Concise syntheses of the cruciferous phytoalexins brassilexin, sinalexin, wasalexins, and analogues: Expanding the scope of the Vilsmeier formylation

Article abstract of DOI:10.1021/jo0479866

(Chemical Equation Presented) Efficient syntheses of the phytoalexins brassilexin, sinalexin, and analogues are demonstrated through the application of the Vilsmeier formylation to indoline-2-thiones followed by a new aqueous ammonia workup procedure. Similarly, a very concise two-pot synthesis of the phytoalexins wasalexins using sequential formylation-amination of indolin-2-ones is described. Remarkably, this novel aqueous ammonia workup allows the sequential one-pot formylation-amination, expanding substantially the scope of the Vilsmeier formylation of both indoline-2-thiones and indolin-2-ones. The examination of the formylation-amination reaction and optimization of conditions, as well as the syntheses and antifungal activities of several brassilexin analogues, are reported.

Full text of DOI:10.1021/jo0479866

Concise Syntheses of the Cruciferous Phytoalexins Brassilexin,
Sinalexin, Wasalexins, and Analogues: Expanding the Scope of the
Vilsmeier Formylation
M. Soledade C. Pedras* and Mukund Jha
Department of Chemistry, University of Saskatchewan, 110 Science Place,
Saskatoon SK S7N 5C9, Canada
soledade.pedras@usask.ca
Received November 12, 2004
Efficient syntheses of the phytoalexins brassilexin, sinalexin, and analogues are demonstrated
through the application of the Vilsmeier formylation to indoline-2-thiones followed by a new aqueous
ammonia workup procedure. Similarly, a very concise two-pot synthesis of the phytoalexins
wasalexins using sequential formylation-amination of indolin-2-ones is described. Remarkably,
this novel aqueous ammonia workup allows the sequential one-pot formylation-amination,
expanding substantially the scope of the Vilsmeier formylation of both indoline-2-thiones and indolin-
2-ones. The examination of the formylation-amination reaction and optimization of conditions, as
well as the syntheses and antifungal activities of several brassilexin analogues, are reported.
SCHEME 1. Vilsmeier-Haack Reaction1
Introduction
Since the first publication appeared in 1925, the
Vilsmeier-Haack reaction has been applied to an im-
mense variety of substrates, from substituted benzenes
to complex heterocycles.1 The Vilsmeier reagent, an
iminium salt with weak electrophilic character, results
from the reaction between an acid chloride (e.g., POCl3,
SOCl2, (COCl)2, COCl2) and an amide, usually DMF
(Scheme 1, eq 1). Further reaction of the Vilsmeier
reagent with a reactive aromatic2 substrate followed by
basic workup affords, in general, acylation products via
electrophilic aromatic substitution (Scheme 1, eq 2).1 If
DMF is used in the reaction, the product of the Vilsmeier
reaction is an aldehyde, thus the Vilsmeier reaction is
often called Vilsmeier formylation.
Among the annulated furans, thiophenes and pyrroles,
indoles display the highest reactivity. In general, the
regiochemistry of the Vilsmeier reaction of indoles (e.g.,
1) is quite predictable, occurring at the 3-positon, unless
this position is substituted, as for example in skatole (2)
(Scheme 2, eqs 3 and 4).1 Furthermore, substrates such
as indolin-2-ones (6-8) with lower reactivity than indole
(1) have been formylated using the Vilsmeier reagent to
afford the corresponding 2-chloro-3-formyl indoles (11)
(Scheme 2, eq 5).1 Until recently, indoline-2-thiones (9)
* Address correspondence to this author. Phone: (306)966-4772.
Fax: (306)966-4730.
(1) (a) Jones, G.; Stanforth, S. P. In Organic Reactions; Paquette L.
A., Ed.; Wiley: New York, 1997; Vol. 49, pp 1-330. (b) Downie, I. M.;
Earle, M. J.; Heaney, H.; Shuhaibar, K. F. Tetrahedron 1993, 49, 4015.
(2) The Vilsmeier formylation has also been applied to alkenes,
acetals, and ketals, see: Smith, M. B.; March, J. Advanced Organic
Chemistry, 5th ed.; J. Wiley & Sons: New York, 2001; p 785 and
references therein.
10.1021/jo0479866 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/03/2005
1828
J. Org. Chem. 2005, 70, 1828-1834

Brassilexin, Sinalexin, Wasalexins, and Analogues
as brassilexin (12),8 sinalexin (13), brassicanate A (14),9
brassicanal A (15), spirobrassinins (16), wasalexins (17),
and cyclobrassinin (18).5 Phytoalexins are secondary
metabolites biosynthesized de novo by plants in response
to diverse forms of stress, including pathogen attack.10
Brassilexin (12) and sinalexin (13) are among the most
potent phytoalexins produced by economically important
cruciferous plants. Sinalexin (13) appears to be involved
in defense mechanisms of white mustard (Sinapis alba),11
whereas brassilexin (12) and wasalexins (17) have simi-
lar roles in brown mustard (Brassica juncea)12 and wasabi
(Wasabia japonica),13 respectively. To investigate phy-
toalexin metabolic pathways in plants and their fungal
pathogens, isotopically labeled phytoalexins are required,
hence it is crucial to design concise and efficient synthe-
ses.
