Chemotaxonomy of the pantropical genus Merremia (Convolvulaceae) based on the distribution of tropane alkaloids

PHYTOCHEMISTRY

Phytochemistry 66 (2005) 1448–1464

www.elsevier.com/locate/phytochem

Chemotaxonomy of the pantropical genus

Merremia (Convolvulaceae) based on the distribution

q

of tropane alkaloids

a

Kristina Jenett-Siems , Robert Weigl , Anke Bohm , Petra Mann ,

a

¨

Britta Tofern-Reblin , Sonja C. Ott , Azar Ghomian , Maki Kaloga , Karsten Siems ,

a

b

c,

d

a,

*

Ludger Witte ^z, Monika Hilker , Frank Muller , Eckart Eich

¨

a

Institut fu¨r Pharmazie (Pharmazeutische Biologie), Freie Universita¨t Berlin, Ko¨nigin-Luise-Str. 2+4, D-14195 Berlin, Germany

b

AnalytiCon Discovery, Hermannswerder Haus 17, D-14473 Potsdam, Germany

c

Institut fu¨r Pharmazeutische Biologie, Technische Universita¨t Braunschweig, Mendelssohnstrabe 1, D-38106 Braunschweig, Germany

d

¨

Institut fu¨r Biologie (Angewandte Zoologie/Okologie der Tiere), Freie Universita¨t Berlin, Haderslebener Str. 9, D-12163 Berlin, Germany

Received 11 February 2005; received in revised form 12 April 2005

Available online 26 May 2005

Abstract

The occurrence and distribution of tropane and biogenetically related pyrrolidine alkaloids in 18 Merremia species of paleo-, neo-,

and pantropical occurrence have been studied. The extensive GC–MS study included members of almost all sections of the genus

and has been carried out with epigeal vegetative parts as well as with roots. It comprises altogether 74 tropanes and 13 pyrrolidines

including nicotine. Along with datumetine known already from a solanaceous species, the study led to the isolation (from M. dis-

secta and M. guerichii, respectively) and structure elucidation (spectral data) of four novel 3a-acyloxytropanes, merresectines A–D:

3a-(4-methoxybenzoyloxy)nortropane (A), 3a-kurameroyloxytropane (B), 3a-nervogenoyloxytropane (C), 3a-[4-(b-^D-glucopyrano-

syloxy)-3-methoxy-5-(3-methyl-2-butenyl)benzoyloxy]tropane (b-^D-glucoside of D). Moreover, the novel 3a,6b-di-(4-meth-

oxybenzoyloxy)tropane (merredissine) has been isolated from M. dissecta and structurally elucidated. In addition the structures

of datumetine and merresectine A could be conﬁrmed by synthesis. Spectral data for two known 3a-acyloxytropanes (merresectine

E b-^D-glucoside, 4⁰-dihydroconsabatine) and one known 3b-acyloxytropane (concneorine) are documented for the ﬁrst time. The

structures of three further merresectines (F-H) have been determined by mass spectrometry. Furthermore, the linkage (2⁰,3- and

2⁰,4-, respectively) of two position isomer N-methylpyrrolidinylhygrines was proven by synthesis. The results of the study contribute

to the solution of infrageneric taxonomic problems. Whereas all species yield pyrrolidine alkaloids without suitably diﬀerentiating

results the diverging occurrence of tropane alkaloids leads to three groups of sections: (1) taxa free of tropanes, (2) taxa with simple

tropanes, and (3) taxa with merresectines in addition to simple tropanes.

Keywords: Merremia; Convolvulaceae; Merremieae; GC–MS; Structure elucidation; Nicotine; Pyrrolidine alkaloids; Tropane alkaloids; Concne-

orine; 4⁰-Dihydroconsabatine; Merredissine; Merresectines; Chemotaxonomy

q

Part 18 in the series ‘‘Phytochemistry and Chemotaxonomy of the

Convolvulaceae’’. For part 17 see Schimming et al. [Phytochemistry 66

(2005) 469–480].

*

Corresponding author. Tel.: +49 30 838 53720; fax: +49 30 838

1. Introduction

53729.

The convolvulaceous genus Merremia Dennst. ex

Endl. comprising about 70 species (Deroin, 2001) is

E-mail address: eckeich@zedat.fu-berlin.de (E. Eich).

