Aliphatic pyrrolidine amides from two tropical convolvulaceous species

Article abstract of DOI:10.1016/S0031-9422(99)00245-9

Seven aliphatic pyrrolidine amides with branched and linear saturated C₁₅-C₁₉ acyl moieties were detected in vegetative plant organs of Ipomoea aquatica and Merremia quinquefolia as well as in seeds of M. quinquefolia by GC-MS analysis. One of the compounds was isolated from both species and characterized as 1-(14-methylhexadecanoyl)pyrrolidine, a new natural product. The presence of 1-hexadecanoylpyrrolidine and 1-octadecanoylpyrrolidine was confirmed by comparison of their GC-MS data with those of synthesized compounds.

Full text of DOI:10.1016/S0031-9422(99)00245-9

Phytochemistry 52 (1999) 1437±1441
Aliphatic pyrrolidine amides from two tropical convolvulaceous
species
p
a
Britta Tofern , Petra Mann , Macki Kaloga , Kristina Jenett-Siems , Ludger Witte ,
a
a
a
b
a,
Eckart Eich *
a
Institut fuÈr Pharmazie II (Pharmazeutische Biologie), Freie UniversitaÈt Berlin, KoÈnigin-Luise-Straûe 2+4, 14195 Berlin, Germany
Institut fuÈr Pharmazeutische Biologie, Technische UniversitaÈt Braunschweig, Mendelssohnstraûe 1, 38106 Braun-schweig, Germany
b
Received 19 November 1998; received in revised form 16 April 1999; accepted 21 April 1999
Abstract
Seven aliphatic pyrrolidine amides with branched and linear saturated C15±C19 acyl moieties were detected in vegetative plant
organs of Ipomoea aquatica and Merremia quinquefolia as well as in seeds of M. quinquefolia by GC±MS analysis. One of the
compounds was isolated from both species and characterized as 1-(14-methylhexadecanoyl)pyrrolidine, a new natural product.
The presence of 1-hexadecanoylpyrrolidine and 1-octadecanoylpyrrolidine was con®rmed by comparison of their GC±MS data
with those of synthesized compounds. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Ipomoea aquatica; Merremia quinquefolia; Convolvulaceae; GC±MS; Amides; Alkamides; Pyrrolidine amides; 1-(14-Methylhexadeca-
noyl)pyrrolidine; 1-Hexadecanoylpyrrolidine; 1-Octadecanoylpyrrolidine
1
. Introduction
Jenett-Siems, Kaloga & Eich, 1993) as well as cyano-
genic glycosides (Nahrstedt, Jensen & Wray, 1989).
Furthermore, di€erent types of amides were isolated:
N-feruloyltyramine (Tseng et al., 1986), serotonin±
hydroxycinnamic acid conjugates of the ipobscurine-
type (Eich, Henn, Kolshorn, Pertz & Schulz, 1989) and
lignanamides (Henrici, Kaloga, & Eich, 1994). The
present study reports on the detection and isolation of
aliphatic pyrrolidine amides from Ipomoea aquatica
FORSK., the leaves of which are used as a vegetable
in Southeast Asia, and Merremia quinquefolia (L.) H.
HALL. f., a pantropical twiner of American origin.
The Convolvulaceae comprise nearly 2000 predomi-
nantly tropical species. A wide variety of low-molecu-
lar N-containing secondary metabolites has been
found in this family over the years: ergolines
(
Hofmann & Tscherter, 1960), pyrrolidines (e. g. cus-
cohygrine) (Evans & Somanabandhu, 1974; Jenett-
Siems & Eich, 1994), lipophilic tropanes (Orecho€ &
Konowalowa, 1933; Weigl, Kaloga & Eich, 1992) and
hydrophilic tropanes (calystegines) (Goldmann et al.,
1
990), indolizidine and pyrrolizidine alkaloids
(
Gourley, Heacock, McInnes, Nikolin & Smith, 1969;
p
2. Results and discussion
Part 9 in the series ``Phytochemistry and Chemotaxonomy of the
Convolvulaceae''. For part 8 see Tofern et al., 1999 [Tofern, B.,
Kaloga, M., Witte, L., Hartmann, T. Eich, E. (1998)
&
The petrol ether and dichloromethane extracts of
the roots and aerial vegetative parts (I. aquatica, M.
quinquefolia ) as well as of the seeds (M. quinquefolia )
were analyzed by GC±MS. Two groups of compounds
were characterized as pyrrolidine amides with satu-
rated aliphatic C ±C acyl moieties by their mass
Phytochemistry, 51, 1177]. Presented in part at the 44th Annual
Congress of the Society for Medicinal Plant Research, 1996, Prague,
Czech Republic and at the IOCD/CYTED International Joint
Symposium, 1997, Panama
Corresponding author. Tel.: +49-30-838-3724; fax: +49-30-838-
729.
Â
, Panama.
*
3
15
19
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(99)00245-9

