Unprecedented vilsmeier formylation: Expedient syntheses of the cruciferous phytoalexins sinalexin and brassilexin and discovery of a new heteroaromatic ring system

Article abstract of DOI:10.1021/ol015691y

Matrix presented A very concise first synthesis of sinalexin was achieved by regioselective formylation of 1-methoxyindoline-2-thione under Vilsmeier conditions followed by unprecedented ammonia workup. Similar formylation of indoline-2-thione yielded brassilexin and a novel pentacyclic heteroaromatic compound resulting from condensation of the Vilsmeier adduct of indoline-2-thione. Both sinalexin and brassilexin displayed strong antifungal activity against several pathogens of crucifers.

Full text of DOI:10.1021/ol015691y

ORGANIC
LETTERS
2001
Vol. 3, No. 8
1213-1216
Unprecedented Vilsmeier Formylation:
Expedient Syntheses of the Cruciferous
Phytoalexins Sinalexin and Brassilexin
and Discovery of a New Heteroaromatic
Ring System
M. Soledad C. Pedras* and Irina L. Zaharia
Department of Chemistry, UniVersity of Saskatchewan, 110 Science Place,
Saskatoon SK, S7N 5C9 Canada
soledade.pedras@usask.ca
Received February 9, 2001
ABSTRACT
A very concise first synthesis of sinalexin was achieved by regioselective formylation of 1-methoxyindoline-2-thione under Vilsmeier conditions
followed by unprecedented ammonia workup. Similar formylation of indoline-2-thione yielded brassilexin and a novel pentacyclic heteroaromatic
compound resulting from condensation of the Vilsmeier adduct of indoline-2-thione. Both sinalexin and brassilexin displayed strong antifungal
activity against several pathogens of crucifers.
The wide scope of the Vilsmeier-Haack formylation renders
it an extremely useful reaction in organic synthesis. Since it
was first reported in 1925, its application to a variety of both
aromatic and heteroaromatic substrates is well-documented.1
Nonetheless, this formylation reaction is only applicable to
substrates more reactive than benzene. For example, the
formylation of indole under Vilsmeier conditions occurred
regioselectively in high yield, whereas the formylation
product of benzo[b]thiophene was obtained in very poor
yield.1 Although the reactivity of substrates such as indolin-
2-one (1) is expected to be lower than that of indole, a
number of indolin-2-ones (1, R ) H, Me, Ph, COPh) have
been formylated to yield the corresponding 2-chloro-3-formyl
indoles (Scheme 1).1
Scheme 1
On the other hand, indoline-2-thiones (2) do not appear
to have been formylated under Vilsmeier conditions, whereas
2-thioethers such as 2-thiomethylindole were formylated in
good yield.2 Significant interest in the formylation of indolin-
(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) Pedras, M. S. C.; Khan, A. K. J. Agric. Food Chem. 1996, 44, 3403.
10.1021/ol015691y CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/21/2001

