Synthesis of new 5-bromo derivatives of indole and spiroindole phytoalexins

Article abstract of DOI:10.1515/chempap-2015-0230

Electrophilic aromatic substitution is one of the most thoroughly studied reactions in organic chemistry. In the present paper, the 5-brominated spirobrassinol methyl ethers VII, VIII were obtained by electrophilic substitution of the aromatic core of indoline at the C-5 position in the presence of various brominating agents. The same products were also prepared from 5-bromoindole (IX) following the sequence for the synthesis 1-methoxyspirobrassinol methyl ether (V) from indoline. In addition, the new related 5-bromospiroindoline derivatives XX-XXIII were synthesised and their biological activity on human tumour cell lines was examined. The presence of bromine in the indole or indoline skeleton at the C-5 position resulted in the partial increase in anticancer activity on leukaemia cell lines (Jurkat, CEM). The structures of the newly prepared products were determined by ¹H and ¹³C NMR spectroscopy, including HSQC, HMBC, COSY, NOESY and DEPT measurements.

Full text of DOI:10.1515/chempap-2015-0230

Chemical Papers
DOI: 10.1515/chempap-2015-0230
ORIGINAL PAPER
Synthesis of new 5-bromo derivatives of indole and spiroindole
phytoalexins
a
a
b
aLucia Očenášová*, Peter Kutschy , Jozef Gonda, Martina Pilátová,
bGabriela Go¨nciová, Ján Mojžiš, Pavel Pazdera
b
c
aDepartment of Organic Chemistry, Institute of Chemical Science, Faculty of Science, P. J. Šafárik University,
Moyzesova 11, 040 01 Košice, Slovakia
bDepartment of Pharmacology, Faculty of Medicine, P. J. Šafárik University, SNP 1, 040 66 Košice, Slovakia
cCentre for Syntheses under Sustainable Conditions and their Management, Department of Chemistry, Masaryk University,
Kotlářská 2, 611 37 Brno, Czech Republic
Received 7 August 2015; Revised 30 September 2015; Accepted 7 October 2015
Electrophilic aromatic substitution is one of the most thoroughly studied reactions in organic
chemistry. In the present paper, the 5-brominated spirobrassinol methyl ethers VII, VIII were ob-
tained by electrophilic substitution of the aromatic core of indoline at the C-5 position in the
presence of various brominating agents. The same products were also prepared from 5-bromoindole
(IX ) following the sequence for the synthesis 1-methoxyspirobrassinol methyl ether (V ) from indo-
line. In addition, the new related 5-bromospiroindoline derivatives XX–XXIII were synthesised and
their biological activity on human tumour cell lines was examined. The presence of bromine in the
indole or indoline skeleton at the C-5 position resulted in the partial increase in anticancer activity
on leukaemia cell lines (Jurkat, CEM). The structures of the newly prepared products were deter-
mined by 1H and 13C NMR spectroscopy, including HSQC, HMBC, COSY, NOESY and DEPT
measurements.
c
ꢀ 2015 Institute of Chemistry, Slovak Academy of Sciences
Keywords: 5-bromobrassinin, spiroindole phytoalexins, bromination, spirocyclisation, anti-proli-
ferative activity
Introduction
ciferae) in response to fungal attack and other forms
of stress (pathogen infection, UV radiation) (Pedras
et al., 2011). Brassinin (I ) has been demonstrated
as exhibiting chemo-preventative activity in preclin-
ical models and this phytoalexin, together with a syn-
thetic derivative, 5-bromobrassinin (II ), have been
classified as bioavailable inhibitors of indoleamine 2,3-
dioxygenase (IDO), a tryptophan-catabolising enzyme
that drives immune escape in cancer (Banerjee et al.,
2008).
The indole skeleton is present in a wide number
of natural products and pharmaceuticals and is one
of the most important structural subunits for the dis-
covery of new drug candidates (Boyd & Sperry, 2011).
In recent decades, the number of new isolated indole
compounds, many of which contain halogen on the
aromatic ring, has increased significantly as a con-
sequence of the increase in their biological activity
(Pauletti et al., 2010).
Since these compounds can serve as lead com-
pounds for the development of new biologically active
agents, research into their synthesis is desirable; this
article reports on the synthesis and anti-proliferative
Brassinin (I, Fig. 1) is an essential indole phy-
toalexin, a class of natural metabolites produced de
novo by plants of the family Brassicaceae (syn. Cru-
*Corresponding author, e-mail: lucia.ocenas@gmail.com
Dr. Peter Kutschy passed away on 11 June 2012.
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in δ relative to TMS. Coupling constants (J) were
obtained by first-order analysis and measured in
Hertz (Hz). IR spectra were recorded on an Avatar
FT-IR 6700 spectrometer using the attenuated to-
tal 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) spec-
trometer at ionisation energy of 70 eV. Microanaly-
ses were performed using a Perkin–Elmer, Model 2400
analyser (USA). The progress of chemical reactions
was monitored by thin layer chromatography, using
Macherey-Nagel plates Alugramꢀ Sil G/UV254 (Slo-
vakia). Detection was carried out with ultraviolet light
(254 nm). Preparative column chromatography was
performed on a Kieselgel 60 Merck Type 9385 (Slo-
vakia) (0.040–0.063 mm). Melting points were deter-
mined on a Koffler micro melting point apparatus and
are uncorrected. The properties and composition of
the corresponding products are summarized in Tables
1 and 2.
Fig. 1. Brassinin (I) and its synthetic derivative 5-bromobras-
sinin (II).
activity of new 5-bromo derivatives of the 1-methoxy
and 1-Boc-spirobrassinol methyl ether III and the re-
lated 5-bromospiroindoline analogues. The retrosyn-
thesis of the target compounds was based on the three
synthetic routes (A, B, C) outlined in Fig. 2.
Experimental
All commercially available reagents were pur-
chased at the highest available purity from Aldrich,
Merck or Acros Organics (all from Slovakia) and were
used without further purification. Solvents were dried
and purified prior to use following standard proce-
dures. 1H and 13C NMR spectra were recorded for
compounds dissolved in CDCl3 and (CD3)2CO at
ambient temperature on a Varian Mercury Plus 400
Cell culture
Jurkat and CEM (acute T-lymphoblastic leukae-
mia), A-549 (non-small cell lung adenocarcinoma),
MCF-7 and MDA-MB-231 (mammary gland ade-
nocarcinoma) and HeLa (cervical adenocarcinoma)
cells were maintained in RPMI 1640 medium with
Glutamax-I supplemented or D-MEM medium with
Glutamax-I and glucose supplemented with 10 mass %
foetal calf serum, penicillin (100 IU mL−1) and strep-
tomycin (100 mg mL−1; all from Invitrogen, UK), in
1
(USA) FT-NMR (400.13 MHz for H and 100.6 MHz
for 13C) spectrometer using tetramethylsilane (TMS)
as the internal reference. Chemical shifts are given
Fig. 2. Retrosynthetic analysis.
