Synthesis and absolute stereochemisty of a cruciferous phytoalexin, (-)-spirobrassinin

Article abstract of DOI:10.1246/cl.2000.886

Synthetic (±)-spirobrassinin (1) was enantioresolved by a chiral auxiliary method giving (+)-1 and natural (-)-1. The absolute configuration was unambiguously determined by X-ray crystallography of a ( 1′S,4′R)-camphanoy] derivative of (+)-1. Consequently

Full text of DOI:10.1246/cl.2000.886

886
Chemistry Letters 2000
Synthesis and Absolute Stereochemisty of a Cruciferous Phytoalexin, (–)-Spirobrassinin
Kenji Monde,* Shuichi Osawa, Nobuyuki Harada, Mitsuo Takasugi,†
Mojmir Suchy,†† Peter Kutschy,*†† Milan Dzurilla,†† and Eva Balentova††
Institute for Chemical Reaction Science, Tohoku University, Aoba-ku, Sendai 980-8577
†Division of Material Science, Graduate School of Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060-0810
††Department of Organic Chemistry, P. J. Safarik University, Moyzesova 11, Kosice 041 67, Slovak Republic
(Received May 16, 2000; CL-000471)
Synthetic ( )-spirobrassinin (1) was enantioresolved by a
chiral auxiliary method giving (+)-1 and natural (–)-1. The
absolute configuration was unambiguously determined by X-
ray crystallography of a (1'S,4'R)-camphanoyl derivative of (+)-
1. Consequently, natural (–)-1 has an S configuration. Their
CD spectra supported this result.
Phytoalexins are defined as antifungal compounds produced
by plants de novo after infection of microorganisms.1 We report-
ed the first cruciferous phytoalexins (e.g., brassinin (2)) from
Pseudomonas cichorii inoculated Chinese cabbage (Brassica
campestris).2 Since cruciferous crops are cultivated worldwide
and they are extremely valuable, various research groups have
investigated the cruciferous phytoalexins and reported more than
twenty phytoalexins3 as well as their interesting biological activi-
ties4 for the last decade. All these compounds are indole or
indole-related compounds possessing one or two sulfur atoms,
except for one case.5 In 1987, we isolated the first oxindole phy-
toalexin, spirobrassinin ((–)-1) from P. cichorii inoculated
Japanese radish (Rhaphanus sativus).6 Although several com-
methylbenzyl isocyanate giving two urea derivatives 4 and 4'.10
As expected, the diastereomeric mixture was separated by sim-
ple SiO2 column chromatography. Following deprotection of 4
and 4' by sodium methoxide gave (+)-1 (91% ee) and (–)-1
(92% ee), respectively (Scheme 1).11,12 Their CD spectra were
complete mirror images, and (–)-1 showed an exciton type split
at 221 (∆ε +25.9) and 204 (∆ε −21.0) nm with a moderate A
value (+46.9). UV data of 3,3-dimethyloxindole (λmax 205 nm,
ε 30000)13 and CH3(CH2)4CH2–N=C(SCH3)2 (5) (λmax 219 nm,
ε 8590)13 suggested that two chromophores in 1 (oxindole and
N=C(S–)SCH3 moiety) would induce the split Cotton effects in
its CD spectrum.14 In the case of natural (–)-(1), two axes of
their transition dipole moments should be clockwise to give pos-
itive chirality leading to the S configuration of (–)-(1) (Figure 1).
pounds including 1 among cruciferous phytoalexin family have
their asymmetric centers, no stereochemical study has been
investigated.
Detailed chiral analysis of natural (–)-1 revealed that it was
not enantiomerically pure. Moreover, enantiomeric excesses of
two natural spirobrassinin fractions separated by non-chiral
chromatography were notably different (98% ee and 83% ee).7
This phenomenon called the enantiomeric enrichment is an
extremely novel example about a natural product. The curious
stereochemical phenomenon and interest in biological studies of
chiral cruciferous phytoalexins strongly push us to confirm the
absolute configuration of 1. Herein, we describe about the
absolute configuration of 1 by the X-ray crystallographic analy-
sis of 1-[(1'S,4'R)-camphanoyl]-(R)-spirobrassinin (6). This is
the first report concerning the absolute stereochemistry of the
cruciferous phytoalexins.
