera, Ján Mojžiš,
cMartina Pilátová
aDepartment of Organic Chemistry, Institute of Chemical Science, Faculty of Science, P. J. Šafárik University,
Moyzesova 11, 040 01 Košice, Slovakia
bCentre for Syntheses under Sustainable Conditions and their Management, Department of Chemistry, Masaryk University,
Kotlářská 2, 611 37 Brno, Czech Republic
cDepartment of Pharmacology, Faculty of Medicine, P. J. Šafárik University, SNP 1, 040 66 Košice, Slovakia
Received 3 October 2012; Revised 27 November 2012; Accepted 1 December 2012
The anti-cancer properties of naturally occurring (2R, 3R)-(–)-1-methoxyspirobrassinol methyl
ether (I ) and their synthetic amino or piperidyl analogues II inspired us to study the synthesis
of new target compounds III with a C—C bond in the 2-position of indole rather than a C—N
or C—O bond (I or II respectively). The goal was achieved via electrophilic–nucleophilic 3,2-di-
functionalisation of 1-methoxybrassinin (IV ) in the presence of bromine and the Grignard reagent
leading to the formation of cis- and trans-C—C analogues of I. Finally, the anti-cancer activities of
the new compounds were measured and compared with I and II in order to show the importance
of a heteroatom in the 2-substituted indole on the anti-cancer activity of spirobrassinols.
c
ꢀ 2013 Institute of Chemistry, Slovak Academy of Sciences
Keywords: phytoalexins, spirocyclisation, Grignard addition, spiroindolines, anti-proliferative ac-
tivity
Introduction
of compounds II show a biological activity higher
than/or comparable to cisplatin on Jurkat cells and
they exhibit a higher activity than etoposide on MDA-
MB-231 cells (Kutschy et al., 2009). Logically, these
promising results invite comparison with alkyl or aryl
derivatives of I. For this reason, we describe here the
original syntheses of III and their anti-cancer activity
screening test.
Indole phytoalexins are the secondary metabolites
isolated from plants of the Cruciferae family; they
represent an important group of natural compounds
due to their importance in the plant protection (Pe-
dras et al., 2011). Spiroindole phytoalexins represent a
special group of these compounds, wherein the indole
nucleus is spiro-attached to the thiazoline in position
C-3 (Kutschy & Mezencev, 2008). Recently, Kutschy
et al. (2009) designed and synthesised aryl-amino or
piperidyl (Mezencev et al., 2008) analogues II of
1-methoxyspirobrassinol methyl ether (I ) as prospec-
tive anti-cancer compounds (Fig. 1).
Experimental
General
All commercially available reagents were pur-
chased in the highest available purity from Sigma–
Aldrich (USA), Merck (Germany), or Acros Organics
(Belgium) and used without further purification. Sol-
The amino analogues II of I were tested for their
cytostatic/cytotoxic activity against selected human
solid tumour and leukaemia cell lines in vitro. Some
*Corresponding author, e-mail: peter.ocenas@gmail.com

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P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
Fig. 1. Natural enantiomer of 1-methoxyspirobrassinol methyl ether (I) and their analogues II and III.
vents were dried and purified prior to use in accor-
dance with standard procedures. 1H NMR and 13C
NMR spectra of compounds dissolved in CDCl3 were
recorded at ambient temperature on a Varian Mer-
cury Plus 400 FT NMR (400.13 MHz for 1H NMR and
100.6 MHz for 13C NMR) or a Varian Premium COM-
PACT 600 (599.87 MHz for 1H NMR and 150.84 MHz
for 13C NMR) (both Varian, USA) spectrometer us-
ing tetramethylsilane (TMS) as the internal reference.
Chemical shifts are given in δ relative to TMS. Cou-
pling constants (J) were obtained by first-order anal-
ysis and measured in Hz. IR spectra were recorded on
an Avatar FT-IR 6700 spectrometer (Thermo Scien-
tific, IK) using the attenuated total reflectance (ATR)
method in the range of 4000–400 cm−1. The EI mass
spectra were recorded on a GC-MS Trio 1000 (Fisons
Instruments, UK) spectrometer at ionisation energy
of 70 eV. Microanalyses were performed with Perkin–
Elmer (USA), model 2400 analyser. The progress of
chemical reactions was monitored by thin layer chro-
matography, using Macherey-Nagel (Germany) plates
Alugramꢀ Sil G/UV254. Detection was carried out
with ultraviolet light (254 nm). Preparative column
chromatography was performed on Kieselgel 60 Merck
type 9385 (0.040–0.063 mm). Melting points were de-
termined on a Koffler micro melting point apparatus
and are uncorrected.
Assessment of cytotoxicity by MTT assay
The cytotoxic effect of the compounds tested was
studied by using a colorimetric microculture assay
with the MTT end-point. The amount of MTT re-
duced to formazan was proportional to the number
of viable cells (Mosmann, 1983). Briefly, 5 × 103 cells
were plated per well in 96-well polystyrene microplates
(Sarstedt, Germany) in 100 µL of the culture medium
containing the tested chemicals at final concentrations
of 10−4–10−6 mol L−1. After 72 h, 10 µL of MTT
(5 mg mL−1) was added to each well. After an addi-
tional 4 h at 37◦C, during which insoluble formazan
was produced, 100 µL of 10 mass % sodium dode-
cylsulphate (Sigma–Aldrich) was added to each well
and another 12 h was afforded for the formazan to
dissolve. The absorbance was measured at 540 nm
and 630 nm – reference wavelength, by the automated
uQuantTM Universal Microplate Spectrophotometer
(Bio-tek, USA). The absorbance of the control wells
was taken as 100 %, and the results were expressed as
a percentage of the control.
Synthesis of 1-methoxybrassinin (IV)
1-Methoxybrassinin (IV ) was syntesised as previ-
ously (Somei & Kawasaki, 1989; Kutschy & Mezencev,
2008).
Cell culture
General procedure for synthesis of Grignard
reagents (V–XII)
A-549 (lung carcinoma), MDA-MB-231, and MCF-
7 (mammary gland adenocarcinoma), Jurkat cells
(acute T-lymphoblastic leukaemia), CEM (acute T-
lymphoblastic leukaemia), and HeLa (cervical carci-
noma) cells were maintained in an RPMI 1640 medium
supplemented with Glutamax-I or a DMEM medium
with Glutamax-I and glucose supplemented with 10
Commercially available halides (2-bromopropane,
0.380 mL; 1-bromobutane, 0.432 mL; bromocyclo-
hexane, 0.493 mL; bromobenzene, 0.421 mL; 2-
bromoanisole, 0.498 mL; 3-bromoanisole, 0.498 mL;
4-bromoanisole, 0.498 mL; 4 mmol) were added drop-
wise over 10 min to a suspension of metallic magne-
sium turnings (0.097 g, 4 mmol) and powder molec-
vol. % foetal calf serum, penicillin (100 IU mL−1
)
and streptomycin (100 µg mL−1) (all from Invitro-
gen, UK), in an atmosphere of 5 % CO2 in humidified
air at 37◦C. Cell viability, estimated by trypan blue
exclusion (Invitrogen), was greater than 95 % prior to
each experiment.
˚
ular sieves (3 A) in dry diethyl ether (3 mL), under
an atmosphere of nitrogen. The resulting mixture was
stirred for 60 min at reflux.

