Synthesis of camalexin

Article abstract of DOI:10.3390/60900716

In this paper we describe a new method for the synthesis of camalexin (1) based on the reaction of 1-(tert-butoxycarbonyl)indole-3-carboxaldehyde with methyl L-cysteinate hydrochloride, followed by oxidation and decarboxylation. Compounds 1, and intermediates 5-7 were identified by elemental analysis, ¹H NMR, ¹³C NMR and mass spectroscopy.

Full text of DOI:10.3390/60900716

Molecules 2001, 6, 716-720
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis of Camalexin
M. Dzurilla*1, P. Kutschy1, J. Zaletova1, M. Ruzinsky1 and V. Kovacik2
1 Department of Organic Chemistry, Faculty of Natural Sciences, P.J. Šafárik University,
Moyzesova 11, 041 67 Košice, Slovak Republic.
E-mail: dzurilla@kosice.upjs.sk, kutschy@kosice.upjs.sk, zaletova@pobox.sk
2 Institute of chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 842 38 Bratislava,
Slovak Republic. E-mail: chemvkov@savba.sk
*Author to whom correspondence should be addressed; E-mail: dzurilla@kosice.upjs.sk
Received: 6 April 2001; in revised form 13 August 2001 / Accepted: 14 August 2001 / Published: 31
August 2001
Abstract.- In this paper we describe a new method for the synthesis of camalexin (1)
based on the reaction of 1-(tert-butoxycarbonyl)indole-3-carboxaldehyde with methyl L-
cysteinate hydrochloride, followed by oxidation and decarboxylation. Compounds 1, and
1
intermediates 5-7 were identified by elemental analysis, H NMR, 13C NMR and mass
spectroscopy.
Keywords: Camalexin, phytoalexins, indoles
Introduction
Camalexin [3-(2’-thiazolyl)indole] (1) is a natural phytoalexin, isolated for the first time from the
leaves of Camelina sativa and elicited by the fungus Alternaria brassicae [1]. Camalexin is also the
principal phytoalexin found in Arabidopsis thaliana [2]. It exhibits antifungal activity similar to the
systemic fungicide thiabendazole (2) [1,3] and also has antitumor activity [4]. In the literature there are
described four methods for synthesis of camalexin, based on the reaction of indolylmagnesium iodide
with 2-bromothiazole [3], heating of indole-3-carboxamide with P2S5 and chloroacetaldehyde diethyl
acetal in ethanol [5], reductive cyclization of 2-formamidophenyl-2´-thiazolylketone upon heating with

Molecules 2001, 6
717
TiCl3 and zinc dust [6] and reaction of 1-sulfonyl-3-iodoindole with active zinc and following Pd
catalyzed arylation with 2-iodothiazole [7]. Recently, it has been suggested that the biosynthesis of
camalexin involves the condensation of indole-3-carboxaldehyde with cysteine followed by a two-step
oxidation and decarboxylation [8,9]. In the presence work we have studied the synthesis of camalexin
according to this biosynthetic scheme.
S
N
N
N
N
H
N
H
S
Camalexin 1
Thiabendazole 2
Results and Discussion
As the first step in the investigated synthesis of camalexin, we have examined the
cyclocondensation of indole-3-carboxaldehyde with methyl L-cysteinate. The product of this reaction
appeared to be unstable and therefore it was decided to use the 1-Boc protected aldehyde 3. The
cyclocondensation of 1-(tert-butoxycarbonyl)indole-3-carboxaldehyde with methyl L-cysteinate (4)
hydrochloride afforded 4´-methoxycarbonyl-1-(tert-butoxycarbonyl)-3-thiazolidine-2´-yl)indole (5) as
a mixture of diastereoisomeres in 85% yield (Scheme 1). The ratio of diastereoisomers was determined
1
to be 57:43 by integration of the signals of proton H-2´ at δ= 5,78 and 6,01 ppm in the H- NMR
spectrum of the crude product.
Scheme 1
COOCH3
COOCH3
c
S
S
COOCH3
CHO
+
N
H
H2N
HS
N
b
a
N
N
N
Boc
Boc
Boc
3
5
4
,
6
,
5
,
1
4
COOCH3
S
S
,
2
4
N
N
,
3
5
3
d
2
6
N
H 1
N
H
7
1
7
a) 1:2 methanol/benzene, (C2H5)3N, 25o C, 3 h. (85%); b) MnO2, benzene/pyridine, 55o C, 1.5 h.,
(44%); c) CH3ONa, methanol, 25o C, 20 min.(59%); d) NaOH, NaHCO3, 25o C, 2 h., (12%).