Racemic dioxibrassinin (3), which was a minor phytoalex-
in related metabolite isolated from cabbage, was synthesized
from isatin by the previously reported method.8 Cyclization of
( )-3 was achieved with thionyl chloride9 or methanesulfonyl
chloride leading to ( )-1. The racemic 1 reacted with (S)-(–)-α-
To confirm this prediction, X-ray crystallographic study has
been performed. Unfortunately, numerous attempts to crystal-
lize (+)-1, (–)-1, 4, and 4' for the X-ray study were unsuccessful.
Therefore, (+)-1 and (–)-1 were examined to derive to crystalline
compounds. Camphanoyl group was chosen as a crystalline
Copyright © 2000 The Chemical Society of Japan

Chemistry Letters 2000
887
8
9
K. Monde, K. Sasaki, A. Shirata, and M. Takasugi, Phytochemistry,
inducing part as well as its advantage having its known absolute
configuration as an internal standard. Compounds (+)-1 and (–)-
1 reacted with (1S,4R)-(–)-camphanoyl chloride to give two crys-
talline compounds 6 and 6', respectively (Scheme 2). Careful
recrystallization of 615 from CH2Cl2–hexane afforded colorless
prisms suitable for the X-ray analysis.
30, 2915 (1991).
K. Monde, M. Takasugi, and T. Ohnishi, J. Am. Chem. Soc., 116,
6650 (1994).
10 Selected data for 4: amorphous solid: [α]D20 +92.4˚ (c 0.17, CH2Cl2);
EI-MS (%) m/z 397 (M+, 10), 250 (100), 203 (20), 177 (30), 149 (8),
1
105 (12); IR (CHCl3): 3320, 1736, 1720 cm–1; H NMR (80 MHz,
CDCl3) δ 8.86 (1H, d, J = 7 Hz), 8.24 (1H, m), 7.57 (8H, m), 5.15
(1H, quintet, J = 7 Hz), 4.72 (1H, d, J = 15 Hz), 4.47 (1H, d, J = 15
Hz), 2.65 (3H, s), 1.69 (3H, d, J = 7 Hz); 13C NMR (75 MHz, CDCl3)
δ 179.1, 163.9, 150.6, 142.9, 139.3, 130.2, 128.8, 128.3, 127.5, 126.0,
125.6, 123.7, 116.6, 75.5, 65.5, 50.1, 22.7, 15.7. 4': amorphous solid:
20
[α]D +26.8˚ (c 0.17, CH2Cl2); EI-MS (%) m/z 397 (M+, 10), 250
(100), 203 (20), 177 (30), 149 (8), 105 (12); IR (CHCl3): 1738, 1726,
1722 cm–1; 1H NMR (80 MHz, CDCl3) δ 8.86 (1H, d, J = 7 Hz), 8.24
(1H, m), 7.37 (8H, m), 5.15 (1H, quintet, J = 7 Hz), 4.78 (1H, d, J =
15 Hz), 4.52 (1H, d, J = 15 Hz), 2.65 (3H, s), 1.62 (3H, d, J = 7 Hz);
13C NMR (75 MHz, CDCl3) δ 179.1, 164.0, 150.6, 142.8, 139.2,
130.1, 128.8, 128.3, 127.5, 126.0, 125.6, 123.7, 116.6, 75.6, 65.5,
50.1, 22.7, 15.7.
11 The enantiomeric excesses were determined by HPLC analysis using
a Sumichiral OA-4700 chiral column (i-PrOH–dichloroethane–hexa-
ne 2:8:90) monitoring by photodiode array and HPLC–CD detectors.