2-ones (1) and indoline-2-thiones (2)3 derives from their
application to the synthesis of a number of tryptophan-
derived alkaloids, in particular, some cruciferous phytoalexins
such as spirobrassinin (4), wasalexins (5), brassicanals (6,
R ) H, Y ) SMe, S(O)OMe), sinalexin (7), and brassilexin
(8).4 Phytoalexins are secondary metabolites biosynthesized
de novo by plants in response to diverse forms of stress,
including pathogen attack.5,6 Sinalexin (7)7 appears to have
a notable role in defense mechanisms of white mustard
(Sinapis alba);8 however, partly because of the extremely
small amounts isolable from plants, the biological activity
and ecological significance of sinalexin (7) remains to be
established. Toward this end, it was of great interest to
develop a chemical synthesis of sinalexin (7).
decomposition of the starting material (2, R ) OMe) was
observed. Subsequently, to avoid decomposition, sulfur was
introduced at a later stage in the synthesis, as shown in
Scheme 3. Thus thioamides 14 (R ) OMe) were prepared
Scheme 3
from 13 (obtained quantitatively by acetylation of enamine
12)10 but in poor yield as a result of extensive decomposi-
tion.11 Eventually, oxidation12 and solvolysis of 14 yielded
sinalexin in low overall yield (Scheme 3).
The rather lengthy route required to obtain sinalexin (7,
Scheme 3) coupled with low overall yield (18%) directed
us to investigate the direct Vilsmeier formylation of 1-meth-
oxyindoline-2-thione (2, R ) OMe) and/or its thiol tau-
tomer.13 Although this key intermediate had not been
described previously, the electron-withdrawing effect of the
MeO substituent at N-1 suggested that its reactivity toward
electrophilic substitution would be significantly lower than
that of indoline-2-thione (2, R ) H). Furthermore, consider-
ing that indoline-2-thione (2, R ) H, pkHA ) 10.0) is
significantly more acidic than indolin-2-one (2, R ) H, pkHA
Here we report the first synthesis of sinalexin (7) through
an unprecedented application of the Vilsmeier formylation
to N-methoxyindoline-2-thione (2, R ) OMe). Most interest-
ingly, application of these conditions to the synthesis of
brassilexin (8) led to the isolation of a new product containing
a heterocyclic aromatic ring system. In addition, the anti-
fungal activity of both sinalexin and brassilexin against four
economically important plant pathogens was determined and
is reported.
Initially, we sought an apparently direct route to sinalexin
(7) based on the convenient biomimetic synthesis of the
structurally related phytoalexin brassilexin (8), as shown in
Scheme 2.9 During this synthesis it was established that the
(3) Indoline-2-thiones are also useful in the preparation of certain 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.
(4) Pedras, M. S. C.; Okanga, F. I.; Zaharia, I. L.; Khan, A. K.
Phytochemistry 2000, 53, 161.
(5) . (a) Bailey, J. A.; Mansfield, J. W., Eds. Phytoalexins; Blackie &
Son: Glasgow, U.K., 1982; p 334. (b) Brooks, C. J. W.; Watson, D. G.
Nat. Prod. Rep. 1985, 427.
Scheme 2
(6) For a recent review see ref 4.
(7) Pedras, M. S. C.; Smith, K. C. Phytochemistry 1997, 46, 833.
(8) Pedras, M. S. C.; Zaharia, I. L.; Gai, Y., Zhou, Y.; Ward, D. E. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 747.
(9) (a) Pedras, M. S. C.; Okanga, F. I. Chem. Commun. 1998, 1565. (b)
Pedras, M. S. C.; Okanga, F. I. J. Agric. Food Chem. 1999, 47, 1196.
(10) Pedras, M. S. C.; Sorensen, J. L.; Okanga, F. I.; Zaharia, I. L. Bioorg.
Med. Chem. Lett. 1999, 9, 3015.
(11) Satisfactory spectroscopic data were obtained for all synthetic
intermediates.
(12) (a) Joule, J. A.; Mills, K.; Smith, G. F. Heterocyclic Chemistry;
Chapman & Hall: London, U.K., 1995; p 404. (b) Goerdeler, J.; Krone, U.
Chem. Ber. 1969, 102, 2273.
(13) Spectroscopic data of 1-methoxyindoline-2-thione (2, R ) OMe).
1H NMR (300 MHz, CDCl3) δ 7.37 (dd, J ) 7.5, 7.5 Hz, 1H), 7.30 (d, J
) 7.5 Hz, 1H), 7.20 (dd, J ) 7.5, 7.5 Hz, 1H), 7.11 (d, J ) 8 Hz, 1H),
4.16 (s, 3H), 4.04 (s, 2H). 13C NMR (75.5 MHz, CDCl3) δ 191.9 (s), 142.8
(s), 128.4 (d), 126.1 (s), 125.1 (d), 124.8 (d), 108.5 (d), 62.3 (q), 47.2 (t).
HREIMS m/z measured 179.0407 (179.0405 calcd for C9H9NOS). EIMS
m/z (% relative abundance) 179 (M+, 100), 149 (69), 148 (60), 121 (32),
117 (23), 104 (13). FTIR νmax 2937, 1620, 1464, 1391, 1364, 1285, 1192,
1138, 1067, 949, 749 cm-1.
required intermediate 9 (R ) H), readily obtained from
reaction of indoline-2-thione (2, R ) H) with NaH and ethyl
formate, could not be obtained from N-methoxyindoline-2-
thione (2, R ) OMe) under similar reaction conditions.
Instead of the expected formylation product, extensive
1214
Org. Lett., Vol. 3, No. 8, 2001