SCHEME 2. Vilsmeier Formylation of Indoles
(Eqs 3 and 4) and Indolin-2-ones (Eq 5)1
Toward this end, we investigated conditions for the
Vilsmeier formylation of indoline-2-thiones (9, 23a-j)
and indolin-2-ones (6-7b). Remarkably, the Vilsmeier
formylation of these substrates followed by a new aque-
ous ammonia workup was shown to have a wider scope
than ever reported, affording the respective 3-(amino)-
methyleneindoline-2-thiones and 3-(amino)methylenein-
dolin-2-ones directly in reasonable yields. Thus, our
aqueous ammonia workup expanded the scope of the
Vilsmeier formylation and allowed for the first time a
very concise two-pot synthesis of wasalexins (17) and
several new brassilexin analogues. Finally, the antifungal
activity of brassilexin (12), sinalexin (13), and analogues
24a-j was determined and is reported.
SCHEME 3. Application of the Vilsmeier
Formylation to the Synthesis of Brassilexin (12)
and Sinalexin (13)
had not been formylated under Vilsmeier conditions,3 but
2-thioethers such as 2-thiomethylindole have been formyl-
ated in good yield.4 Despite the lack of precedent, the
most efficient synthesis of sinalexin (13) used the Vils-
meier formylation of 1-methoxyindoline-2-thione (10)
followed by a new aqueous ammonia workup procedure
(Scheme 3). Similarly, although several syntheses of
brassilexin (12) have been published to date,5 the most
efficient preparation was also accomplished under Vils-
meier conditions (Scheme 3).3
Our interest in the formylation of indolin-2-ones (6, 7)6
and indoline-2-thiones (9, 10)7 derives from the potential
application of these substrates to the synthesis of tryp-
tophan-derived alkaloids, in particular phytoalexins such
Results and Discussion
The most efficient preparation of brassilexin (12) is a
two-pot synthesis,3 starting with a Vilsmeier formylation
of readily available indoline-2-thione (9)14 and an un-
precedented aqueous ammonia workup, followed by
quantitative oxidation of 3-(amino)methyleneindoline-2-
(8) Pedras, M. S. C.; Okanga, F. I. J. Agric. Food Chem. 1999, 47,
1196.
(9) Pedras, M. S. C.; Montaut, S.; Suchy, M. J. Org. Chem. 2004,
69, 4471.
(3) Pedras, M. S. C.; Zaharia, I. L. Org. Lett. 2001, 3, 1213.
(4) Pedras, M. S. C.; Khan, A. Q. J. Agric. Food Chem. 1996, 44,
3403.
(5) For recent reviews on cruciferous phytoalexins see: (a) Pedras,
M. S. C.; Okanga, F. I.; Zaharia, I. L.; Khan, A. Q. Phytochemistry
2000, 53, 161. (b) Pedras, M. S. C.; Jha, M.; Ahiahonu, P. W. K. Curr.
Org. Chem. 2003, 7, 1635.
(6) Indolin-2-ones have been used to prepare of GABAergic agents;
see, for example: Sarges, R.; Howard, H. R.; Koe, B. K.; Weissman, A.
J. Med. Chem. 1989, 32, 437.
(7) Indoline-2-thiones are also useful in the preparation of tyrosine
kinase inhibitors, i.e., the corresponding 3-substituted 2,2′-dithiobis-
(1H-indoles); see, for example: Palmer, B. D.; Rewcastle, G. W.;
Thompson, A. M.; Boyd, M.; Showalter, H. D. H.; Sercel, A. D.; Fry, D.
W.; Kraker, A. J.; Denny, W. A. J. Med. Chem. 1995, 38, 58.
(10) (a) Bailey, J. A.; Mansfield, J. W., Eds. Phytoalexins; Blackie
& Son: Glasgow, UK, 1982; p 334. (b) Brooks, C. J. W.; Watson, D. G.
Nat. Prod. Rep. 1985, 427.
(11) Pedras, M. S. C.; Zaharia, I. L.; Gai, Y.; Zhou, Y.; Ward, D. E.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 747.
(12) (a) Rouxel, T.; Kollmann, A.; Boulidard, L.; Mithen, R. Planta
1991, 184, 271. (b) Pedras, M. S. C.; Nycholat, C. M.; Montaut, S.; Xu,
Y.; Khan, A. Q. Phytochemistry 2002, 59, 611.
(13) Pedras, M. S. C.; Chumala, P. B.; Suchy, M. Phytochemistry
2003, 64, 949.
(14) Indoline-2-thione was prepared following a general procedure
for conversion of the carbonyl group to the thione group: Scheeren, J.
W.; Ooms, P. H. J.; Nivard, R. J. F. Synthesis 1973, 149.
J. Org. Chem, Vol. 70, No. 5, 2005 1829

Pedras and Jha
TABLE 1. Synthesis of Brassilexin (12) (50 mg scale)
% productsb
entry
POCl3 (equiv)
formylation conditionsa (in DMF)
brassilexin (12)
20
1
2
3
4
5
6
7
8
0.5
1
rt, 60 min
0
93c
rt, 60 min
30c
60
2
rt, 60 min
60c
30
0.5
1
0 °C, 7 h
20 (72)d
56 (55)d
80 (48)d
54c
<5
<5
<5
35c
<5
0 °C, 7 h
2
0 °C, 7 h
1
40-50 °C, 25 min
40-50 °C, 25 min
2
71c
a Ammonia workup at 0 °C. b Percentage yields are averages of at least four reactions. c No recovery of starting material. d Based on
recovered starting material, values in parentheses represent % of recovered material. Similar yields were obtained using 100-200 mg of
9.
thione (19)15 to brassilexin (12) using I2 in pyridine
(Scheme 3). Because the overall yield of this synthesis is
determined by the yield of the Vilsmeier formylation,3 it
was of interest to investigate the Vilsmeier reaction
conditions used to formylate indoline-2-thione (9). Three
procedures were used: the thione 9 dissolved in DMF
(50 mg scale) was added slowly by syringe to the
Vilsmeier reagent (POCl3/DMF) prepared in situ, or the
solid thione was added by cannula, or the Vilsmeier
reagent dissolved in CHCl3 was added by syringe to a
DMF solution of the thione. The reaction mixture was
monitored by TLC and after the thione 9 was consumed,
ice-cold ammonium hydroxide (excess) was added, fol-
lowed by extraction. Direct addition of the Vilsmeier
reagent to the thione resulted in a lower yield of 19 than
adding the thione to the Vilsmeier reagent, whereas the
yields were similar whether the thione was added in
solution or as a solid. When the solvent was changed from
DMF to CHCl3 and the formylating agent was oxalyl
chloride/DMF the product yields were ca. 15% lower.