P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
633
General procedure for synthesis of trans- and
cis-diastereoisomers of 2-alkyl- and 2-aryl-
analogues of 1-methoxyspirobrassinol methyl
ether (XIIIa–XXa and XIIIb–XXb)
and XVb were obtained by using cyclohexylmagne-
sium bromide (2.0 mL, 1.125 mmol) and triethylamine
(0.26 mL, 1.875 mmol) after separation on silica gel
(hexane/acetone, ϕr = 3 : 1) to afford 0.054 g (41 %)
of compounds XVa and XVb as a colourless oil.
A freshly prepared solution of bromine (0.96 mL,
0.413 mmol; stock solution prepared by dissolv-
ing 0.04 mL of bromine in 1.76 mL of anhydrous
dichloromethane) was added to a magnetically stirred
solution of 1-methoxybrassinin (IV, 0.1 g, 0.375 mmol)
trans- and cis–1-Methoxy–2-phenyl–2ꢀ-
(methylsulphanyl)spiro{indoline-3,5ꢀ-[4ꢀ,5ꢀ]di-
hydrotiazole} (XVIa and XVIb)
˚
with powder molecular sieves (3 A) in anhydrous
Following the general procedure, products XVIa
and XVIb were obtained by using 3 M solution of
phenylmagnesium bromide (0.25 mL, 0.751 mmol) and
triethylamine (0.52 mL, 3.75 mmol) after separation
on silica gel (hexane/acetone, ϕr = 3 : 1) to afford
0.081 g (63 %) of compounds XVIa and XVIb as a
colourless oil.
dichloromethane (4.5 mL) at ambient temperature un-
der an atmosphere of nitrogen. After stirring for 1 min,
a freshly prepared solution of organometallic reagent
(n-butyllithium with triethylamine, VIII, IX were
commercially available) was added. After 5 min (after
15 min in the case of n-butyllithium) triethylamine
was added to the reaction mixture. 5 min later the
reaction mixture was diluted with dichloromethane
(15 mL), successively washed with a saturated solu-
tion of ammonium chloride (20 mL), brine (20 mL),
and dried over an anhydrous Na2SO4. The residue ob-
tained after solvent removal in vacuum was submit-
ted to chromatography on silica gel to afford products
XIIIa–XXa and XIIIb–XXb.
trans- and cis–1-Methoxy–2–(4-chlorophenyl)–
2ꢀ-(methylsulphanyl)spiro{indoline-3,5ꢀ-
[4ꢀ,5ꢀ]dihydrotiazole} (XVIIa and XVIIb)
Following the general procedure, products XVIIa
and XVIIb were obtained by using 1 M solution of
4-chlorophenylmagnesium bromide (1.875 mL, 1.875
mmol), CuCl (0.019 g, 0.188 mmol), and triethy-
lamine (0.52 mL, 3.75 mmol) after separation on sil-
ica gel (hexane/acetone, ϕr = 3 : 1) to afford 0.085 g
(60 %) of compounds XVIIa and XVIIb as a colourless
oil.
trans- and cis–1-Methoxy–2-isopropyl–2ꢀ-
(methylsulphanyl)spiro{indoline-3,5ꢀ-[4ꢀ,5ꢀ]di-
hydrotiazole} (XIIIa and XIIIb)
Following the general procedure, products XIIIa
and XIIIb were obtained by using freshly prepared
isopropylmagnesium bromide (1.4 mL, 1.875 mmol)
and triethylamine (0.52 mL, 3.75 mmol) after sepa-
ration on silica gel (hexane/acetone, ϕr = 3 : 1) to
afford 0.07 g (60 %) of compounds XIIIa and XIIIb as
a colourless oil.
trans- and cis–1-Methoxy–2–(2-methoxy-
phenyl)–2ꢀ-(methylsulphanyl)spiro{indoline-
3,5ꢀ-[4ꢀ,5ꢀ]dihydrotiazole} (XVIIIa and XVI-
IIb)
Following the general procedure, products XVIIIa
and XVIIIb were obtained by using 2-methoxyphenyl-
magnesium bromide (2.0 mL, 1.125 mmol) and tri-
ethylamine (0.26 mL, 1.875 mmol) after separation on
silica gel (hexane/acetone, ϕr = 5 : 1) to afford 0.055 g
(39 %) of compounds XVIIIa and XVIIIb as a colour-
less oil.
trans- and cis–1-Methoxy–2-butyl–2ꢀ-
(methylsulphanyl)spiro{indoline-3,5ꢀ-[4ꢀ,5ꢀ]di-
hydrotiazole} (XIVa and XIVb)
Following the general procedure, products XIVa
and XIVb were obtained by using the commercially
available n-butyllithium (0.77 mL, 1.238 mmol) with
triethylamine (0.26 mL, 1.875 mmol) or freshly pre-
pared butyl-1-magnesium bromide (2.0 mL, 1.125
mmol) and triethylamine (0.26 mL, 1.875 mmol) after
separation on silica gel (hexane/acetone, ϕr = 3 : 1) to
afford 0.065 g (n-butyllithium, 54 %) or 0.03 g (butyl-
1-magnesium bromide, 24 %) of compounds XIVa and
XIVb as a colourless oil.
trans- and cis–1-Methoxy–2–(3-methoxy-
phenyl)–2ꢀ-(methylsulphanyl)spiro{indoline-
3,5ꢀ-[4ꢀ,5ꢀ]dihydrotiazole} (XIXa and XIXb)
Following the general procedure, products XIXa
and XIXb were obtained by using 3-methoxyphenyl-
magnesium bromide (2.0 mL, 1.125 mmol) and tri-
ethylamine (0.26 mL, 1.875 mmol) after separation on
silica gel (hexane/acetone, ϕr = 5 : 1) to afford 0.074 g
(53 %) of compounds XIXa and XIXb as a colourless
oil.