Figure 2 shows an ORTEP drawing of 6 determined by the
single-crystal X-ray diffraction analysis.16 The absolute config-
uration of 6, derived from (+)-1, was doubly confirmed as R by
the internal reference method and by the Bijvoet method using
the heavy atom (sulfur) effect. Consequently, the absolute con-
figuration of natural (–)-1 was unambiguously concluded as S,
which is consistent with the result predicted by CD data.
20
12 (+)-1: colorless needles: mp 143–145 ˚C; [α]D +142.7˚ (c 0.25,
CH2Cl2); UV (EtOH) λmax (ε) 218.0 (28800), 260.0 (sh, 6480), 295.6
(1550) nm; CD (EtOH) λext (∆ε) 204.4 (21.0), 221.0 (–25.9), 240.0
(2.6), 248.8 (–1.0), 263.6 (5.9), 308.2 (5.1) nm. (–)-1: colorless nee-
20
dles: mp 142–144 ˚C; [α]D –143.6˚ (c 0.25, CH2Cl2); UV (EtOH)
λmax (ε) 218.0 (28800), 260.0 (sh, 6480), 295.6 (1550) nm; CD
(EtOH) λext (∆ε) 204.4 (–21.0), 221.0 (25.9), 240.0 (–2.6), 248.8
(1.0), 263.6 (–5.9), 308.2 (–5.1) nm.
13 3,3-Dimethyloxindole was prepared by a published method, see: D.
Döpp and H. Weiler, Chem. Ber., 112, 3950 (1979). Compound 5 was
synthesized from n-hexylamine by two steps. Detailed experimental
1
conditions and its data will be published elsewhere. 5: H NMR (400
MHz, CDCl3) δ 3.39 (2H, t, J = 7.1 Hz), 2.54 (3H, s), 2.36 (3H, s),
1.65 (2H, q, J = 7.1 Hz), 1.40 (2H, m), 1.32 (4H, m), 0.89 (3H, t, J =
7.0 Hz); 13C NMR (100 MHz, CDCl3) δ 156.5 (C=N), 53.0 (CH2),
31.6 (CH2), 30.6 (CH2), 27.1 (CH2), 22.6 (CH2), 14.5 (CH3), 14.4
(CH3), 14.0 (CH3).
14 a) N. Harada and K. Nakanishi, "Circular Dichroic Spectroscopy –
Exciton Coupling in Organic Stereochemistry," University Science
Books, Mill Valley (1983). b) K. Nakanishi and N. Berova, in
"Circular Dichroism –Principles and Applications," ed. by K.
Nakanishi, N. Berova, and R. W. Woody, VCH Publishers, New
York (1994), p. 361.
In summary, we have succeeded in the determination of the
absolute configuration of natural (–)-spirobrassinin. This is the
first report concerning the absolute stereochemistry of crucifer-
ous phytoalexin family. Further investigation on chiral proper-
ties of 1 and its analog is in progress and the results will be
reported in detail elsewhere.