Molecules 2001, 6
718
Oxidation of thiazolidine 5 to thiazole 6 by oxidizing reagents such as pyridinium chlorochromate
(PCC), p-chloranil, 2,3-dichloro-5,6-dicyano-p-benzochinone (DDQ) and FeCl3 lead to decomposition
products. The desired oxidation was achived by using 25 equivalents of activated MnO2 in dry benzene
[10]. Elimination of the tert-butoxycarbonyl protective group was realized using 16 equivalents of
sodium methoxide in methanol at ambient temperature. Subsequent hydrolysis and decarboxylation of
the resulting 4´-methoxycarbonyl-3-(thiazole-2´-yl)indole (7) with an aqueous solution of NaOH and
NaHCO3 gave camalexin (1) in 12% yield. The spectral data and melting point of 1 are identical with
the literature data [1,3,6].
Conclusions
In this contribution we report a biomimethic synthesis of camalexin (1) according to the proposed
biosynthetic scheme. The formation of the thiazole ring involves only one oxidation step followed by
decarboxylation to camalexin.
Acknowledgments
We thank the Grant Agency for Science of the Slovak Republic (grant No. 1/6080/99) for financial
support of this work.
Experimental
General
Melting points were measured on a Koffler hot stage apparatus and are uncorrected. Purity of
compounds was confirmed by elemental analysis on a Perkin-Elmer, model 2400 analyzer. The
reaction course was monitored by TLC on Silufol (Kavalier) and Alumina 60 F
neutral (Merck)
254
TLC plates. Preparative column chromatography was performed on Kavalier 40/100 µm silica gel and
Merck Kieselgel 60 F25. The infrared absorption spectra of compounds 1, and 5-7 were measured in
CHCl3 on an IR75 (Zeiss Jena) spectrometer in the region 400-4000 cm-1. The 1H-NMR spectrum of 5
was measured on a TESLA BS 487 A (80 MHz), 1H- and 13C-NMR spectra of compounds 1, 6, 7 on a
Varian Gemini 2000 (300 MHz) in deuterochloroform, using tetramethylsilane as an internal standard.
The electron impact mass spectra of 7 were recorded on a Finnigan SSQ 700 spectrometer at an
ionization energy of 70 eV. Methyl L-cysteinate hydrochloride, pyridinium chlorochromate (PCC), p-
chloranil and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) from Fluka, Merck and Avocado were
used as obtained without further purification. 1-(tert-Butoxycarbonyl)indole-3-carboxaldehyde (3) was
prepared according to the described procedure [11].

