Synthesis, absolute configuration, and enantiomeric enrichment of a cruciferous oxindole phytoalexin, (S)-(-)-spirobrassinin, and its oxazoline analog

Article abstract of DOI:10.1021/jo0155052

Stereochemical studies of a cruciferous oxindole phytoalexin, (S)-(-)-spirobrassinin [(-)-4], and its oxazoline analogue, spirooxazoline (11), were carried out. Racemic spirobrassinin [(±)-4] was synthesized by SOCl_2- or MsCl-mediated cyclization of dioxibrassinin [(±)-8]. Treatment of (3-hydroxyoxindol-3-yl)methylammonium chloride [(±)-9] with CSCl₂ and subsequent methylation of the obtained spirooxazolidinethione (±)-10 afforded spirooxazoline [(±)-11]. Enantioresolution of (±)-4 and (±)-11 was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate (12), chromatographic separation of diastereomeric amides 13, 14 or 15, 16, and their cleavage with CH₃ONA. Absolute configuration of the stereogenic center in natural (S)-(-)-4 was derived from the exciton, calculated via CD methods, and unequivocally confirmed by X-ray crystallographic analyses of 1-[1′S,4′R-(-)-camphanoyl] derivatives [(-)-19 and (-)-20] of (+)- and (-)-4. Novel enantiomeric enrichment phenomena of 4 and 11 were discovered during their chromatographic separations under achiral HPLC conditions. Screening of antifungal activity against the fungus Bipolaris leersiae revealed no significant dependence of this activity on absolute configuration.

Full text of DOI:10.1021/jo0155052

3940
J . Org. Chem. 2001, 66, 3940-3947
Syn th esis, Absolu te Con figu r a tion , a n d En a n tiom er ic En r ich m en t
of a Cr u cifer ou s Oxin d ole P h ytoa lexin , (S)-(-)-Sp ir obr a ssin in , a n d
Its Oxa zolin e An a log
Mojm´ır Suchy´,† Peter Kutschy,*,† Kenji Monde,*,‡ Hitoshi Goto,§ Nobuyuki Harada,|
Mitsuo Takasugi,| Milan Dzurilla,† and Eva Balentova´†
Department of Organic Chemistry, P. J . Sˇ afa´rik University, Moyzesova 11,
041 67 Kosˇice, Slovak Republic, Division of Material Science, Graduate School of
Environmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060-0810, J apan, Knowledge-based
Information Engineering, Toyohashi University of Technology, Toyohashi, 441-8580, J apan, and
Institute for Chemical Reaction Science, Tohoku University, Aoba-ku, Sendai 980-8577, J apan
kutschy@kosice.upjs.sk
Received J anuary 5, 2001
Stereochemical studies of a cruciferous oxindole phytoalexin, (S)-(-)-spirobrassinin [(-)-4], and
its oxazoline analogue, spirooxazoline (11), were carried out. Racemic spirobrassinin [(()-4] was
synthesized by SOCl2- or MsCl-mediated cyclization of dioxibrassinin [(()-8]. Treatment of
(3-hydroxyoxindol-3-yl)methylammonium chloride [(()-9] with CSCl2 and subsequent methylation
of the obtained spirooxazolidinethione (()-10 afforded spirooxazoline [(()-11]. Enantioresolution
of (()-4 and (()-11 was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate (12),
chromatographic separation of diastereomeric amides 13, 14 or 15, 16, and their cleavage with
CH3ONa. Absolute configuration of the stereogenic center in natural (S)-(-)-4 was derived from
the exciton, calculated via CD methods, and unequivocally confirmed by X-ray crystallographic
analyses of 1-[1′S,4′R-(-)-camphanoyl] derivatives [(-)-19 and (-)-20] of (+)- and (-)-4. Novel
enantiomeric enrichment phenomena of 4 and 11 were discovered during their chromatographic
separations under achiral HPLC conditions. Screening of antifungal activity against the fungus
Bipolaris leersiae revealed no significant dependence of this activity on absolute configuration.
In tr od u ction
hitherto isolated cruciferous phytoalexins is the presence
of indole or an indole-related nucleus possessing a side
chain or another heterocycle containing one or two sulfur
atoms.3a Typical representatives are brassinin (1), meth-
oxybrassinin (2), and cyclobrassinin (3) isolated from
Pseudomonas cichorii-inoculated Chinese cabbage (Bras-
sica campestris).4
In responding to biological, physical, or chemical stress,
plants produce antimicrobial secondary metabolites,
called phytoalexins, de novo.1 These compounds play a
significant role in the induced defense mechanisms of
plants against microbial microrganisms.2 An important
group of stress metabolites represented by phytoalexins
has been reported from the plant family cruciferae,3
which includes many important crops cultivated world-
wide. A common structural feature of more than 20
* To whom correspondence should be addressed. Tel: +421-95-62-
22610, ext. 192; Fax: +421-95-62-22124.
† P. J . Sˇafa´rik University.
‡ Tohoku University. Tel: +81-22-217-5634; Fax; +81-22-217-5667.
E-mail: kmonde@icrs.tohoku.ac.jp.
§ Toyohashi University of Technology.
| Hokkaido University.
(1) Purkayastha, R. P. In Handbook of Phytoalexins metabolism and
Action; Daniel, M., Purkayastha, R. P., Eds.; Marcel Dekker: New
York, 1995; pp 1-39.
(2) (a) Bailey, J . A.; Mansfield, J . W. Phytoalexins; Blackie & Sons
Ltd.: Glasgow, 1982; pp 1-334. (b) Lamb, C. J .; Ryals, J . A.; Ward, R.
E.; Dixon, R. A. Biotechnology 1992, 10, 1436-1445. (c) Hain, R.; Reif,
H.-J .; Krause, J .; Langebartels, R.; Kindl, H.; Vornam, B.; Wiese, W.;
Schmelzer, E.; Schreier, P. H.; Sto¨cker, R. H.; Stenzel, K. Nature 1993,
361, 153-156. (d) Yoshikawa, M.; Tsuda, M.; Takeuchi, Y. Naturwis-
senschaften 1993, 80, 417-420. (e) Afek, U.; Carmeli, S.; Aharoni, R.
Phytochemistry 1995, 39, 1347-1350. (f) Glazerbrook, J .; Zook, M.;
Mert, F.; Kagan, I.; Rogers, E. E.; Crute, I. R.; Holub, E. B.;
Hammerschmidt, R.; Ausubel, F. M. Genetics 1997, 146, 381-392.
(3) For a recent reviews of indole phytoalexins, see: (a) Pedras, M.
S. C.; Okanga, F. I.; Zaharia, I. L.; Khan, A. Q. Phytochemistry 2000,
53, 161-176. (b) Rouxel, T.; Kollman, A.; Belesdent, M.-H. In Handbook
of Phytoalexins metabolism and Action; Daniel, M., Purkayastha, R.
P., Eds.; Marcel Dekker: New York, 1995; pp 229-261. (c) Gross, D.
J . Plant. Dis. Prot. 1993, 100, 433-442.
In the past decade, interesting biological activities of
the cruciferous phytoalexins have been reported from
various research groups, including antimicrobial,3a,c an-
titumor,5 and oviposition-stimulant6 activities. With re-
spect to the examinations of various biological activities
(4) (a) Takasugi, M.; Katsui, N.; Shirata, A. J . Chem. Soc., Chem.
Commun. 1986, 1077-1078. (b) Takasugi, M.; Monde, K.; Katsui, N.;
Shirata, A. Bull. Chem. Soc. J pn. 1988, 61, 285-289.