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Table 1. Spectral data of newly prepared compounds
iii
Compound
Spectral data
II
IR, ν˜/cm−1: 3349, 2925, 2849, 1446, 1371, 1087
1H NMR (400 MHz, (CD3)2CO)), δ: 10.41 (s, 1H, NH), 9.20 (s, 1H, NH—CH2), 7.86 (d, 1H, J = 1.9 Hz, H-4), 7.48
(d, 1H, J = 2.2 Hz, H-2), 7.39 (d, 1H, J = 8.6 Hz, H-7), 7.24 (dd, 1H, J = 8.6 Hz, J = 1.9 Hz, H-6), 5.09 (d, 2H,
J = 5.2 Hz, CH2), 2.58 (s, 3H, SCH3)
13C NMR (100 MHz, (CD3)2CO), δ: 196.3 (CS), 133.9 (C-7a), 127.4 (C-3a), 125.1 (C-2), 122.8 (C-6), 120.0 (C-4),
111.9 (C-7), 110.6 (C-5), 109.3 (C-3), 40.4 (CH2), 15.6 (SCH3)
MS, m/z (Ir/%): 316 (27) (M + H)+, 208 (100), 129 (72), 102 (37), 43 (28)
XIIIa
XIIIb
XIVa
XIVb
IR, ν˜/cm−1: 2917, 2904, 2848, 1560, 1460, 1136
1H NMR (400 MHz, CDCl3), δ: 7.36 (dd, 1H, J = 8.3 Hz, J = 1.9 Hz, H-6), 7.33 (d, 1H, J = 1.9 Hz, H-4), 6.82 (d,
1H, J = 8.3 Hz, H-7), 4.62 (s, 1H, H-2), 4.46 (d, 1H, J = 15.4 Hz, H-4a’), 4.31 (d, 1H, J = 15.4 Hz, H-4b’), 3.93 (s,
3H, CH3O—N), 3.71 (s, 3H, CH3O—C), 2.57 (s, 3H, SCH3)
13C NMR (100 MHz, CDCl3), δ: 166.3 (C-2’), 146.4 (C-7a), 132.8 (C-6), 131.4 (C-3a), 126.1 (C-4), 116.1 (C-5),
114.3 (C-7), 105.0 (C-2), 73.1 (C-4’), 69.8 (C-3), 63.8 (CH3O—N), 59.7 (CH3O—C), 15.2 (SCH3)
MS, m/z (Ir/%): 376 (9) (M + H)+, 345 (55), 313 (32), 72 (30), 43 (100)
IR, ν˜/cm−1: 2926, 2892, 2848, 1576, 1461, 1137
1H NMR (400 MHz, CDCl3), δ: 7.39 (d, 1H, J = 1.9 Hz, H-4), 7.35 (dd, 1H, J = 8.3 Hz, J = 1.9 Hz, H-6), 6.81 (d,
1H, J = 8.3 Hz, H-7), 4.95 (d, 1H, J = 15.4 Hz, H-4b’), 4.92 (s, 1H, H-2), 3.92 (s, 3H, CH3O—N), 3.89 (d, 1H, J =
15.4 Hz, H-4a’), 3.69 (s, 3H, CH3O—C), 2.58 (s, 3H, SCH3)
13C NMR (100 MHz, CDCl3), δ: 163.5 (C-2’), 147.0 (C-7a), 132.6 (C-6), 130.3 (C-3a), 126.9 (C-4), 116.0 (C-5),
114.5 (C-7), 108.6 (C-2), 69.7 (C-4’), 68.1 (C-3), 63.9 (CH3O—N), 59.8 (CH3O—C), 15.1 (SCH3)
MS, m/z (Ir/%): 376 (17) (M + H)+, 345 (77), 313 (50), 149 (100), 72 (82)
IR, ν˜/cm−1: 2955, 2926, 2869, 1741, 1677, 1550, 1493, 1265, 1093
1H NMR (400 MHz, CDCl3), δ: 7.71 (d, 1H, J = 9.0 Hz, H-7), 7.53 (d, 1H, J = 9.0 Hz, H-6), 7.44 (s, 1H, H-4), 5.14
(s, 1H, H-2), 4.35 (d, 1H, J = 15.1 Hz, H-4a’), 3.91 (d, 1H, J = 15.1 Hz, H-4b’), 3.53 (s, 3H, OCH3), 2.59 (s, 3H,
SCH3), 1.59 (s, 9H, C(CH3)3)
13C NMR (100 MHz, CDCl3), δ: 167.7 (C-2’), 145.6 (CO), 132.4 (C-7a), 132.3 (C-3a), 130.8 (C-6), 128.8 (C-7),
126.8 (C-4), 116.0 (C-5), 96.0 (C-2), 82.5 (C(CH3)3), 74.8 (C-4’), 70.0 (C-3), 58.5 (OCH3), 28.3 (C(CH3)3), 15.2
(SCH3)
IR, ν˜/cm−1: 2957, 2917, 2849, 1713, 1680, 1552, 1472, 1261, 1093
1H NMR (400 MHz, CDCl3), δ: 7.43 (d, 1H, J = 1.8 Hz, H-4), 7.40–7.31 (m, 2H, H-6, H-7), 5.46 (s, 1H, H-2), 4.83
(d, 1H, J = 15.8 Hz, H-4b’), 4.29 (d, 1H, J = 15.8 Hz, H-4a’), 3.50 (s, 3H, OCH3), 2.58 (s, 3H, SCH3), 1.59 (s, 9H,
C(CH3)3)
13C NMR (100 MHz, CDCl3), δ: 165.0 (C-2’), 148.8 (CO), 140.1 (C-7a), 132.6 (C-6), 132.3 (C-3a), 128.6 (C-7),
126.6 (C-4), 115.6 (C-5), 99.0 (C-2), 82.7 (C(CH3)3), 71.4 (C-3), 66.6 (C-4’), 58.0 (OCH3), 28.3 (C(CH3)3), 15.2
(SCH3)
XVIII
XIX
XX
IR, ν˜/cm−1: 2987, 2940, 2829, 1541, 1434, 1075
1H NMR (400 MHz, CDCl3), δ: 7.71 (d, 1H, J = 0.8 Hz, H-4), 7.31 (m, 2H, H-6, H-7), 7.24 (d, 1H, J = 3.4 Hz,
H-2), 6.29 (d, 1H, J = 3.4 Hz, H-3), 4.06 (s, 3H, OCH3)
13C NMR (100 MHz, CDCl3), δ: 130.4 (C-7a), 125.8 (C-3a), 125.2 (C-6), 124. 0 (C-2), 123.6 (C-4), 113.1 (C-5),
109.6 (C-7), 97.6 (C-3), 66.1 (OCH3)
MS, m/z (Ir/%): 227 (15) (M + H)+, 149 (43), 115 (29), 58 (27), 43 (100)
IR, ν˜/cm−1: 3099, 2942, 2819, 1652, 1449, 1363, 1050
1H NMR (400 MHz, CDCl3), δ: 9.92 (s, 1H, CHO), 8.47 (d, 1H, J = 1.8 Hz, H-4), 7.88 (s, 1H, H-2), 7.47 (dd, 1H,
J = 8.6 Hz, J = 1.8 Hz, H-6), 7.35 (d, 1H, J = 8.6 Hz, H-7), 4.19 (s, 3H, OCH3)
13C NMR (100 MHz, CDCl3), δ: 183.8 (CHO), 132.3 (C-2), 131.3 (C-7a), 127.7 (C-6), 124.8 (C-4), 123.0 (C-3a),
117.1 (C-5), 113.4 (C-3), 110.2 (C-7), 67.1 (OCH3)
MS, m/z (Ir/%): 255 (47) (M + H)+, 115 (52), 88 (22), 58 (29), 43 (100)
IR, ν˜/cm−1: 3314, 3166, 2994, 1510, 1453, 1229, 1112
1
—
—
H NMR (400 MHz, CDCl3), δ: 11.06 (s, 0.8H, OH major), 10.83 (s, 0.2H OH minor), 8.33 (s, 0.8H, CH N major),
—
8.28 (s, 0.2H, CH N minor), 7.87 (d, 1H, J = 1.8 Hz, H-4), 7.66 (s, 1H, H-2), 7.35 (d, 1H, J = 8.6 Hz, H-7), 7.27
—
(dd, 1H, J = 8.6 Hz, J = 1.8 Hz, H-6)
13
—
—
—
C NMR (100 MHz, CDCl3), δ: 144.3 (CH N minor), 138.3 (C-2), 133.6 (C-7a), 131.7 (CH N major), 128.1
—
(C-3a major), 127.9 (C-3a minor), 124.5 (C-6), 120.3 (C-4), 113.1 (C-7), 112.9 (C-5), 109.5 (C-3 minor), 105.9 (C-3
major)
MS, m/z (Ir/%): 240 (30) (M + H)+, 142 (30), 114 (21), 58 (30), 43 (100)
XXI
IR, ν˜/cm−1: 3227, 3126, 2937, 1513, 1439, 1226, 1050
1
—
—
—
H NMR (400 MHz, CDCl3), δ: 8.41 (s, 0.5H, CH N), 8.25 (s, 0.5H, H-4), 8.23 (s, 0.5H, CH N), 7.91 (s, 0.5H,
—
H-4), 7.75 (s, 0.5H, H-2), 7.45 (s, 0.5H, H-2), 7.44–7.24 (m, 2H, H-6, H-7), 4.15 (s, 1.5H, OCH3), 4.11 (s, 1.5H,
OCH3)
13
—
—
—