trans- and cis–1-Methoxy–2-cyclohexyl–2ꢀ-
(methylsulphanyl)spiro{ indoline-3,5ꢀ-[4ꢀ,5ꢀ]-
dihydrotiazole} (XVa and XVb)
Following the general procedure, products XVa

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P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
Table 1. Spectral data of newly prepared compounds
Compound
Spectral dataa
XIIIa
XIIIb
XIVa
1H NMR (600 MHz, CDCl3), δ: 1.11 (d, 3H, J = 6.9 Hz, CH3-iPr), 1.21 (d, 3H, J = 7.1 Hz, CH3-iPr), 2.27 (m,
1H, CH-iPr), 2.56 (s, 3H, SCH3), 3.62 (d, 1H, J = 3.5 Hz, H-2), 3.90 (s, 3H, OCH3), 3.93 (d, 1H, J = 15.4 Hz, Ha),
4.64 (d, 1H, J = 15.4 Hz, Hb), 7.06 (m, 2H, H-5, H-7), 7.24 (dd, 1H, J = 7.3 Hz, J = 8.0 Hz, H-6), 7.31 (d, 1H, J
= 7.3 Hz, H-4)
13C NMR (100 MHz, CDCl3), δ: 15.0 (SCH3), 19.2 (CH3-iPr), 22.2 (CH3-iPr), 27.4 (CH-iPr), 62.4 (OCH3), 70.7
(C-3), 70.7 (CH2), 83.1 (C-2), 114.2 (C-7), 123.4 (C-4), 124.4 (C-5), 129.2 (C-6), 131.7 (C-3a), 151.6 (C-7a), 165.7
—
(C N)
—
MS, m/z (Ir/%): 308 (25) (M+), 277 (100), 204 (60), 160 (66)
1H NMR (400 MHz, CDCl3), δ: 1.11 (d, 3H, J = 6.8 Hz, CH3-iPr), 1.20 (d, 3H, J = 7.0 Hz, CH3-iPr), 2.27 (m, 1H,
CH-iPr), 2.57 (s, 3H, SCH3), 3.37 (d, 1H, J = 3.8 Hz, H-2), 3.91 (s, 3H, OCH3), 4.29 (d, 1H, J = 15.5 Hz, Hb), 4.59
(d, 1H, J = 15.5 Hz, Ha), 7.04 (dd, 2H, J = 7.3 Hz, J = 7.8 Hz, H-5, H-7), 7.22 (dd, 2H, J = 7.2 Hz, J = 7.7 Hz,
H-4, H-6)
13C NMR (100 MHz, CDCl3), δ: 15.1 (SCH3), 19.3 (CH3-iPr), 22.1 (CH3-iPr), 29.4 (CH-iPr), 62.5 (OCH3), 71.4
(C-3), 76.6 (CH2), 80.8 (C-2), 113.9 (C-7), 122.7 (C-4), 124.7 (C-5), 129.3 (C-6), 134.1 (C-3a), 151.0 (C-7a), 166.0
—
(C N)
—
MS, m/z (Ir/%): 308 (29) (M+), 277 (100), 204 (76), 161 (58)
IR, ν˜/cm−1: 1578 (C N)
—
—
1H NMR (400 MHz, CDCl3), δ: 0.95 (t, 3H, J = 7.4 Hz, H-4), 1.44 (m, 2H, H-3), 1.64 (m, 2H, H-2), 2.56 (s, 3H,
SCH3), 3.76 (td, 2H, J = 6.5 Hz, J = 9.5 Hz, H-1), 3.86 (d, 1H, J = 15.3 Hz, Ha), 3.94 (s, 3H, OCH3), 4.99 (d, 1H,
J = 15.3 Hz, Hb), 5.00 (s, 1H, H-2), 6.93 (d, 1H, J = 7.9 Hz, H-7), 7.01 (dd, 1H, J = 1.0 Hz, J = 7.5 Hz, H-5), 7.23
(dd, 1H, J = 1.0 Hz, J = 7.5 Hz, H-6), 7.29 (d, 1H, J = 7.5 Hz, H-4)
13C NMR (100 MHz, CDCl3), δ: 13.8 (C-4), 14.9 (SCH3), 19.1 (C-3), 32.2 (C-2), 63.9 (OCH3), 68.9 (C-3), 70.0
(CH2), 72.0 (C-1), 108.1 (C-2), 112.8 (C-7), 123.6 (C-5), 123.9 (C-4), 127.7 (C-3a), 129.5 (C-6), 148.1 (C-7a), 163.1
—
(C N)
—
MS, m/z (Ir/%): 322 (1.5) (M+), 307 (75), 251 (100), 150 (54)
IR, ν˜/cm−1: 1567 (C N)
—
XIVb
—
1H NMR (400 MHz, CDCl3), δ: 0.95 (t, 3H, J = 7.4 Hz, H-4), 1.46 (m, 2H, H-3), 1.67 (m, 2H, H-2), 2.55 (s, 3H,
SCH3), 3.73 (td, 2H, J = 6.3 Hz, J = 8.7 Hz, H-1), 3.95 (s, 3H, OCH3), 4.32 (d, 1H, J = 15.3 Hz, Hb), 4.51 (d, 1H,
J = 15.3 Hz, Ha), 4.68 (s, 1H, H-2), 6.94 (d, 1H, J = 7.7 Hz, H-7), 7.01 (dd, 1H, J = 1.1 Hz, J = 7.5 Hz, H-5), 7.24
(d, 1H, J = 7.5 Hz, H-4), 7.28 (dd, 1H, J = 1.1 Hz, J = 7.5 Hz, H-6)
13C NMR (100 MHz, CDCl3), δ: 13.9 (C-4), 15.1 (SCH3), 19.1 (C-3), 32.1 (C-2), 63.9 (OCH3), 70.2 (C-3), 71.9
(C-1), 73.2 (CH2), 104.8 (C-2), 112.7 (C-7), 123.1 (C-4), 123.8 (C-5), 128.3 (C-3a), 129.9 (C-6), 147.8 (C-7a), 166.7
—
(C N)
—
MS, m/z (Ir/%): 338 (6) (M + O)+, 307 (69), 251 (100), 150 (54)
IR, ν˜/cm−1: 1578 (C N)
—
XVa
—
1H NMR (400 MHz, CDCl3), δ: 1.25–2.02 (m, 10H, cyclohexyl ring), 2.56 (s, 3H, SCH3), 3.74 (dt, 2H, J = 4.3 Hz,
J = 8.8 Hz, H-1), 3.86 (d, 1H, J = 15.2 Hz, Ha), 3.95 (s, 3H, OCH3), 5.02 (d, 1H, J = 15.2 Hz, Hb), 5.10 (s, 1H,
H-2), 6.92 (d, 1H, J = 7.9 Hz, H-7), 7.00 (dd, 1H, J = 1.1 Hz, J = 7.6 Hz, H-5), 7.23 (dd, 1H, J = 1.1 Hz, J =
7.6 Hz, H-6), 7.29 (d, 1H, J = 7.6 Hz, H-4)
13C NMR (100 MHz, CDCl3), δ: 15.0 (SCH3), 23.8, 23.9, 25.6, 32.7, 32.8 (5 × Cꢀ-cyclohexyl ring), 64.2 (OCH3),
69.1 (C-3), 70.0 (CH2), 79.3 (C-1), 106.1 (C-2), 112.8 (C-7), 123.5 (C-5), 124.0 (C-4), 127.5 (C-3a), 129.5 (C-6),
—
148.2 (C-7a), 163.0 (C N)
—
MS, m/z (Ir/%): 364 (2) (M + O)+, 251 (100), 178 (19), 150 (20)
IR, ν˜/cm−1: 1596 (C N)
—
XVb
—
1H NMR (400 MHz, CDCl3), δ: 1.26–2.12 (m, 10H, cyclohexyl ring), 2.55 (s, 3H, SCH3), 3.63–3.72 (m, 1H, H-1),
3.94 (s, 3H, OCH3), 4.32 (d, 1H, J = 15.3 Hz, Hb), 4.48 (d, 1H, J = 15.3 Hz, Ha), 4.79 (s, 1H, H-2), 6.92 (d, 1H, J