10.1021/jo0155052 CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/27/2001

Stereochemical Studies of Spirobrassinin
J . Org. Chem., Vol. 66, No. 11, 2001 3941
Sch em e 1
of the cruciferous phytoalexins as well as the dietary
importance of brassicaceous vegetables in cancer chemo-
prevention,7 it is quite important to investigate synthetic
approaches to the indole phytoalexins4,8,9 and their
analogues. In 1987, the first oxindole phytoalexin, spiro-
brassinin [(-)-4] was isolated from P. cichorii-inoculated
J apanese radish (Raphanus sativus).10 However, no stud-
ies regarding the stereochemistry of 4 have been reported
to date. Moreover, stereochemical studies of the other
three spiroindoline[3,5′]thiazolidine-type phytoalexins,
methoxyspirobrassinin (5),11 methoxyspirobrassinol (6),
and methoxyspirobrassinol methyl ether (7),12 have not
been carried out.3a Preliminary experiments have re-
vealed that partially enantioenriched spirobrassinin (4)
shows an enantiomeric-enrichment phenomenon during
nonchiral chromatographic separation.13 This interesting
feature is extremely novel in a natural product. Because
of this curious stereochemical phenomenon and interest
in biological studies of chiral cruciferous phytoalexins,
we have begun stereochemical studies of spirobrassinin
(4) and its oxazoline analogue, spirooxazoline (11). Pre-
liminary studies have revealed that natural (-)-4 has an
S configuration based on X-ray crystallography of a
(1′S,4′R)-camphanoyl derivative of (+)-4.14 Herein, we
describe our detailed stereochemical studies of spiro-
brassinin (4) and its analogue, spirooxazoline (11) by
means of chiral syntheses, the exciton chirality and
calculated CD methods, and X-ray crystallography, as
well as their enantiomeric-enrichment phenomena and
biological activities.
sequent cyclization of (()-8 with thionyl chloride17 or
methanesulfonyl chloride in pyridine afforded racemic
(()-4. Spectroscopic data of (()-4 were fully identical to
those of the natural spirobrassinin.10 In contrast, (()-9
was treated with thiophosgene to give (()-10, with the
desired spiro ring system, in 92% yield. The subsequent
methylation of (()-10 afforded racemic spirooxazoline
[(()-11], an isosteric analogue of 4. The structure of 11
was confirmed by NMR, including NOE, DQF-COSY,
HMQC, and HMBC experiments18 at the stage of (R)-
(+)-11 as well as other spectroscopic data.
Resu lts a n d Discu ssion
To enantioresolve (()-4 and (()-11, a chiral auxiliary
method was applied. Each racemate was allowed to react
with (S)-(-)-1-phenylethyl isocyanate (12) with the ad-
dition of triethylamine for reaction acceleration to pro-
duce urea derivatives, respectively (Scheme 2).
Syn th esis a n d En a n tior esolu tion . The common
starting material for the syntheses of racemic spiro-
brassinin [(()-4] and spirooxazoline [(()-11] was (()-(3-
hydroxyoxindol-3-yl)methylammonium chloride15 [(()-9]
(Scheme 1).
Compound 9 was converted to racemic dioxibrassinin
[(()-8], a minor phytoalexin-related metabolite isolated
from cabbage, by a previously reported method.16 Sub-
As expected, the obtained diastereomeric pairs of
amides (+)-13, (+)-14 and (+)-15, (+)-16 could be sepa-
rated by simple silica gel chromatography. Removal of
the chiral auxiliary by treatment with sodium methoxide
afforded (+)-4 ([R]20 +142.7, c 0.25, CH2Cl2, 91% ee),
D
(5) (a) Mehta, R. G.; Liu, J .; Constantinou, A.; Hawthorne, M.;
Pezzuto, J . M.; Moon, R. C.; Moriarty, R. M. Anticancer Res. 1994, 14,
1209-1214. (b) Mehta, R. G.; Liu, J .; Constantinou, A.; Thomas, C.
F.; Hawthorne, M.; You, M.; Gerha¨user, C.; Moon, R. C.; Moriarty, R.
M. Carcinogenesis 1995, 16, 399-404.
(6) Baur, E.; Sta¨dler, K.; Monde, K.; Takasugi, M. Chemoecology
1998, 8, 163-168.
(7) Verhoeven, D. T. H.; Verhagen, H.; Goldbohm, R. A.; van den
Brandt, P. A.; van Poppel, G. Chem. Biol. Interact. 1997, 103, 79-
129, and cited references.
(8) Yamada, F.; Kobayashi, K.; Shimizu, A.; Aoki, N.; Somei, M.
Heterocycles 1993, 36, 2783-2804.
(9) Kutschy, P.; Dzurilla, M.; Takasugi, M.; To¨ro¨k, M.; Achbergerova´,
I.; Homzova´, R.; Ra´cova´, M. Tetrahedron 1998, 54, 3549-3566.
(10) Takasugi, M.; Monde, K.; Katsui, N.; Shirata, A. Chem. Lett.
1987, 1631-1632.
(-)-4 ([R]20 -143.6, c 0.25, CH2Cl2, 92% ee), and (+)-11
D
([R]20D +31.7, c 1.37, CHCl3, 95% ee), (-)-11 ([R]20D -34.1,
c 0.89, CHCl3, 83% ee), respectively. The chiral analysis
was carried out by HPLC using a Sumichiral OA-4700
chiral column (i-PrOH/dichloroethane/hexane), which
was monitored by a photodiode array and on-line HPLC-
CD detectors.18 It should be noted that the online HPLC-
CD detector proved to be a powerful tool to verify the
chirality of each enantiomer during the analysis.
CD Stu d ies. To predict the absolute configuration of
natural (-)-spirobrassinin [(-)-4], we carried out CD
studies of (+)-4, (-)-4, (+)-11, and (-)-11. CD spectra of
enantiomeric pairs are completely mirror images.18 Enan-
tiomer (-)-4 showed an exciton-type split at 221 (∆ꢀ
+25.9) and 204 (∆ꢀ -21.0) nm with a moderate A value
(+46.9) in the short-wavelength region and two negative
Cotton effects at 308 (∆ꢀ -5.1) and 264 nm (∆ꢀ -5.9) in
the long-wavelength region (Figure 1).
(11) Gross, D.; Porzel, A.; Schmidt, J . Z. Naturforsch. 1994, 49c,
281-285.
(12) Monde, K.; Takasugi, M.; Shirata, A. Phytochemistry 1995, 39,
581-586.
(13) Monde, K.; Harada, N.; Takasugi, M.; Kutschy, P.; Suchy´, M.;
Dzurilla, M. J . Nat. Prod. 2000, 63, 1312-1314.
(14) The details of the X-ray analyses of compounds (+)-15, (-)-19,
and (-)-20, see Supporting Information. Preliminary communication
[compound (-)-19]: Monde, K.; Osawa, S.; Harada, K.; Takasugi, M.;
Suchy´, M.; Kutschy, P.; Dzurilla, M.; Balentova´, E. Chem. Lett. 2000,
886-887.
To assign this split type to the Cotton effect, a model
compound, dimethyl N-hexyldithiocarbonimidate (17),
(15) Conn, W. R.; Lindwall, H. G. J . Am. Chem. Soc. 1936, 58, 1236-
1239; for
Supporting Information.
a modified procedure for the preparation of (()-9, see
(17) Monde, K.; Takasugi, M.; Ohnishi, T. J . Am. Chem. Soc. 1994,
116, 6650-6657.
(18) See Supporting Information.
(16) Monde, K.; Sasaki, K.; Shirata, A.; Takasugi, M. Phytochemistry
1991, 30, 2915-2917.

3942 J . Org. Chem., Vol. 66, No. 11, 2001
Suchy´ et al.
F igu r e 2. A structure of natural (S)-(-)-4. Two arrows on
the molecule indicate axes of transition dipole moments.
F igu r e 1. Experimental UV and CD spectra of (-)-4, 17, and
3,3-dimethyloxindole. Solid line: UV and CD spectra of (-)-4
(a major natural enantiomer). Dotted line: UV spectrum of
17. Alternate long and short dash line: UV spectrum of 3,3-
dimethyloxindole.
