Alcoholysis of naturally occurring imides: Misleading interpretation of antifungal activities

Article abstract of DOI:10.1021/np1003092

The frequent presence of the sulfur-containing amide penangin (10) in leaf extracts of Glycosmis species turned out to be the result of decomposition of imides generated by extraction and storage in MeOH. Reinvestigation of Glycosmis mauritiana and G. cf. puberula with acetone revealed the presence of six imides. In addition to penimides A (1) and B (2) and ritigalin (6), three new derivatives, krabin (4), isokrabin (5), and methoxypenimide B (3), were isolated and identified by spectroscopic methods. All six imides were shown to be susceptible to different rates of methanolic cleavage, leading to their corresponding methyl esters and sulfur-containing amides. Whereas the decomposition products penangin (10), isopenangin (11), and sinharin (14) are known, the corresponding cleavage of methyl N-methylthiocarbamate (7) from ritigalin (6), monitored in situ by ¹H NMR spectroscopy, is described here for the first time. Its structure was further confirmed by GC-MS coupling. HPLC-UV comparison of many different samples of G. mauritiana, extracted with MeOH, revealed considerable chemical variations in sulfur-containing amides, strongly correlated with different antifungal potency. The lack of activity of many methanolic crude extracts can be explained by a preponderance of the inactive decomposition product penangin (10), whereas the corresponding naturally occurring imides penimides A (1) and B (2) and methoxypenimide B (3), extracted with acetone, showed high fungitoxic properties.

Full text of DOI:10.1021/np1003092

J. Nat. Prod. 2010, 73, 1389–1393
1389
Alcoholysis of Naturally Occurring Imides: Misleading Interpretation of Antifungal Activities⊥
Thomas Pacher,† Adriane Raninger,† Eberhard Lorbeer,‡ Lothar Brecker,*,‡ Paul Pui-Hay But,§ and Harald Greger*,†
ComparatiVe and Ecological Phytochemistry, Faculty Center of Botany, UniVersity of Vienna, Rennweg 14, A-1030 Wien, Austria, Institute of
Organic Chemistry, UniVersity of Vienna, Wa¨hringerstrasse 38, A-1090 Wien, Austria, and Department of Biology, Chinese UniVersity of Hong
Kong, Shatin, N.T. Hong Kong, People’s Republic of China
ReceiVed May 7, 2010
The frequent presence of the sulfur-containing amide penangin (10) in leaf extracts of Glycosmis species turned out to
be the result of decomposition of imides generated by extraction and storage in MeOH. Reinvestigation of Glycosmis
mauritiana and G. cf. puberula with acetone revealed the presence of six imides. In addition to penimides A (1) and B
(2) and ritigalin (6), three new derivatives, krabin (4), isokrabin (5), and methoxypenimide B (3), were isolated and
identified by spectroscopic methods. All six imides were shown to be susceptible to different rates of methanolic cleavage,
leading to their corresponding methyl esters and sulfur-containing amides. Whereas the decomposition products penangin
(10), isopenangin (11), and sinharin (14) are known, the corresponding cleavage of methyl N-methylthiocarbamate (7)
1
from ritigalin (6), monitored in situ by H NMR spectroscopy, is described here for the first time. Its structure was
further confirmed by GC-MS coupling. HPLC-UV comparison of many different samples of G. mauritiana, extracted
with MeOH, revealed considerable chemical variations in sulfur-containing amides, strongly correlated with different
antifungal potency. The lack of activity of many methanolic crude extracts can be explained by a preponderance of the
inactive decomposition product penangin (10), whereas the corresponding naturally occurring imides penimides A (1)
and B (2) and methoxypenimide B (3), extracted with acetone, showed high fungitoxic properties.