= 8.0 Hz, H-7), 7.00 (dd, 1H, J = 1.0 Hz, J = 7.5 Hz, H-5), 7.23–7.28 (m, 2H, H-4, H-6)
13C NMR (100 MHz, CDCl3), δ: 15.1 (SCH3), 24.0–32.7 (5 × Cꢀ-cyclohexyl ring), 64.1 (OCH3), 70.3 (C-3), 72.7
(CH2), 79.4 (C-1), 102.8 (C-2), 112.5 (C-7), 123.1 (C-4), 123.6 (C-5), 127.6 (C-3a), 129.8 (C-6), 148.0 (C-7a), 163.8
—
(C N)
—
MS, m/z (Ir/%): 364 (2) (M + O)+, 251 (100), 178 (20), 149 (29)
XVIa
1H NMR (600 MHz, CDCl3), δ: 2.26 (s, 3H, SCH3), 3.73 (d, 1H, J = 15.3 Hz, Ha), 3.83 (s, 3H, OCH3), 4.38 (d, 1H,
J = 15.3 Hz, Hb), 4.78 (s, 1H, H-2), 7.09 (dd, 1H, J = 7.5 Hz, J = 7.5 Hz, H-5), 7.11 (d, 1H, J = 7.9 Hz, H-7), 7.31
(dd, 1H, J = 7.8 Hz, J = 7.8 Hz, H-6), 7.35–7.41 (m, 4H, H-4, H-3, H-4, H-5), 7.67 (d, 2H, J = 7.7 Hz, H-2, H-6)
13C NMR (100 MHz, CDCl3), δ: 14.7 (SCH3), 64.1 (OCH3), 71.1 (CH2), 71.6 (C-3), 83.7 (C-2), 113.9 (C-7), 124.0
(C-5), 124.1 (C-4), 127.9 (C-2, C-6), 128.0 (C-3a), 128.2 (C-3, C-5), 128.4 (C-4), 129.5 (C-6), 135.4 (C-1), 151.7
—
(C-7a), 164.4 (C N)
—
MS, m/z (Ir/%): 342 (15) (M+), 311 (100), 238 (65), 204 (57)

P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
635
Table 1. (continued)
Compound
Spectral dataa
XVIb
1H NMR (400 MHz, CDCl3), δ: 2.34 (s, 3H, SCH3), 3.75 (s, 3H, OCH3), 4.36 (d, 1H, J = 15.2 Hz, Hb), 4.53 (s,
1H, H-2), 4.65 (d, 1H, J = 15.2 Hz, Ha), 7.07–7.10 (m, 2H, H-5, H-7), 7.29–7.41 (m, 5H, H-4, H-6, H-3, H-4, H-5),
7.52 (d, 2H, J = 7.7 Hz, H-2, H-6)
13C NMR (100 MHz, CDCl3), δ: 15.2 (SCH3), 64.3 (OCH3), 73.1 (C-3), 73.2 (CH2), 82.5 (C-2), 113.6 (C-7), 123.1
(C-4), 124.4 (C-5), 128.1 (C-3, C-5), 128.7 (C-4), 129.8 (C-2, C-6), 129.9 (C-6), 130.5 (C-3a), 136.4 (C-1), 151.3
—
(C-7a), 166.3 (C N)
—
MS, m/z (Ir/%): 342 (15) (M+), 311 (100), 238 (68), 204 (58)
IR, ν˜/cm−1: 1599 (C N), 738 (C—Cl)
—
—
XVIIa
1H NMR (400 MHz, CDCl3), δ: 2.28 (s, 3H, SCH3), 3.73 (d, 1H, J = 15.4 Hz, Ha), 3.82 (s, 3H, OCH3), 4.35 (d,
1H, J = 15.4 Hz, Hb), 4.72 (s, 1H, H-2), 7.07–7.13 (m, 2H, H-5, H-7), 7.32 (dd, 1H, J = 7.2 Hz, J = 7.8 Hz, H-6),
7.35–7.42 (m, 3H, H-4, H-3, H-5), 7.62 (d, 2H, J = 8.7 Hz, H-2, H-6)
13C NMR (100 MHz, CDCl3), δ: 15.0 (SCH3), 64.4 (OCH3), 71.2 (CH2), 71.8 (C-3), 83.5 (C-2), 114.3 (C-7), 124.3
(C-4), 124.4 (C-5), 127.8 (C-3a), 128.6 (C-3, C-5), 129.3 (C-2, C-6), 129.9 (C-6), 134.3 (C-4), 134.6 (C-1), 151.8
—
(C-7a), 165.0 (C N)
—
MS, m/z (Ir/%): 376 (13) (M+), 345 (100), 272 (55)
IR, ν˜/cm−1: 1597 (C N), 754 (C—Cl)
—
—
XVIIb
1H NMR (400 MHz, CDCl3), δ: 2.36 (s, 3H, SCH3), 3.74 (s, 3H, OCH3), 4.34 (d, 1H, J = 15.2 Hz, Hb), 4.50 (s,
1H, H-2), 4.65 (d, 1H, J = 15.2 Hz, Ha), 7.05–7.14 (m, 2H, H-5, H-7), 7.29–7.39 (m, 4H, H-4, H-6, H-3, H-5), 7.46
(d, 2H, J = 8.5 Hz, H-2, H-6)
13C NMR (100 MHz, CDCl3), δ: 15.0 (SCH3), 64.1 (OCH3), 72.6 (C-3), 73.0 (CH2), 81.9 (C-2), 113.5 (C-7), 123.0
(C-6), 124.3 (C-5), 128.0 (C-3, C-5), 128.7 (C-3a), 129.8 (C-4), 130.8 (C-2, C-6), 134.3 (C-4), 134.6 (C-1), 151.1
—
(C-7a), 166.0 (C N)
—
MS, m/z (Ir/%): 376 (12) (M+), 345 (100), 272 (54)
XVIIIa 1H NMR (400 MHz, CDCl3), δ: 2.36 (s, 3H, SCH3), 3.76 (s, 3H, N—OCH3), 3.83 (d, 1H, J = 15.0 Hz, Ha), 3.84
(s, 3H, C—OCH3), 4.24 (d, 1H, J = 15.0 Hz, Hb), 5.52 (s, 1H, H-2), 6.90 (d, 1H, J = 8.3 Hz, H-3), 6.99 (t, 1H, J
= 7.5 Hz, H-5), 7.04 (dt, 1H, J = 1.0 Hz, J = 7.5 Hz, H-5), 7.08 (d, 1H, J = 7.5 Hz, H-7), 7.27–7.32 (m, 2H, H-4,
H-6), 7.34 (d, 1H, J = 7.5 Hz, H-4), 7.70 (d, 1H, J = 7.5 Hz, H-6)
13C NMR (100 MHz, CDCl3), δ: 15.0 (SCH3), 55.5 (C—OCH3), 64.1 (N—OCH3), 71.4 (CH2), 71.5 (C-3), 75.2
(C-2), 110.8 (C-3), 113.9 (C-7), 120.2 (C-5), 124.0 (C-5), 124.1 (C-4), 124.8 (C-1), 129.3 (C-6), 129.4 (C-4), 129.6
—
(C-6), 131.7 (C-3a), 151.5 (C-7a), 158.7 (C-2), 165.2 (C N)
—
MS, m/z (Ir/%): 372 (17) (M+), 341 (100), 268 (28)
XVIIIb IR, ν˜/cm−1: 1601 (C N)
—
—
1H NMR (400 MHz, CDCl3), δ: 2.36 (s, 3H, SCH3), 3.76 (s, 3H, N—OCH3), 3.82 (s, 3H, C—OCH3), 4.59 (s, 2H,
Ha, Hb), 5.22 (s, 1H, H-2), 6.89 (d, 1H, J = 8.3 Hz, H-3), 7.00–7.10 (m, 3H, H-5, H-7, H-5), 7.25–7.35 (m, 3H, H-4,
H-6, H-4), 7.78 (d, 1H, J = 7.5 Hz, H-6)
13C NMR (100 MHz, CDCl3), δ: 15.0 (SCH3), 55.3 (C—OCH3), 64.0 (N—OCH3), 73.0 (CH2), 73.7 (C-3), 74.2