Sch em e 2
F igu r e 3. UV and CD spectra of (-)-4 and (-)-11. Solid line:
UV and CD spectra of (-)-11. Dotted line: UV and CD spectra
of (-)-4.
(Figure 2). The CD spectrum of (-)-11 fully corresponds
to the spectrum of (-)-4 (Figure 3), and (+)-11 to (+)-4,
suggesting that (-)-11 also has an S configuration.
To more accurately predict the absolute configuration,
the theoretical CD calculation was examined. The geom-
etries of spirobrassin (4) for s-cis/trans conformations on
a S-C-S-CH3 side-chain were optimized using the
Gaussian 98 program.21 Using these two conformers, both
oscillator and rotational strengths in the dipole length
and velocity expressions corresponding to 30 excitation
energies were calculated by the Dalton program.22 The
calculated UV and CD spectra of s-cis and s-trans forms
(20) (a) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy
- Exciton Coupling in Organic Stereochemistry; University Science
Books: Mill Valley, CA, 1983. (b) Nakanishi, K.; Berova, N. In Circular
Dichroism - Principles and Application; Nakanishi, K., Berova, N.,
Woody, R. W., Eds.; VCH Publishers: New York, 1994; pp 361-398.
(21) Frisch, M. J .; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J . R.; Zakrzewski, V. G.; Montgomery, J . A.,
J r.; Stratmann, R. E.; Burant, J . C.; Dapprich, S.; Millam, J . M.;
Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J .;
Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo,
C.; Clifford, S.; Ochterski, J .; Petersson, G. A.; Ayala, P. Y.; Cui, Q.;
Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J . B.; Cioslowski, J .; Ortiz, J . V.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.;
Fox, D. J .; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.;
Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; J ohnson, B.; Chen, W.;
Wong, M. W.; Andres, J . L.; Gonzalez, C.; Head-Gordon, M.; Replogle,
E. S.; Pople, J . A. Gaussian 98 (Revision A.7); Gaussian, Inc.:
Pittsburgh, PA, 1998.
was synthesized from n-hexylamine. The UV spectrum
of 17 shows a moderate absorption at 219 nm (ꢀ 8590),
while 3,3-dimethyloxindole shows a stronger absorption
at 205 nm (ꢀ 30000).19 These data suggest that two
chromophores in (-)-4 (oxindole and NdC(SCH3)2 moiety)
would induce the split of the Cotton effects in its CD
spectrum.20 In the case of natural (-)-4, two axes of their
transition dipole moments should be clockwise to give
positive chirality leading to the S configuration of (-)-4
(19) 3,3-Dimethyloxindole was prepared by the published method:
Do¨pp, D.; Weiler, H. Chem. Ber. 1979, 112, 3950-3955.

Stereochemical Studies of Spirobrassinin
J . Org. Chem., Vol. 66, No. 11, 2001 3943
Sch em e 3
F igu r e 4. Experimental UV and CD spectra of (-)-4 and
calculated UV and CD spectra of (S)-4. Solid line: Experi-
mental UV and CD spectra of (-)-4 (a major natural enantio-
mer). Dotted line: Calculated UV and CD spectra of s-trans-
(S)-4. Alternate long and short dash line: Calculated UV and
CD spectra of s-cis-(S)-4.
column separation lasting more than 1 h, the original
spirobrassinin (4) was recovered. Rapid separation, in
less than 1 h, gave sufficient yields of (-)-19 (22%) and
(-)-20 (22%). The diastereomers of (-)-19 and (-)-20
were assigned to derivatives from (+)-4 and (-)-4 by a
direct comparison of products obtained from the reactions
of (+)-4 and (-)-4 with 18, respectively (Scheme 3). The
diastereomeric amides (-)-19 and (-)-20 afforded suit-
able single crystals from dichloromethane/hexane which
were submitted to X-ray analysis.14 The absolute config-
uration of (-)-20, derived from (-)-4, was confirmed as
S by the internal reference method. Thus, the natural
spirobrassinin [(-)-4] has an S-configuration. Further-
more, the X-ray analysis of (-)-19 confirmed the R-
configuration of (+)-spirobrassinin [(+)-4]14 by both the
internal reference method and the Bijvoet method using
the heavy-atom (sulfur) effect. These conclusions are
consistent with the result predicted by the CD data.
En a n tiom er ic En r ich m en t. To investigate its enan-
tiomeric purity, natural (S)-(-)-4, was newly isolated
from P. cichorii - inoculated turnip roots (B. campestris
L. ssp. Rapa).13 A short separation by nonchiral HPLC
gave two spirobrassinin fractions. Surprisingly, their
enantiomeric excesses were considerably different (earlier-
eluted fraction 98% ee, later-eluted fraction 83% ee) as
determined by the chiral HPLC. Preliminary experiments
revealed that this phenomenon, called enantiomeric
enrichment, was caused by natural (S)-(-)-4 not being
enantiomerically pure. In addition, partially enantio-
enriched (R)-(+)-4 (47% ee, 7.7 mg) showed a more
dramatic change in its enantiomeric excesses during
separation by nonchiral HPLC (Figure 5, I). This phe-
nomenon of 4 was observed even in the case of a very
small injection amount (0.1 mg, Figure 5, II). To inves-
tigate the related mechanisms in more detail, spiro-
oxazoline 11 was examined. As expected, partially enan-
tioenriched (R)-(+)-11 (48% ee) also showed the same
phenomenon.18 (Figure 5, III). This phenomenon has
previously been observed in several cases;24 however, this
is a novel example of the enantiomeric-enrichment
phenomenon in a natural product in the case of 4.25 As
of (S)-4 show reasonable agreement with the experimen-
tal spectrum of (-)-4, especially at 221 and 308 nm
(Figure 4).23 This comparison suggests that the configu-
ration of (-)-4 should be S, which is consistent with the
result from the exciton chirality method. Almost the same
result was obtained in the calculation for (S)-11.18
X-r a y Cr ysta llogr a p h y. Although all of the prepared
enantiomers were crystalline compounds, none of them
afforded single crystals suitable for X-ray analysis to
allow determination of the absolute stereochemistry. Of
the diastereomeric amides (+)-13 - (+)-16, only (+)-15 is
crystalline, and the others are amorphous. Fortunately,
compound (+)-15 afforded well-developed single crystals
from dichloromethane/hexane, and X-ray analysis re-
vealed its configuration at the spiro atom to be R by the
internal reference method.14 Consequently, we can con-
clude that spirooxazoline (+)-11 has an R configuration
and therefore that (-)-11 has an S configuration. The
absolute configuration of natural spirobrassinin [(-)-4]
was clearly solved by derivatization with (1S,4R)-(-)-
camphanoyl chloride (18), including a crystalline-induc-
ing portion (Scheme 3).
In addition, the use of compound 18 as an internal
standard was advantageous as its absolute configuration
is known. Racemic 4 was allowed to reacted with (1S,4R)-
(-)-18 to give two amides. Flash chromatographic sepa-
ration of diastereomers (-)-19 and (-)-20 was somewhat
complicated by their instability on silica gel. After a
(22) Helgaker, T.; J ensen, H. J . Aa.; J orgensen, P.; Olsen, J .; Ruud,
K.; Agren, H.; Andersen, T.; Bak, K. L.; Bakken, V.; Christiansen, O.;
Dahle, P.; Dalskov, E. K.; Enevoldsen, T.; Fernandez, B.; Heiberg, H.;
Hettema, H.; J onsson, D.; Kirpekar, S.; Kobayashi, R.; Koch, H.;
Mikkelsen, K. V.; Norman, P.; Packer, M. J .; Saue, T.; Taylor, P. R.;
Vahtras, O. Dalton - An electronic structure program, Release 1.0,
1997.
(23) The caluculated CD curve of s-cis-(S)-4 is more compatible with
the experimental CD curve of (-)-4. However, a major conformer
calculated Gaussian 98 and MOPAC-AM1 is s-trans. See also Sup-
porting Information.

3944 J . Org. Chem., Vol. 66, No. 11, 2001
Suchy´ et al.