Eventually, we settled on the procedure technically easier
to carry out, addition of the solid thione 9 by cannula to
POCl3/DMF, followed by aqueous ammonia workup.
Subsequently, to optimize the formylation yields, the
conditions for the Vilsmeier reaction were modified as
summarized in Table 1. Importantly, it was determined
that the use of 0.5 equiv of POCl3 (with respect to the
thione 9) led to the exclusive formation of 5H-thiopyrano-
[2,3-b:6,5-b′]diindole (20) in 93% yield (Table 1, entry 1),
whereas the use of 1 equiv of POCl3 resulted in the
formation of both 20 and brassilexin (12) in 70% and 30%
yield, respectively (Table 1, entry 2). When the amount
of POCl3 was further increased (2 equiv), the yield of
brassilexin (12) increased to 60% and that of 20 was
reduced to 30% (Table 1, entry 3); however, an additional
increase of POCl3 did not improve the yield of brassilexin
(12). The temperature of the Vilsmeier reaction affected
differently the formation of enamine 19 (through its
precursor N,N-dimethylenamine 21) and 20. For ex-
ample, at 0 °C the rates of both reactions decreased
substantially, but the formation of 20 was slower than
that of 12 (Table 1, entries 4-6).16 The best yield of 12
was obtained within a temperature range of 40-50 °C
(Table 1, entries 7 and 8); reactions carried out at higher
temperatures (90-100 °C) resulted in the formation of
multiple side products. The reaction of indoline-2-thione
(9) with 2 equiv of POCl3 in DMF at 40-50 °C afforded
the best yield of brassilexin (12, 71%) (Table 1, entry 8).17
To determine if the aqueous ammonia workup was
promoting the formation of undesired products, the
Vilsmeier intermediate 3-(N,N-dimethylamino)methyl-
eneindoline-2-thione (21) was treated with an excess of
ammonia and extracted, the extract was concentrated to
dryness, and the residue was subjected to I2 oxidation to
yield brassilexin (12) in very good yield (88%).18 This
result confirmed that the ammonia workup did not
promote formation of 20 or other side products.
Next, we examined the pathway for the formation of
5H-thiopyrano[2,3-b:6,5-b′]diindole (20).3 A solution of
indoline-2-thione (9) and 3-(N,N-dimethylamino)meth-
yleneindoline-2-thione (21) in DMF was allowed to react
under acidic conditions at room temperature (HCl gas
bubbled in) to yield compound 20 in 77% yield. This result
together with those reported in Table 1 indicated that
20 results from condensation of the Vilsmeier adduct 21
with the thione/thiol 9/9a. The proposed stepwise forma-
tion is summarized in Scheme 4. The classic Vilsmeier
formylation step leading to 21 might be followed by
nucleophilic attack of ene-thiol 9a, leading to unstable
intermediates I1 and I2 or equivalents. This I2 intermedi-
ate could rapidly undergo rearomatization with elimina-
tion of H2S to yield the novel heteroaromatic 5H-
thiopyrano[2,3-b:6,5-b′]diindole (20). When aqueous
ammonia is used for workup of the Vilsmeier formylation,
dimethylamine 21 is transformed into enamine 19.19
After having improved the formylation-amination
yield of indoline-2-thione (9), we examined the Vilsmeier
(16) Although the results shown in Table 1 entry 6 represented an
improvement in the yield of 12, due to the amount of starting material
remaining in the reaction mixture, the additional chromatographic
separation required rendered this preparation less efficient.
(17) A number of undetermined products were formed including
2-chloroindole-3-carbaldeheyde (11) in ca. 10%.
(18) Compound 21 was synthesized using the conditions of entry 8,
Table 1 followed by water workup instead of ammonia.
(19) For complete spectroscopic data of 19, see ref 8.
(15) Enamine 19 was previously prepared by reduction of 2-mer-
captoindole-3-carboxaldehyde oxime using TiCl3/NaBH3CN, as reported
in ref 8; 19 oxidizes slowly to 12 (<5%, 24 h) on standing in CH2Cl2.
1830 J. Org. Chem., Vol. 70, No. 5, 2005

Brassilexin, Sinalexin, Wasalexins, and Analogues
was carried out using phosphorus pentasulfide14 in THF
to yield indoline-2-thiones 23f-i (Table 2, entries 6-9).
1-Methylindoline-2-thione (23j) and 1-methoxyindoline-
2-thione (10) were prepared by thiation of 1-methylin-
dolin-2-one (7) and 1-methoxyindolin-2-one (10, R ) OMe,
Y ) O).14
SCHEME 4. Proposed Stepwise Formation of
5H-Thiopyrano[2,3-b:6,5-b′]diindole (20)
As shown in Table 3, the presence of electron-
withdrawing or electron-donating substituents at C-5 or
C-6 did not appear to affect noticeably the reactivity of
the indoline-2-thiones toward the Vilsmeier formylation.
Similar to the formylation of indoline-2-thione (9), the
formylation of indoline-2-thiones 23a-j was completed
within 20-30 min at 40-50 °C (Table 3, entries 1-9).
