A facile method for the synthesis of indole phytoalexin rutalexin

Article abstract of DOI:10.1016/j.tetlet.2015.05.001

Novel methods for the preparation of the indole phytoalexin rutalexin (8) in high yields are presented. The synthesis of rutalexin (8) was achieved from previously synthesized 9-tert-butoxycarbonyl-2-methoxy-4-oxo-[1,3]thiazino[6,5-b]indole (5) by its hydrolysis, methylation, and deprotection. A second method starting from 9-Boc-cyclobrassinin (9) involved oxidation, methylation, and deprotection.

Full text of DOI:10.1016/j.tetlet.2015.05.001

Tetrahedron Letters 56 (2015) 3945–3947
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A facile method for the synthesis of indole phytoalexin rutalexin
a,
⇑
a
a
b
b
Mariana Budovská , Zuzana Kudli cˇ ková , Peter Kutschy , Martina Pilátová , Ján Moj zˇ iš
a
Institute of Chemical Sciences, Faculty of Science, P. J. Šafárik University, Moyzesova 11, 040 01 Košice, Slovak Republic
Department of Pharmacology, Faculty of Medicine, P. J. Šafárik University, SNP 1, 040 66 Košice, Slovak Republic
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel methods for the preparation of the indole phytoalexin rutalexin (8) in high yields are presented.
The synthesis of rutalexin (8) was achieved from previously synthesized 9-tert-butoxycarbonyl-2-meth-
oxy-4-oxo-[1,3]thiazino[6,5-b]indole (5) by its hydrolysis, methylation, and deprotection. A second
method starting from 9-Boc-cyclobrassinin (9) involved oxidation, methylation, and deprotection.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 12 January 2015
Revised 11 April 2015
Accepted 2 May 2015
Available online 8 May 2015
Keywords:
Indoles
Phytoalexins
Rutalexin
Natural products
Phytoalexins are defined as antimicrobial low molecular weight
secondary metabolites, that are produced by plants upon exposure
to physical, biological, or chemical stress.1 About 40 indole
phytoalexins have been isolated from economically and dietary
S
O
SCH3
N
H
H
Cl
N
R
N
H
important plants of the family Cruciferae, which are cultivated
2
worldwide.
Among them, several 1,3-thiazino[6,5-b]indole
1
2
derivatives such as cyclobrassinin (3a) from the Chinese cabbage
3a
Cyclization
(
Brassica campestris L. ssp. pekinensis), and sinalbin B (3b) from
O
3b
the white mustard (Sinapis alba) have been described. Indole
N
N
phytoalexins demonstrate in vitro cytotoxic/cytostatic activities
2
c,e
SCH3
OCH3
against human solid tumor and leukemia cell lines.
Indole
S
S
N
R
N
H
phytoalexins have also been shown to exhibit cancer
chemopreventive activity in 7,12-dimethylbenz[a]anthracene
3
4
1
a, 3a: R = H; 1b, 3b: R = OCH3
(
DMBA)-induced precancerous lesions in mouse mammary gland
2
f
organ cultures. Compounds 3a and 3b have been prepared by
oxidative bromocyclization of the corresponding indole
phytoalexins brassinin (1a) or 1-methoxybrassinin (1b) using
pyridinium tribromide (3a, 34%), NBS (3a, 35%; 3b, 41% ),
dioxane dibromide (3a, 45%)3 or phenyltrimethylammonium
tribromide (3a, 59%, Scheme 1).3e
Scheme 1.
3a
3c
3b
synthetic product 4 showed an identical mass spectrum to the
d
natural product, it was poorly soluble in CDCl3 and its 1H NMR
spectrum in DMSO-d exhibited differences in the chemical shifts
6
4
b
4a
4b
In 1994 Gross et al. described the isolation and characterization
of a new phytoalexin from kohlrabi (Brassica oleracea var. gongy-
lodes) which was assigned as structure 4 and named cyclobrassi-
3
for OMe (d 4.18 vs d 3.55 in CDCl ) and NH (d 12.69 vs d
4
a
8.56 in CDCl ) which we attributed to the different solvent
used. Although an original sample of the natural product isolated
from kohlrabi was not available for comparison, we correctly
assigned the structure of synthetic product 4 by H NMR,
3
non. However only the 1H NMR (CDCl
4
a
) and mass spectrum
3
4a
1
13
were published and a melting point was not given.