F igu r e 5. Enantiomeric enrichment of spirobrassinin (4) and spirooxazoline (11) by nonchiral HPLC system. Partially
enantioenriched 4 and 11 was separated by HPLC on YMC SIL06 column (300 × 10 mm) using i-PrOH/CHCl3 at speed of 3
mL/min. The peak of 4 or 11 was divided into several fractions (one fraction volume is ca. 0.5 mL). Enantioexcesses of each
fraction were analyzed by the chiral HPLC analysis system. I: (+)-4 (47% ee, 7.7 mg) was injected to the HPLC (1% i-PrOH-
CHCl3) and a peak (tR 25.0-30.0 min) was divided into 30 fractions. II: (+)-4 (43% ee, 0.1 mg) was injected to the HPLC (1%
i-PrOH-CHCl3) and a peak (tR 33.6-35.6 min) was divided into 12 fractions. III: (+)-11 (48% ee, 0.1 mg) was injected to the
HPLC (5% i-PrOH-CHCl3) and a peak (tR 16.2-17.6 min) was divided into 9 fractions. (a): On-line CD detection at 308 or 307
nm. (b): UV detection at 308 or 307 nm. (c): Enantioexcesses of each fraction and total elution curve calculated from absorption
areas of each enantiomer at 230 nm.
other authors have mentioned, this phenomenon is likely
caused by a diastereomeric difference between homo-
chiral and heterochiral associations on the surface of the
stationary phase. At this stage, we have concluded that
the enantiomeric excess of natural (S)-(-)-4 is 95% from
the average calculation of both fractions. The composite
nature of 4 suggests to us a difference in biological
activity between the racemate and both of the enanti-
omers. Therefore, the biological activities of these com-
pounds were examined.
An tifu n ga l Activity. An examination of the antifun-
gal activity of 4 and 11, following the previously described
procedure,9 against the fungus Bipolaris leersiae revealed
only moderate activity, compared to brassinin (1), with
little or no difference between the enantiomers and
racemates of 4 and 11 (Table 1).
(24) (a) Cundy, K. C.; Crooks, P. A. J . Chromatogr. 1983, 281, 17-
33. (b) Charles, R.; Gil-Av, E. J . Chromatogr. 1984, 298, 516-512. (c)
Tsai, W.-L.; Hermann, K.; Hug, E.; Rohde, B.; Dreiding, A. S. Helv.
Chim. Acta 1985, 68, 2238-2243. (d) Dobashi, A.; Motoyama, Y.;
Kinoshita, K.; Hara, S. Anal. Chem. 1987, 59, 2209-2211. (e) Matusch,
R.; Coors, C. Angew. Chem., Int. Ed. Engl. 1989, 28, 626-627. (f)
Carman, R. M.; Klika, K. D. Aust. J . Chem. 1991, 44, 895-896. (g)
Diter, P.; Taudien, S.; Samuel, O.; Kagan, H. B. J . Org. Chem. 1994,
59, 370-373. (h) Nicoud, R.-M.; J aubert, J .-N.; Rupprecht, I.; Kinkel,
J . Chirality 1996, 8, 234-243. (i) Kosugi, H.; Abe, M.; Hatsuda, R.;
Uda, H.; Kato, M. Chem. Commun. 1997, 1857-1858.
Con clu sion
Racemic spirobrassinin [(()-4] and its oxazoline ana-
logue, spirooxazoline [(()-11], were synthesized and
enantioresolved by a chiral auxiliary method. The abso-
lute configurations of natural (-)-4 and (-)-11 were
(25) To the best of our knowledge, only two examples of enantiomeric
enrichment in the case of natural products have been reported. One is
14C-labeled nicotine,24a and the other is a urinary metabolite of the
female Australian brushtail possum.24f

Stereochemical Studies of Spirobrassinin
J . Org. Chem., Vol. 66, No. 11, 2001 3945
Ta ble 1. In h ibition by 4 a n d 11 of F u n ga l Gr ow th of
(100 MHz, acetone-d6, COM, DEPT) δ 189.3 (C), 174.0 (C),
143.5 (C), 132.5 (CH), 127.0 (C), 126.0 (CH), 123.9 (CH), 111.4
(CH), 85.5 (C), 51.8 (CH2); UV (EtOH) λmax (ꢀ) 211.4 (26900),
244.6 (20200), 302.8 (1670) nm; EIMS m/z (%) 220 (M+, 20),
191 (25). 177 (10), 164 (50), 148 (60), 89 (22), 60 (100). Anal.
Calcd for C10H8N2O2S2: C, 54.53; H, 3.66; N, 12.72. Found:
C, 54.62; H, 3.57 N, 12.70.
(()-2′-(Meth ylsu lfa n yl)sp ir o{in d olin e-3,5′-([4′,5′]d ih y-
d r ooxa zol}-2-on e [(()-11]. To a stirred suspension of pow-
dered K2CO3 (307 mg, 2.25 mmol) in dry acetone (27 mL) was
added (()-10 (451 mg, 2.05 mmol) and CH3I (862 mg, 0.38 mL,
6.13 mmol). After being stirred for 7.5 h, the reaction mixture
was poured into cold water (300 mL) and extracted with
AcOEt. The organic layer was dried over Na2SO4 and concen-
trated in vacuo. Crystallization from methanol afforded (()-
11 as white crystals (370 mg, 77%): mp 174-176 °C; IR, NMR,
and EIMS data are identical with those for (R)-(+)-11. Anal.
Calcd for C11H10N2O2S: C, 56.39; H, 4.30; N, 11.96. Found:
C, 56.60; H, 4.57; N, 12.13.
P r ep a r a tion of Dia ster eom er ic Am id es (+)-13 a n d (+)-
14. To a solution of (S)-(-)-1-phenylethyl isocyanate (12, 98
mg, 0.66 mmol) in dry acetone (3 mL) was added (()-4 (150
mg, 0.6 mmol). Then, triethylamine (65 mg, 0.09 mL, 0.6
mmol) was added, and the reaction mixture was set aside for
68 h. The solvent was evaporated in vacuo, and the residue
was submitted to column chromatography (CH2Cl2). The first
fraction gave (+)-13 (110 mg, 46%) and second fraction afforded
(+)-14, contaminated with (+)-13. Repeated chromatography
of the second fraction afforded pure (+)-14 (55 mg, 23%). (+)-
N1-[(1S)-1-P h en ylet h yl]-1-[(R)-sp ir ob r a ssin in ]ca r b oxa -
m id e [(+)-13]: an amorphous pale yellow solid, mp 43-46 °C;
Rf (CH2Cl2) 0.24; [R]20D +92.4 (c 0.17, CH2Cl2); IR (CHCl3) 3320,
1736, 1720 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.84 (1H, br d,
J ) 7.3 Hz, NH), 8.21 (1H, d, J ) 8.3 Hz,), 7.37 (6H, m), 7.28
(1H, m), 7.21 (1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 5.12 (1H,
quintet, J ) 7.1 Hz), 4.69 (1H, d, J ) 15.4 Hz), 4.48 (1H, d,
J ) 15.4 Hz), 2.63 (3H, s), 1.58 (3H, d, J ) 7.1 Hz); 13C NMR
(100 MHz, CDCl3, COM, DEPT) δ 179.1(C), 163.9 (C), 150.6
(C), 142.9 (C), 139.2(C), 130.2 (CH), 128.8 (CH), 128.3 (C),
127.5 (CH), 126.0 (CH), 125.6 (CH), 123.7 (CH), 116.6 (CH),
75.5 (CH2), 65.5 (C), 50.1 (CH), 22.7 (CH3), 15.7 (CH3); UV
(EtOH) λmax (ꢀ) 196.2 (48400), 216.8 (31500) nm; CD (EtOH)
Bipola r is leer sia ea
concentration (mmol‚L-1
0.1 0.25 0.5
)
compound
0.05
1
(()-4
(+)-4
(-)-4
(()-10
(+)-10
(-)-10
1
-
-
-
-
+
+
+
-
-
-
-
-
-
-
-
-
-
+
++
++
+
++
++
+
-
-
+
-
-
-
++
+++
+++
+++
+++
a
Intensity of antifungal activity was scored and classified into
four grades: -, normal growth; +, a small reduction in growth;
++, about half of the normal growth; +++, no growth.
determined to be S by the X-ray crystallographic analyses
and their CD studies. The HPLC separation under
achiral conditions of nonracemic mixtures of (R)-(+)-4
and (S)-(-)-4 as well as (R)-(+)-11 and (S)-(-)-11 showed
a novel enantiomeric-enrichment phenomenon.