20
15 Selected data for 6: colorless prisms: mp 177–179 °C; [α]D –16.9˚
(c 0.20, CH2Cl2); EI-MS (%) m/z 430 (M+, 58), 402 (10), 383 (12),
357 (100), 329 (19), 249 (38); UV (EtOH) λmax (ε) 205.6 (30700),
237.2 (sh, 14900), 269.4 (sh, 5290), 297.0 (sh, 1340) nm; CD (EtOH)
λext (∆ε) 203.4 (6.2), 213.6 (–15.7), 226.4 (2.1), 240.2 (–6.2), 258.2
(4.3), 274.8 (5.7), 306.2 (–1.3); IR (CHCl3) 1787, 1772, 1718 cm–1;
1H NMR (400 MHz, CDCl3) δ 7.52 (1H, d, J = 8.1 Hz), 7.39 (1H, dd,
J = 7.4 and 1.3 Hz), 7.27 (1H, ddd, J = 8.1, 7.6, and 1.3 Hz), 7.17
(1H, ddd, J = 7.6, 7.4, and 1.0 Hz), 4.82 (1H, d, J = 15.4 Hz), 4.40
(1H, d, J = 15.4 Hz), 2.92 (1H, ddd, J = 13.5, 9.4, and 4.4 Hz), 2.55
(3H, s), 2.27 (1H, ddd, J = 13.5, 10.8, and 4.4 Hz), 1.89 (1H, ddd, J =
12.8, 10.8, and 4.4 Hz), 1.72 (1H, ddd, J = 12.8, 9.4, and 4.4 Hz),
1.22 (3H, s), 1.04 (3H, s), 0.90 (3H, s); 13C NMR (100 MHz, CDCl3)
δ 177.4 (C), 175.1(C), 171.1 (C), 163.9 (C), 138.2 (C), 129.9 (CH),
129.4 (C), 125.9 (CH), 124.6 (CH), 113.7 (CH), 92.9 (C), 76.0 (CH2),
65.7 (C), 57.3 (C), 54.5 (C), 30.4 (CH2), 29.5 (CH2), 17.6 (CH3), 16.6
(CH3), 15.6 (CH3), 9.7 (CH3).
P. K. thanks the Grant Agency for Science of the Slovak
Republic (No. 1/6080/99) for financial support.
References and Notes
1
2
a) J. A. Bailey and J. W. Mansfield, "Phytoalexins," Blackie & Son,
Glasgow (1982). b) C. J. W. Brooks and D. G. Watson, Nat. Prod.
Rep., 8, 427 (1991).
a) M. Takasugi, N. Katsui, and A. Shirata, J. Chem. Soc., Chem.
Commun., 1986, 1077. b) M. Takasugi, K. Monde, N. Katsui, and A.
Shirata, Bull. Chem. Soc. Jpn., 61, 285 (1988).
16 Crystal data for 6: C21H22N2O4S2, M = 430.54, colorless prisms (0.42
× 0.28 × 0.20 mm), orthorhombic, space group P212121 (#19), a =
16.076(3), b = 17.194(4), c = 7.554(1) Å, V = 2088(7) Å3, Z = 4, Dc =
1.369 g cm–3, Dm = 1.359 g cm–3 by flotation using a CCl4–hexane
solution, radiation Cu Kα (1.54178 Å), unique data Fο > 3σ(Fο),
2040. The skeletal structure was solved by the direct method and suc-
cessive Fourier syntheses. All hydrogen atoms were found by the dif-
ference Fourier syntheses. Absorption correction and full matrix
least-squares refinement of positional and thermal parameters, includ-
ing anomalous scattering factors of sulfur, oxygen, nitrogen, and car-
3
4
Recent Review: M. S. C. Pedras, F. I. Okanga, I. L. Zaharia, and A.
Q. Khan, Phytochemistry, 53, 161 (2000).
For antimicrobial activity, see ref. 3. For antitumor activity, see R. G.
Mehta, J. Liu, A. Constantinou, M. Hawthorne, J. M. Pezzuto, R. C.
Moon, and R. M. Moriarty, Anticancer Res., 14, 1209 (1994). For
oviposition stimulant activity, see R. Baur, E. Städler, K. Monde, and
M. Takasugi, Chemoecology, 8, 163 (1998).
5
6
M. S. C. Pedras and J. L. Sorensen, Phytochemistry, 49, 1959 (1998).
M. Takasugi, K. Monde, N. Katsui, and A. Shirata, Chem. Lett., 1987,
1631.
bon atoms, led to the final convergence with R = 0.0330 and Rw
=
0.0472, while R = 0.0450 and Rw = 0.0648 for the mirror image struc-
ture. The absolute configuration was also determined by measurement
of the Bijvoet pairs.
7
K. Monde, N. Harada, M. Takasugi, P. Kutschy, M. Suchy, and M.
Dzurilla, J. Nat. Prod., submitted (2000).