C NMR (100 MHz, CDCl3), δ: 144.8 (CH N), 139.0 (C-2), 131.3 (C-7a), 129.5 (CH N), 129.4 (C-7a), 126.7
—
(C-6), 126.3 (C-6), 125.6 (C-2), 125.0 (C-4), 124.7 (C-3a), 122.6 (C-3a), 121.2 (C-4), 114.9 (C-5), 114.8 (C-5), 110.0
(C-7), 109.8 (C-7), 105.3 (C-3), 101.4 (C-3), 66.6 (OCH3), 66.5 (OCH3)
MS, m/z (Ir/%): 270 (4) (M + H)+, 114 (2), 58 (26), 43 (100)
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Table 1. (continued
Compound
Spectral data
XXII
IR, ν˜/cm−1: 3456, 2980, 1720, 1450, 1368, 1155
H NMR (400 MHz, CDCl3), δ: 8.63 (s, 0.4H, CH N minor), 8.28 (d, 0.6H, J = 1.9 Hz, H-4 major), 8.25 (s, 0.6H,
1
—
—
—
CH N major), 8.08 (d, 0.4H, J = 8.8 Hz, H-7 minor), 8.02 (d, 0.6H, J = 8.4 Hz, H-7 major), 7.84 (d, 0.4H, J =
—
1.7 Hz, H-4 minor), 7.74 (s, 0.6H, H-2 major), 7.70 (s, 0.4H, H-2 minor), 7.46 (dd, 1H, J = 8.8 Hz, J = 2.0 Hz, H-6),
1.69 (s, 3.6H, C(CH3)3 minor), 1.68 (s, 5.4H, C(CH3)3 major)
C NMR (100 MHz, CDCl3), δ: 149.0 (CO minor), 148.9 (CO major), 144.6 (CH N major), 138.4 (C-2 minor),
13
—
—
—
134.6 (C-7a major), 133.2 (C-7a minor), 132.4 (CH N minor), 130.3 (C-3a minor), 128.7 (C-2 major), 128.6 (C-3a
—
major), 128.3 (C-6), 125.3 (C-4 major), 121.2 (C-4 minor), 117.0 (C-5 minor), 116.9 (C-5 major), 116.5 (C-7), 113.6
(C-3 major), 108.9 (C-3 minor), 85.1 (C(CH3)3), 28.1 (C(CH3)3)
XXIII
XXIV
IR, ν˜/cm−1: 3308, 2918, 2850, 1497, 1387, 1087
1H NMR (400 MHz, CDCl3), δ: 7.73 (s, 1H, H-4), 7.38–7.29 (m, 3H, H-7, H-6, H-2), 7.02 (s, 1H, NH), 4.96 (d, 2H,
J = 4.3 Hz, CH2), 4.08 (s, 3H, OCH3), 2.65 (s, 3H, SCH3)
13C NMR (100 MHz, CDCl3), δ: 198.6 (CS), 130.8 (C-7a), 126.0 (C-6), 124.5 (C-3a), 123.8 (C-7), 121.6 (C-4), 113.7
(C-5), 110.0 (C-2), 106.0 (C-3), 66.3 (OCH3), 42.4 (CH2), 18.2 (SCH3)
IR, ν˜/cm−1: 3260, 2918, 1740, 1447, 1366, 1149, 1054
1H NMR (400 MHz, CDCl3), δ: 7.99 (d, 1H, J = 8.8 Hz, H-7), 7.68 (s, 1H, H-4), 7.61 (s, 1H, H-2), 7.42 (dd, 1H,
J = 8.8 Hz, J = 1.9 Hz, H-6), 7.11 (s, 1H, NH), 4.97 (d, 2H, J = 4.4 Hz, CH2), 2.65 (s, 3H, SCH3), 1.66 (s, 9H,
C(CH3)3)
13C NMR (100 MHz, CDCl3), δ: 199.2 (CS), 149.1 (CO), 134.3 (C-7a), 130.8 (C-3a), 127.8 (C-6), 126.2 (C-2), 121.7
(C-4), 116.9 (C-7), 116.4 (C-5), 114.8 (C-3), 84.6 (C(CH3)3), 42.1 (CH2), 28.2 (C(CH3)3), 18.3 (SCH3)
XXV
IR, ν˜/cm−1: 3194, 2922, 1711, 1574, 1463, 1290, 1149
1H NMR (CDCl3), δ: 8.64 (s, 1H, NH), 7.46 (d, 1H, J = 1.7 Hz, H-4), 7.39 (dd, 1H, J = 8.3 Hz, J = 1.7 Hz, H-6),
6.81 (d, 1H, J = 8.3 Hz, H-7), 4.66 (d, 1H, J = 15.2 Hz, H-4b’), 4.50 (d, 1H, J = 15.2 Hz, H-4a’), 2.63 (s, 3H, SCH3)
13C NMR (100 MHz, CDCl3), δ: 177.7 (C-2), 164.0 (C-2’), 138.3 (C-7a), 133.3 (C-3a), 132.6 (C-6), 127.6 (C-4),
116.2 (C-5), 111.9 (C-7), 75.1 (C-4’), 64.5 (C-3), 15.8 (SCH3)
XXVI
IR, ν˜/cm−1: 2915, 1729, 1579, 1462, 1289, 1148
1H NMR (400 MHz, CDCl3), δ: 7.47 (d, 1H, J = 1.7 Hz, H-4), 7.35 (dd, 1H, J = 8.7 Hz, J = 1.7 Hz, H-6), 6.87 (d,
1H, J = 8.7 Hz, H-7), 4.66 (d, 1H, J = 15.2 Hz, H-4b’), 4.47 (d, 1H, J = 15.2 Hz, H-4a’), 4.07 (s, 3H, OCH3), 2.63
(s, 3H, SCH3)
13C NMR (100 MHz, CDCl3), δ: 170.2 (C-2), 163.8 (C-2’), 137.9 (C-7a), 132.7 (C-6), 130.9 (C-3a), 127.5 (C-4),
116.7 (C-5), 109.3 (C-7), 74.7 (C-4’), 64.0 (OCH3), 62.6 (C-3), 15.8 (SCH3)
XVIIa
IR, ν˜/cm−1: 3414, 2975, 2929, 1701, 1561, 1471, 1367, 1143, 1092
1H NMR (400 MHz, CDCl3), δ: 7.46–7.70 (m, 1H, H-7), 7.47 (d, 1H, J = 2.0 Hz, H-4), 7.37 (dd, 1H, J = 8.6 Hz,
J = 2.0 Hz, H-6), 5.60 (s, 1H, H-2), 4.38 (d, 1H, J = 15.1 Hz, H-4b’), 4.03 (d, 1H, J = 15.1 Hz, H-4a’), 2.59 (s, 3H,
SCH3), 1.60 (s, 9H, C(CH3)3)
13C NMR (100 MHz, CDCl3), δ: 166.0 (C-2’), 152.0 (CO), 138.5 (C-7a), 132.6 (C-6), 129.7 (C-3a), 127.1 (C-4),
116.3 (C-7), 115.8 (C-5), 88.5 (C-2), 83.3 (C(CH3)3), 75.5 (C-4’), 72.4 (C-3), 28.3 (C(CH3)3), 15.0 (SCH3)
XVIIb
IR, ν˜/cm−1: 3402, 2975, 2928, 1701, 1561, 1474, 1367, 1159, 1063
1H NMR (400 MHz, CDCl3), δ: 7.50–7.90 (m, 1H, H-7), 7.47 (s, 1H, H-4), 7.37 (d, 1H, J = 8.4 Hz, H-6), 5.90 (s, 1H,
H-2), 5.04 (d, 1H, J = 15.7 Hz, H-4b’), 4.26 (d, 1H, J = 15.6 Hz, H-4a’), 2.58 (s, 3H, SCH3), 1.60 (s, 9H, C(CH3)3)
13C NMR (100 MHz, CDCl3), δ: 164.1 (C-2’), 153.9 (CO), 138.5 (C-7a), 132.7 (C-6), 129.9 (C-3a), 127.0 (C-4),
116.2 (C-7), 115.7 (C-5), 91.8 (C-2), 83.3 (C(CH3)3), 69.6 (C-3), 67.9 (C-4’), 28.4 (C(CH3)3), 15.2 (SCH3)
XXVIII
IR, ν˜/cm−1: 2918, 2849, 1762, 1727, 1591, 1463, 1288, 1148
1H NMR (400 MHz, CDCl3), δ: 7.76 (d, 1H, J = 8.6 Hz, H-7), 7.50 (d, 1H, J = 2.1 Hz, H-4), 7.47 (dd, 1H, J =
8.6 Hz, J = 2.1 Hz, H-6), 4.71 (d, 1H, J = 15.3 Hz, H-4b’), 4.49 (d, 1H, J = 15.3 Hz, H-4a’), 2.63 (s, 3H, SCH3),
1.63 (s, 9H, C(CH3)3)
13C NMR (100 MHz, CDCl3), δ: 173.8 (C-2), 163.6 (C-2’), 148.6 (CO), 137.4 (C-7a), 132.9 (C-6), 131.4 (C-3a),
127.1 (C-4), 118.2 (C-5), 116.9 (C-7), 85.4 (C(CH3)3), 76.1 (C-4’), 64.6 (C-3), 28.0 (C(CH3)3), 15.7 (SCH3)
an atmosphere of 5 vol. % of CO2 in humidified air at
37◦C. Cell viability, estimated by trypan blue exclu-
sion, was greater than 95 % prior to each experiment.