Exp er im en ta l Section
Gen er a l Meth od s. Melting points are uncorrected. Optical
rotations were measured at room temperature in either 1-cm
or 1-dm tubes, using J ASCO DIP-1000 and Messtechnik
POLAR L-mp digital polarimeters; specific rotations are given
in 10-1 deg cm2 g-1. UV and CD spectra were obtained using
a J ASCO V-570 and a J ASCO J -720 spectrometer, respectively.
IR spectra were recorded on a Zeiss IR-75 spectrometer. The
NMR spectra were measured on a J EOL Lambda 400 spec-
trometer with TMS as an internal standard. Mass spectra were
taken on a Finigan SSQ 700 spectrometer at ionization energy
70 eV. X-ray crystallographic data were collected with a Mac
Science single-crystal diffractometer MXC18SH. Microanalyses
were performed with a Perkin-Elmer, Model 2400 analyzer.
HPLC separations were performed on a J ASCO 900S instru-
ment equipped with a J ASCO MD-1515 multiwavelength
detector, a J ASCO-Borwin data processing system and a
J ASCO CD-1595 on-line CD detector using an YMC-Pack
SIL-06 column (10 × 300 mm, YMC Co., Ltd.). The reaction
course was monitored by thin-layer chromatography, using
Kavalier Silufol plates. The preparative column chromatog-
raphy (flash chromatography) was performed over the Kiesel-
gel Merck Type 9385, 230-400 mesh.
(()-Sp ir obr a ssin in [(()-4]. To a stirred solution of dioxi-
brassinin16 [(()-8, 400 mg, 1.48 mmol] in dry pyridine (4 mL)
was added SOCl2 (320 mg, 0.195 mL, 2.68 mmol). Alterna-
tively, MsCl (133 mg, 0.09 mL, 1.16 mmol) was added to a
solution of (()-8 (100 mg, 0.37 mmol) in dry pyridine (3 mL).
After being stirred for 1 h (SOCl2), or 5.5 h and setting aside
for 18 h (MsCl), the reaction mixture was neutralized with 1
N HCl, and extracted with AcOEt. Organic layer was dried
over Na2SO4, filtered on charcoal and concentrated in vacuo.
Crystallization of the residue from acetone/cyclohexane af-
forded (()-4 as colorless crystals (245 mg, 66%), using SOCl2
or (45 mg, 49%), using MsCl: mp 160-162 °C, ref10 158-159
°C. Spectral data of (()-4 are identical with natural (-)-
spirobrassinin.10 Anal. Calcd for C11H10N2OS: C, 52.77; H,
4.03; N, 11.19. Found: C, 52.90; H, 4.21, N, 10.92.
λ
ext (∆ꢀ) 196.8 (-17.0), 208.0 (+24.8), 230.2 (-3.6), 245.4 (+3.6),
259.2 (+3.6), 306.4 (+1.3) nm. EIMS m/z (%) 397 (M+, 10), 250
(100), 203 (20), 177 (30), 145 (10), 105 (30). Anal. Calcd for
C
20H19N3O2S2: C, 60.43; H, 4.82; N, 10.57. Found: C, 60.85;
H, 4.43; N, 10.97. (+)-N1-[(1S)-1-P h en yleth yl]-1-[(S)-sp ir o-
br a ssin in ]ca r boxa m id e [(+)-(14)]: an amorphous pale yel-
low solid, mp 48-51 °C; Rf (CH2Cl2) 0.18; [R]20 +26.8 (c 0.17,
D
CH2Cl2); IR (CHCl3) 3322, 1738, 1726 cm-1
;
1H NMR (400
MHz, CDCl3) δ 8.83 (1H, br d, J ) 7.1 Hz, NH), 8.21 (1H, d,
J ) 8.1 Hz,), 7.37 (6H, m), 7.28 (1H, m), 7.21 (1H, ddd, J )
7.6, 7.6, and 1.0 Hz), 5.12 (1H, quintet, J ) 7.1 Hz), 4.73 (1H,
d, J ) 15.4 Hz), 4.50 (1H, d, J ) 15.4 Hz), 2.63 (3H, s), 1.59
(3H, d, J ) 7.1 Hz); 13C NMR (100 MHz, CDCl3, COM, DEPT)
δ 179.1 (C), 164.0(C), 150.6 (C), 142.8 (C), 139.2(C), 130.2 (CH),
128.8 (CH), 128.3 (C), 127.5 (CH), 126.0 (CH), 125.6 (CH),
123.7 (CH), 116.6 (CH), 75.6 (CH2), 65.5 (C), 50.1 (CH), 22.7
(CH3), 15.7 (CH3); UV (EtOH) λmax (ꢀ) 195.8 (48600), 216.4
(30300) nm; CD (EtOH) λext (∆ꢀ) 218.0 (-21.6), 234.2 (+22.8),
299.4 (-2.4) nm. EIMS m/z (%) 397 (M+, 10), 250 (100), 203
(20), 177 (28), 145 (11), 105 (30). Anal. Calcd for C20H19N3O2-
S2: C, 60.43; H, 4.82; N, 10.57. Found: C, 60.79; H, 4.48; N,
10.19.
(()-Spir o[in dolin e-3,5′-oxa zolid in ]-2-on e-2′-th ion e [(()-
10]. To a solution of (()-915 (645 mg, 3 mmol) in water (9 mL)
was added CSCl2 (384 mg, 0.225 mL, 3.3 mmol) in CH2Cl2 (15
mL). Then, a solution of 5% Na2CO3 (12 mL) was added to the
mixture under vigorous stirring within 15 min, and the
reaction mixture was stirred for another 20 min. Resulting
precipitate was filtrated, washed with water, to gave (()-10
(610 mg, 92%) as colorless crystals: mp 204-206 °C (acetone/
cyclohexane); IR (CHCl3) 3360, 3185, 1728 cm-1; 1H NMR (400
MHz, acetone-d6) δ 9.67 (1H, br s), 9.04 (1H, br s), 7.57 (1H,
d, J ) 7.6 Hz), 7.38 (1H, ddd, J ) 7.8, 7.6, and 1.2 Hz), 7.12
(1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 6.99 (1H, d, J ) 7.8 Hz),
4.16 (1H, d, J ) 10.7 Hz), 4.10 (1H, d, J ) 10.7 Hz); 13C NMR
P r ep a r a tion of Dia ster eom er ic Am id es (+)-15 a n d (+)-
16. Derivatization of (()-11 (178 mg, 0.8 mmol) in dry acetone
(6 mL), following the above procedure gave (+)-15 and (+)-
16. (+)-N1-[(1S)-1-P h en yleth yl]-1-[(3R)-2′-(m eth ylsu lfan yl)-
sp ir o{in d olin e-3,5′-[4′,5′]d ih yd r ooxa zol)}-2-on e]ca r box-
a m id e [(+)-(15)] was given as colorless crystals (110 mg,
36%), mp 132 °C (CH2Cl2/hexane): Rf (CH2Cl2) 0.39; [R]20
D
+58.0 (c 0.20, CH2Cl2); IR (CHCl3) 3340, 1742, 1710 cm-1; 1H
NMR (400 MHz, CDCl3) δ 8.70 (1H, br d, J ) 7.3 Hz, NH),
8.23 (1H, dd, J ) 8.1 and 1.0 Hz), 7.37 (6H, m), 7.28 (1H, m),
7.23 (1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 5.12 (1H, quintet,

3946 J . Org. Chem., Vol. 66, No. 11, 2001
Suchy´ et al.