However, in the case of thiones 23j (R ) Me, R1 ) R2 )
H) and 10 (R ) OMe, Y ) O), used for synthesis of
1-methylbrassilexin (24j) and sinalexin (13), respectively,
temperatures higher than 25 °C resulted in the formation
of multiple undetermined products, whereas at 25 °C the
formylation reactions proceeded in reasonable yields
(Table 3, entries 10 and 12). The pentacyclic products
25a-i were formed in every reaction except where
substitution at N - 1 of the starting material prevented
their formation (23j and 10). Products 25a-i were
characterized through their methyl derivatives due to
poor solubility of the parent compounds in NMR deuter-
ated solvents.23
After successful application of the Vilsmeier formyla-
tion to indoline-2-thiones, we have examined the formyl-
ation of indolin-2-ones, useful starting materials for the
synthesis of wasalexins (17) and analogues. Indolin-2-
ones 7,25 7a,26 and 7b27 (Table 4, entries 2-4) were
synthesized as previously reported. Similar to the formyl-
ation of thiones 23a-j, the Vilsmeier formylation of
indolin-2-ones was carried out in DMF, but using only
1.2 equiv of POCl3 (Table 4). After completion of the
reaction, the reaction mixture was cooled to 0 °C,
quenched with ice-cold aqueous ammonia, and allowed
to warm to room temperature under continuous stirring
to yield, after extraction, enamines 26a-c in good yields.
Enamine 26d was not formed, instead the acetyl group
was hydrolyzed during the ammonia workup to yield the
deprotected enamine 26a. Alternatively, the Vilsmeier
reaction mixture was quenched with ice-cold H2O and
extracted to yield dimethylenamines 27a-d (Table 4).28
Next, enamines 26a-c were allowed to react with NaH
and CS2 in THF, followed by treatment with MeI and
workup to yield directly wasalexins (17) and analogue
17a in good yields (Scheme 5). This new synthesis of
wasalexins is a concise and fast two-pot preparation
followed by one chromatographic separation, that affords
wasalexins in 68% overall yield. This procedure repre-
sents a substantial improvement to our previous synthe-
sis of wasalexins (17) from 1-methoxyindolin-2-one (7a),
which was carried out in four steps and 54% overall
yield.13 In addition, it is particularly worthy to note that
TABLE 2. Synthesis of 5- and 6-Substituted
Indoline-2-thiones 23a-j
starting
entry material product 23
R
R1
R2
yield (%)
1
2
22a
22b
22c
22d
22e
22f
22 g
22h
22i
7
a
b
c
d
e
f
H
OCH3
CH3
F
H
H
H
H
H
82
93
90
95
78
82
81
92
74
94
H
3
H
4
H
Cl
5
H
Br
H
6
H
OCH3
F
7
g
h
i
H
H
8
H
H
Cl
9
H
H
Br
10
j
CH3
H
H
formylation of substituted indoline-2-thiones. 5-Substi-
tuted indoline-2-thiones 23a-e were synthesized by
thiation14 of indolin-2-ones 22a-e (Table 2, entries 1-5),
which were prepared from the corresponding 5-substi-
tuted isatins using a modification of the Wolff-Kishner
reduction,20 as described in the Supporting Information.
6-Substituted indoline-2-thiones 23f-i were synthesized
in four steps starting from substituted R-halonitroben-
zenes, using an excess of dimethylmalonate in DMF and
t-BuOK.21-23 Thiation of the purified indolin-2-ones 22f-i
(23) Described in the Supporting Information.
(24) Compound 24a was previously prepared using a different
procedure and undisclosed yield; see: Tempete, C.; Devys, M.; Barbier,
M. Z. Naturforsch., C: J. Biosci. 1991, 46, 706.
(25) Crestini, C.; Saladino, R. Synth. Commun. 1994, 24, 2835.
(26) Thompson, M. E. J. Org. Chem. 1984, 49, 1700.
(27) Robertson, D. W.; Krushinski, J. H.; Kau, D. J. Labelled Compd.
Radiopharm. 1986, 23, 343.
(20) Huang, M. J. Am. Chem. Soc. 1949, 71, 3301.
(21) Goetz, F. J.; Hirsch, J. A.; Augustine, R. L. J. Org. Chem. 1983,
48, 2468.
(28) Compound 27d was prepared previously using Vilsmeier condi-
tions in 30% yield; see: Andreani, A.; Bonazzi, D.; Rambaldi, M.;
Mungiovino, G.; Greci, L. Farmaco 1978, 33, 781.
(22) Quallich, G. J.; Morrissey, P. M. Synthesis 1993, 51.
J. Org. Chem, Vol. 70, No. 5, 2005 1831

Pedras and Jha
TABLE 3. Syntheses of Brassilexin Analogues via Vilsmeier Formylation (50 mg scale)a
entry
starting material
main product
R
R1
R2
yield (%)
1
2
23a
23b
23c
23d
23e
23f
23g
23h
23i
23j
9
24a24
24b
24c
24d
24e
24f
H
H
H
H
H
H
H
H
H
OCH3
CH3
F
H
H
H
H
H
67
52
64
54
40
47
68
58
45
65
71
67
3
4
Cl
Br
H
5
6
OCH3
F
7
24g
24h
24i
H
8
H
Cl
9
H
Br
H
10a
11b
12a
24jb
12c
CH3
H
H
H
H
10
13b
OCH3
H
H
a Reactions were carried out at 40 °C, except for 23i and 10 which were carried out at 25° C. b Minor product 25 is not formed. c Minor
product 20 is formed.