Molecules 2001, 6
719
1-(tert-Butoxycarbonyl)-4´-methoxycarbonyl-3-(thiazolidin-2´-yl)indole (5).
To a suspension of methyl L-cysteinate hydrochloride (595 mg, 3.47 mmol) in 1:2
methanol/benzene (6mL) was added 1-(tert-butoxycarbonyl)indole-3-carboxaldehyde (490 mg, 2
mmol) and triethylamine (672 mg, 6.6 mmol). The reaction mixture was stirred for 3 hours at room
temperature, the solvent was evaporated and the oily residue purified by column chromatography, using
a mixture of cyclohexane/acetone (2:1) as eluent. Yield 615 mg (85%), yellow oil; For C18H22N2O4S
(362.50) calculated: 59.65% C, 6.12% H, 7.73% N; found: 59.60% C, 6.07% H, 7.65% N; IR: 3323
1
(NH), 1720 a 1730 (C=O); H-NMR (ppm): 1,66 s, 9H [(CH3)3], 2,96 s 1H (NH), 3,09-4,33 m, 3H
(SCH2CH), 3,79 s, 3H (OCH3), 5,78 s a 6,01 s 57:43, 1H (CH) , 7,13-8,22 m, 5H (H-arom.).
1-(tert-Butoxycarbonyl)-4´-methoxycarbonyl-3-(thiazol-2´-yl) indole (6).
To a stirred suspension of activated MnO2 [10] (1000 mg, 11.5 mmol) in a mixture of dry benzene
(10mL) and pyridine (0.050 mL) was added a solution of thiazolidine 5 ( 400 mg, 1.10 mmol) in dry
benzene (2 mL). The reaction mixture was stirred for 1.5 hour at 55o C. After cooling the insoluble
material was removed by filtration, washed with benzene, the solvent was evaporated and the solid
residue crystallized from a mixture of diethylether/hexane. Yield 175 mg (44%), M.p. 128-130o C; For
C18H18N2O4S (359.48) calculated: 60.14% C, 5.05% H, 7.79% N; found: 60.23% C, 5.21% H, 7.29%
N; IR: 3323 (NH), 1720 a 1730 (C=O); 1H-NMR (ppm): 1,66 s, 9H [(CH3)3], 3.79 s, 3H (OCH3), 8,10
s, 1H (H-2), 8,25 s, 1H (H-5´), 7.13-8,22 m, 4H (H-arom.).
4´-Methoxycarbonyl-3-( thiazol-2´-yl) indole (7).
To a suspension of thiazole 6 (150 mg, 0.42 mmol) in dry methanol (12mL) was added sodium
methoxide (330 mg, 6.11 mmol) during 5 min. The reaction mixture was poured into cold water (60
mL), extracted with chloroform (3x10 mL), dried over Na2SO4, the solvent evaporated and the product
crystallized from a mixture of diethylether/hexane. Yield 64 mg (59%), M.p. 166-168o C; For
C13H10N2O2S (259.50) calculated: 60.17% C, 3.88% H, 10.80% N; found: 60.28% C, 3.99% H, 10.95%
1
N; IR: 3200 (NH), 1720 a 1730 (C=O); H-NMR (ppm): 3,79 s, 3H (OCH3), 8.10 d, 1H (H-2), J=2,55
13
Hz, 8,25 s, 1H (H-5´), 7.13-8.22 m, 4H (H-arom.), 10,85 s, 1H (NH). C NMR (ppm): 52,32 (CH3),
113.05, 121.66, 122.04, 123.83, 125.50, 125.72, 127.41, 127.58, 137.99, 147.78 (C arom.), 162.61
(C=N), 164.75 (C=O). MS, m/z (%), : 258 (M+, 100), 200 (24), 160 (24), 142 (24), 115 (12), 57 (12).
Camalexin (1).
To a solution of NaOH (22.0 mg, 5.6 mmol) in water (2 mL) was added a solution of thiazole 7
(120 mg, 0.46 mmol) in methanol (2 mL) and the reaction mixture was refluxed for 1 hour. After
cooling and evaporation of the methanol, NaHCO3 (660 mg, 7.86 mmol) was added and the reaction
mixture was refluxed for 1 hour. The product separated after cooling and was collected on filter paper
and dried. Crystallization from a mixture of diethylether/hexane yielded 10 mg (12%); M.p. 140-141o

Molecules 2001, 6
720
C; For C11H8N2S (200.10) calculated: 66.00% C, 4.00% H, 14.00% N; found: 65.80% C, 4.00% H,
1
13.50% N; IR, H-, 13C-NMR and mass spectra were identical with previously described data for
camalexin [1, 3, 6].
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Sample availability: Samples of compounds 1, 5 and 6 are available from the authors.
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