(C-2), 110.2 (C-3), 113.4 (C-7), 119.6 (C-5), 122.8 (C-4), 124.0 (C-5), 124.9 (C-1), 129.2 (C-4), 129.4 (C-6), 130.7
—
(C-6), 130.9 (C-3a), 151.3 (C-7a), 157.8 (C-2), 165.1 (C N)
—
MS, m/z (Ir/%): 372 (17) (M+), 341 (100), 268 (27)
IR, ν˜/cm−1: 1600 (C N)
—
XIXa
—
1H NMR (400 MHz, CDCl3), δ: 2.29 (s, 3H, SCH3), 3.73 (d, 1H, J = 15.3 Hz, Ha), 3.85 (s, 3H, C—OCH3), 3.86 (s,
3H, N—OCH3), 4.39 (d, 1H, J = 15.3 Hz, Hb), 4.77 (s, 1H, H-2), 6.90 (d, 1H, J = 7.5 Hz, H-4), 7.06 (ddd, 1H, J =
1.0 Hz, J = 5.5 Hz, J = 8.5 Hz, H-5), 7.10 (d, 1H, J = 8.5 Hz, H-7), 7.19 (m, 1H, H-2), 7.30 (dd, 1H, J = 1.0 Hz, J
= 7.5 Hz, H-4), 7.39 (dd, 3H, J = 1.0 Hz, J = 7.5 Hz, H-6, H-5, H-6)
13C NMR (100 MHz, CDCl3), δ: 14.7 (SCH3), 55.2 (C—OCH3), 64.1 (N—OCH3), 71.2 (CH2), 71.7 (C-3), 83.7
(C-2), 112.9 (C-2), 113.9 (C-7), 114.5 (C-4), 120.1 (C-6), 124.0 (C-5), 124.1 (C-4), 128.0 (C-3a), 129.2 (C-5), 129.5
—
(C-6), 137.1 (C-1), 151.6 (C-7a), 159.4 (C-3), 164.3 (C N)
—
MS, m/z (Ir/%): 372 (18) (M+), 341 (100), 268 (33)
IR, ν˜/cm−1: 1568 (C N)
—
XIXb
—
1H NMR (400 MHz, CDCl3), δ: 2.38 (s, 3H, SCH3), 3.78 (s, 3H, N—OCH3), 3.85 (s, 3H, C—OCH3), 4.34 (d, 1H,
J = 15.1 Hz, Hb), 4.51 (s, 1H, H-2), 4.66 (d, 1H, J = 15.1 Hz, Ha), 6.92 (dd, 1H, J = 0.9 Hz, J = 8.2 Hz, H-4),
7.05–7.10 (m, 4H, H-5, H-7, H-2, H-6), 7.29 (m, 3H, H-4, H-6, H-5)
13C NMR (100 MHz, CDCl3), δ: 14.9 (SCH3), 55.3 (C—OCH3), 64.1 (N—OCH3), 69.9 (C-3), 72.9 (CH2), 81.7
(C-2), 113.4 (C-7), 114.1 (C-4), 115.0 (C-2), 121.9 (C-6), 122.8 (C-5), 124.2 (C-5), 129.0 (C-4), 129.6 (C-6), 130.8
—
(C-3a), 137.8 (C-1), 150.7 (C-7a), 159.2 (C-3), 166.3 (C N)
—
MS, m/z (Ir/%): 372 (16) (M+), 341 (100), 268 (32)

636
P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
Table 1. (continued)
Compound
Spectral dataa
XXa
IR, ν˜/cm−1: 1594 (C N)
—
—
1H NMR (400 MHz, CDCl3), δ: 2.29 (s, 3H, SCH3), 3.74 (d, 1H, J = 15.3 Hz, Ha), 3.82 (s, 3H, C—OCH3), 3.83 (s,
3H, N—OCH3), 4.37 (d, 1H, J = 15.3 Hz, Hb), 4.73 (s, 1H, H-2), 6.93 (d, 2H, J = 8.7 Hz, H-3, H-5), 7.09 (m, 2H,
J = 1.1 Hz, H-5, H-7), 7.28–7.33 (m, 1H, H-6), 7.38 (d, 1H, J = 7.7 Hz, H-4), 7.57 (d, 2H, J = 8.7 Hz, H-2, H-6)
13C NMR (100 MHz, CDCl3), δ: 14.7 (SCH3), 55.2 (N—OCH3), 64.1 (C—OCH3), 71.0 (CH2), 71.5 (C-3), 83.1
(C-2), 113.6 (C-3, C-5), 113.8 (C-7), 123.9 (C-5), 124.1 (C-4), 127.3 (C-1), 128.1 (C-3a), 129.0 (C-2, C-6), 129.4
—
(C-6), 151.6 (C-7a), 159.8 (C-4), 164.4 (C N)
—
MS, m/z (Ir/%): 372 (6) (M+), 341 (100), 268 (39)
IR, ν˜/cm−1: 1568 (C N)
—
XXb
—
1H NMR (400 MHz, CDCl3), δ: 2.37 (s, 3H, SCH3), 3.74 (s, 3H, N—OCH3), 3.84 (s, 3H, C—OCH3), 4.30 (d, 1H,
J = 15.1 Hz, Hb), 4.46 (s, 1H, H-2), 4.64 (d, 1H, J = 15.1 Hz, Ha), 6.92 (d, 2H, J = 8.7 Hz, H-3, H-5), 7.03–7.08
(m, 2H, H-5, H-7), 7.27–7.32 (m, 2H, H-4, H-6), 7.43 (d, 1H, J = 8.7 Hz, H-2, H-6)
13C NMR (100 MHz, CDCl3), δ: 14.9 (SCH3), 55.2 (C—OCH3), 64.0 (N—OCH3), 72.8 (CH2), 73.0 (C-3), 81.6
(C-2), 113.2 (C-3, C-5), 113.3 (C-7), 122.8 (C-4), 124.1 (C-5), 128.1 (C-1), 129.6 (C-6), 130.7 (C-3a), 130.8 (C-2,
—
C-6), 150.9 (C-7a), 159.7 (C-4), 166.0 (C N)
—
MS, m/z (Ir/%): 372 (6) (M+), 341 (100), 268 (38)
XXI
1H NMR (400 MHz, CDCl3), δ: 1.44 (d, 6H, J = 7.2 Hz, 2 × CH3-iPr), 2.64 (s, 3H, SCH3), 3.39 (m, 1H, CH-iPr),
4.09 (s, 3H, OCH3), 5.02 (d, 2H, J = 4.1 Hz, CH2), 6.83 (s, 1H, NH), 7.14 (dd, 1H, J = 1.0 Hz, J = 8.0 Hz, H-5),
7.23 (dd, 1H, J = 1.0 Hz, J = 8.0 Hz, H-6), 7.40 (d, 1H, J = 8.1 Hz, H-7), 7.52 (d, 1H, J = 7.8 Hz, H-4)
13C NMR (100 MHz, CDCl3), δ: 18.1 (SCH3), 22.2 (2 × CH3-iPr), 25.1 (CH-iPr), 42.3 (CH2), 65.3 (OCH3), 100.7
—
(C-3), 108.2 (C-7), 118.0 (C-4), 120.4 (C-5), 122.2 (C-6), 123.5 (C-3a), 131.3 (C-7a), 141.6 (C-2), 197.7 (C S)
—
MS, m/z (Ir/%): 308 (5) (M+), 277 (100), 202 (51)
IR, ν˜/cm−1: 3187 (NH), 1085 (C S)
—
XXII
—
1H NMR (400 MHz, CDCl3), δ: 2.61 (s, 3H, SCH3), 3.70 (s, 3H, OCH3), 5.06 (d, 2H, J = 4.2 Hz, CH2), 6.96 (s,
1H, NH), 7.20 (dd, 1H, J = 7.5 Hz, J = 7.4 Hz, H-5), 7.33 (dd, 1H, J = 7.5 Hz, J = 7.5 Hz, H-6), 7.43–7.55 (m,
4H, H-7, H-2ꢀ, H-4ꢀ, H-6ꢀ), 7.57 (d, 2H, J = 7.0 Hz, H-3ꢀ, H-5ꢀ), 7.62 (d, 1H, J = 7.5 Hz, H-4)
13C NMR (100 MHz, CDCl3), δ: 18.4 (SCH3), 43.0 (CH2), 64.9 (OCH3), 104.5 (C-3), 109.1 (C-7), 119.2 (C-4),