J ) 7.1 Hz), 4.36 (1H, d, J ) 14.2 Hz), 4.11 (1H, d, J ) 14.2
Hz), 2.56 (3H, s), 1.59 (3H, d, J ) 6.8 Hz); 13C NMR (100 MHz,
CDCl3, COM, DEPT) δ 176.6 (C), 165.8 (C), 150.4 (C), 142.9
(C), 140.4 (C), 131.4 (CH), 128.8 (CH), 127.5 (CH), 126.1 (C),
126.0 (CH), 125.6 (CH), 123.8 (CH), 116.8 (CH), 84.7 (C), 65.4
(CH2), 50.1 (CH), 22.7 (CH3), 14.7 (CH3); UV (EtOH) λmax (ꢀ)
198.6 (41600), 211.6 (36300), 216.2 (36100) nm; CD (EtOH)
λext (∆ꢀ) 204.4 (+57.0), 219.8 (-51.3), 236.0 (+6.9) nm. EIMS
m/z (%) 381(M+, 10), 234 (100), 187 (35), 159 (28), 105 (55).
Anal. Calcd for C20H19N3O3S: C, 62.97; H, 5.02; N, 11.02.
Found: C, 62.75; H, 5.19; N, 10.81. (+)-N1-[(1S)-1-P h en yl-
eth yl]-1-[(3S)-2′-(m eth ylsu lfa n yl)sp ir o{in d olin e-3,5′-[4′,5′]-
d ih yd r ooxa zol)}-2-on e]ca r boxa m id e [(+)-16] was given as
a white amorphous solid (140 mg, 46%), mp 26-28 °C: Rf (CH2-
H-5/C-5: H-7/C-7: H-11a/C-11: H-11b/C-11; HMBC correla-
tion (CDCl3) H-4/C-6, C-7a: H-6/C-4, C-7a: H-5/C-4a, C-6:
H-7/C-5, C-4a: H-11a/C-4a, C-9(CdN), C-2(CdO): H-11b/C-
4a, C-9(CdN), C-2(CdO): SCH3/C-9(CdN); UV (EtOH) λmax
(ꢀ) 213.8 (28100), 252.4 (4880), 267.4 (sh, 2920) 301.8 (1520)
nm; CD (EtOH) λext (∆ꢀ) 202.4 (22.3), 221.2 (-34.1), 249.8 (5.9),
307.8 (1.9) nm; EIMS m/z (%) 234 (M+, 70), 219 (5), 206 (16),
187 (100), 159 (70), 87 (65). Anal. Calcd for C11H10N2O2S: C,
56.39; H, 4.30; N, 11.96. Found: C, 56.18; H, 4.10; N, 12.09.).
(S)-(-)-2′-(Meth ylsu lfa n yl)sp ir o{in d olin e-3,5′-([4′,5′]d ih y-
d r ooxa zol}-2-on e [(S)-(-)-11]: colorless needles, mp 180-
182 °C (CH3OH); [R]22 -34.1 (c 0.89, CHCl3); 84% ee; UV
D
(EtOH) λmax (ꢀ) 214.2 (28300), 251.8 (4670), 267.2 (sh, 2660),
303.4 (1340) nm; CD (EtOH) λext (∆ꢀ) 202.2 (-23.4), 221.0
(35.7), 249.8 (-6.2), 306.4 (-2.0) nm. IR, NMR and EIMS data
are identical with (R)-(+)-11. Anal. Calcd for C11H10N2O2S: C,
56.39; H, 4.30; N, 11.96. Found: C, 56.45; H, 4.32; N, 12.24.
Dim eth yl N-Hexyld ith ioca r bon im id a te (17). To a mix-
ture of hexylamine (317 mg, 3.13 mmol), dry triethylamine
(0.44 mL, 3.13 mmol), and dry pyridine (3 mL) was added
carbon disulfide (239 mg, 0.19 mL, 3.13 mmol) at 0 °C. The
mixture was kept at 0 °C for 2 h, treated with methyl iodide
(488 mg, 0.21 mL, 3.44 mmol), and kept at 0 °C for 14 h. The
reaction mixture was poured into 2 M HCl and extracted with
AcOEt twice. The combined organic solution was washed with
2 M HCl, saturated NaHCO3, and water, and dried over Na2-
SO4. The residue obtained after solvent removal in vacuo was
submitted to the silica gel flash column chromatography
(hexane/ethyl acetate 10:1) to give an oily compound (methyl
N-hexyldithiocarbamate, 536 mg, 90%). IR (neat) 3222, 2955,
2927, 2857, 1509, 1465, 1389, 1335, 1303, 1203, 1151, 1065,
Cl2) 0.19; [R]20 +7.0 (c 0.20, CH2Cl2); IR (CHCl3) 3340, 1750,
D
1
1710 cm-1; H NMR (400 MHz, CDCl3) δ 8.69 (1H, br d, J )
7.3 Hz, NH), 8.23 (1H, dd, J ) 8.2 and 1.0 Hz,), 7.37 (6H, m),
7.28 (1H, m), 7.23 (1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 5.11
(1H, quintet, J ) 7.1 Hz), 4.40 (1H, d, J ) 14.2 Hz), 4.14 (1H,
d, J ) 14.2 Hz), 2.55 (3H, s), 1.59 (3H, d, J ) 6.8 Hz); 13C
NMR (100 MHz, CDCl3, COM, DEPT) δ 176.6 (C), 165.9 (C),
150.4 (C), 142.8 (C), 140.4 (C), 131.4 (CH), 128.8 (CH), 127.5
(CH), 126.1 (C), 126.0 (CH), 125.7 (CH), 123.8 (CH), 116.8
(CH), 84.7 (C), 65.5 (CH2), 50.1 (CH), 22.7 (CH3), 14.7 (CH3);
UV (EtOH) λmax (ꢀ) 195.8 (46600), 211.4 (34500) nm; CD
(EtOH) λext (∆ꢀ) 207.6 (-15.9), 227.0 (+17.8), 253.8 (-2.1) nm.
EIMS m/z (%) 381(M+, 10), 234 (100), 187 (30), 159 (25), 105
(40). Anal. Calcd for C20H19N3O3S: C, 62.97; H, 5.02; N, 11.02.
Found: C, 62.58; H, 4.71; N, 10.63.
En a n tiom er s of Sp ir obr a ssin in [(R)-(+)-4, (S)-(-)-4]. To
a stirred solution of (+)-13 or (+)-14 (110 mg, 0.277 mmol) in
dry CH3OH (7 mL) was added a solution of CH3ONa (151 mg,
2.8 mmol) in dry CH3OH (2 mL) within 20 min at -10 °C.