We have previously studied the synthesis of thiazino[6,5-b]in-
C
NMR, and IR spectra. This assignment was also based on the
dole derivative 4 and its analogs (Scheme 1).4
b–d
Although our
experience we had gained from previous syntheses of analogous
e,f
2
-substituted-4H-benzo[4,5]thieno[2,3-e]-1,3-thiazin-4-ones.4 It
was later shown that the structure of the natural product
cyclobrassinon had originally been misassigned, thus accounting
⇑
Corresponding author. Tel.: +421 552341196.
E-mail address: mariana.budovska@upjs.sk (M. Budovská).
http://dx.doi.org/10.1016/j.tetlet.2015.05.001
040-4039/Ó 2015 Elsevier Ltd. All rights reserved.
0

3
946
M. Budovská et al. / Tetrahedron Letters 56 (2015) 3945–3947
O
O
N
NH
N
N
b
g
f
OCH3
O
SCH3
SCH3
S
S
S
S
N
N
Boc
N
Boc
N
H
Boc
5
6
9
3a
a (Ref.4b
e (Ref.3e
)
)
c
S
O
N
O
O
CH3
CH3
SCH3
N
N
N
H
d
OCH3
O
O
S
S
S
N
H
N
N
H
N
H
unnatural
Boc
9
H-2-methoxy-4-oxo-
[
1,3]thiazino[6,5-b]indole 4
7
natural rutalexin 8
1a
Scheme 2. Reagents and conditions: (a) 165–170 °C, 40 min, 70%; (b) HCl/H
2
O (1:1, cat.), acetone, rt, overnight, 80%; (c) DBU, CH
3 2
I, THF, under N , rt, 2 h, 95%; (d) 165–170 °C,
3
0 min., quant.; (e) PhMe NBr , Et N, CH Cl , rt, 1 min, 59%; (f) Boc-anhydride, DMAP, THF, 5 °C, 1 h, 88%; (g) PCC, CH
3
3
3
2
2
2
Cl , 24 h, 66%.
2
Table 1
Antiproliferative activities of cyclobrassinin (3a), rutalexin (8), 9H-2-methoxy-4-oxo-[1,3]thiazino[6,5-b]indole (4), and 9-Boc-cyclobrassinin (9)
Compound
Cell line, IC50
MDA-MB-231
(
l
mol  LÀ1
)
Jurkat
MCF-7
HeLa
CCRF-CEM
A-549
Cyclobrassinin (3a)
29.5
50
>100
54.0
12
72.0
>100
37
100
11.4
10.9
48.3
>100
>100
100
14.7
21.2
57.2
47
>100
100
7.7
26.2
63
>100
47.2
4.4
70.3
>100
>100
100
12.2
14.3
9
H-2-Methoxy-4-oxo-[1,3]thiazino[6,5-b]indole (4)
Rutalexin (8)
-Boc-cyclobrassinin (9)
9
Cisplatin
VP-16 (Etoposide)
1.2
3.9
1.1
The potency of compounds was determined using the MTT (Thiazolyl Blue Tetrazolium Bromide) assay after 72 h incubation of cells and presented as IC50.7
for the spectroscopic discrepancies between the synthetic and
natural products (see below).
methylation of 6 proceeded smoothly in high yield (Scheme 2).9
Finally the Boc group was removed from 7 by heating without sol-
1
0
In 2004 Pedras et al. isolated the phytoalexin rutalexin (8) from
vent to yield rutalexin (8), whose data were identical with those
rutabaga tubers (Brassica napus L. ssp. rapifera).5 Because the
1
H
of the described natural product. Interestingly the melting points
5
NMR spectroscopic data were similar to those reported for phy-
toalexin 4, they also investigated phytoalexins produced in kohl-
rabi, again isolating rutalexin (8). To unambiguously prove the
of compounds 7 and 8 are identical, which is rationalized during
heating of 7, deprotection occurred before melting. This caused
problems during the measurement of the C NMR spectrum of 7,
1
3
structures, they synthesized the proposed structures of rutalexin
where due to its insolubility, heating to 60 °C was required.
Deprotection also occurred at this time and the C spectrum of
5
13
(
(
8) and cyclobrassinon (4). Comparison of the spectroscopic data
1
1
13
H NMR in CDCl
3
,
H and C NMR in DMSO-d
6
) of the isolated
7 could not be measured. A second method for the preparation of
rutalexin (8) based on the biomimetic synthesis from the indole
phytoalexin brassinin (1a) and proceeding via phytoalexin cyclo-
brassinin (3a) was examined. Cyclobrassinin (3a) was obtained
by the bromocyclization of brassinin (1a) using phenyltrimethy-
phytoalexins (from kohlrabi and rutabaga) with synthetic rutalexin
8) and synthetic cyclobrassinon (4) showed that the structure of
(
the natural product first isolated from kohlrabi and named cyclo-
brassinon was identical to rutalexin isolated from rutabaga.