The amides in the genus Glycosmis Correˆa of the family Rutaceae
deserve attention. They are not derived from cinnamic acid and
thus differ from other amides in the family, including those of the
closely related genus Clausena Burm. f. Instead, sulfur-containing
acid moieties, most likely derived from the amino acid cysteine,
play a prominent role in the amide formation in many Glycosmis
species. The amine parts are mostly represented by phenethyl or
phenethenyl (styryl) groups that can be further linked to various
prenyloxy and geranyloxy groups in the para position.1,2 These
amides can also be oxidized to sulfones and sulfoxides1,3,4 or
shortened by ꢀ-oxidation.5 The accumulation of sulfur-containing
amides, otherwise rarely reported in the plant kingdom,6 was shown
to represent an important chemical character for an infrageneric
grouping in this genus.7 Apart from their chemotaxonomic value,8
many amides exhibited pronounced biological activities in various
bioassays.9,10
In the course of our broad-based phytochemical comparison of
many different samples of Glycosmis mauritiana (Lam.) Tanaka,
collected in Sri Lanka and Thailand, a considerable chemical
Figure 1. Structures of penimides A (1) and B (2), methoxypen-
imide B (3), krabin (4), isokrabin (5), ritigalin (6), and methyl
N-methylthiocarbamate (7).
variation of accumulated sulfur-containing amides was observed,
even among single individuals of the same population.7,9 The
numerous collections from Sri Lanka showed remarkable differ-
ences in amide patterns, which were strongly correlated with
variations in fungitoxic properties. The highly active antifungal
amides methylillukumbins A (8) and B (9)7 were detected only in
a few collections, whereas the inactive penangin (10)11 was shown
to be more widespread as the major component. In view of the
expected protective role of 8 and 9 against fungal attack, the
predominance of the latter was surprising. This seemingly functional
inconsistency can now be explained by using different solvents for
extraction. Our findings demonstrated that the inactive penangin
(10) was actually an artifact obtained by conventional MeOH
extraction, but could not be detected in acetone. In acetone the
amide penangin (10) was replaced by the imide penimide A (1). In
fact, subsequent tests with pure penimide A (1) dissolved in MeOH
showed a complete transformation into penangin (10) after ∼40 h
at room temperature.12 In contrast to the inactive penangin (10),
penimide A (1) displayed pronounced antifungal activity.12,13
However, due to the small amounts of penimide obtained in the
original crude extract, this effect has not yet been determined.11
In order to get an overview about the possible occurrence of
other transformation products, generated during MeOH extraction,
freshly harvested leaves from two Glycosmis species, cultivated in
the Botanical Garden of the University of Vienna, were dried and
extracted separately with acetone and MeOH. The chemical profiles
of both extracts were compared by HPLC-UV analyses. The results
revealed that the Z/E pair of the imides krabin (4) and isokrabin
(5) was detected in the acetone extracts together with the known
imides penimides A (1) and B (2)11 and ritigalin (6).5 Furthermore,
a small amount of the undescribed Z-isomeric methoxypenimide
B (3) was isolated (Figure 1). The present paper reports on the
⊥ Dedicated to the memory of Prof. Otmar Hofer.
* To whom correspondence should be addressed. (L.B.) Tel: +43 1 4277
52131. Fax: +43 1 4277 9521. E-mail: lothar.brecker@univie.ac.at. (H.G.)
Tel: +43-1-4277-54072. Fax: +43-1-4277-9541. E-mail: harald.greger@
univie.ac.at.
† Comparative Phytochemistry, University of Vienna.
‡ Organic Chemistry, University of Vienna.
§ Department of Biology, Chinese University of Hong Kong.
10.1021/np1003092  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/11/2010

1390 Journal of Natural Products, 2010, Vol. 73, No. 8
Pacher et al.
Figure 2. Methanolic cleavage of imides to corresponding methyl esters and amides.
isolation and structure elucidation of the new imides and describes
the MeOH-induced cleavage of all imides (1-6) into their corre-
sponding amides and methyl esters, as well as the associated
changes in antifungal activities.