After being stirred at the same temperature for 40 min, the
reaction mixture was diluted with water (1 mL) and neutral-
ized with 1 N HCl. After removal of CH3OH, the product was
extracted with AcOEt, and AcOEt solution was dried over Na2-
SO4 and evaporated in vacuo. Purification of the residue by
flash chromatography (cyclohexane/acetone 2:1) afforded (R)-
(+)-4 [35 mg, 51% from (+)-13] and (S)-(-)-4 [31 mg, 45% from
(+)-14]. (R)-(+)-Spir obr assin in [(R)-(+)-4]: Colorless needles,
1
954, 727 cm-1; H NMR (400 MHz, CDCl3) δ 7.01 (1H, br s,
NH), 3.73 (2H, q, J ) 7.3 Hz, NH-CH2-), 2.64 (3H, s, SCH3),
1.65 (2H, m, NHCH2CH2), 1.32 (6H, m), 0.89 (3H, t, J ) 6.7
Hz, CH3). The spectrum showed minor signals (1/2 intensity
of the major signal) due to a rotamer at δ 7.79 (br s, NH), 3.43
(q, J ) 6.8 Hz, NHCH2), 2.68 (3H, s, SCH3), 1.65 (m
NHCH2CH2), 1.32 (m), 0.89 (t, J ) 6.7 Hz, CH3); 13C NMR (100
MHz, CDCl3, COM, DEPT) δ 198.7 (CdS), 47.4 (CH2), 31.4
(CH2), 28.3 (CH2), 26.5 (CH2), 22.5 (CH2), 18.1 (CH3), 13.9
(CH3). The spectrum showed minor signal due to a rotamer at
δ 201.7 (CdS), 46.3 (CH2), 31.2 (CH2), 28.6 (CH2), 26.4 (CH2),
22.5 (CH2), 18.8 (CH3), 13.9 (CH3); UV (EtOH) λmax 269.2 (ꢀ
12200), 252.6 (11300), 222.4 (10300) nm. EIMS m/z (%) 191
(M+, 100), 176 (3), 144 (52), 115 (87), 110 (27), 100 (9), 91 (22),
85 (49). Anal. Calcd for C8H17NS2: C, 50.21; H, 8.95; N, 7.32;
S, 33.51. Found: C, 50.07; H, 8.69; N, 7.22; S, 33.50. Methyl
N-hexyldithiocarbamate (302 mg, 1.58 mmol) was dissolved
in CH3OH (3 mL) and treated with CH3I (2.28 g, 1 mL, 16
mmol) and K2CO3 (0.5 g, 3.62 mmol) at 0 °C. After being kept
at room temperature for 3 h, the reaction mixture was filtered
with Celite and evaporated. The reaction mixture was ex-
tracted with Et2O, and the organic fraction was washed with
water. The residue obtained after solvent removal in vacuo
was submitted to silica gel flash column chromatography
(hexane) to give an oily compound 17 (159 mg, 50%). IR (neat)
2955, 2925, 2856, 1584, 1465, 1428, 1351, 1308, 1120, 1017,
963, 906, 724 cm-1; 1H 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, COM, DEPT) δ 156.5 (CdN), 53.0 (CH2),
31.6 (CH2), 30.6 (CH2), 27.1 (CH2), 22.6 (CH2), 14.5 (CH3), 14.4
(CH3), 14.0 (CH3); UV (EtOH) λmax 231.0 (sh, ꢀ 7630), 219.1
(8590) nm. EIMS m/z (%) 205 (M+, 2), 190 (9), 158 (100), 85
(18), 74 (77). Anal. Calcd for C9H19NS2: C, 52.63; H, 9.32; N,
6.82; S, 31.23. Found: C, 52.50; H, 9.11; N, 6.76; S, 31.04.
1-[(1′S,4′R)-(-)-Ca m p h a n oyl] Der iva tives of (R)-(+)-4
a n d (S)-(-)-4 [(-)-19, (-)-20]. To a stirred suspension of
sodium hydride (64 mg of 60% suspension in mineral oil) in
anhydrous acetonitrile (3 mL) was added (()-4 (100 mg, 0.40
mmol) and after 5 min (1S,4R)-(-)-camphanoyl chloride [(-)-
18] (173 mg, 0.8 mmol). After being stirred for 30 min, the
reaction mixture was poured into cold water (30 mL), and the
product was extracted with AcOEt. The organic solvent was
dried over Na2SO4 and concentrated in vacuo, and the residue
20
mp 143-145 °C (acetone/cyclohexane); [R]D +142.7 (c 0.25,
CH2Cl2); 91% ee; CD (EtOH) λext (∆ꢀ) 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.
IR, NMR, UV and EIMS data are identical with those of
natural spirobrassinin.10 Anal. Calcd for C11H10N2OS2: C,
52.77; H, 4.03; N, 11.19. Found: C, 52.63; H, 4.12, N, 11.04.
(S)-(-)-Sp ir obr a ssin in [(S)-(-)-4]: Colorless needles, mp
142-144 °C (acetone/cyclohexane); [R]20D -143.6 (c 0.25, CH2-
Cl2); 92% ee; CD (EtOH) λext (∆ꢀ) 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. IR,
NMR, UV, and EIMS data are identical with those of natural
spirobrassinin.10 Anal. Calcd for C11H10N2OS2: C, 52.77; H,
4.03; N, 11.19. Found: C, 52.81; H, 3.89, N, 11.35.
En a n tiom er s of Sp ir ooxa zolin e [(R)-(+)-11, (S)-(-)-11].
Deprotection of (+)-15 and (+)-16 (162 mg, 0.424 mmol)
following the above protocol afforded (R)-(+)-11 [50 mg, 53%
from (+)-15] and (S)-(-)-11 [45 mg, 48% from (+)-16]. (R)-
(+)-2′-(Meth ylsu lfan yl)spir o{in dolin e-3,5′-([4′,5′]dih ydr oox-
a zol}-2-on e [(R)-(+)-11]: colorless needles, mp 186-188 °C
(CH3OH); [R]22 +31.7 (c 1.37, CHCl3); 95% ee; IR (CHCl3)
D
3455, 1738 cm-1; 1H NMR (CDCl3) δ 9.13 (1H, br s, NH), 7.35
(1H, d, J ) 7.3 Hz, H-4), 7.32 (1H, ddd, J ) 7.8, 7.6, 1.2 Hz,
H-6), 7.09 (1H, ddd, J ) 7.6, 7.3, 1.0 Hz, H-5), 6.94 (1H, d,
J ) 7.8 Hz, H-7), 4.37 (1H, d, J ) 13.9 Hz, H-11a), 4.14 (1H,
d, J ) 13.9 Hz, H-11b), 2.55 (3H, s, SCH3); 13C NMR (COM,
DEPT, CDCl3) δ 176.5 (C-2, CdO), 166.0 (C-9, CdN), 140.9
(C-7a), 131.0 (C-6), 127.9 (C-4a), 124.5 (C-4), 123.6 (C-5), 110.8
(C-7), 84.5 (C-3), 64.4 (C-11), 14.6 (SCH3). The full assignment
of NMR was performed by the following data: Difference NOE
spectra (CDCl3) irr. at δ 9.13 (NH) enhanced signal δ 6.94
(6.8%, H-7), irr. at δ 4.14 (H-11b) enhanced signal δ 4.37
(26.1%, H-11a), 7.35 (4.2%, H-4); DQF COSY correlation
(CDCl3) H-4/H-5, H-6: H-6/H-7, H-5, H-4: H-5/H-7, H-6, H-4:
H-7/H-5, H-6; HMQC correlation (CDCl3) H-4/C-4: H-6/C-6:

Stereochemical Studies of Spirobrassinin
J . Org. Chem., Vol. 66, No. 11, 2001 3947
was subjected to flash chromatography (cyclohexane/AcOEt
5:1, the duration of the separation must not exceed 60 min).