121.3 (C-5), 123.5 (C-2), 123.6 (C-6), 128.7 (C-3a), 129.0 (C-2ꢀ, C-6ꢀ), 129.1 (C-4ꢀ), 130.0 (C-3ꢀ, C-5ꢀ), 132.6 (C-1ꢀ),
—
135.9 (C-7a), 198.2 (C S)
—
MS, m/z (Ir/%): 342 (0.8) (M+), 311 (100), 236 (47), 204 (53)
XXIII
1H NMR (400 MHz, CDCl3), δ: 2.63 (s, 3H, SCH3), 3.70 (s, 3H, OCH3), 5.04 (d, 2H, J = 4.2 Hz, CH2), 6.97 (s,
1H, NH), 7.20 (dd, 1H, J = 7.4 Hz, J = 7.5 Hz, H-5), 7.34 (dd, 1H, J = 7.3 Hz, J = 7.9 Hz, H-6), 7.46–7.54 (m,
5H, H-7, H-2ꢀ, H-3ꢀ, H-5ꢀ, H-6ꢀ), 7.61 (d, 1H, J = 7.9 Hz, H-4)
13C NMR (100 MHz, CDCl3), δ: 18.2 (SCH3), 42.6 (CH2), 64.7 (OCH3), 104.7 (C-3), 108.9 (C-7), 119.0 (C-4),
121.2 (C-5), 123.2 (C-3a), 123.7 (C-6), 126.9 (C-4ꢀ), 129.1 (C-3ꢀ, C-5ꢀ), 131.0 (C-2ꢀ, C-6ꢀ), 132.4 (C-7a), 134.3 (C-2),
ꢀ
—
135.0 (C-1 ), 198.1 (C S)
—
MS, m/z (Ir/%): 376 (0.9) (M+), 345 (100), 270 (96), 204 (98)
a) For atom numbering see Fig. 1a.
trans- and cis–1-Methoxy–2–(4-methoxy-
phenyl)–2ꢀ-(methylsulphanyl)spiro{indoline-
3,5ꢀ-[4ꢀ,5ꢀ]dihydrotiazole} (XXa and XXb)
completed, the reaction mixture was diluted with
dichloromethane (15 mL), successively washed with
a saturated solution of ammonium chloride (20 mL),
brine (20 mL), and dried over an anhydrous Na2SO4.
The residue obtained after solvent removal in vacuum
was submitted to chromatography on silica gel to af-
ford products XXI from XIII and XXII from XVI.
Following the general procedure, products XXa
and XXb were obtained by using 4-methoxyphenyl-
magnesium bromide (1.4 mL, 1.875 mmol) and tri-
ethylamine (0.52 mL, 3.75 mmol) after separation on
silica gel (hexane/acetone, ϕr = 5 : 1) to afford 0.062 g
(44 %) of compounds XXa and XXb as a colourless oil.
Method B
p-Toluenesulphonic acid (0.028 g, 0.146 mmol for
XVIa and XVIb, 0.025 g, 0.133 mmol for XVIIa and
XVIIb) or magnesium bromide (0.075 g, 0.292 mmol
for XVIa and XVIb, 0.097 g, 0.375 mmol for the
mixture of XVI and XVII ) was added to a freshly
prepared stirred reaction mixture of trans- and cis-
diastereoisomers (XVIa and XVIb, XVIIa, and XVIIb
from 0.375 mmol of IV ) or pure diastereoisomers
(XVIa, XVIb, XVIIa, XVIIb, 0.1 g) in anhydrous
General procedure for synthesis of 2-alkyl- and
2-aryl-1-methoxybrassinin (XXI–XXIII)
Method A
In the spirocyclisation reaction (see procedure for
XIII, XVI ), 1-methoxybrassinin (0.1 g, 0.375 mmol)
was stirred for a prolonged time of 24 h. When

P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
Table 2. Characterisation data of newly prepared compounds
637
wi(calc.)/%
wi(found)/%
Yield
Compound
Formula
Mr
C
H
N
%
27
33
32
22
31
10
20
43
30
30
25
14
18
35
24
20
42
63
88
XIIIa
XIIIb
XIVa
XIVb
XVa
C15H20N2OS2
C15H20N2OS2
C16H22N2OS2
C16H22N2OS2
C18H24N2OS2
C18H24N2OS2
C18H18N2OS2
C18H18N2OS2
C18H17ClN2OS2
C18H17ClN2OS2
C19H20N2O2S2
C19H20N2O2S2
C19H20N2O2S2
C19H20N2O2S2
C19H20N2O2S2
C19H20N2O2S2
C15H20N2OS2
C18H18N2OS2
C18H17ClN2OS2
308.46
308.46
322.49
322.49
348.53
348.53
342.48
342.48
376.92
376.92
372.50
372.50
372.50
372.50
372.50
372.50
308.46
342.48
376.92
58.41
58.29
58.41
58.24
59.59
59.30
59.59
59.27
62.03
61.88
62.03
61.75
63.13
63.01
63.13
62.96
57.36
57.04
57.36
57.13
61.26
61.03
61.26
60.95
61.26
61.10
61.26
60.97
61.26
61.12
61.26
61.08
58.41
58.33
63.13
63.01
57.36
57.07
6.54
6.47
6.54
6.38
6.88
6.71
6.88
6.64
6.94
6.73
6.94
6.68
5.30
5.19
5.30
5.09
4.55
4.36
4.55
4.29
5.41
5.36
5.41
5.27
5.41
5.18
5.41
5.23
5.41
5.35
5.41
5.16
6.54
6.49
5.30
5.04
4.55
4.28
9.08
8.99
9.08
8.89
8.69
8.45
8.69
8.42
8.04
7.81
8.04
7.90
8.18
8.10
8.18
8.02
7.43
7.15
7.43
7.28
7.52
7.44
7.52
7.31
7.52
7.26
7.52
7.31
7.52
7.40
7.52
7.23
9.08
8.89
8.18
7.95
7.43
7.18
XVb
XVIa
XVIb
XVIIa
XVIIb
XVIIIa
XVIIIb
XIXa
XIXb
XXa
XXb
XXI
XXII
XXIII
dichloromethane (3 mL for pure diastereoisomers)
at ambient temperature. Once conversion was com-
plete, the reaction mixture was diluted with dichloro-
methane (15 mL), successively washed with a satu-
rated solution of ammonium chloride (20 mL), brine
(20 mL), and dried over an anhydrous Na2SO4. The
residue obtained after solvent removal in vacuum was
submitted to chromatography on silica gel to afford
products XXII from XVI and XXIII from XVII.