For use as authentic markers in chromatographic comparisons
and for bioassays all isolated pure compounds were stored at -20
°C in MeOH solution. However, after three days at room temper-
ature, penimide A (1) could not be recovered from the MeOH
solution in HPLC-UV profiles. Instead, penangin (10) was detected
as the main peak together with an additional small peak identified
as methyl phenylacetate. These findings suggested methanolysis
of penimide A (1), as shown in Figure 2, and explained the frequent
presence of the inactive penangin (10) as the major component in
many crude extracts stored in MeOH solution. Parallel experiments
with penimide A (1) using EtOH, propanol, 2-propanol, or BuOH
as solvents also resulted in the formation of penangin (10) and the
corresponding alkyl phenylacetates. Penangin (10) was shown to
be accompanied by small amounts of its Z-isomer isopenangin (11),
suggesting the presence of the corresponding imide penimide B
(2) (Figure 2).11
Results and Discussion
During our first field trips in Sri Lanka in 1991-1992, we
collected G. mauritiana. In order to avoid formation of artifacts
by drying the plant material and subsequent transport to Vienna,
all samples were freshly chopped at the place of collection and
immediately soaked and stored in MeOH. The lipophilic CHCl3
fractions of the methanolic crude extracts of leaves, stem, and root
bark were later investigated separately by HPLC-UV analyses. The
results showed that amides and/or imides were accumulated mostly
in the leaves. Parallel bioautographic tests on TLC plates sprayed
with a spore suspension of the fungus Cladosporium herbarum
(Persoon & Fries) Link exhibited clear inhibition spots for extracts
containing methylillukumbins A (8) and B (9), ritigalin (6), sinharin
(14), and methylsinharin (16) as active compounds, whereas those
dominated by penangin (10) did not display notable antifungal
activity. Penangin (10) was originally isolated and identified from
a plant collected in Penang Island in Malaysia, erroneously named
G. cf. chlorosperma (Bl.) Spreng. and later identified as G. cf.
puberula Lindl.14 The HPLC profile of this collection was
characterized by a preponderance of penangin (10) along with small
amounts of two isomeric imides, penimides A (1) and B (2).11
A new type of imide was found to dominate in the acetone extract
of the leaves of G. mauritiana collected in south Thailand. Referring
to the place of collection near the city of Krabi, this imide was
designated as krabin (4). Its structure is similar to those of
methylsinharin (16)15 and could formally be described as “N-
formylsinharin”. The N-(2-phenylethyl)- and the (E)-N-3-(meth-
ylthio)prop-2-en-1-onyl moieties of krabin (4) were easily deter-
mined by comparison of their NMR data with those of methylsinharin
(16).15 The presence of the N-formyl group was determined by mass
spectrometry and by 1H and 13C NMR chemical shifts. Additionally,

Alcoholysis of Naturally Occurring Imides
Journal of Natural Products, 2010, Vol. 73, No. 8 1391
the 3JH-C long-range couplings between the formyl proton and the
N-bound 13C atoms in the two other moieties indicated the presence
of the new imide structure. Methanolysis of krabin (4) led to methyl
formate and sinharin (14), as determined by HPLC (Figure 2).
Krabin (4) was shown to be accompanied by small amounts of its
Z-isomer, named isokrabin (5). The difference between the two
structures was determined by 3JH-H coupling constants of the double
bound. The E-isomer krabin (4) was identified by the large coupling
constant of 14.5 Hz, while isokrabin (5) showed the intermediate
coupling constant of 9.7 Hz, typical for 3-thioprop-2-(Z)-enone
moieties.15,16
of an electron-withdrawing carboxyl group, which replaces the
sulfur atom and leads to reasonably higher electrophilicity of the
imide carbonyl group. Isokrabin (5) and methoxypenimide B (3),
however, also contain an electron-donating thioether moiety and,
hence, quite likely underwent the same methanolysis as the imides
1, 2, and 4 (Figure 2).
Different rates of methanolysis were observed in the imides 1-6
by HPLC-UV comparison, exhibiting a relatively quick transfor-
mation of penimide A (1) into penangin (10) within ∼40 h at room
temperature, whereas ritigalin (6) and krabin (4) were still present
in large amounts after ∼60 h. In the latter compound an elimination
of methyl formate led to the formation of sinharin (14), the first
sulfur-containing amide reported for Glycosmis (Figure 2).16,26 The
structures of the decomposition products penangin (10), isopenangin
(11), and sinharin (14) have been reported previously. The
corresponding formation of methyl N-methylthiocarbamate (7) from
ritigalin (6) has not yet been described. This methanolysis is
interesting, as it does not lead to release of the amide, CO2, and
CH3SH, but to the S-methyl-N-methylthiocarbamate (7) and methyl
phenylacetate (Figure 2). Therefore, the methanolysis of ritigalin
A small amount of an unknown p-methoxy derivative of
penimide B (2), named methoxypenimide B (3), was isolated from
the acetone extract of G. cf. puberula Lindley, collected in Khao
Soi Dao Wildlife Sanctuary in southeast Thailand. Its structure was
identified from 1H NMR spectra, indicating a methoxy group being
in the para position of the phenyl ring. A Z-configuration of the
3
double bond similar to penimide B (2) was indicated by a JH-H
coupling constant of 9.5 Hz. The UV spectrum and retention time
in the HPLC-UV analysis also indicated the presence of the
corresponding E-isomer methoxypenimide A in small amounts.