proportional to the number of viable cells (Mosmann,
1983). In brief, 5 × 103 cells were plated per well in
96-well polystyrene microplates (Sarstedt, Germany)
in the culture medium containing the tested chemicals
at final concentrations of 10−4–10−6 mol L−1. After
72 h, 10 mL of MTT (5 mg mL−1) was added to each
well. After an additional 4 h at 37◦C, during which in-
soluble formazan was produced, 100 mL of 10 mass %
of sodium dodecylsulphate were added to each well
and a further 12 h were allowed for the formazan to
Assessment of cytotoxicity by MTT assay
The cytotoxic effect of the tested compounds was
studied using a colorimetric microculture assay with
the MTT (thiazolyl blue tetrazolium bromide) end-
point. The amount of MTT reduced to formazan was
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v
+ +
cis-( )- and trans-( )-5-Bromo-1-methoxy-
dissolve. The absorbance was measured at 540 nm us-
ing the automated MRX microplate reader (Dynatech
laboratories UK). The absorbance of the control wells
was taken as 100 %, and the results were expressed as
a percentage of control.
−
−
+
spirobrassinol methyl ether cis-( )-XIIIa and
−
+
trans-( )-XIIIb
−
Following general procedure A, products ( )-XIIIa
and ( )-XIIIb were obtained using the mixture of di-
astereoisomer cis-( )-XIa and trans-( )-XIb (0.030 g,
0.101 mmol) and by method; a) after stirring the re-
action mixture for 40 min, washing with 1 M HCl
(2 × 5 mL) and brine (5 mL) and isolated on silica gel
(hexane–diethyl ether, ϕr = 1 : 1). Yield: ( )-XIIIa
(0.019 g, 50 %), ( )-XIIIb (0.0185 g, 49 %); b) af-
ter stirring the reaction mixture for 40 min, adding
triethylamine (0.030 mL, 0.022 g, 0.222 mmol), wash-
ing with brine (2 × 5 mL) and isolated on silica gel
(hexane–diethyl ether, ϕr = 1 : 1). Yield: ( )-XIIIa
(0.019 g, 50 %), ( )-XIIIb (0.0185 g, 49 %); c) after
stirring the reaction mixture for 40 min, adding tri-
ethylamine (0.030 mL, 0.022 g, 0.222 mmol), washing
with 1 M HCl (5 mL) and brine (5 mL), and isolated
on silica gel (hexane–diethyl ether, ϕr = 1 : 1). Yield:
( )-XIIIa (0.019 g, 50 %), ( )-XIIIb (0.017 g, 45 %);
d) after stirring the reaction mixture for 60 min,
adding triethylamine (0.099 mL, 0.072 g, 0.707 mmol),
and isolated on silica gel (hexane–diethyl ether, ϕr =
1 : 1). Yield: ( )-XIIIa (0.018 g, 47 %), ( )-XIIIb
(0.0184 g, 48 %).
Following general procedure B, products ( )-
XIIIa and ( )-XIIIb were obtained using brassinin
IX (0.050 g, 0.188 mmol), a solution of bromine
(1.307 mL, 0.564 mmol) and triethylamine (0.105 mL,
0.076 g, 0.752 mmol) and isolated on silica gel
(hexane–diethyl ether, ϕr = 1 : 1). Yield: ( )-XIIIa
(0.031 g, 44 %), ( )-XIIIb (0.034 g, 49 %).
Following general procedure C, products ( )-XIIIa
and ( )-XIIIb were obtained using brassinin XXIII
(0.050 g, 0.145 mmol) and isolated on silica gel
(hexane–diethyl ether, ϕr = 1 : 1). Yield: ( )-XIIIa
(0.021 g, 39 %), ( )-XIIIb (0.022 g, 41 %).
General procedures for synthesis of 5-bromi-
nated spirobrassinol methyl ether XIIIa–XIVb
Procedure A: to a solution of a mixture of
the corresponding diastereoisomers XIa and XIb
(Kutschy et al., 2002) or XIIa and XIIb (Budovská
et al., unpublished results; 0.101 mmol) in dry
dichloromethane (1.8 mL) was added; a) a freshly pre-
pared solution of bromine (0.006 mL, 0.111 mmol)
and pyridine (0.042 mL, 0.505 mmol) in anhydrous
dichloromethane (0.26 mL); b) N-bromosuccinimide
(0.020 g, 0.111 mmol); c) a solution of bromine
(0.257 mL, 0.111 mmol) in anhydrous dioxane
(0.26 mL); d) phenyltrimethylammonium tribromide
(0.228 g, 0.606 mmol). The reaction mixture was
stirred at ambient temperature and, after dilution of
the reaction mixture with dichloromethane (5 mL),
treatment of the individual mixtures and drying of
the organic layer over Na2SO4, the residue obtained
after evaporation of the solvent was subjected to chro-
matography on silica gel to afford products XIIIa–
XIVb.
Procedure B: to a stirred solution of the cor-
responding brassinin XI (Kutschy et al., 2009), X
(Kutschy, 1998) (0.188 mmol) in a mixture of anhy-
drous dichloromethane–methanol (2.3 mL : 0.3 mL)
at ambient temperature a freshly prepared solu-
tion of bromine (stock solution prepared by dis-
solving 0.04 mL of bromine in 1.76 mL of an-
hydrous dichloromethane) was added. After stir-
ring for 1 h, triethylamine was added. After stir-
ring for 5 min, the reaction mixture was diluted
with dichloromethane (20 mL), washed with brine
(2 × 10 mL) and dried over Na2SO4. The residue
obtained after evaporation of the solvent was sub-
jected to chromatography on silica gel to afford XIIIa–
XIVb.
+
+
cis-( )- and trans-( )-5-Bromo-1-(tert-
−
−
butoxycarbonyl)spirobrassinol methyl ether
+
+
cis-( )-XIVa and trans-( )-XIVb
−
−
Procedure C: to a stirred solution of the cor-
responding 5-bromo-1-substituted brassinin XXIII,
XXIV (0.145 mmol) in a mixture of anhydrous
dichloromethane–methanol (1.4 mL : 0.1 mL) at
ambient temperature a freshly prepared solution of
bromine (0.369 mL, 0.159 mmol; stock solution ob-
tained by dissolving 0.04 mL of bromine in 1.76 mL
anhydrous dichloromethane) was added. After stir-
ring for 30 min, triethylamine (0.044 mL, 0.032 g,
0.319 mmol) was added. After stirring for 5 min, the
reaction mixture was diluted with dichloromethane
(20 mL), washed with brine (2 × 10 mL) and dried
over Na2SO4. The residue obtained after evaporation
of the solvent was subjected to chromatography on
silica gel to afford XIIIa–XIVb.
Following general procedure A, products ( )-
XIVa and ( )-XIVb were obtained using a mix-
ture of diastereoisomer cis-( )-XIIa and trans-( )-
XIIb (0.037 g, 0.101 mmol) and by method b) af-
ter stirring for 8 h, adding triethylamine (0.056 mL,
0.041 g, 0.404 mmol), washing the reaction mixture
with brine (2 × 5 mL), and isolated on silica gel
(dichloromethane). Yield: ( )-XIVa (0.0175 g, 39 %),
( )-XIVb (0.0185 g, 41 %).