) 18.5; R ) Me, pkHA ) 18.5) or 1-acetylindolin-2-one (2,
R ) Ac, pkHA ) 13.7),14 it was suspected that formylation
of 1-methoxyindoline-2-thione (2, R ) OMe) might occur
at both C-3 and S with potential ring opening and decom-
position. Preliminary attempts to convert 1-methoxyindoline-
2-thione (2, R ) OMe) under mild Vilsmeier conditions
(POCl3/DMF, rt, 1 h, followed by NaOH work up at rt)
yielded enamine thione 15 (R ) OMe) in 15-25% yield
along with multiple undetermined products. When the work
up was carried out with boiling NaOH, 15 (R ) OMe) (in
similar yield) and traces of the desired aldehyde 9 (R )
OMe) were obtained.
Scheme 4
obtained directly from reaction of aldehydes with ammonia.15
In this procedure the imine 11 (R ) OMe) is formed by
reaction of enamine 15 (R ) OMe) with ammonia, thus
avoiding formation of an unstable aldehyde and the need
for two additional steps to prepare sinalexin (7) (oximation
and oxime reduction shown in Scheme 2).
To extend the application of this method to additional and
useful substrates, the formylation of indoline-2-thione (2, R
) H) was carried out under reaction conditions similar to
those described for sinalexin (7). The expected enamine/
imine intermediate 11 (R ) H) was isolated in good yield
(65-70%) along with a significant amount of an undeter-
mined orange product (30-35%). Oxidation of 3-(amino)-
methylene-2-thione (11, R ) H) with iodine gave brassilexin
(8, Scheme 4) quantitatively. The structure of the orange
product was established from its spectroscopic data to be
the novel heterocycle 17.16 Compound 17 represents a new
heteroaromatic ring system of 22 π-electrons with two
equivalent tautomeric structures 17a and 17b. Reaction of
17 with MeI yielded quantitatively the expected methyl
derivative 18, whose spectroscopic data corroborated the
structure of 17. The formation of product 17 is rationalized
in Scheme 5 (structures inside brackets are possible reactive
intermediates); 17 may be formed by condensation of the
Vilsmeier adduct 15 (R ) H) and subsequent elimination of
hydrogen sulfide or equivalent. Subsequently, we established
that the crude reaction mixture containing both thione 11
(R ) H) and 17 could be directly oxidized to brassilexin (8,
R ) H) without affecting its overall yield.
Subsequently, we reasoned that the product of a Vilsmeier
formylation with POCl3/DMF is an iminium salt, e.g. 16,
that could be converted into groups other than aldehydes.
For example, treatment of the Vilsmeier iminium salt 16 (Ar
) pyrrole) with hydroxylamine1 yielded oximes, whereas
treatment of 16 (Ar ) indolizine) with hydrogen sulfide1
lead to thioaldehydes.1 Thus we sought a new workup
procedure that might lead directly to the desired intermediate
3-(amino)methylene-1-methoxyindoline-2-thione (11, R )
OMe). Thione 2 (R ) OMe) dissolved in DMF was first
treated with POCl3 (1 h at 40 °C), and then the reaction
mixture was cooled to 0 °C, basified with aqueous NH3 (pH
> 11), and extracted and concentrated in the usual way. The
resulting reaction mixture was dissolved in pyridine and
oxidized (I2)12 to yield, after FCC fractionation, sinalexin
(7) identical in all respects to the natural product (Scheme
4, 60-65% overall yield).
Hence the regioselective formylation of 1-methoxyindo-
line-2-thione (2, R ) OMe) under Vilsmeier conditions
followed by ammonia workup is the key to a very concise
synthesis of the phytoalexin sinalexin (7). This unprecedented
workup procedure1 is extremely useful in the preparation of
primary imines/enamines, since these products cannot be
In conclusion, the ammonia workup procedure applied to
the Vilsmeier formylation provides an extremely simple and
unique two-step synthesis (with only one chromatographic
separation) of two important cruciferous phytoalexins.
Table 1. Antifungal Activitya of Sinalexin (7) and Brassilexin (8) against Pathogens of Crucifers: Alternaria brassicae (isolate AB 11,
7-day incubation), Phoma lingam (perfect stage ) Leptosphaeria maculans) (isolate BJ 125, 4-day incubation), Rhizoctonia solani
(isolate group AG 2-1, 2-day Incubation), and Sclerotinia sclerotiorum (clone 33, 2-day incubation)
concentration
A. brassicae
R. solani
S. sclerotiorum
P. lingam
compound
(in growth medium)
B 11
AG 2-1
clone 33
BJ 125
sinalexin (7)
5 × 10-4
1 × 10-4
2 × 10-5
5 × 10-4
1 × 10-4
2 × 10-5
M
M
M
M
M
M
100%
31 ( 8%
n. i.b
100%
42 ( 10%
n. i.
100%
100%
33 ( 4%
100%
100%
100%
100%
37 ( 4%
100%
100%
60 ( 11%
25 ( 9%
n. i.
100%
81 ( 4%
n. i.
brassilexin (8)
66 ( 7%
86 ( 4%
a Percent of inhibition ) 100 - [(growth on medium containing compound/growth on control medium) × 100)] ( standard deviation; incubation time
(fungal growth on control medium) depends on each species. b n.i. ) no inhibition (growth on control medium and on medium containing compound is
similar).
Org. Lett., Vol. 3, No. 8, 2001
1215