However, due to limited plant material, no detailed spectroscopic
analysis could be carried out.
1
(6) was monitored in methanol-d4 over several days by in situ H
NMR.27,28 In Figure 3, stack plots of two different spectrum regions
are shown, with indicative signals of the substrate ritigalin (6) as
well as of the resulting products methyl phenylacetate and methyl
N-methylthiocarbamate (7). The time course indicates the pseudo-
first-order kinetics of the methanolysis and of the concomitant
formation of the reaction products. H/D exchange in the R-position
to the carbonyl groups was detected in negligible amounts (<5%
after 400 h) in ritigalin (6) as well as in methyl phenylacetate. The
slightly diminished formation of methyl N-methylthiocarbamate (7),
as compared to methyl phenylacetate, might be caused by further
decomposition of 7 or indicated by quite long longitudinal relaxation
times of the two methyl groups.
Methanolysis of penimides A (1) and B (2) and krabin (4) led
to the cleavage of the saturated acyl moieties and resulted in their
corresponding methyl esters. A similar cleavage has been reported
for coniothyriomycin [N-(3-chloro-4-hydroxyphenylacetyl)fumarate]
in MeOH.17 This acyclic imide was isolated from the fungus
Coniothyrium and showed reasonably good stability in MeOH and
MeOH/H2O mixtures at neutral pH. However, addition of catalytic
amounts HCl led to its complete methanolysis within a few days.17
The lability of imides against hydrolysis in alkaline environment
has been investigated in detail, for example, in the hydrolysis of
substituted N-methylphthalimides18 and maleimides.19 Even poly[N-
(4-sulfophenyl) dimethacrylamide], an imide that is stable under
neutral and acidic conditions, is hydrolyzed at pH 12.3.20 Addition-
ally, transition metal ions further catalyze hydrolysis and metha-
nolysis of amide bonds when the amide nitrogen is incorporated
into their coordination. Such catalytic effects have been described
in ꢀ-lactam hydrolysis21 and were recently investigated with Cu2+
ions as a potential synthetic tool.22 Mechanisms of the hydrolytic
cleavage of C-N bonds in amides have been studied extensively23,24
and are likely very similar to those of corresponding cleavages of
the C-N bonds of imides.
The stability and structure of 7 were studied in further detail
directly from the reaction mixture in methanol-d4. HMBC indicated
3
the N- and S-bound methyl groups to have JC-H couplings to the
carbonyl function. Mass spectra taken from GC-MS coupling
showed two compounds with the molecular mass of m/z ) 153
and m/z ) 105 for the methyl phenylacetate with a CD3 moiety in
both the ester function and 7, respectively. Fragments of the latter
compound were m/z ) 75 and m/z ) 58, which indicated the loss
of -NH-CH3 and of -S-CH3, respectively. Hence, methyl
N-methylthiocarbamate (7) was identified as the stable product
formed by methanolysis of ritigalin (6).
The methanolysis of compounds 1, 2, and 4 in our crude and
partly purified plant extracts is probably not caused just by the
influence of the solvents. Small amounts of co-dissolved alkaloids
or plant acids could slightly raise or lower pH values, respectively,
which enhanced the methanolysis. The presence of transition metal
ions in the plant extracts could not be excluded and might have
further catalyzed the methanolysis of the imides, which have a
reasonable potential to form complex structures with those ions.
A hypothetical biosynthetic route of sulfur-containing amides
has been proposed.9 It suggests that the sulfur-containing acid
moieties are derived from the amino acid cysteine, whereas
phenylalanine is the precursor of the amine moieties. An elimination
of a C-2 unit by ꢀ-oxidation of the acid moiety leads to the
formation of methylthiocarbonic acid amides. As shown in Figure
4, methylsinharin (16) possibly assumes a central position and is
transformed either to methylillukumbin (8, 9) by dehydration or to
dehydroniranin (12, 13)9 by dehydration and ꢀ-oxidation. Oxidation
of the double bond next to the aromatic ring in methylillukumbin
(8, 9) and dehydroniranin (12, 13), or the N-methyl group of
methylsinharin (16), probably results in the formation of the imides
1, 4, and 6 (Figure 4).