Following general procedure B, products ( )-
XIVa and ( )-XIVb were obtained using brassinin
X (0.063 g, 0.188 mmol), a solution of bromine
(4.358 mL, 1.88 mmol) and triethylamine (0.288 mL,
0.209 g, 2.068 mmol) and isolated on silica gel (petrol
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vi
L. Očenášová et al./Chemical Papers
Table 2. Characterisation data of newly prepared compounds
wi(calc.)/%
wi(found)/%
Yield
Compound
Formula
Mr
C
H
N
%
50
50
49
40
51
81
77
94
93
II
C11H11BrN2S2
C13H15BrN2O2S2
C13H15BrN2O2S2
C17H21BrN2O3S2
C17H21BrN2O3S2
C9H8BrNO
315.25
375.30
375.30
445.39
445.39
226.07
254.08
239.07
269.09
41.91
41.86
3.52
3.43
8.89
8.67
XIIIa
XIIIb
XIVa
XIVb
XVIII
XIX
41.60
41.51
4.03
3.97
7.46
7.39
41.60
41.39
4.03
3.91
7.46
7.22
45.84
45.63
4.75
4.67
6.29
6.18
45.84
45.79
4.75
4.81
6.29
6.22
47.82
47.69
3.57
3.61
6.20
6.07
C
10H8BrNO2
47.27
47.17
3.17
3.11
5.51
5.32
XX
C9H7BrN2O
45.22
44.98
2.95
2.84
11.72
11.58
XXI
C10H9BrN2O2
44.63
44,56
3.37
3.24
10.41
10.48
XXII
XXIII
C
14H15BrN2O3
339.18
345.28
415.37
329.24
359.26
431.37
431.37
429.35
49.57
49.48
4.46
4.36
8.26
8.21
90
75
66
62
29
25
40
75
C12H13BrN2OS2
C16H19BrN2O2S2
C11H9BrN2OS2
41.74
41.66
3.79
3.82
8.11
8.01
XXIV
46.27
46.19
4.61
4.49
6.74
6.67
XXV
40.13
40.26
2.76
2.93
8.51
8.67
XXVI
C12H11BrN2O2S2
C16H19BrN2O3S2
C16H19BrN2O3S2
C16H17BrN2O3S2
40.12
40.22
3.09
3.14
7.80
7.94
XXVIIa
XXVIIb
XXVIII
44.55
44.63
4.44
4.37
6.49
6.38
44.55
44.51
4.44
4.45
6.49
6.39
44.76
44.75
3.99
3.93
6.52
6.48
ether–ethyl acetate, ϕr = 5 : 1) to afford a mixture
of products XIVa and XIVb. Individual isomers XIVa
(0.024 g, 29 %) and XIVb (0.043 g, 51 %) were ob-
tained by a further separation using chromatography
on silica gel (dichloromethane).
Following general procedure C, products ( )-XIVa
and ( )-XIVb were obtained using brassinin XXIV
(0.060 g, 0.145 mmol) and isolated on silica gel
(hexane–acetone, ϕr = 5 : 1) to afford a mixture of
isomers XIVa and XIVb. The individual cis-isomer
XIVa (0.026 g, 40 %) and trans-isomer XIVb (0.031 g,
49 %) were obtained by a further separation using
chromatography on silica gel (dichloromethane).
bromoindoline (XVII; 0.3 g, 1.515 mmol) in methanol
(6 mL) and the mixture was stirred under cooling at –
20◦C (Chandra & Brown, 2005). An aqueous solution
of hydrogen peroxide (30 mass %; 1.44 mL, 13.1 mmol)
in methanol (1.5 mL) was added drop-wise over 30 min
at –20◦C and the stirring was continued at –10–0◦C for
30 min. Potassium carbonate (1.67 g, 12.08 mmol) and
dimethyl sulphate (0.375 mL, 0.497 g, 3.939 mmol)
were added and the reaction mixture was stirred at
–10–0◦C for 1 h, then the mixture was poured into
water (15 mL). After extraction with diethyl ether
(1 × 50 mL and 2 × 30 mL), the combined organic
phase was washed with brine (50 mL), dried with
anhydrous Na2SO4, filtered and concentrated under
vacuum. The residue was purified by chromatography
on silica gel (hexane–dichloromethane–ethyl acetate,
ϕr = 5 : 2 : 1) to afford indole XVIII (0.276 g, 81 %)
as a pale yellow oil.
5-Bromo-1-methoxy-1H-indole XVIII
Sodium tungstate dihydrate (0.084 g, 0.273 mmol)
in water (0.555 mL) was added to a solution of 5-
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vii
5-Bromo-1-methoxy-1H-indole-3-
carboxaldehyde XIX
(0.942 g, 90 %) was obtained using aldehyde XVI
(1.0 g, 3.085 mmol) as a mixture of (E)- and (Z)-
isomers at a 60 : 40 ratio.
To DMF (0.318 mL), phosphorus oxychloride
(0.312 mL, 0.524 g, 3.415 mmol) was added drop-wise
at 0◦C. The mixture was stirred for 10 min and then
a solution of 5-bromo-1-methoxy-1H-indole (XVIII;
0.234 g, 1.035 mmol) in DMF (0.43 mL) was added.
After the mixture was stirred at ambient temperature
for 2 h, ice was added followed by a 16 mass % solu-
tion of sodium hydroxide (3.27 mL). The precipitated
product was extracted with diethyl ether (1 × 30 mL
and 2 × 15 mL), the combined organic layers were
washed with brine (20 mL), dried with Na2SO4, fil-
tered and concentrated under vacuum. The residue
was allowed to crystallise from diethyl ether–hexane
to yield aldehyde XIX (0.202 g, 77 %) as a crystalline.
General procedure for synthesis of brassinins
II, XXIII and XXIV
To a solution of NiCl2 ·6H2O (0.349 g, 1.468 mmol)
in methanol (20 mL) was added the corresponding
oxime XX, XXII (1.474 mmol) in methanol (15 mL)
followed by NaBH4 (0.556 g, 14.74 mmol) in a sin-
gle portion with stirring and cooling under flowing
cold water. After 5 min, the methanol in the reaction
mixture was evaporated to 1/4 of its original volume
and the mixture was poured into a saturated solution
of NH4OH (37 mL). In the case of brassinin XXIII,
sodium cyanoborohydride (0.926 g, 14.74 mmol) and
NH4OAc (1.25 g, 16.21 mmol) were added to a cooled
solution at 0◦C of oxime XXI (1.474) in methanol
(8.75 mL). To this mixture, a solution (4.083 mL) of
TiCl3 (30 mass % in 2 M HCl) neutralised with 2 M
NaOH (2.916 mL) was added. After stirring for 15 min
at 0◦C, the reaction mixture was poured into water
(23 mL) containing 26 mass % NH4OH (4.43 mL).
The black precipitate was collected by suction fil-
tration and washed with water (30 mL), methanol
(30 mL) and ethyl acetate (30 mL). After extraction
of the products with ethyl acetate (1 × 50 mL and
2 × 30 mL), drying the extract with Na2SO4 and
evaporation of the solvent, the crude amines obtained
were immediately dissolved in methanol (12 mL) and
triethylamine (0.616 mL, 0.447 g; 4.422 mmol) and
carbon disulphide (0.267 mL, 0.337 g; 4.422 mmol)
were added. After stirring for 5 min, methyl iodide
(0.276 mL, 0.627 g, 4.422 mmol) was added and stir-
ring was continued for 20 min at ambient tempera-
ture. The solvent was evaporated and the residue ob-
tained after evaporation of the solvent was subjected
to chromatography on silica gel (hexane–acetone, ϕr =
2 : 1) (II ), (hexane–dichloromethane–ethyl acetate,
ϕr = 5 : 2 : 1) (XXIII ) and (hexane–acetone, ϕr =
3 : 1) (XXIV ).