Scheme 5
Although several synthesis of brassilexin (8) have been
should facilitate future biosynthetic and other metabolic
studies of sinalexin (7) and brassilexin (8).4
published,6 the present synthesis represents a very significant
improvement in both overall yield and simplicity. Further-
more, our facile synthesis of intermediates 11 (R ) OMe or
H), potential biosynthetic precursors of 7 and 8, respectively,9
will allow an efficient one-carbon isotopic labeling and
The antifungal activities of sinalexin and brassilexin
against four of the major pathogens of crucifers, i.e.,
Alternaria brassicae, Phoma lingam (perfect stage ) Lep-
tosphaeria maculans), Rhizoctonia solani, and Sclerotinia
sclerotiorum were determined as previously described.17 As
shown in the Table, brassilexin (8, R ) H) caused complete
inhibition of fungal mycelium growth in all four species at
a concentration of 5 × 10-4 M for the duration of the
experiment (up to 3 weeks), whereas sinalexin (7, R ) OMe)
showed less growth inhibition (60%) to P. lingam at this
concentration. In addition, both sinalexin and brassilexin
inhibited the growth of S. sclerotiorum and R. solani at 1 ×
10-4 M. These results confirm a possible role for sinalexin
in the resistance of white mustard to A. brassicae.8 The
availability of synthetic sinalexin and brassilexin should
allow further investigation on their mechanism of action
against economically important cruciferous pathogens.
(14) Bordwell, F. G.; Fried, H. E. J. Org. Chem. 1991, 56, 4218.
(15) The reaction of ammonia with aldehydes leads to spontaneous
polymerization of the resulting imines. (a) 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.
(16) Spectroscopic data for compound 17: 1H NMR (500 MHz, (CD3)2-
SO) δ 13.14 (br s, 1H), 9.48 (s, 1H), 8.24 (d, J ) 7.5 Hz, 2H), 7.66 (d, J
) 8 Hz, 2H), 7.43 (dd, J ) 7.5, 7.5 Hz, 2H), 7.35 (dd, J ) 7.5, 7.5 Hz,
2H). 13C NMR (125.8 MHz, (CD3)2SO) δ128.1 (d), 125.3 (d), 125.2 (d),
121.2 (2×, d), 119.2 (2×, d), 114.9 (2×, br d). HREIMS m/z measured
274.0564 (274.0566 calcd for C17H10N2S). EIMS m/z (% relative abundance)
274 (M+, 100), 137 (11). FTIR νmax 3400 (br), 3060, 2923, 2692, 1598,
1449, 1387, 1364, 1308, 1247, 1186, 1132, 744 cm-1. Spectroscopic data
for compound 18: 1H NMR (500 MHz, CD2Cl2) δ 8.83 (s, 1H), 8.09 (d, J
) 7.5 Hz, 1H), 8.05 (d, J ) 7.5 Hz, 1H), 7.74 (d, J ) 8 Hz, 1H), 7.51 (d,
J ) 7.5 Hz, 1H), 7.48 (m, 2H), 7.42 (dd, J ) 8, 8 Hz, 1H), 7.33 (dd, J )
7.5, 7.5 Hz, 1H), 3.85 (s, 3H). 13C NMR (125.8 MHz, CD2Cl2) δ 156.7 (s),
154.2 (s), 141.1 (s), 139.8 (s), 127.2 (d), 126.8 (s), 126.6 (d), 125.2 (s),
124.8 (d), 123.2 (s), 122.4 (d), 121.9 (d), 120.1 (d), 118.8 (2×, d), 111.2
(s), 110.0 (d), 31.4 (q). HREIMS m/z measured 288.0716 (288.0721 calcd
for C18H12N2S). EIMS m/z (% relative abundance) 288 (M+, 100), 273 (14),
144 (11). FTIR νmax 2926, 1593, 1392, 1383, 1307, 1255, 1126, 1170, 744
cm-1.
Acknowledgment. We thank the Natural Sciences and
Engineering Research Council of Canada (research grant to
M.S.C.P.) and the University of Saskatchewan (graduate
fellowship to I.L.Z.) for financial support. Isolates of A.
brassicae, P. lingam, S. sclerotiorum, and R. solani were
obtained from G. Se´guin-Swartz, P. R. Verma, and D.
Makenzie, Agriculture and Agri-Food Canada, Saskatoon
Research Centre, Saskatoon SK.
(17) Bioassays against Phoma lingam and Rhizoctonia solani were
conducted as described in ref 9b but utilizing 4-well plates and liquid instead
solid medium (400 µL medium); bioassays against Alternaria brassicae
and Sclerotinia sclerotiorum were conducted similarly to assays with R.
solani. Different incubation times were used for each pathogen: A.
brassicae, 7-day incubation; P. lingam, 4-day incubation; R. solani, 2-day
incubation; and S. sclerotiorum, 2-day incubation (if complete inhibition
was observed the assay plates were kept for 3 weeks).
OL015691Y
1216
Org. Lett., Vol. 3, No. 8, 2001