Therefore thiazino[6,5-b]indole derivative 4 is not a natural
product and phytoalexin 8, produced by kohlrabi and rutabaga,
named rutalexin is the correct structure.5
In the present Letter we report the synthetic relationship
between synthetic thiazino[6,5-b]indole derivative 4 and the natu-
ral product rutalexin (8) by removing the methyl group from the
oxygen and introducing it to the imide-type nitrogen atom of the
3
e
11
lammonium tribromide. Next, 9-Boc-cyclobrassinin (9) was
prepared by the reaction of cyclobrassinin (3a) with Boc anhydride
in 88% yield. Oxidation of compound 9 using PCC gave of 9-Boc-
1,3-thiazino[6,5-b]indole-2,4-dione (6) in moderate yields (66%,
Scheme 2).
The antiproliferative activity of natural phytoalexins rutalexin
(8) and cyclobrassinin (3a) as well as 9H-2-methoxy-4-oxo-
[1,3]thiazino[6,5-b]indole (4) and 9-Boc-cyclobrassinin (9) was
tested on selected human cancer cell lines; Jurkat (acute T-lym-
phoblastic leukemia), MCF-7 and MDA-MB-231 (mammary gland
adenocarcinomas), HeLa (cervical adenocarcinoma), CCRF-CEM
(acute T-lymphoblastic leukemia) and A-549 (non-small cell lung
cancer). Results of this investigation are shown in Table 1, which
also includes IC50 values for conventional anticancer agents etopo-
side and cisplatin for comparison. The highest antiproliferative
1
,3-thiazine ring. In the first step 9-Boc-derivative 5, which was
4
b
prepared according to the previously reported procedure, was
selectively hydrolyzed to 9-Boc-1,3-thiazino[6,5-b]indole-2,
8
4
-dione (6, Scheme 2). Next, the methylation of nitrogen was exam-
ined using literature conditions for related compounds. Methyl
6
a,b
iodide in alcoholic sodium hydroxide,
dimethyl sulfate in
6
c
6e
aqueous or methanolic sodium hydroxide and diazomethane in
6
a,d
diethyl ether
were all not suitable for methylation of 6 due to
its low solubility and hydrolysis in strongly basic media.
It was found, that when using DBU (1,8-diazabicyclo[5.4.0]un-
dec-7-ene) as a base and methyl iodide as an electrophile, the
effect was noted with cyclobrassinin (3a) where measured IC50 val-
À1
ues of 26.2–72
lmol  L were obtained depending on the cell
line, with leukemic cells being the most sensitive. Rutalexin (8)

M. Budovská et al. / Tetrahedron Letters 56 (2015) 3945–3947
3947
exhibited selective antiproliferative activity against the MCF-7
breast cancer cell line and synthetic 9H-2-methoxy-4-oxo-[1,3]thi-
azino[6,5-b]indole (4) and 9-Boc-cyclobrassinin (9) demonstrated
moderate antiproliferative activity on Jurkat, CEM, and HeLa cancer
cell lines. Based on our results we postulate that the presence of
keto groups in both positions 2 and 4 and the methyl group on
the thiazine-2,4-dione nitrogen is responsible for the selective
effect of rutalexin (8) on MCF cells.
In summary, we have developed two novel, efficient methods
for the preparation of the indole phytoalexin rutalexin (8) starting
from 9-tert-butoxycarbonyl-2-methoxy-4-oxo-[1,3]thiazino[6,5-
b]indole (5) and 9-Boc-cyclobrassinin (9). The advantages of the
present methods in terms of facile manipulation, high yields,
simple methodology, and simple work-up procedures, should
make this protocol a valuable alternative to the existing method.
(c) Mehta, R. G.; Liu, J.; Constantinou, A.; Thomas, C. F.; Hawthorne, M.; You, M.;
Gerhäuser, C.; Pezzuto, J. M.; Moon, R. C.; Moriarty, R. M. Carcinogenesis 1995,
1
6, 399–404; (d) Kutschy, P.; Such y´ , M.; Monde, K.; Harada, N.; Marušková, R.;
Curillová, Z.; Dzurilla, M.; Miklošová, M.; Mezencev, R.; Moj zˇ iš, J. Tetrahedron
Lett. 2002, 43, 9489–9492; (e) Csomós, P.; Fodor, L.; Sohár, P.; Bernáth, G.
Tetrahedron 2005, 61, 9257–9262.