General procedure for synthesis of oximes
XX–XXII
To a stirred solution of the corresponding alde-
hyde XV (Wang et al., 2001), XVI (Ishiyama et al.,
2012) and XIX (3.085 mmol) in ethanol (5.799 mL)
a solution of hydroxylammonium chloride (0.321 g,
4.627 mmol) and sodium carbonate (0.228 g,
2.159 mmol) in water (0.438 mL) was added. The mix-
ture was stirred for 40 min at 0◦C (XX and XXI ) or
heated at 50◦C for 15 min (XXII ). After evaporation
of the ethanol and the addition of water (15.6 mL),
the product was extracted with ethyl acetate (XX
and XXI ) or diethyl ether (XXII ) (1 × 30 mL and
1 × 15 mL) and after drying with anhydrous Na2SO4
the solvent was evaporated. The residue was allowed
to crystallise from ethyl acetate–hexane (XX, XXI )
and dichloromethane–hexane (XXII ) to yield the cor-
responding oxime.
5-Bromo-1H-indole-3-carboxaldehyde oxime
XX
Following the general procedure, product XX
(0.693 g, 94 %) was obtained using aldehyde XV
(0.691 g, 3.085 mmol) as a mixture of (E)- and (Z)-
isomers at a 80 : 20 ratio.
5-Bromobrassinin II
Following the general procedure, product II
(0.232 g, 50 %) was obtained using oxime XX (0.352 g,
1.474 mmol) as a crystalline. According to the lit-
erature, 5-bromobrassinin was previously synthesised
by the Advanced Synthesis Group, Newark, DE, USA
(Banerjee et al., 2008), but its synthesis and spectral
data have not yet been published.
5-Bromo-1-methoxy-1H-indole-3-
carboxaldehyde oxime XXI
Following the general procedure, product XXI
(0.772 g, 93 %) was obtained using aldehyde XIX
(0.784 g, 3.085 mmol) as a mixture of (E)- and (Z)-
isomers at a 50 : 50 ratio.
5-Bromo-1-methoxybrassinin XXIII
5-Bromo-1-(tert-butoxycarbonyl)indole-3-
carboxaldehyde oxime XXII
Following the general procedure, product XXIII
(0.382 g, 75 %) was obtained as a colourless oil us-
ing oxime XXI (0.397 g, 1.474 mmol).
Following the general procedure, product XXII
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viii
L. Očenášová et al./Chemical Papers
Fig. 3. Synthesis of target compounds XIII and XIV by synthetic routes A and B. Reagents and conditions: i) Br2, CH2Cl2/MeOH
(ϕr = 9 : 1), Et3N, ambient temperature, 20 min, XI, 89 %, XII, 72 %; ii) bromination agent (Table 1); iii) Br2,
CH2Cl2/MeOH (ϕr = 9 : 1), Et3N, ambient temperature, 60 min, XIII, 93 %, XIV, 80 %.
5-Bromo-1-(tert-butoxycarbonyl)brassinin
brassinin II (0.050 g, 0.159 mmol).
XXIV
+
XXVI
+
( )-5-Bromo-1-methoxyspirobrassinin ( )-
−
−
Following the general procedure, product XXIV
(0.404 g, 66 %) was obtained as a crystalline using
oxime XXII (0.5 g, 1.474 mmol).
Following the general procedure, product ( )-
XXVI (0.013 g, 29 %) was obtained as a pale yellow
oil using brassinin XXIII (0.055 g, 0.159 mmol).
General procedure for synthesis
of spirobrassinins ( )-XXV and ( )-XXVI
+
+
−
−
+
+
cis-( )- and trans-( )-5-Bromo-1-(tert-
butoxycarbonyl)spirobrassinol cis-( )-XXVIIa
and trans-( )-XXVIIb
−
−
+
To a stirred solution of the corresponding brassinin
II, XXIII (0.159 mmol) in dichloromethane (2.69 mL)
at ambient temperature was added pyridinium chloro-
chromate (0.239 g, 1.113 mmol) and the reaction
mixture was stirred vigorously for 1 h (XXV ) and
for 2 h (XXVI ). After diluting the mixture with
dichloromethane (5 mL) and adding a small amount
of silica gel, the solvent was evaporated and the
residue obtained subjected to chromatography on sil-
ica gel (hexane–ethyl acetate, ϕr = 1 : 1) (XXV ) and
(hexane–ethyl acetate, ϕr = 1 : 1) (XXVI ).
−
+
−
To a solution of brassinin XXIV (0.084 g,
0.202 mmol) in a mixture of dichloromethane–water
(2.25 mL : 0.25 mL) at ambient temperature was
added a freshly prepared stock solution of Br2
(0.515 mL, 0.222 mmol), obtained by dissolving
bromine (0.04 mL) in dichloromethane (1.76 mL).
The reaction mixture was stirred for 30 min at
ambient temperature and triethylamine (0.062 mL,
0.045 g, 0.0444 mmol) was added. Stirring contin-
ued for 5 min, then the reaction mixture was di-
luted with dichloromethane (15 mL) and washed with
brine (2 × 15 mL). The organic layer was dried over
Na2SO4 and the residue obtained after evaporation
+
+
( )-5-Bromospirobrassinin ( )-XXV
−
−
Following the general procedure, product ( )-XXV
(0.032 g, 62 %) was obtained as a pale yellow oil using
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ix
Table 3. Electrophilic aromatic substitution of compounds IX–XII in the presence of various brominating agents produced by
Fig. 3
Starting material
Brominating agent/eq.
Time/min
Products
Yield/%
(
(
(
(
)-XIa, ( )-XIb
)-XIa, ( )-XIb
)-XIa, ( )-XIb
)-XIa, ( )-XIb
)-XIIa, ( )-XIIb
IX
Br2 (1.1)/pyridine (5)
40
40
40
60
480
60
(
(
(
(
(
(
(
)-XIIIa, ( )-XIIIb
)-XIIIa, ( )-XIIIb
)-XIIIa, ( )-XIIIb
)-XIIIa, ( )-XIIIb
)-XIVa, ( )-XIVb
)-XIIIa, ( )-XIIIb
)-XIVa, ( )-XIVb
99
99
95
95
80
93
80
NBS (1.1)
DDB (1.1)
Phenyltrimethylammonium tribromide (6)
(
NBS (3)
Br2 (3)
Br2 (10)
X
60
of the solvent subjected to chromatography on sil-
ica gel (dichloromethane), affording a mixture of di-
astereoisomers XXVIIa and XXVIIb. The individual
isomers XXVIIa (0.022 g, 25 %) and XXVIIa (0.035
g, 40 %) were obtained by a further separation us-
ing chromatography on silica gel (dichloromethane–
acetone, ϕr = 30 : 1).
ane dibromide (DDB), phenyltrimethylammonium tri-
bromide; Fig. 3). Table 3 shows that the bromina-
tion of mixtures XIa and XIb in the first three cases
was carried out using 1.1 eq. of reagent for 40 min
while up to 6 eq. were required when phenyltrimethy-
lammonium tribromide was used; the bromination
was incomplete with a lower quantity of this reagent.
The compounds XIVa and XIVb were synthesised by
bromination of mixtures XIIa and XIIb in the pres-
ence of 3 eq. of NBS in anhydrous dichloromethane
for 8 h. Interestingly, the amount of the brominat-
ing agent is probably dependent on the character of
the group bonding onto the indoline nitrogen. The
bulky tert-butoxycarbonyl group displays electron-
acceptor properties towards the spiroindoline skele-
ton, unlike the methoxy group which exhibits electron-
donor characteristics. The brominated diastereoiso-
mers ( )-XIIIa and ( )-XIIIb thus prepared were sep-
arated by chromatography on silica gel using hexane–
diethyl ether, ϕr = 1 : 1 as eluent and diastereoisomers
( )-XIVa and ( )-XIVb using dichloromethane.
The target products ( )-XIIIa, ( )-XIIIb, ( )-
XIVa, ( )-XIVb were also prepared by the direct
spirocyclisation reaction (synthetic route B, Fig. 3)
of 1-methoxybrassinin (IX ) (Kutschy et al. (2009) or
1-(tert-butoxycarbonyl)brassinin (X ), (Kutschy et al.