1
438
B. Tofern et al. / Phytochemistry 52 (1999) 1437±1441
fragmentation pattern (Table 1), both in I. aquatica
and M. quinquefolia. The compounds classi®ed as alka-
loid MQ-A ±alkaloid MQ-A (1, 2, 4, 5, 7) showed
1
5
regular di€erences of approximately 100 units in their
retention indices (RI) and of 14 mass units in their
molecular ions, indicating a set of homologous com-
pounds. Additionally, two compounds named alkaloid
MQ-B and MQ-B (3, 6) were detected, which were,
2
4
+
according to their [M] at m/z 309 and 337, isomeric
to alkaloid MQ-A (2) and alkaloid MQ-A (5). Since
2
4
3
and 6 had higher retention indices than 2 and 5, it
could be postulated that 3 and 6 have acyl moieties,
which are linear or at least less branched than those of
2
and 5. It could be shown by synthesis and GC±MS
analysis of 1-hexadecanoylpyrrolidine and 1-octadeca-
noylpyrrolidine that these synthetical compounds have
retention indices identical with those of 3 and 6; there-
fore the alkaloid MQ-B and MQ-B possess a linear
2
4
acyl moiety.
The tentative identi®cation of 1, 2, 4, 5 and 7 as pyr-
rolidine amides linked with branched saturated fatty
acids could be con®rmed by isolation of 4 from the
roots of I. aquatica and the epigeal vegetative parts of
M. quinquefolia. Surprisingly, this compound showed a
positive reaction with Dragendor€'s reagent on TLC.
Its structure was elucidated by EIMS, FABMS,
1
1
1
13
HRMS, H NMR, H, H COSY, C NMR, DEPT
and HETCOR. The EIMS showed a molecular ion
peak at m/z 323, which was con®rmed by FABMS
(
positive mode). The molecular formula was deter-
mined as C H NO by HRMS. Additionally, the
21
41
EIMS showed a peak at m/z 70, indicative of a pyrroli-
dine moiety, a base peak at m/z 113, caused by
McLa€erty rearrangement, as well as characteristic
peaks at m/z 266 [M±C H ], m/z 294 [M±C H ] and
4
9
2
5
m/z 308 [M±CH ], indicating the presence of a
3
1
3
branched alkyl chain, as depicted in Fig. 1. The
C
NMR and DEPT spectra revealed the presence of two
methyl groups, one methine group, the carbonyl group
of an amide, eight methylene groups and additionally
a cluster of methylene groups, which could not be
1
1
1
clearly distinguished. In the H NMR and H,
COSY, two methylene groups of the pyrrolidine moi-
H
ety at d 1.88 and 1.98 (2 H each, m, 2 Â H-3' and H-
4
3
') coupled with two methylene groups at d 3.43 and
.48 (2 H each, t, 2  H-2' and H-5'). The di€ering
shifts of H-2'/H-5' as well as H-3'/H-4' and of the cor-
responding carbons were probably caused by restricted
rotation of the N±C bond of the amide moiety due to
partial double-bond character, which led to cis- and
trans-location of these protons. A similar phenomenon
has been observed e.g. for N-formyl-2-methylpiperidine
and its methyl group before (LaPlanche & Rogers,
1
963). The appearance of one secondary methyl group
at d 0.86 (3 H, d, J = 6.5 Hz, H-17) and one primary
methyl group at d 0.88 (3 H, t, J = 7.3 Hz, H-16) as