The first fraction gave (-)-19 (37.8 mg, 22%), while the second
fraction afforded (-)-20, contaminated with (-)-19. Repeated
chromatography of the second fraction afforded pure (-)-20
(37.2 mg, 22%). Alternatively, (-)-19 (50%) was prepared from
(R)-(+)-4, and (-)-20 (50%) from (S)-(-)-4, following the above
procedure. In these cases, however, the pure products were
given by crystallization of the residue obtained after evapora-
tion of AcOEt. (-)-1-[(1′S,4′R)-Ca m p h a n oyl]-(R)-sp ir o-
br a ssin in [(-)-19]: colorless prisms, mp 177-179 °C (CH2Cl2/
hexane); Rf (cyclohexane/AcOEt 5:1) 0.54; [R]20D -16.9 (c 0.20,
Calcd for C21H22N2O4S2: C, 58.58; H, 5.15; N, 6.51. Found: C,
58.43; H, 5.29; N, 6.62. (-)-1-[(1′S,4′R)-Ca m p h a n oyl]-(S)-
sp ir obr a ssin in [(-)-20]: colorless prisms, mp 207-209 °C
(CH2Cl2/hexane); Rf (cyclohexane/AcOEt 5:1) 0.46; [R]D20-39.6
1
(c 0.20, CH2Cl2); IR (CHCl3) 1788, 1770, 1710 cm-1; H NMR
(400 MHz, CDCl3) δ 7.63 (1H, d, J ) 7.8 Hz), 7.45 (1H, dd,
J ) 7.6 and 0.7 Hz), 7.35 (1H, ddd, J ) 7.8, 7.6, and 1.2 Hz),
7.24 (1H, ddd, J ) 7.6, 7.6, and 1.0 Hz), 4.76 (1H, d, J ) 15.3
Hz), 4.54 (1H, d, J ) 15.3 Hz), 2.96 (1H, ddd, J ) 13.5, 9.3,
and 4.4 Hz), 2.62 (3H, s), 2.35 (1H, ddd, J ) 13.5, 10.7, and
4.4 Hz), 1.96 (1H, ddd, J ) 13.0, 10.7, and 4.4 Hz), 1.80 (1H,
ddd, J ) 13.0, 9.3, and 4.4 Hz), 1.29 (3H, s), 1.11 (3H, s), 0.96
(3H, s); 13C NMR (100 MHz, CDCl3, COM, DEPT) δ 177.3 (C),
175.3 (C), 171.5 (C), 163.7 (C), 137.8 (C), 130.0 (CH), 129.9
(C), 126.1 (CH), 124.5 (CH), 113.8 (CH), 92.8 (C), 75.1 (CH2),
63.8 (C), 57.3 (C), 54.5 (C), 30.6 (CH2), 29.5 (CH2), 17.6 (CH3),
16.6 (CH3), 15.7 (CH3), 9.7 (CH3); UV (EtOH) λmax (ꢀ) 206.8
(30100), 229.8 (sh, 16700), 267.4 (sh, 5950), 297.6 (sh, 1210)
nm; CD (EtOH) λext (∆ꢀ) 203.4 (-5.2), 210.8 (+3.0), 224.0
(-17.1), 243.4 (+9.9), 265.0 (-3.7), 291.4 (-2.8), 314.4 (+0.4)
nm. EIMS m/z (%) 430 (M+, 58), 402 (10), 383 (12), 357 (100),
329 (19), 249 (38). Anal. Calcd for C21H22N2O4S2: C, 58.58; H,
5.15; N, 6.51. Found: C, 58.32; H, 5.37; N, 6.70.
CH2Cl2); IR (CHCl3) 1787, 1772, 1718 cm-1 1H NMR (400
;
MHz, CDCl3) δ 7.52 (1H, d, J ) 8.1 Hz, H-7), 7.39 (1H, dd,
J ) 7.4 and 1.3 Hz, H-4), 7.27 (1H, ddd, J ) 8.1, 7.6, and 1.3
Hz, H-6), 7.17 (1H, ddd, J ) 7.6, 7.4, and 1.0 Hz, H-5), 4.82
(1H, d, J ) 15.4 Hz, H-11a), 4.40 (1H, d, J ) 15.4 Hz, H-11b),
2.92 (1H, ddd, J ) 13.5, 9.4, and 4.4 Hz, H-6′R), 2.55 (3H, s,
SCH3), 2.27 (1H, ddd, J ) 13.5, 10.8, and 4.4 Hz, H-6′â), 1.89
(1H, ddd, J ) 12.8, 10.8, and 4.4 Hz, H-5′â), 1.72 (1H, ddd,
J ) 12.8, 9.4, and 4.4 Hz, H-5′R), 1.22 (3H, s, H-11′, 7′-CH3),
1.04 (3H, s, H-9′, 4′-CH3), 0.90 (3H, s, H-10′, 7′-CH3); 13C NMR
(100 MHz, CDCl3, COM, DEPT) δ 177.4 (C-3′, CdO), 175.1
(C-2, CdO), 171.1 (C-8′, CdO), 163.9 (C-9, CdN), 138.2 (C-
7a), 129.9 (C-6), 129.4 (C-4a), 125.9 (C-5), 124.6 (C-4), 113.7
(C-7), 92.9 (C-1′), 76.0 (C-11), 65.7 (C-3), 57.3 (C-4′), 54.5 (C-
7′), 30.4 (C-6′), 29.5 (C-5′), 17.6 (C-11′), 16.6 (C-10′), 15.6
(SCH3), 9.7 (C-9′). The full assignment of NMR was performed
by the following data: Difference NOE spectra (CDCl3, 400
MHz) irr. at δ 4.40 (H-11b), enhanced signal δ 4.82 (34.4%,
H-11a), 7.39 (7.0%, H-4); irr. at δ 1.22 (H-11′, 7′-CH3),
enhanced signal δ 2.27 (6.0%, H-6′â), 1.89 (4.8%, H-5′â); irr.
at δ 1.04 (H-9′, 4′-CH3) enhanced signal δ 1.89 (2.5%, H-5′â),
1.72 (1.5%, H-5′R); irr. at δ 0.90 (H-10′, 7′-CH3) enhanced signal
δ 7.52 (4.3%, H-7); HMQC correlations (CDCl3) H-7/C-7: H-4/
C-4: H-6/C-6: H-5/C-5: H-6′R/C-6′: H-11a/C-11: H-11b/C-
11: H-6′â/C-6′: H-5′R/C-5′; H-5′â/C-5′: H-SCH3/SCH3: H-11′/
C-11′: H-9′/C-9: H-10′/C-10′; HMBC correlations (400 MHz,
CDCl3) H-7/C-4a, C-5, C-7a: H-4/C-3, C-5, C-6, C-7a: H-6/C-
4, C-7, C-7a: H-5/C-4, C-7: H-11a/C-2 (CdO), C-4a, C-9(Cd
N): H-11b/C-2 (CdO), C-4a, C-9(CdN): H-6′R/C-1′, C-4′, C-5′,
C-7′:H-SCH3/C-9(CdN): H-6′â/C-5′, C-7′: H-5′R/C-3′(CdO),
C-6′, C-9′, C-7′: H-5â/C-3′(CdO), C-6′, C-4′, C-7′: H-11′/C-1′,
C-4′, C-7′, C-10′: H-9′/C-3′ (CdO), C-4′, C-5′, C-7′: H-10′/C-1′,
C-4′, C-7′, C-11′; UV (EtOH) λmax (ꢀ) 297.0 (sh, 1340), 269.4
(sh, 5290), 237.2 (sh, 14900), 205.6 (30700) nm; CD (EtOH)
λext (∆ꢀ) 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) nm; EIMS m/z (%) 430
(M+, 58), 402 (10), 383 (12), 357 (100), 329 (19), 249 (38). Anal.
Ack n ow led gm en t. The authors are grateful to
Assoc. Professor V. Milata and Dr. J . Salon, Slovak
Technical University Bratislava, for their cooperation
with a new synthesis of amine (()-9. We wish to thank
the Grant Agency for Science of the Slovak Republic
(project No. 1/6080/99) for their financial support. This
research was partially supported by the Ministry of
Education, Science, Sports, and Culture, Grant-in-Aid
for Encouragement of Young Scientists, 11780413, 2000.
The authors would also like to thank Dr. Shuichi
Osawa, Tohoku University, for his helpful discussion
regarding the computational analyis of (S)-4.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
1
cedure for the synthesis of (()-9; copies of H and 13C NMR
spectra of (+)-13, (+)-14, (+)-16 and 2D-NMR spectra for (R)-
(+)-11; CD spectra of (R)-(+)-4, (S)-(-)-4, (R)-(+)-11, and (S)-
(-)-11; calculated UV and CD data for (S)-4 and (S)-11; the
chiral HPLC analyses with the chiral column of (()-4 and (()-
11; X-ray data for (+)-13, (-)-19 and (-)-20. This material is
available free of charge via the Internet at http://pubs.acs.org.
J O0155052