On the basis of EC50 values of germ-tube inhibition tests against
the fungus Cladosporium herbarum and the rice pathogen Pyricu-
laria grisea (Cooke) Sacc., significant antifungal activities were
observed. Penimide A (1) showed the highest activity, with an EC50
value of 0.9 µg/mL against C. herbarum and 3 and 5 µg/mL
respectively against two different strains of P. grisea.13 Even though
the values of some imides and amides could not be determined
explicitly, because nonsigmoid dose-response relationships im-
paired estimation, comparative bioautographic tests on TLC plates
against C. herbarum12 indicated that all sulfur-containing imides
In penimides A (1) and B (2) and krabin (4), the R,ꢀ-unsaturated
acyl moieties did not cleave and remained as part of the formed
N-alkyl amides. Such chemoselectivity also occurs in the compa-
rable acid-catalyzed methanolysis of coniothyriomycin.17 The rate-
determining step of C-N bond hydrolysis in amides is the
deterioration of the conjugated system between these two atoms,
caused by sp2 to sp3 hybridization changes of the nitrogen atom.25
This makes the carbon atom of the remaining carbonyl group a
better target for a nucleophilic attack. In imides 1, 2, and 4 the two
carbonyl groups are distinguished by their conjugation. The carbonyl
group of the conjugated system is held in the sp2 hybridization by
the formation of a push-pull system with the electron-donating
sulfur atom. The unconjugated carbonyl group, however, is a good
target for the nucleophilic attack. The reduced chemoselectivity in
the methanolysis of coniothyriomycin17 is caused by the presence

1392 Journal of Natural Products, 2010, Vol. 73, No. 8
Pacher et al.
Figure 3. (A) Methanolysis of ritigalin (6) to methyl phenylacetate and methyl N-methylthiocarbamate (7) together with indication of
protons showing indicative signals. (B) Corresponding stack plots of in situ 1H NMR data monitored in methanol-d4 for a time interval of
5 days. (C) Reaction progress over 400 h (ritigalin (6): solid diamonds; methyl phenylacetate: open squares; methyl N-methylthiocarbamate
(7): open triangles).
component together with methylsinharin (16) in some samples of
G. mauritiana, collected in Sri Lanka, it cannot be excluded that
sinharin (14) also represents an independent natural compound of
its own, besides formation by decomposition of krabin (4).
On the basis of leaf extracts of two Glycosmis species, as
compared by HPLC-UV analyses, it became evident that imides
mostly co-occur with varying amounts of their corresponding sulfur-
containing amides, a phenomenon supporting the biosynthetic
hypothesis shown in Figure 4. Moreover, there may be special
chemotaxonomic possibilities in view of the vicarious accumulation
of two different types of sulfur-containing amides, characterized
either by the above-mentioned amide/imide series or by methyl-
sulfonylpropenoic acid amides with para-positioned prenyloxy or
geranyloxy side chains of the aromatic ring.1,2,30 In fact, HPLC-
UV profiles showed a clear mutual exclusion of these two types of
amides. However, due to the great difficulties of an infrageneric
grouping of Glycosmis,31,32 a clear correlation to a particular species
was not always possible.14 Further investigations will have to show
to what extent these different accumulation trends of amide
formation can be used as chemical markers and/or interpreted as
an ecological function.
Figure 4. Supposed oxidation pathway from amides to imides.
have pronounced antifungal characteristics. Marked antifungal
Experimental Section
properties of acyclic imides were reported for fungi.17,29 In the
General Experimental Procedures. NMR: Bruker DRX-400
AVANCE spectrometer. GC-MS: GC 8000 series gas chromatograph
present investigation no significant differences in activity were
observed between imides and their structurally related amides.
(Fisons Instruments, Milano, Italy); DB-5 fused silica column (25 m
However, the widely distributed transformation product penangin
× 0.32 mm, film thickness 0.25 µm, J & W Scientific); carrier gas
(10) clearly deviated with an EC50 value of >200 µg/mL.9,13 This
helium (50 kPa); injection temperature 230 °C; temperature program
lack of antifungal activity can probably be explained by the lack
40 °C (2 min isotherm), 5 °C/min up to 200 °C. MS 800 quadrupole
of an aromatic system. Since sinharin (14) was detected as the major
mass spectrometer (Fisons Instruments): ionization energy 70 eV, ion

Alcoholysis of Naturally Occurring Imides
Journal of Natural Products, 2010, Vol. 73, No. 8 1393
source temperature 150 °C. EI-MS: MAT95. ESI-MS: MAT900.