(1998) in dry dichloromethane using bromine (3 eq.
for compound IX and 10 eq. for compound X ) in the
presence of methanol (10 vol. %). The reaction mix-
ture was stirred for 60 min at ambient temperature
and triethylamine was added subsequently to trap the
hydrogen bromide liberated during the reaction. The
+
( )-5-Bromo-1-(tert-butoxycarbonyl)
−
+
spirobrassinin ( )-XXVIII
−
To a solution of a mixture of diastereoisomers
XXVIIa and XXVIIb (0.037 g, 0.086 mmol) in acetic
acid (2.6 mL) was added CrO3 (0.043 g, 0.43 mmol)
and the reaction mixture was vigorously stirred at
ambient temperature for 1 h. After pouring the mix-
ture into water (11 mL), the product was extracted
with diethyl ether (2 × 5 mL), washed with 10
mass % NaOH (2 × 5 mL), dried with anhydrous
Na2SO4, filtered and concentrated under vacuum. The
residue was purified by chromatography on silica gel
(hexane–acetone, ϕr = 3 : 1) and allowed to crystallise
from dichloromethane–hexane to afford spirobrassinin
XXVIII (0.027 g, 75 %) as a crystalline.
Results and discussion
The known mixture of cis-( ) and trans-( )-
diastereoisomers of 1-methoxyspirobrassinol methyl
ether (XIa, XIb) (Kutschy et al. (2002) and 1-(tert-
butoxycarbonyl)spirobrassinol methyl ether (XIIa,
XIIb; Budovská et al., unpublished results), as the
starting materials for the first synthetic route A, was
prepared via bromine-mediated spirocyclisation of the
respective 1-substituted brassinins IX (Kutschy et al,
2009), X (Kutschy et al., 1998) in the presence of
methanol (10 vol. %) as nucleophile following the pro-
cedure previously reported.
The new 5-bromo-1-methoxyspirobrassinol methyl
ethers (XIIIa, XIIIb) and 5-bromo-1-(tert-butoxy-
carbonyl)spirobrassinol methyl ethers (XIVa, XIVb)
were synthesised via electrophilic aromatic substi-
tution of the indoline skeleton at the C-5 posi-
tion in the presence of various brominating agents
(bromine/pyridine, N-bromosuccinimide (NBS), diox-
1
ratio of diastereoisomers was determined by H NMR
spectra of the crude reaction products obtained af-
ter dilution with dichloromethane, washing with 1 M
solution of hydrochloric acid and brine, drying and
evaporation of the solvent. The ratios of the prod-
ucts were confirmed by the integration ꢀof separated
ꢀ
signals corresponding to the H-2, H-4a , H-4b pro-
tons. Diastereoisomers cis-( )-XIIIa and trans-( )-
XIIIb were isolated by column chromatography with a
93 % yield in a 47 : 53 ratio, and isomers ( )-XIVa and
( )-XIVb were obtained with an 80 % yield (Table 3)
after two chromatographic separations in a 37 : 63 ra-
tio favouring the trans-isomer.
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L. Očenášová et al./Chemical Papers
Fig. 4. Synthesis of target compounds XIII and XIV by synthetic route C. Reagents and conditions: i) POCl3, DMF, 0◦C, 1 h,
87 %; ii) (Boc)2O, THF, DMAP, 0◦C, 1 h, 85 %; iii) NaBH3CN, AcOH, 15◦C, 3 h, 91 %; iv) Na2WO4 · 2H2O, 30 mass %
H2O2, MeOH/H2O, –20◦C, 1 h; v) K2CO3, (CH3O)2SO2, 0◦C, 1 h, 81 %; vi) POCl3, DMF, 0◦C, 2 h, 77 %; vii) NH2OH
· HCl, Na2CO3, EtOH/H2O, XX, 0◦C, 40 min, 94 %, XXI, 0◦C, 40 min, 93 %, XXII, 50◦C, 15 min, 90 %; viii) II, XXIV,
NiCl2 · 6H2O, NaBH4, MeOH/H2O, XXIII, NaBH3CN, AcONH4, 30 % TiCl3, MeOH/H2O, 0◦C, 15 min; ix) Et3N, CS2,
MeOH, CH3I, at, 20 min, II, 50 %, XXIII, 75 %, XXIV, 66 %; x) Br2, CH2Cl2–MeOH (ϕr = 9 : 1), Et3N, at, 20 min,
XIII, 80 %, XIV, 89 %.
5-Bromo-1H-indole (VIII ), which was used as a
starting compound of the third synthetic route C,
was reduced by a reaction with sodium cyanoboro-
hydride in acetic acid to afford 5-bromoindoline
(XVII ) as previously described (Chandra & Brown,
2005). Indoline XVII was first oxygenated to 5-bromo-
1-hydroxyindole by the Somei’s tungstate method
with a 30 mass % solution of hydrogen peroxide
in the presence of sodium tungstate as a catalyst
and 5-bromo-1-methoxyindole (XVIII ) was then ob-
tained by the methylation of 5-bromo-1-hydroxyindole
with dimethyl sulphate using potassium carbonate
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xi
Fig. 5. Synthesis of 5-brominated spirobrassinins XXV, XXVI and XXVIII. Reagents and conditions: i) PCC, CH2Cl2, at, XXV,
1 h, 62 %, XXVI, 2 h, 29 %; ii) Br2, CH2Cl2–H2O (ϕr = 9 : 1), Et3N, at, 20 min, 65 %; iii) CrO3, CH3COOH, at, 1 h,
75 %.
as a base (Somei & Kawasaki, 1989). Aldehydes
XV (Wang et al., 2001) and XIX were synthesised
with good yields by the Vilsmeier–Haack reaction
(Acheson et al., 1978) of the respective indolesVIII
and XVIII with phosphorus oxychloride in dimethyl-
formamide. 5-Bromo-1-(tert-butoxycarbonyl)indole-3-
carboxaldehyde (XVI ) was prepared by the proce-
dure previously described (Wang et al., 2001). The
treatment of aldehydes XV, XVI and XIX with hy-
droxylamine hydrochloride provided the correspond-
ing oximes XX–XXII, which were obtained as mix-
tures of (Z)- and (E)-isomers with high yields (Hanley
et al., 1990). The reduction of oximes XX and XXII
with sodium borohydride using catalytic nickel boride
(Kutschy et al., 1998) or the reduction of 5-bromo-1-
methoxyindole-3-carboxaldehyde oxime (XXI ) in the
presence of sodium cyanoborohydride and titanium
catalyst (Pedras & Zaharia, 2000) provided the un-
stable oily amines which were immediately used as
crude products in the next reaction. The treatment
of amines with carbon disulphide and iodomethane
in methanol in the presence of triethylamine afforded
5-bromobrassinins II, XXIII and XXIV in sufficient
amounts (Fig. 4).
Subsequently, the iminium ion B (R = OCH3, Boc)
reacted with the nucleophile (methanol) to afford the
target products XIIIa, XIIIb with an 80 % yield in
a 44 : 56 ratio and products XIVa, XIVb with an
89 % yield in a 29 : 71 ratio (Fig. 4). Due to the
low reactivity of the spiroindoleninium intermediate B
(R = H) with the nucleophile (Kutschy et al., 2002),
5-bromospirobrassinol methyl ethers were not pre-
pared.
In order to obtain the novel 5-bromo-substituted
spirocyclic compounds, 5-bromobrasinnin (II ) and
5-bromo-1-methoxybrassinin (XXIII ) were oxidised in
a reaction with 7 eq. pyridinium chlorochromate in
dichloromethane to afford the racemic products XXV
and XXVI with 62 % and 29 % yields, respectively
(Pedras et al., 2006) (Fig. 5). However, the treatment
of 5-bromo-1-Boc-brassinin (XXIV ) with PCC led to
the oxidation of sulphur to form S-oxid. With the aim
of obtaining 5-bromo-1-Boc-spirobrassinin (XXVIII ),
the spirocyclisation of brassinin XXIV using bromine
in the presence of water at ambient temperature
was first performed to afford the diastereoisomers
of 5-bromo-1-Boc-spirobrassinol cis-( )-XXVIIa and
trans-( )-XXVIIb with a 65 % yield in a 23 : 77 ratio.
The target compound XXVIII was subsequently pre-
pared by the oxidation of diastereoisomers XXVIIa
and XXVIIb with CrO3 in acetic acid with a 75 %
yield (Fig. 5).
Having prepared brassinins II, XXIII and XXIV,
the key spirocyclisation was performed with 1.1 eq.
of bromine in dichloromethane in the presence of
methanol as a nucleophile. As previously suggested,
the reaction probably started at the thiocarbamoyl
functionality with the formation of sulphenyl bromide
A, in which the electrophilic sulphur attacked the 3-
position of the indole skeleton to afford a spiroin-
doleninium intermediate B (Kutschy et al., 2002).