TABLE 4. Synthesis of
de Not.), the so-called blackleg fungus, one of the major
fungal pathogens of cruciferous crops.30 As shown in
Table 5, the antifungal activity of the naturally occurring
compounds 12 and 13 against P. lingam/L. maculans is
quite similar. In general, the results suggest that the
position of the substituent has stronger influence on the
activity of the analogues than the nature of the substitu-
ent itself. For example, the 6-F, 6-Cl, and 6-Br derivatives
display stronger activity than the 5-substituted com-
pounds, except for the 5- and 6-OMe derivatives which
appear to display similar antifungal activity.
3-(Amino)methyleneindolin-2-ones 26a-c and
3-(N,N-Dimethylamino)methyleneindolin-2-ones 27a-d
via Vilsmeier Formylation (100 mg reaction scale)
productb of productb of NH3/
starting
entry material
formylation
conditionsa
H2O workup NH4OH workup
at 0 °C (%)
at 0 °C (%)
Conclusion
1
2
3
4
6
40 °C, 30 min
40 °C, 60 min
40 °C, 60 min
40 °C, 120 min
27a (88)
27b (81)
27c (86)
27d (85)
26a (82)
7
26b (83)
The unprecedented ammonia workup of the Vilsmeier
formylation reaction is crucial for the preparation of
primary imine/enamines of both methyleneindoline-2-
thiones and methyleneindolin-2-ones, because these prod-
ucts cannot be obtained directly from reactions of the
corresponding aldehydes with ammonia.31 Several ami-
nomethyleneindoline-2-thiones were prepared for the
first time using this method and were used to prepare
several new brassilexin analogues (24b-j). Furthermore,
the formylation-amination of indolin-2-ones allowed the
most concise synthesis of wasalexins (17). This process
could be used to prepare 5- or 6-substituted wasalexins.
Importantly, these procedures will allow more efficient
syntheses of isotopically labeled phytoalexins and inter-
mediates that have immediate application in the inves-
tigation of plant and fungal metabolic pathways.
The antifungal activity of brassilexin (12), sinalexin
(13), and analogues 24a-j toward the blackleg fungus
(L. maculans/P. lingam) was determined. Because this
fungus is able to detoxify brassilexin (12) via an efficient
but undetermined pathway,8 compounds 24a-j will be
valuable tools to probe this detoxification pathway as well
as the selectivity of enzyme(s) involved in it.
7a
7b
26c (86)
only 26a (80)
a 1.2 equiv of POCl3 in DMF. b Mixture of E/Z isomers.
SCHEME 5. Synthesis of Wasalexins (17) and
Analogue 17a
imines 26a-c were obtained directly in a one-pot syn-
thesis for the first time.29 Interestingly, formylation of
the N - 1 of 6 occurred under mild Vilsmeier conditions
to yield 27a, whereas ammonia workup yielded the
desired enamine 26a in good yield.
Antifungal Activity. To establish the antifungal
activity of the new phytoalexin analogues, bioassays were
carried out against Phoma lingam (Tode ex Fr.) Desm.
(perfect stage Leptosphaeria maculans (Desm.) Ces. et
(29) Enamines 26a-c exist in CH3CN solution as a mixture of E/Z
isomers (spectroscopic data in the Supporting Information).
(30) For recent work on this pathogen see, for example: Pedras, M.
S. C.; Chumala, P. B. Org. Lett. 2004,
1832 J. Org. Chem., Vol. 70, No. 5, 2005

Brassilexin, Sinalexin, Wasalexins, and Analogues
TABLE 5. Antifungal Activitya of Phytoalexins and
Analogues against Phoma lingam (perfect stage )
Leptosphaeria maculans) (5 days incubation)
identical with previously reported data.33 HRMS m/z measured
149.0298 (149.0299 calculated for C8H7NS).
Brassilexin (12). Indoline-2-thione (9, 50 mg, 0.33 mmol)
was added to a mixture of freshly distilled POCl3 (64 µL, 0.70
mmol) in DMF (350 µL) at 45 °C and the temperature of the
reaction mixture was kept at 45 °C. After 20 min, the reaction
mixture was cooled to 0 °C on an ice bath, and aq NH4OH
(29%, 8 mL) was added dropwise with constant stirring. The
resulting solution was allowed to warm to room temperature
and extracted with Et2O, and the organic extracts were
combined, dried over Na2SO4, and concentrated to dryness. The
crude product was dissolved in pyridine (1 mL) and I2 (42 mg,
0.33 mmol) was added to the reaction mixture. After being
stirred for 60 min at room temperature, the reaction mixture
was acidified (1.5 M H2SO4, 10 mL), the resulting solution was
extracted with Et2O, and the organic extracts were combined,
dried over Na2SO4 and concentrated to dryness. The crude
reaction mixture was subjected to flash column chromatogra-
phy (silica gel, EtOAc- hexane, 20:80) to afford 42 mg of 12
(71%); mp 140-142 °C.
Brassilexin (12) from 3-(N,N-Dimethylamino)methyl-
eneindoline-2-thione (21). Indoline-2-thione 21 (20 mg, 0.14
mmol) was dissolved in DMF (300 µL) and aq ammonium
hydroxide (2 mL) was added. After being stirred for 10 min,
the mixture was diluted with H2O (10 mL) and extracted with
Et2O, and the organic extracts were combined, dried over Na2-
SO4, and concentrated to dryness. I2 (34 mg, 0.14 mmol) was
added to the crude product dissolved in pyridine (1 mL) and
the reaction mixture was stirred for 60 min at room temper-
ature. After acidification (H2SO4,10 mL) the resulting solution
was extracted with Et2O, and the organic extracts were
combined, dried over Na2SO4, and concentrated to dryness. The
crude reaction mixture was subjected to flash column chro-
matography (silica gel, EtOAc-hexane, 20:80) to afford 15 mg
of 12 (88%); mp 140-142 °C.