HRMS: MAT900. IR: Perkin-Elmer 16PC FT-IR. HPLC: Agilent 1100,
UV diode array detection at 230 nm, column Hypersil BDS-C18 250
× 4.6 mm, 5 µm, mobile gradient either MeOH 20-100% or
acetonitrile 20-60% in aqueous buffer (0.015 M H3PO4, 0.0015 M
tetrabutylammonium hydroxide), flow rate 1 mL/min.
1454w, 1432w, 1384w, 1342w, 1330m, 1302m, 1158s, 1082w, 1038w,
1
984w, 838w, 698m cm-1; H NMR (400 MHz, CDCl3, δH) 9.24 (1H,
s, CH-11), 7.35 (1H, d, J ) 9.7 Hz, CH-2), 7.28 (2H, m, CH-8), 7.23
(2H, m, CH-9), 7.23 (1H, m, CH-10), 6.28 (1H, d, J ) 9.7 Hz, CH-3),
3.93 (2H, m, CH2-5), 2.86 (2H, m, CH2-6), 2.45 (3H, s, CH3-1); 13C
NMR (100 MHz, CDCl3, δC) 166.6 (C-4), 161.3 (C-11), 156.8 (C-2),
137.8 (C-7), 128.9 (C-8), 128.6 (C-9), 126.6 (C-10), 109.7 (C-3), 41.5
(C-5), 37.7 (C-6), 19.9 (C-1); EIMS m/z 249 [M]+, 202, 130, 104, 101
(100), 91, 83.
NMR Analyses. For NMR spectroscopy compound mixtures were
dissolved in methanol-d4 or CDCl3 (∼0.5-5.0 mg in 0.7 mL) and
transferred into 5 mm high-precision NMR sample tubes. CHD2OD or
1
CHCl3 was used as internal standard for H (δH 3.31 or 7.24) and 13C
Methyl N-methylthiocarbamate (7). In situ from reaction: 1H NMR
(400 MHz, CD3OD, δH) 2.65 (3H, s, CH-3), 2.18 (3H, s, CH-1); 13C
NMR (100 MHz, CD3OD, δC) 170.1 (C-2), 27.4 (C-1), 13.1 (C-3);
GC-MS, phenylacetic acid methyl ester/methyl N-methylthiocarbamate
) 20:1 (15, rt )16 min); MS m/z 105 (20%, M+, C3H7ONS), 75 (5%,
M+ - NHCH3), 58 (100%, M+ - SCH3).
(δC 49.0 or 77.1) measurements. All spectra were measured at 300 (
0.1 K at 400.13 MHz (1H) or 100.61 MHz (13C) and performed using
the Bruker Topspin 2.1 software. 1D spectra were recorded by
acquisition of 32k data points and after zero filling to 64k data points,
and Fourier transformation spectra were performed with a range of 7200
Hz (1H) and 20 000 Hz (13C). To determine the 2D COSY, TOCSY,
NOESY, HMQC, and HMBC spectra 128 experiments with 1024 data
points each were recorded and Fourier transformed to 2D spectra with
a range of 4000 Hz and 20 000 Hz for 1H and 13C, respectively. In situ
1H NMR measurements were directly taken from the sample at regular
intervals. The reaction was performed in the magnet and temporarily
kept in a temperature-controlled water bath (300 ( 0.5 K).
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Plant Material. Fresh leaves of G. mauritiana and G. cf. puberula
were picked from plants cultivated in the Botanical Garden of the
University of Vienna (HBV). These plants were originally collected
from south Thailand and were kept in the greenhouse during winter.
More specifically, G. mauritiana came from (i) Satun Province, Tarutao
Island (HG 330, HG 331), and (ii) limestone in open deciduous forest
near Krabi (HG 464). G. cf. puberula (HG 395, HG 397) came from
Khao Soi Dao, southeast Thailand. Voucher specimens were identified
by H.G. and deposited at the Herbarium of the Institute of Botany,
University of Vienna (WU).