The structure of the individual target products
was unequivocally confirmed by NMR studies, in-
cluding 2D HSQC, HMBC, COSY and NOESY ex-
periments. In the cis-diastereoisomer, the sulphur of
the thiazoline ring and the 2-methoxy or 2-hydroxy
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L. Očenášová et al./Chemical Papers
Table 4. Anti-proliferative activity of target compounds
Cell line, IC50/( mol L−1
)
Compound
R1
R2
R3
Jurkat
A-549
MCF-7
MDA
HeLa
CEM
I
II
IX
XXIII
X
XXIV
H
Br
H
Br
H
> 100
67.5
37.5
59.8
17.8
NT
> 100
86.6
100
85.0
21.4
NT
> 100
85.6
100
85.6
23.0
> 100
90.5
100
79.7
21.4
47.9
> 100
> 100
100
> 100
16.9
90.2
29.4
63.5
36.1
19.6
NT
H
–
–
–
OCH3
Boc
Br
> 100
75.7
cis-( )-Xia
cis-( )-XIIIa
trans-( )-XIb
trans-( )-XIIIb
H
Br
H
57.4
97.1
30.2
72.5
100
> 100
100
100
> 100
100
100
> 100
100
53.2
> 100
48.9
100
69.0
100
OCH3
Boc
OCH3
OCH3
OH
Br
> 100
94.5
> 100
> 100
51.1
cis-( )-XIIa
cis-( )-XIVa
trans-( )-XIIb
trans-( )-XIVb
H
Br
H
43.4
48.0
37.3
57.0
96.3
> 100
70.5
100
41.1
70.2
97.7
> 100
87.0
77.6
> 100
74.3
41.9
34.2
37.9
33.2
Br
> 100
> 100
> 100
89.3
cis-( )-XXIXa
cis-( )-XXVIIa
trans-( )-XXIXb
trans-( )-XXVIIb
H
Br
H
29.8
29.2
34.0
32.1
100
NT
100
NT
100
31.0
100
95.0
70.0
82.8
75.6
93.6
65.7
78.0
42.2
27.1
NT
30.6
NT
Boc
H
Br
42.9
(
(
)-XXX
)-XXV
H
Br
H
Br
Br
63.4
> 100
41.1
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
NT
(
(
)-XXXI
)-XXVI
)-XXVIII
O
OCH3
Boc
(
Cisplatin
VP-16 (Etoposide)
12.0
1.2
12.2
14.3
11.4
10.9
14.7
21.2
7.7
3.9
4.4
1.1
The potency of the compounds was determined using the MTT (thiazolyl blue tetrazolium bromide) assay after 72 h incubation of
cells and presented as IC50 values (concentration of a given compound that decreased the amount of viable cells to 50 % relative
to untreated control cells). NT – not tested.
Fig. 6. NOESY interactions confirming cis- and trans-diastereoisomeric structure.
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xiii
groups are located on the same sides of the indo-
line ring, while, in the trans-diastereoisomer, the sul-
phur and 2-methoxy or 2-hydroxy groups are on the
opposite sides. Inꢀ the NOESY spectra, the interac-
tion between H-4b and H-2 protons confirms the cis-
configuration, whereas the interactions between H-4b
and methoxy or hydroxy protons correspond to the
trans-diastereoisomers structures (Fig. 6).
tant for the anticancer activity of brassinins II and
XXIII. 5-Bromobrassinin (II ) displayed the highest
potency of anti-proliferative/cytotoxic activity espe-
cially on the CEM cancer cell line with an IC50 value
of 29.4 mol L−1 relative to brassinin (I ), where the
IC50 value was 90.2 mol L−1. Further, 5-bromo-1-
methoxybrassinin (XXIII ) demonstrated a higher ac-
tivity with an IC50 value of 36.1 mol L−1 than the
non-brominated brassinin IX with an IC50 = 63.5
mol L−1 on the CEM cell line. The 5-bromo-1-
substituted spirobrassinins XXV, XXVI and XXVIII
tested did not exhibit any anti-proliferative/cytotoxic
activity. Ultimately, the newly prepared compounds
were not as effective as the standard chemotherapeu-
tic agents, cisplatin and etoposide, respectively.
ꢀ
The newly synthesised compounds were provided
for testing the biological activity on a panel of six hu-
man tumour cell lines Jurkat (acute T-lymphoblastic
leukaemia), A-549 (non-small cell lung cancer), MCF-
7 (mammary gland adenocarcinoma), MDA-MB-231
(mammary gland adenocarcinoma), HeLa (cervical
adenocarcinoma) and CEM (acute T-lymphoblastic
leukaemia). The potency of these compounds was de-
termined using the MTT (thiazolyl blue tetrazolium
bromide) assay after 72 h incubation of cells and is
presented in Table 4 as IC50 values (concentration of
a given compound that decreased the amount of vi-
able cells to 50 % relative to untreated control cells
(Mosmann, 1983)). For comparison, Table 4 also in-
cludes IC50 values for non-brominated brassinins I,
X (Kutschy et al., 1998), IX (Kutschy et al., 2009),
diastereoisomers of 1-methoxyspirobrassinol methyl
ether cis-( )-XIa, trans-( )-XIb (Kutschy et al.,
2002), 1-Boc-spirobrassinol methyl ether cis-( )-XIIa,
trans-( )-XIIb (Budovská et al., unpublished results)
and 1-Boc-spirobrassinol cis-( )-XXIXa, trans-( )-
XXIXb (Kutschy et al., 2002), and spirobrassinins
XXX (Pedras et al., 2006), XXXI (Monde et al., 2005)
whose syntheses were published previously, except for
the synthesis of products XII. Next, the biological ac-
tivities of the standard chemotherapeutic agents, cis-
platin and etoposide are reported.
Conclusions
New 5-brominated spirobrassinol methyl ethers
and related 5-bromo derivatives of indole and spiroin-
doline phytoalexins were designed and synthesised.
Their synthesis was based on either the electrophilic
substitution of the aromatic core of indoline in the
presence of various brominating agents or the prod-
ucts were obtained from 5-bromoindole following the
sequence for the preparation of 1-methoxyspirobras-
sinol methyl ether from indoline. The anti-proliferative
activity of the synthesised compounds against selected
human tumour cell lines was examined. Substance
XXVIIa displayed the highest anti-proliferative activ-
ity on Jurkat cells and compound II on the CEM can-
cer cell line. The presence of bromine on the aromatic
ring of indole or indoline at the C-5 position resulted
in the partial increase in anticancer activity relative to
non-brominated compounds on leukaemia cell lines.
The present study sought to compare the rela-
tionship between the structure and biological activ-
ity of the new 5-bromo-substituted compounds on
the aromatic ring of indole or indoline with the
non-brominated derivatives previously discussed. The
tested cis- and trans-diastereoisomers of 5-bromo-1-
methoxyspirobrassinol methyl ether ( )-XIIIa and
( )-XIIIb exhibited a 50 % higher potency of anti-
proliferative activity over the non-brominated di-
astereoisomers ( )-XIa and ( )-XIb on CEM cell
line. Unfortunately, these diastereoisomers ( )-XIIIa
and ( )-XIIIb did not demonstrate any activity on
the other cancer cell lines. The cis- and trans-
diastereoisomers of 5-bromo-1-(tert-butoxycarbonyl)
spirobrassinol methyl ether ( )-XIVa and ( )-XIVb
exhibited approximately the same potency as non-
brominated diastereoisomers ( )-XIIa and ( )-XIIb
on the CEM cell line. Of all the compounds tested in
this report, cis-( )-5-bromo-1-(tert-butoxycarbonyl)
spirobrassinol ( )-XXVIIa exhibited the highest anti-
proliferative activity on Jurkat cells with an IC50 value
of 29.2 mol L−1 (Table 4).
Acknowledgements. The authors wish to express their
gratitude to the Slovak Scientific Grant Agency (grant nos.
1/0954/12, 1/0322/14 and 1/398/14) and the Slovak Research
and Development Agency (contract no. APVV-14-0883) for
their financial support of this work. This work was also sup-
ported by the State NMR Programme no. 2003SP200280203
and VVGS PF 2012-31.
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