ˇ
4
.
(a) Gross, D.; Porzel, A.; Schmidt, J. Z. Naturforsch., C 1994, 49, 281–285; (b)
Such y´ , M.; Kutschy, P.; Dzurilla, M.; Ková cˇ ik, V.; Andreani, A.; Alföldi, J.
Tetrahedron Lett. 2001, 42, 6961–6963; (c) Kutschy, P.; Such y´ , M.; Andreani, A.;
Dzurilla, M.; Rossi, M. Tetrahedron Lett. 2001, 42, 9281–9283; (d) Kutschy, P.;
Such y´ , M.; Andreani, A.; Dzurilla, M.; Ková cˇ ik, V.; Alföldi, J.; Rossi, M.;
Gramatová, M. Tetrahedron 2002, 58, 9029–9039; (e) Kutschy, P.; Imrich, J.;
Bernát, J. Synthesis 1983, 929–931; (f) Kutschy, P.; Imrich, J.; Bernát, J.; Kristian,
P.; Fedoriková, I. Collect. Czech. Chem. Commun. 1986, 51, 2002–2012.
Pedras, M. S. C.; Montaut, S.; Such y´ , M. J. Org. Chem. 2004, 69, 4471–4476.
(a) Kretov, A. E.; Bespalyi, A. S. J. Gen. Chem. USSR (Engl. Transl.) 1963, 33, 206–
209; (b) Kretov, A. E.; Bespalyi, A. S. J. Gen. Chem. USSR (Engl. Transl.) 1964, 34,
5
6
.
.
991–993; (c) Warrener, R. N.; Cain, E. N. Tetrahedron Lett. 1966, 7, 3225–3229;
(
d) Winterfeldt, E.; Nelke, J. M. Chem. Ber. 1967, 100, 3671–3678; (e) Cain, E. N.;
Warrener, R. N. Aust. J. Chem. 1970, 23, 51–72.
Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.
7.
Acknowledgements
8. Data for 6: mp 283–285 °C (THF/n-hexane); Found: C, 56.44; H, 4.61; N, 8.55.
C
1
15
H
14
N
2
O
4
S requires C, 56.59; H, 4.43; N, 8.80;
m
max (KBr) 1723, 1680, 1647,
2
) 12.43 (1H, s, NH-D O
À1
440, 1347, 1213, 1140 cm ; d
H
(400 MHz, DMSO-d
6
We would like to thank the Slovak Grant Agency for Science
exchangeable), 8.19 (1H, dd, J = 2.2, 6.9 Hz), 8.06 (1H, dd, J = 7.26, 1.5 Hz), 7.45
(1H, ddd, J = 7.4, 7.2, 1.8 Hz), 7.41 (1H, ddd, J = 7.3, 6.9, 1.3 Hz), 1.70 (9H, s,
(
Grant Nos. 1/0954/12 and 1/0322/14) for financial support of this
C(CH
25.4, 124.6, 120.0, 114.9, 106.3, 87.3, 27.5; EIMS: m/z (%): 318 (M , 5), 262
26), 218 (30), 175 (100), 146 (40), 120 (42), 57 (60).
16 2 4
Data for 7: mp 310–311 °C; Found: C, 58.01; H, 4.62; N, 8.62. C16H N O S
3 3 C 6
) ); d (100 MHz, DMSO-d ) 164.2, 160.9, 148.9, 138.9, 135.5, 125.9,
work.
+
1
(
9.
Supplementary data
requires C, 57.82; H, 4.85; N, 8.43;
m
max (KBr) 1720, 1647, 1567, 1347,
À1
1140 cm
;
d
H
(400 MHz, DMSO-d
6
)
8.26 (1H, d, J = 7.6 Hz), 8.09 (1H, d,
Supplementary data (experimental procedures and characteri-
J = 7.9 Hz), 7.53-7.44 (2H, m), 3.38 (3H, s, CH ), 1.71 (9H, s, C(CH ) ); EIMS: m/z
3
3
3
1
13
+
zation data for compounds 6–9 and copies of H and C NMR spec-
tra for compounds 4 and 8) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.
(%): 332 (M , 5), 276 (26), 232 (30), 175 (100), 146 (40), 120 (35), 57 (67).