Extraction and Isolation. Dried leaves were ground and extracted
with acetone at room temperature for 3 days, filtered, and concentrated,
and the remaining residues were extracted with CHCl3 from the aqueous
solution. The CHCl3 fractions were evaporated to dryness, and the
lipophilic crude extracts were roughly separated by CC (Merck silica
gel 60, 0.2-0.5 mm) with solvent mixtures of n-hexane, Et2O, and
MeOH. Further separation was achieved by preparative MPLC (400
× 40 mm column, Merck LiChroprep silica gel 60, 25-40 µm) with
mixtures of EtOAc in n-hexane as mobile phase.
From 60 g of dried leaves of three collections of G. mauritiana (HG
330, HG 331, HG 464) the CHCl3 fraction (1000 mg) was roughly
separated by CC. The combined imide-containing fractions (208 mg),
monitored by HPLC and TLC, were eluted with 50% Et2O in n-hexane
and 100% Et2O and further separated by preparative MPLC with 10%
EtOAc in n-hexane to afford 46 mg of krabin (4) and 9 mg of isokrabin
(5). From 5 g of dried leaves of G. cf. puberula (HG 395, HG 397) the
CHCl3 fraction (220 mg) was separated in the same way, affording 2.5
mg of crystals of methoxypenimide B (3).
Methoxypenimide B (3): colorless crystals; mp 94-95 °C (Et2O/
n-hexane); UV (MeOH) λmax 213sh, 238sh, 318 nm; IR (CCl4) νmax
3007w, 2927w, 2837w, 1701s, 1681s, 1621w, 1556s, 1521s, 1472w,
1432w, 1392w, 1292 m, 1257s, 1187m, 1108s, 1083m, 1058w cm-1
;
1H NMR (400 MHz, CDCl3, δH) 7.22 (1H, d, J ) 9.9 Hz, CH-2), 7.14
(2H, m, CH-8), 6.83 (2H, m, CH-9), 6.59 (1H, d, J ) 9.9 Hz, CH-3),
4.01 (2H, m, CH2-6), 3.77 (3H, s, O-CH3), 3.22 (3H, s, CH3-11), 2.40
(3H, s, CH3-1); FAB-MS m/z 279 [M + H]+.
Krabin (4): colorless oil; UV (MeOH) λmax 208sh, 237sh, 301 nm;
IR (CCl4) νmax 3028w, 2928w, 2858w, 1718s, 1662s, 1568s, 1498w,
1454w, 1432w, 1342m, 1328s, 1256m, 1158s, 1140w, 972w, 942w,
890w, 700m cm-1; 1H NMR (400 MHz, CDCl3, δH) 9.27 (1H, s, CH-
11), 7.97 (1H, d, J ) 14.5 Hz, CH-2), 7.30 (2H, m, CH-8), 7.22 (2H,
m, CH-9), 7.22 (1H, m, CH-10), 6.03 (1H, d, J ) 14.5 Hz, CH-3),
3.94 (2H, m, CH2-5), 2.86 (2H, m, CH2-6), 2.37 (3H, s, CH3-1); 13C
NMR (100 MHz, CDCl3, δC) 165.6 (C-4), 162.5 (C-11), 151.5 (C-2),
138.2 (C-7), 128.9 (C-8), 128.6 (C-9), 126.6 (C-10), 110.8 (C-3), 42.1
(C-5), 34.6 (C-6), 15.0 (C-1); EIMS m/z 249 [M]+, 202, 130, 104, 101
(100), 91, 73; ESI-MS m/z 250 [M + H]+; HRMS (ESI) calc
(C13H16O2NS) 250.0902, found 250.0904 ( 5 ppm.
(26) Greger, H.; Hofer, O.; Ka¨hlig, H.; Wurz, G. Tetrahedron 1992, 48,
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(29) Maruyama, H. B.; Suhara, Y.; Suzuki-Watanabe, J.; Maeshima, Y.;
Shimizu, N.; Ogura-Hamada, M.; Fujimoto, H.; Takano, K. J. Antibiot.
1975, 28, 636–647.
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Phytochemistry 2000, 54, 207–213.
(31) Stone, B. C. Proc. Acad. Nat. Sci. Phila. 1985, 137, 1–27.
(32) Stone, B. C. Garden Bull. Singapore 1994, 46, 113–119.
Isokrabin (5): colorless oil; UV (MeOH) λmax 208sh, 235sh, 314
nm; IR (CCl4) νmax 3028w, 2926m, 2856w, 1712s, 1654s, 1546s, 1498w,
NP1003092