1-Methoxy–2–phenylbrassinin (XXII)
Following the general procedure (Method A), prod-
ucts XXII were obtained after separation on silica gel
(hexane/acetone, ϕr = 3 : 1) to afford 0.073 g (57 %) of
compounds XXII as a crystalline (M.p. = 116–118◦C).
Following the general procedure (Method B), prod-
ucts XXII were obtained by using p-toluenesulphonic
acid for 4 h or magnesium bromide (mixture of XVI
for 3 h, pure diastereoisomers XVIa or XVIb for 7
h) after separation on silica gel (hexane/acetone, ϕr
= 3 : 1) to afford 0.06 g (60 % from XVIa with
p-toluenesulphonic acid), 0.063 g (63 % from XVIb
with p-toluenesulphonic acid), 0.067 g (52 % from mix-
ture of XVIa and XVIb with magnesium bromide),
0.045 g (45 % from XVIa with magnesium bromide),
1-Methoxy–2–isopropyl–brassinin (XXI)
Following the general procedure (Method A), prod-
ucts XXI were obtained after separation on silica gel
(hexane/acetone, ϕr = 3 : 1) to afford 0.049 g (42 %)
of compounds XXI as a colourless oil.

638
P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
Fig. 2. Retrosynthetic analysis.
Fig. 3. Spirocyclisation of IV.
0.055 g (55 % from XVIb with magnesium bromide) of
compounds XXII as a crystalline (M.p. = 116–118◦C).
ture of XVIIa and XVIIb with magnesium bromide)
of compounds XXIII as a colourless oil.
1-Methoxy–2–(4-chlorophenyl)brassinin
(XXIII)
Results and discussion
Retrosynthetic analysis (Fig. 2) reveals that the
target 2-alkyl- and 2-aryl analogues III of 1-methoxy-
spirobrassinol methyl ether (I ) can be synthesised via
spirocyclisation of IV as a key compound of the syn-
thesis, which itself can be prepared from commercially
available indoline (XXV ) as a starting material in sev-
eral subsequent reaction steps.
Following the general procedure (Method B), prod-
ucts XXIII were obtained by using p-toluenesulphonic
acid for 4 h or magnesium bromide for 3 h af-
ter separation on silica gel (hexane/acetone, ϕr
=
3 : 1) to afford 0.06 g (60 % from XVIIa with
p-toluenesulphonic acid), 0.088 g (88 % from XVIIb
with p-toluenesulphonic acid), 0.057 g (40 % from mix-
The synthesis of a key compound, (IV ) was pre-

P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
Table 3. Screening of spirocyclisation reactions with phenylmagnesium bromide
Reaction conditions
639
Entry
Reagent
eq.
Et3Na
eq.
Temperature
◦C
Time
min
Yield
%
Additive
1
2
3
4
5
6
7
8
9
2
2
5
2
2
2
2
2
5
5
5
5
2
2
–
–
–
–
5
5
5
5
–
3
3
3
10
10
10
10
10
10
–20
a.t.
a.t.
40
15
5
35
20
24
33
25
f
–
–
Et3Nd
a.t.
a.t.
–20
–20
a.t.
a.t.
a.t.
a.t.
a.t.
a.t.
overnight
f
–
–
–
5
5
5
5
5
15
5
5
20
24
f
–
CuCle
–
–
f
10b
11b
12b,c
13b,c
14b,c
20
15
31
38
63
CuCle
CuCle
CuCle
–
5
a) Triethylamine was used as reaction-quenching reagent; b) performed with commercially available phenylmagnesium bromide; c)
reactions were performed with double dilution; d) 3.0 eq. used; e) 0.5 eq. used; f) decomposition of starting material; a.t. – ambient
temperature.
viously described as a six-step transformation of in-
doline XXV. Indoline XXV was first transformed to
1-methoxyindole (XXIV ) by Somei’s tungstate method
(Somei & Kawasaki, 1989), then to 1-methoxyindole-
3-carboxaldehyde (Acheson et al., 1978) and finally
to its corresponding oxime (Hanley et al., 1990). Re-
duction of the oxime provided unstable (1-H-indole-3-
yl)methylamine (Pedras & Zaharia, 2000) which, after
treatment with carbon disulphide and iodomethane,
afforded IV (Kutschy & Mezencev, 2008) in a suffi-
cient amount. IV, upon its treatment with bromine,
is prone to electrophilic spirocyclisation. The reac-
tion proceeds, in the first step, via the formation of
sulphenyl bromide XXVI which undergoes a sponta-
neous cyclisation to 1-methoxyspiroindoleninium in-
termediate XXVII (Kutschy et al., 2009). The highly
reactive intermediate XXVII was trapped by various
alkyllithium, alkyl or aryl Grignard reagents affording
products XIIIa–XXa and XIIIb–XXb (Fig. 3). Grig-
nard reagents are the organometallic reagents most
extensively used due to their ease of preparation and
great reactivity (Seyferth, 2009; Entemann & John-
son, 1933).
Grignard reagents were used as nucleophiles, ei-
ther freshly prepared from activated metallic mag-
nesium and organic halide (V, VIb, VII, VIII, X,
XI, XII ), or commercially available organometallic
reagents (VIa, VIII, IX ). Most of them are easily
prepared in a dry ethereal solution of diethyl ether
or THF (Smith, 1999), with the preference being
for diethyl ether due to the higher reaction rate.
The spirocyclisation in the presence of phenylmag-
nesium bromide was studied. cis- and trans-Isomers
XVIa and XVIb were isolated with only a moderate,
20 % yield. Consequently, reaction screening was per-
formed to increase the efficiency of the reaction (Ta-
ble 3).
The screening results showed that the highest yield
of cis- and trans-diastereoisomers was obtained in
Entry 14 (Table 3) and the total yield of the mix-
ture of products XVIa and XVIb was 63 %. We pre-
sumed that these reaction conditions were optimal
for spirocyclisation reactions with a range of Grig-
nard reagents. However, we found that, in the case
of 4-chlorophenylmagnesium bromide, the same reac-
tion condition resulted only in decomposition of the
starting material (Table 4, Entry 11) Further screen-
ing was required to increase the yield of the reaction
with 4-chlorophenylmagnesium bromide (Table 4). We
were finally able to improve the total yield of products
XVIIa and XVIIb to 60 %. The results revealed the
difficulty of preparing the required diastereoisomeric
products under the general procedure.
In addition, o-, m-, and p-methoxy-substituted
phenylmagnesium bromides X–XII were used in the
spirocyclisation reactions. Diastereoisomeric products
XVIIIa–XXa and XVIIb–XXb were obtained with
yields in a range of 39–53 %. The yields reported
here are the results obtained in accordance with
the previously optimised procedure after multiple at-
tempts. The effects of temperature, quantity of Grig-
nard reagent, and reaction time were monitored. In
general, the best results were obtained when 3 eq. of
X and XI or 5 eq. of XII were applied.