Sinalexin (13). Preparation was the same as reported for
brassilexin (12) but the temperature of the reaction was kept
at room temperature. The crude reaction mixture was sub-
jected to flash column chromatography (silica gel, EtOAc-
hexane, 20:80) to afford 38 mg of 13 (67%); mp 36-37 °C.
3-(N,N-Dimethylamino)methyleneindoline-2-thione
(21). Indoline-2-thione (9, 200 mg, 1.34 mmol) was added to a
mixture of freshly distilled POCl3 (260 µL, 2.8 mmol) in DMF
(1.7 mL) at 45 °C, and the reaction mixture was kept at 45
°C. After 90 min the reaction mixture was cooled to 0 °C on
an ice bath and diluted withice-cold water (20 mL), then the
precipitate formed was filtered off. The filtrate was extracted
with Et2O, and the organic extracts were combined, dried over
Na2SO4, and concentrated to dryness. The crude product was
subjected to flash column chromatography (silica gel, CH2Cl2)
to yield 168 mg of 21 (60%). 1H NMR (500 MHz, CD3CN) δ 9.8
(br s, 1 H), 8.41 (s, 1 H), 7.49 (d, J ) 7.5 Hz, 1 H), 7.08 (d, J
) 3.5 Hz, 1H), 7.04 (m, 2 H), 3.46 (s, 6 H). 13C NMR (500 MHz,
CD3CN, 50 °C) δ 186.2 (br s), 157.5, 139.8, 125.5, 123.5, 121.6,
120.3, 109.6, 108.4, 45.1 (br s, 2 CH3). FTIR νmax (KBr): 3089
(br), 2932, 1626, 1447, 1312, 1184, 745 cm-1. HRMS m/z
measured 204.0171 (204.0721 calculated for C11H12N2S).
5-Methoxybrassilexin (24a). Preparation was the same
as reported for brassilexin (12). The crude reaction mixture
was subjected to flash column chromatography (silica gel,
EtOAc-hexane, 20:80) to afford 39 mg of 24a (67%); mp 169-
172 °C. 1H NMR (500 MHz, CD3CN) δ 9.71 (br s, 1 H, D2O
exchangeable), 8.67 (s, 1 H), 7.48 (m, 2 H), 6.97 (dd, J ) 9, 2.5
Hz, 1H), 3.87 (s, 3 H). 13C NMR (500 MHz, CD3CN) δ 159.7,
154.9, 147.7, 139.3, 127.6, 120.9, 113.1, 112.9, 103.2, 55.7. FTIR
νmax (KBr) 3150, 1623, 1468, 1212, 1068, 789 cm-1. HRMS m/z
measured 204.0357 (204.0356 calculated for C10H8N2OS).
EIMS m/z (% relative abundance) 204 (M+, 100), 189 (59), 161
(23), 134 (9).
inhibition (%)
compd
concn (M)
( standard deviationa
12
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
5 × 10-4
2 × 10-4
1 × 10-4
c.i.b
62 ( 1
8 ( 1
c.i.
13
69 ( 4
24 ( 7
92 ( 4
40 ( 4
14 ( 1
c.i.
24a
24b
24c
24d
24e
24f
24g
24h
24i
24j
c.i.
70 ( 7
98 ( 4
32 ( 9
24 ( 19
96 ( 5
66 ( 7
n.i.c
72 ( 10
66 ( 12
60 ( 10
98 ( 4
25 ( 3
n.i.
c.i.
c.i.
64 ( 1
c.i.
c.i.
69 ( 6
c.i.
c.i.
c.i.
c.i.
72 ( 1
38 ( 1
a Percent of inhibition ) 100 - [(growth on medium containing
compound/growth on control medium) × 100)] ( standard devia-
tion. b c.i. ) complete inhibition. c n.i. ) no inhibition (growth on
control medium and on medium containing compound is similar).
Experimental Section
Antifungal Bioassays. Bioassays with L. maculans/P.
lingam (isolate BJ-125) were performed using potato dextrose
agar as previously described.32
Indoline-2-thione (9).14 A mixture of 2-oxindole (500 mg,
3.76 mmol) and P2S5 (1.0 g, 2.3 mmol) in THF (25 mL) was
allowed to stir for 10 min at room temperature. Sodium
bicarbonate (631 mg, 7.5 mmol) was added portionwise to the
above mixture and the reaction mixture was allowed to stir
for 4 h at room temperature. The reaction mixture was filtered
(gravity) and the excess of THF was removed under reduced
pressure. Ice-cold water (50 mL) was added to the residue with
vigorous stirring to afford a light yellow precipitate that was
filtered and air-dried; yield 516 mg (92%); mp 144-147 °C.
1H NMR (500 MHz, CDCl3) δ 10.68 (br s, 1 H, D2O exchange-
able), 7.28 (m, 2 H), 7.14 (dd, J ) 7.5, 7.5 Hz, 1 H), 7.03 (d, J
) 8 Hz, 1H), 4.09 (s, 2 H). 13C NMR (500 MHz, CDCl3) δ 203.7,
144.27, 130.3, 128.0, 124.2, 124.1, 110.0, 49.0. IR data were
(31) The reaction of ammonia with aldehydes leads to spontaneous
polymerization of the resulting imines. (a) Smith, M. B.; March, J.
Advanced Organic Chemistry, 5th ed.; J. Wiley & Sons: New York,
2001; p 1186. (b) Hull, W. E.; Sykes, B. D.; Babior, B. M. J. Org. Chem.
1973, 38, 2931.
(32) Pedras, M. S. C.; Montaut, S. Bioorg. Med. Chem. 2003, 11,
3115.