B. Tofern et al. / Phytochemistry 52 (1999) 1437±1441
1439
3
Fig. 1. Structure and EIMS fragmentation pattern of MQ-A (4).
1
3
well as the corresponding C NMR signals at d 11.8
C-16) and 19.6 (C-17) corroborated the branched
and B are substituted at C-2' and possess a shorter
acyl moiety, though.
(
structure of the acyl moiety. Therefore, 4 was charac-
terized as 1-(14-methylhexadecanoyl)pyrrolidine, a new
natural product.
To the best of our knowledge, no pyrrolidine amides
with a branched saturated acyl moiety have been
described as natural products before. Even linear 1-
hexadecanoylpyrrolidine (3) and 1-octadecanoylpyrro-
lidine (6) have only been characterized by GC±MS
analysis of Piper amalago L. (Piperaceae), so far
3. Experimental
3.1. General
1
13
H NMR and C NMR spectra were recorded on a
Bruker DPX 400 spectrometer with TMS as internal
standard. EIMS, HRMS and FABMS spectra were
obtained with Varian MAT CH A, Finnigan MAT
711 and Finnigan MAT CH DF spectrometers, re-
5
spectively. TLC and prep. TLC: precoated silica gel
plates 60 F254 (0.2 mm); Et O±petrol (3:1) or cyclohex-
(
È
Achenbach, Fietz, Worth, Waibel, & Portecop, 1986).
However, the occurrence of aliphatic pyrrolidine
amides does not seem to be a general feature for con-
volvulaceous species, since such compounds could not
be detected in various other species of the genera
Argyreia, Falkia, Ipomoea, Merremia, Odonellia,
Operculina and Turbina (Mann, 1997; Tofern, 1999).
Furthermore, this is the ®rst report on the occur-
rence of alkamides in the Convolvulaceae in general.
To date, alkamides like isobutylamides, isopentyla-
mides, pyrrolidine amides and piperidine amides have
been found in the plant kingdom. The pyrrolidine
amides are mainly restricted to the Asteraceae and the
Piperaceae (Greger, 1984; Greger, Hofer, & Werner,
7
2
ane±CHCl ±MeOH (5:4.5:0.5) (solvent systems I and
3
II), detection with Dragendor€'s reagent.
3.2. Plant material
Aerial vegetative parts and roots were obtained
from plants cultivated in the greenhouse of the Institut
fu
È
r
Universita
Pharmazie (Pharmazeutische Biologie), Freie
t Berlin, Germany, where voucher specimens
È
1
987; Greger, Zdero, & Bohlmann, 1987; Parmar et
are deposited. The plants were grown from seeds col-
lected near Don Muang/Thailand (I. aquatica ) and
near Guayaquil/Ecuador (M. quinquefolia ).
al., 1997; Singh, Dhar, & Atal, 1971). In contrast to
the pyrrolidine amides found in I. aquatica and M.
quinquefolia most of those compounds have ole®nic or
acetylenic acyl moieties (Greger, 1984). While the sec-
ondary metabolism of the families mentioned so far
shows no close resemblance to that of the
Convolvulaceae, the detection of the pyrrolidine
amides conioidine A and B in Chamaesaracha con-
ioides (DUN.) BRITT. (Solanaceae) (Chan et al.,
3.3. Extraction for GC±MS analysis
Ground dried plant material (50 g) was extracted
with MeOH (3 Â 400 ml) at room temperature under
protection against daylight. After evaporation the resi-
due was dissolved in 2% aq. tartaric acid (250 ml) and
successively extracted with petrol (3 Â 150 ml) and
CH Cl (3 Â 150 ml). After evaporation in vacuo these
1993) is chemotaxonomically quite remarkable, since
both families belong to the Solanales. In contrast to
the compounds described in this paper conioidine A
2
2
extracts were used for the pyrrolidine amide analysis.