As with the aryl Grignard reagents, alkyl-reagents
(V, VIa, VIb, VII ) were applied equally. Products
XIIIa–XVa and XIIIb–XVb were prepared following
the general procedure. In the case of n-butyllithium,
5 eq. of triethylamine was added. n-Butyllithium is a
strong base, but a milder nucleophile. We presumed

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P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
Table 4. Screening of spirocyclisation reactions with 4-chlorophenylmagnesium bromide
Reaction conditions
Entry
Reagent
eq.
Et3Na
eq.
Temperature
◦C
Time
min
Yield
%
Additive
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
2.0
2.0
5.0
2.0
2.0
2.0
2.5
2.5
2.0
2.0
2.0
2.0
2.0
5.0
5.0
5.0
–
–
5
5
–
–
–
–
–
–
10
10
10
10
10
10
–
–20
a.t.
–20
–20
a.t.
–20
–70–0
–70
a.t.
a.t.
a.t.
–20
a.t.
a.t.
35
35
30
5
5
5
70
5
5
5
5
24
20
–
CuClb
CuClb
CuClb
CuClb
Et3Nc
–
f
28
f
–
f
–
f
–
f
–
CuClb
CuClb
–
–
f
f
–
f
–
f
–
5
5
5
5
–
CuClb
CuClb
TMEDAd
Et3Ne
45
60
50
H a.t.
a.t.
f
10
5
–
a) Triethylamine was used as reaction-quenching reagent; b) 0.5 eq. used; c) 2.5 eq. used; d) 5.0 eq. used; e) 1.0 eq. used; f)
decomposition of starting material; a.t. room temperature.
Fig. 4. Synthesis of 2-alkyl- and 2-aryl-derivatives of IV: i) Br2 then Nu-MgBr, CH2Cl2, 24 h; ii) MgBr2 or p-toluenesulphonic
acid, CH2Cl2.
that triethylamine reduced its basicity and increased
its nucleophilicity. The reaction performed under the
triethylamine-free condition only exhibited an 11 %
overall yield of products XIVa, XIVb, while the simul-
taneous addition of n-butyllithium and triethylamine
improved the yield to 54 %.
during the isomerisation of diastereoisomers of spiro-
cyclisation (Kutschy et al., 2009).
The formation of by-products XXI–XXIII was ob-
served during the reaction, resulting in a decreased
overall yield of isolated products XIII, XVI, and
XVII. The isolated by-products XXI–XXIII were iden-
tified spectroscopically as 2-substituted derivatives of
IV (Fig. 4). Careful analysis of the reaction con-
ditions revealed the influence of the reaction time
on the yield of by-products XXI–XXIII; in general,
a longer reaction time leads to an increased quan-
tity of by-products while the quantity of the isolated
products decreases proportionally. Thus, the high-
est yields of spirobrassinines XIII, XIV, and XVII
were obtained when the reaction was completed in
5 min, and yields of products XXI, XXII, XXIII de-
creased to the minimum (15–18 %). By contrast, af-
ter 24 h of reaction time, products XXI and XXII
In all cases, both diastereoisomers were isolated
1
and their ratio (Fig. 3) was determined by H NMR
spectra of the crude reaction products obtained af-
ter dilution with dichloromethane, washing with a
saturated solution of ammonium chloride, brine, dry-
ing, and evaporation of the solvent. The ratio of di-
astereoisomers was determined by integration of the
separated signals corresponding to the H-2, Ha, Hb,
OCH3, and SCH3 protons. No significant diastereos-
electivity was observed in any case. Marked diastere-
oselectivity was noted in the case of spirocyclisation
using bulky chiral alcohols (Čurillová et al., 2007) and

P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
641
Fig. 5. NOESY interactions confirming new products.
Table 5. Anti-proliferative activity of newly prepared compounds
Anti-proliferative activities (IC50)/(µmol L−1
)
Compounds
Jurkat
A-549
MCF-7
MDA
HeLa
CEM
XIIIa
XIIIb
XIVa
XIVb
XVa
XVb
XVIa
XVIb
XVIIa
XVIIb
XVIIIa
XVIIIb
XIXa
XIXb
XXa
XXb
XXI
XXII
XXIII
Ia
Ib
XXVIIIa
XXVIIIb
XXIXa
XXIXb
Cisplatin
Etoposide
55.8
88.1
27.0
> 100
77.7
50.0
63.7
66.1
15.0
26.5
22.0
16.0
37.5
52.5
27.0
36.0
85.4
31.0
33.3
30.2
57.4
69.4
34.8
100.0
30.7
12.0
1.2
67.5
94.6
53.5
> 100
> 100
51.0
94.9
> 100
44.0
44.0
45.0
81.5
87.0
> 100
80.0
> 100
94.8
87.5
63.1
100.0
100.0
100.0
73.9
64.5
> 100
49.0
> 100
> 100
42.5
83.4
> 100
38.5
38.5
42.0
79.5
87.0
> 100
> 100
93.5
> 100
> 100
55.5
> 100
> 100
53.0
93.2
> 100
46.5
> 100
77.0
39.0
97.4
> 100
38.0
25.5
37.0
64.5
71.5
> 100
56.0
> 100
> 100
54.5
74.4
48.9
32.1
78.3
32.0
85.0
82.0
25.5
29.7
46.9
27.0
27.0
22.0
19.5
44.5
59.0
38.5
39.0
67.1
35.5
25.1
100.0
100.0
15.5
10.0
100.0
37.4
4.4
47.5
55.0
> 100
> 100
> 100
74.0
> 100
> 100
66.0
> 100
78.0
> 100
> 100
84.5
60.7
57.3
100.0
100.0
100.0
100.0
28.5
100.0
11.4
10.9
100.0
100.0
64.0
100.0
20.7
100.0
14.7
21.2
53.2
100.0
81.5
22.3
44.8
22.3
85.9
12.2
14.3
7.7
3.9
1.1
were isolated with 42 % and 57 % yields, respec-
tively.
drous magnesium bromide acts as a mild Lewis acid
with an affinity to sulphur’s lone pairs and thus can
catalyse the decyclisation of spirobrassinines. Conse-
quently, the influence of the excess of magnesium bro-
mide on the decyclisation of spirobrassinines was stud-
ied. The results confirmed that the presence of Lewis
acid leads to the formation of 2-substituted brassinines
The formation of magnesium bromide, during the
bromine-initiated spirocyclisation followed by addi-
tion of the Grignard reagent, plays a crucial role in
the formation of acyclic products XXI–XXII rather
than spirocyclised brassinines XIII and XVI. Anhy-

642
P. Očenáš et al./Chemical Papers 67 (6) 631–642 (2013)
with a moderate yield (40–55 %). Similarly, we per-
formed the reaction in the presence of Br¨onsted acid
(p-toluenesulphonic acid, 0.5 eq.) and the reaction pro-
ceeded in favour of acyclic derivatives XXII and XXIII
(60–88 %, Fig. 4).
Acknowledgements. We wish to thank the Slovak Re-
search and Development Agency (grant Nos. 1/0954/12 and
1/0406/10) for the financial support for this work. This work
was also financially supported by State NMR Programme no.
2003SP200280203.
The structure of individual diastereoisomers and
2-substituted 1-methoxybrassinins was unequivocally
proven by NMR studies, including HSQC, HMBC,
and NOESY experiments. Thus, the NOE effect ob-
served between Hb and H-2 protons confirms the
cis-configuration, whereas the interactions between
Hb and H-1’ or H-2’ protons correspond to trans-
diastereoisomer structures (Fig. 5).
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