(33) Rewcastle, G. W.; Janosik, T.; Bergman, J. Tetrahedron 2001,
57, 7185.
J. Org. Chem, Vol. 70, No. 5, 2005 1833

Pedras and Jha
5-Methylbrassilexin (24b). Preparation was the same as
reported for brassilexin (12). The crude reaction mixture was
subjected to flash column chromatography (silica gel, EtOAc-
hexane, 20:80) to afford 30 mg of 24b (52%); mp 171-174 °C.
1H NMR (500 MHz, CD3CN) δ 9.76 (br s, 1 H, D2O exchange-
able), 8.66 (s, 1 H), 7.71 (s, 1 H), 7.44 (d, J ) 8.5 Hz, 1H), 7.16
(d, J ) 8 Hz, 1 H), 2.47 (s, 3 H). 13C NMR (500 MHz, CD3CN)
δ 159.5, 147.7, 142.9, 130.3, 127.4, 125.3, 120.5, 120.0, 112.1,
20.8. FTIR νmax (KBr) 3120, 3051, 2892, 2276, 1513, 1473, 1244,
788 cm-1. HRMS m/z measured 188.0408 (188.0403 calculated
for C10H8N2S). EIMS m/z (% relative abundance) 188 (M+, 100),
187 (35), 155 (34).
5-Fluorobrassilexin (24c). Preparation was the same as
reported for brassilexin (12). The crude reaction mixture was
subjected to flash column chromatography (silica gel, EtOAc-
hexane, 20:80) to afford 38 mg of 24c (64%); mp 198-200 °C.
1H NMR (500 MHz, CD3CN) δ 9.97 (br s, 1 H, D2O exchange-
able), 8.68 (s, 1 H), 7.65 (dd, J ) 9.5, 2.5 Hz, 1 H), 7.55 (dd, J
) 9, 4.5 Hz, 1 H), 7.12 (ddd, J ) 9.5, 9, 2.5 Hz, 1 H). 13C NMR
(500 MHz, CD3CN) δ 160.7, 158.2 (d, JC-F ) 233 Hz), 147.8,
141.0, 127.3 (d, JC-F ) 6), 126.7 (d, JC-F ) 10 Hz), 113.4 (d,
JC-F ) 10 Hz), 111.6 (d, JC-F ) 27 Hz), 105.8 (d, JC-F ) 25
Hz). FTIR νmax (KBr) 3143, 3075, 2968, 2894, 1588, 1473, 1364,
1182, 801 cm-1. HRMS m/z measured 192.0157 (192.0150
calculated for C9H5N2SF). EIMS m/z (% relative abundance)
192 (M+, 100), 160 (16), 149 (24), 137 (13).
5-Chlorobrassilexin (24d). Preparation was the same as
reported for brassilexin (12). The crude reaction mixture was
subjected to flash column chromatography (silica gel, EtOAc-
hexane, 20:80) to afford 35 mg of 24d (60%); mp 226-227 °C.
1H NMR (500 MHz, CD3CN) δ 10.0 (br s, 1 H, D2O exchange-
able), 8.7 (s, 1 H), 7.95 (d, J ) 2 Hz, 1 H), 7.59 (d, J ) 9 Hz,
1 H), 7.33 (dd, J ) 8.5, 1.5 Hz, 1 H). 13C NMR (500 MHz, CD3-
CN) δ 160.5, 147.8, 142.9, 126.8, 125.8, 123.9 121.3, 119.8,
113.7. FTIR νmax (KBr) 3130, 3047, 2953, 2887, 1505, 1462,
1234, 786 cm-1. HRMS m/z measured 207.9861 (207.9861
calculated for C9H5N2SCl). EIMS m/z (% relative abundance)
210 (34), 208 (M+, 100), 146 (10), 103 (10).
5-Bromobrassilexin (24e). Preparation was the same as
reported for brassilexin (12). The crude reaction mixture was
subjected to flash column chromatography (silica gel, EtOAc-
hexane, 20:80) to afford 23 mg of 24e (40%); mp 222-223 °C.
1H NMR (500 MHz, CD3CN) δ 9.98 (br s, 1 H, D2O exchange-
able), 8.69 (s, 1 H), 8.1 (s, 1 H), 7.51 (d, J ) 8.5 Hz, 1 H), 7.45
(d, J ) 8.5 Hz, 1 H). 13C NMR (500 MHz, CD3CN) δ 160.3,
147.9, 143.2, 126.6, 124.9, 123.0, 121.9, 114.9, 113.2. FTIR νmax
(KBr) 3125, 2946, 2886, 1506, 1459, 1233, 786 cm-1. HRMS
m/z measured 253.9336 (253.9330 calculated for C9H5N2SBr).
EIMS m/z (% relative abundance) 254 (M+, 100), 252 (98), 173
(74), 146 (18).
1H NMR (500 MHz, CD3CN) δ 9.93 (br s, 1 H, D2O exchange-
able), 8.67 (s, 1 H), 7.89 (dd, J ) 8.5, 8.5 Hz, 1 H), 7.35 (dd, J
) 10, 2.5 Hz, 1 H), 7.03 (ddd, J ) 9.5, 9, 2.5 Hz, 1 H). 13C
NMR (500 MHz, CD3CN) δ 160.5 (d, JC-F ) 235 Hz), 159.9,
147.5, 144.8 (d, JC-F ) 12.5 Hz), 127.2, 121.2 (d, JC-F ) 10
Hz), 117.0, 108.6 (d, JC-F ) 25 Hz), 99.1(d, JC-F ) 26 Hz). FTIR
νmax (KBr) 3130, 2993, 2909, 1627, 1589, 1508, 1368, 1138, 961
cm-1. HRMS m/z measured 192.0157 (192.0158 calculated for
C9H5N2SF). EIMS m/z (% relative abundance) 192 (M+, 100),
164 (14), 160 (16), 137 (16).