1
440
B. Tofern et al. / Phytochemistry 52 (1999) 1437±1441
3.4. GC±MS analysis
containing 4 were further puri®ed twice by prep. TLC
(1) solvent system I (developed twice); (2) solvent sys-
(
A GC equipped with a 30 m  0.32 mm fused-silica
tem II; elution of the zone containing 4 with CHCl3±
acetone (9:1), respectively) to give 4 (13 mg), which
was similarly obtained from epigeal vegetative parts of
M. quinquefolia, as well.
capillary column (DB-1) was used. Conditions: injector
2
6
508C; split ratio 1:20; temp. programme 70±3008C,
�
1
8C min ; carrier gas He 0.5 bar. The capillary col-
umn was directly coupled to the quadrupole mass
spectrometer Finnigan MAT 4515. EI mass spectra
were recorded at 40 eV. Retention indices (RI): Kovats
indices were calculated with reference to a set of coin-
jected n-alkanes (C ±C ) (Kovats, 1958).
3.9. 1-(14-Methylhexadecanoyl)pyrrolidine (4)
(=MQ-A3)
2
0
R 0.22 (I), 0.50 (II). [a] +58 (CHCl3, c 0.4). IR
9
28
f
D
�
1
(
KBr) nmax (cm ): 1628, 3438. EIMS 70 eV, m/z (rel.
int. %): 323 (5), 308 (1), 294 (2), 266 (1), 126 (15), 113
100), 70 (11), 55 (15), 43 (33). (+)-FABMS m/z: 324
3.5. Synthesis of the pyrrolidine amides 3 and 6
(
+
2.85 g pyrrolidine and 5.6 g hexadecanoyl chloride
[M + H] . HRMS 80 eV, m/z: 323.3189 (C H NO,
21 41
as well as 2.85 g pyrrolidine and 6.0 g octadecanoyl
chloride were dissolved in 5 ml pyridine, respectively,
and left for 3 h at room temperature. Then the sol-
ution was added to 50 ml 10% aq. HCl and extracted
with EtOAc (5 Â 25 ml). The organic layer was dried
with Na SO and evaporated in vacuo; after crystalli-
calc. 323.3188), 308.2955 (C H NO, calc. 308.2953),
20 38
294.2798 (C H NO, calc. 294.2797), 266.2484
36
19
(C H NO, calc. 266.2484), 126.0919 (C H NO, calc.
32
1
7
7
12
1
126.0919), 113.0841 (C H NO, calc. 113.0841).
6
H
11
NMR (400 MHz, CDCl ): d 0.86 (3 H, d, J = 6.5 Hz,
3
3 Â H-17), 0.88 (3 H, t, J = 7.3 Hz, 3 Â H-16), 1.27
2
4
zation from a MeOH±H O mixture 4.5 g of 3 and 4.7
2
g of 6 were obtained, resp.
(23 H, m, CH + 11 Â CH ), 1.66 (2 H, m, 2 Â H-3),
2
1.88 (2 H, m, 2 Â H-3'/2 Â H-4'), 1.98 (2 H, m, 2 Â H-
3
'/2 Â H-4'), 2.27 (2 H, t, J = 7.5 Hz, 2 Â H-2), 3.43
3.6. 1-Hexadecanoylpyrrolidine (3)
(2 H, t, J = 6.8 Hz, 2 Â H-2'/2 Â H-5'), 3.48 (2 H, t,
13
J = 6.9 Hz, 2 Â H-2'/2 Â H-5').
C NMR (100.6
MHz, CDCl ): d 11.8 (q, C-16), 19.6 (q, C-17), 24.8 (t,
1
H NMR (400 MHz, CDCl ): d 0.91 (3 H, t,
3
3
J = 6.7 Hz, CH ), 1.28 (24 H, m, 12 Â CH ), 1.66 (2
C-3'/C-4'), 25.4 (t, C-3), 26.6 (t, C-3'/C-4'), 27.5 (t, C-
15), 29.9-30.0 (t, C-4±C-12), 34.8 (d, C-14), 35.3 (t, C-
2), 37.0 (t, C-13), 46.0 (t, C-2'/C-5'), 47.0 (t, C-2'/C-
5'), 172.3 (s, C-1).
3
2
H, m, 2 Â H-3), 1.88 (2 H, m, 2 Â H-3'/2 Â H-4'), 1.96
(
2 H, m, 2 Â H-3'/2 Â H-4'), 2.27 (2 H, t, J = 7.7 Hz,
2
3
 H-2), 3.43 (2 H, t, J = 6.7 Hz, 2  H-2'/2  H-5'),
.49 (2 H, t, J = 6.9 Hz, 2 Â H-2'/2 Â H-5').
3.7. 1-Octadecanoylpyrrolidine (6)
Acknowledgements
1
H NMR (400 MHz, CDCl ): d 0.90 (3 H, t,
3
The authors are indebted to Mrs. J. Schulz, Institut
J = 6.8 Hz, CH ), 1.28 (28 H, m, 14 Â CH ), 1.66 (2
3
2
fu
technical assistance and to Mrs. U. Ostwald, Institut
fur Organische Chemie, Freie Universitat Berlin, for
recording the FABMS and HRMS spectra. We are
also grateful to Mrs. E. Baumel-Eich, Berlin, for essen-
È
È
r Pharmazie, Freie Universitat Berlin, for skilful
H, m, 2 Â H-3), 1.88 (2 H, m, 2 Â H-3'/2 Â H-4'), 1.96
(
2 H, m, 2 Â H-3'/2 Â H-4'), 2.27 (2 H, t, J = 7.6 Hz,
È
È
2
3
 H-2), 3.43 (2 H, t, J = 6.8 Hz, 2  H-2'/2  H-5'),
.48 (2 H, t, J = 6.8 Hz, 2 Â H-2'/2 Â H-5').
È
tial support in exploring the plants and in collecting
the plant material in Ecuador and Thailand.
3.8. Isolation of MQ-A (4) from I. aquatica
3
Dried ground roots (102 g) were extracted with 3.2 l
MeOH at room temperature under protection against
daylight. After evaporation the residue was dissolved
in 0.4 l 2% aq. tartaric acid and extracted with 0.9 l
petrol and 0.9 l CH Cl , successively. These two
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