8 2 2
0. Data for 8: mp 310–311 °C; Found: C, 56.84; H, 3.45; 12.03. C11H N O S
1
requires C, 56.88; H, 3.47; N, 12.06;
m
max (KBr) 3215, 2925, 1728, 1668, 1633,
12.54 (1H, br s, NH-D
); d
6
) 162.8, 159.9, 136.9, 135.0, 125.1, 123.6, 122.1, 119.5,
À1
1463, 1232 cm
;
d
H
(400 MHz, DMSO-d
6
)
2
O
C
2
015.05.001.
exchangeable), 8.09 (1H, m), 7.53 (1H, m), 7.28 (2H, m), 3.37 (3H, s, CH
100 MHz, DMSO-d
11.8, 101.4, 28.2; EIMS: m/z (%): 232 (M , 26), 175 (100), 147 (18), 120 (21).
(400 MHz, CDCl ) 8.57 (1H, br s, NH-D O exchangeable), 8.31 (1H, m), 7.38
3H, m), 3.55 (3H, s, CH ); Data published for 8 (Ref. 5) d (500 MHz, DMSO-d
2.57 (1H, br s, NH-D O exchangeable), 8.09 (1H, m), 7.54 (1H, m), 7.28 (2H,
m), 3.37 (3H, s, CH ); d (125 MHz, DMSO-d ) 162.8, 160.0, 136.9, 135.0, 125.1,
23.6, 122.1, 119.5, 111.8, 101.5, 28.2; d (500 MHz, CDCl ) 8.52 (1H, br s, NH-
O exchangeable), 8.28 (1H, m), 7.40 (3H, m), 3.57 (3H, s, CH ); Data
3
(
1
+
d
H
3
2
References and notes
(
3
H
6
)
1
2
1
2
.
.
Purkayastha, R. P. In Handbook of Phytoalexin Metabolism and Action; Daniel, M.,
Purkayastha, R. P., Eds.; Progress in Phytoalexin Research During the Past 50
Years; Marcel Dekker: New York, 1995; pp 1–39.
For a recent reviews on cruciferous indole phytoalexins see: (a) Pedras, M. S. C.;
Okanga, F. I.; Zaharia, I. L.; Khan, A. Q. Phytochemistry 2000, 53, 161–176; (b)
Pedras, M. S. C.; Jha, M.; Ahiahonu, P. W. K. Curr. Org. Chem. 2003, 7, 1635–
3
C
6
1
H
3
D
2
3
published for 8 (Ref. 4a) d
H
(CDCl
3
) 8.56 (1H, s), 8.31 (1H, m), 7.41 (1H, m),
7
.37–7.32 (2H, m), 3.55 (3H, s, CH
3
).
11. Data for 9: mp 92–93 °C (dichloromethane/n-hexane); Found: C, 57.33; H, 5.41;
1
647; (c) Mezencev, R.; Moj zˇ iš, J.; Pilátová, M.; Kutschy, P. Neoplasma 2003, 50,
N, 8.42. C16
H
18
N
2
O
2
S
2
requires C, 57.46; H, 5.42; N, 8.38;
m
max (KBr) 3049, 2978,
) 8.07 (1H,
2
39–245; (d) Pedras, M. S. C.; Yaya, E. E.; Glawischnig, E. Nat. Prod. Rep. 2011,
À1
2840, 1716, 1626, 1448, 1364, 1249, 1109 cm ; d
H
(400 MHz, CDCl
3
2
8, 1381–1405; (e) Pilátová, M.; Šarišsk y´ , M.; Kutschy, P.; Miroššay, A.;
d, J = 7.5 Hz, H-8), 7.41–7.38 (1H, m, H-5), 7.29–7.22 (2H, m, H-7, H-6), 5.01
2H, s, CH ), 2.53 (3H, s, SCH ), 1.70 (9H, s, C(CH ); d (100 MHz, CDCl ) 155.5,
49.7, 136.4, 127.6, 124.3, 124.0, 123.1, 117.0, 115.0, 108.4, 85.5, 47.8, 28.2,
Mezencev, R.; Cˇ urillová, Z.; Such y´ , M.; Monde, K.; Miroššay, L.; Moj zˇ iš, J.
(
2
3
)
3 3
C
3
Leukemia Res. 2005, 29, 415–421; (f) Mehta, R. G.; Liu, J.; Constantinou, A.;
Hawthorne, M.; Pezzuto, J. M.; Moon, R. C.; Moriarty, R. M. Anticancer Res. 1994,
1
1
+
5.1; EIMS: m/z (%): 334 (M , 24), 161 (100).
1
4, 1209–1213.
(a) Takasugi, M.; Monde, K.; Katsui, N.; Shirata, A. Bull. Chem. Soc. Jpn. 1988, 61,
85–289; (b) Pedras, M. S. C.; Zaharia, I. L. Phytochemistry 2000, 55, 213–216;
3
.
2