6-Chlorobrassilexin (24h). Preparation was the same as
reported for brassilexin (12). The crude reaction mixture was
subjected to flash column chromatography (silica gel, EtOAc-
hexane, 20:80) to afford 34 mg of 24h (58%); mp 184-186 °C.
1H NMR (500 MHz, CD3CN) δ 9.93 (br s, 1 H, D2O exchange-
able), 8.68 (s, 1 H), 7.86 (d, J ) 8.5 Hz, 1 H), 7.59 (d, J ) 1.5
Hz, 1 H), 7.22 (dd, J ) 8.5, 1.5 Hz, 1 H). 13C NMR (500 MHz,
CD3CN) δ 160.1, 147.8, 144.8, 129.1, 127.1, 121.2, 121.0, 119.0,
112.3. FTIR νmax (KBr) 3107 (br), 2964, 2886, 1564, 1501, 1458,
1239, 1056, 851, 795 cm-1. HRMS m/z measured 207.9861
(207.9856 calculated for C9H5N2SCl). EIMS m/z (% relative
abundance) 210 (34), 208 (M+, 100), 176, (45), 153 (12), 103
(8).
6-Bromobrassilexin (24i). Preparation was the same as
reported for brassilexin (12). The crude reaction mixture was
subjected to flash column chromatography (silica gel, EtOAc-
hexane, 20:80) to afford 25 mg of 24i (45%); mp 182-185 °C.
1H NMR (500 MHz, CD3CN) δ 9.93 (br s, 1H, D2O exchange-
able), 8.7 (s, 1 H), 7.84 (d, J ) 8.5, 1H), 7.64 (d, J ) 1.5, 1H),
7.38 (dd, J ) 8.5, 1.5, 1H). 13C NMR (500 MHz, CD3CN) δ
160.1, 147.9, 145.2, 127.2, 123.8, 121.6, 119.4, 116.6, 115.3.
FTIR νmax (KBr) 3101 (br), 1641, 1570, 1502, 1423, 1367, 1237,
846 cm-1. HRMS m/z measured 253.9336 (253.9336 calculated
for C9H5N2SBr). EIMS m/z (% relative abundance) 254 (M+,
100), 252 (98), 173 (81), 146 (14).
1-Methylbrassilexin (24j). Preparation was the same as
reported for brassilexin (12) but the temperature of the
reaction was kept at room temperature. The crude reaction
mixture was subjected to flash column chromatography (silica
gel, EtOAc-hexane, 20:80) to afford 37 mg of 13 (65%); mp
1
67-69 °C. H NMR (500 MHz, CD3CN) δ 8.71 (s, 1 H), 7.93
(d, J ) 8 Hz, 1 H), 7.48 (d, J ) 8 Hz, 1 H), 7.4 (dd, J ) 8, 7.5
Hz, 1H), 7.26 (dd, J ) 7.5, 7.5 Hz, 1 H), 3.87 (s, 3 H). 13C NMR
(500 MHz, CD3CN) δ 162.1, 148.3, 145.1, 125.9, 123.9, 120.8,
120.4, 120.3, 110.3, 33.1. FTIR νmax (KBr) 1490, 1464, 1319,
1261,912,743cm-1.HRMSm/zmeasured188.04082(188.040773
calculated for C10H8N2S). EIMS m/z (% relative abundance)
188 (M+, 100), 155 (15), 146 (11).
Acknowledgment. Financial support from the Natu-
ral Sciences and Engineering Research Council of
Canada (NSERC Discovery and Strategic Grants to
M.S.C.P) and the University of Saskatchewan (teaching
assistantship to M.J.) is gratefully acknowledged.
6-Methoxybrassilexin (24f). Preparation was the same as
reported for brassilexin (12). The crude reaction mixture was
subjected to flash column chromatography (silica gel, EtOAc-
hexane, 20:80) to afford 27 mg of 24f (47%); mp 174-176 °C.
1H NMR (500 MHz, CD3CN) δ 9.75 (br s, 1 H, D2O exchange-
able), 8.62 (s, 1 H), 7.79 (d, J ) 8.5 Hz, 1 H), 7.12 (d, J ) 2.5
Hz, 1H), 6.87 (dd, J ) 8.5, 2.5 Hz, 1 H), 3.87 (s, 3 H). 13C NMR
(500 MHz, CD3CN) δ 158.7, 157.7, 147.2, 145.7, 127.5, 120.7,
114.3, 109.7, 96.4, 55.5. FTIR νmax (KBr) 3130, 2993, 2909,
1627, 1589, 1508, 1368, 1138, 961 cm-1. HRMS m/z measured
204.035735 (204.035439 calculated for C10H8N2OS). EIMS m/z
(% relative abundance) 204 (M+, 100), 189 (91), 161 (15), 100
(9).
Note Added after ASAP Publication. Table 1
contained coding errors in the version published ASAP
February 3, 2005, and a Review-Only Supporting Infor-
mation file was posted. The corrected version was pub-
lished ASAP February 4, 2005.
Supporting Information Available: General experimen-
tal and characterization data for compounds 23a-j, 25a-i,
and 27a-d. This material is available free of charge via the
Internet at http://pubs.acs.org.
6-Fluorobrassilexin (24g). Preparation was the same as
reported for brassilexin (12). The crude reaction mixture was
subjected to flash column chromatography (silica gel, EtOAc-
hexane, 20:80) to afford 39 mg of 24g (68%); mp 168-170 °C.
JO0479866
1834 J. Org. Chem., Vol. 70, No. 5, 2005