Deceased.

z

doi:10.1016/j.phytochem.2005.04.027

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

1449

nested together with, e.g. Hewittia, Operculina within

2. Results and discussion

the tribal clade Merremieae s.s. according to a phylo-

genetic classiﬁcation based on a molecular cladistic

analysis and aligned with the traditional point of view

2.1. Tropane alkaloids

´

(Stefanovic et al., 2003). Most of the Merremia species

sampled (Stefanovic et al., 2002) form a moderately

2.1.1. Isolation and structure elucidation of novel

aromatic 3a-acyloxytropanes

´

supported subclade including M. dissecta, M. aegyptia,

M. vitifolia, however with several segregates (e.g. M.

umbellata, M. peltata). Unfortunately, the relation-

ships between the Merremia subclade and the Merre-

mia segregates are largely unresolved. This is also

one reason why a tribal clade deﬁnition has not yet

been provided. The number of species included in

the cladistic analysis has been rather low.

On the other hand, the classical infrageneric divi-

sion into ﬁve sections is based on morphological/ana-

tomical characters as established by Hallier (1893,

1894) and afterwards modiﬁed by Van Ooststroom

(1939, 1953). Furthermore, the classical section Strept-

andra has been divided – at least for the species

occurring in the Americas – into three novel sections,

Cissoides, Halliera, Schizips by OÕDonell (1941). La-

ter, members from section Schizips have been segre-

gated into Vitifolia allies and Tuberosa allies

(Austin, 1998a,b) as have from section Halliera into

a novel genus, Xenostegia (Austin and Staples, 1980).

However, this latter segregation is still disputed (Der-

oin, 2001).

Complex resin glycosides, unique constituents of

the Convolvulaceae, have also been detected in some

Merremia spp., e.g. M. mammosa Choisy (Kitagawa

et al., 1988), M. tuberosa (L.) Rendle sub nom. Ipo-

moea tuberosa L. (Ono et al., 1993), Merremia hungai-

ensis Lingelish. and Borza (Noda et al., 1994).

However, at present there is little knowledge about

low-molecular secondary metabolites of the genus

Merremia as well as of the other genera of the tribe

Merremieae s.s. though phytochemical data should

be able to contribute to the solution of taxonomic

problems in both taxa. Single occurrences are reported

in literature, e.g. long-chained caﬀeates have been de-

tected in M. dissecta and M. tuberosa, respectively

(Garcia-Argaez et al., 1999), cyanogenic glycosides

have been isolated from M. dissecta (Nahrstedt

et al., 1989, 1990) and from M. vitifolia (Jenett-Siems,

1996), pyrrolidine and simple tropane alkaloids have

been found in M. gemella as well as in M. tuberosa

(Jenett-Siems and Eich, 1994). The aim of our phyto-

chemical study on Merremia spp. has been to examine

if the occurrence and distribution of these both types

of related alkaloids might contribute to the infragen-

eric classiﬁcation. For this purpose 18 species from

all parts of the tropics have been included (Table 2).

The study led to the discovery and structure elucida-

tion of ﬁve novel tropane alkaloids; two of them rep-

resent the ﬁrst glycosidic tropanes at all.

Isolation of alkaloids from M. dissecta roots was

achieved by column chromatography on silica gel and

preparative HPLC or TLC if necessary. In addition to

the known alkaloid datumetine (1) which was identiﬁed

by comparison with data from the literature (Siddiqui et

al., 1986) and authentic synthesized material, four previ-

ously unknown alkaloids 2, 3, 4, and 5 could be isolated.

The structures have been elucidated by spectroscopic

1

means (EIMS, HRMS, FABMS, H NMR,¹³C NMR,

1

DEPT, H-¹H-COSY, H-¹³C-COSY, NOE).

In the EIMS, compound 2 gave a molecular ion

peak at m/z 261 and signiﬁcant peaks at m/z 110 (base

peak), 135, and 152. From the HRMS spectrum of 2,

its molecular formula could be established as

C₁₅H₁₉NO₃, corresponding to a molecular weight of

261.1368, 14 mass units less than datumetine (1). Tak-

ing into account the base peak at m/z 110 in contrast

to the usual base peak at m/z 124 of tropan-3-ol es-

ters, 2 should be the N-desmethyl derivative of

datumetine or its 3b-isomer. The ¹H NMR and ¹³C

NMR spectra indeed were very similar to those of

1, with the exception of the N-methyl region. The ste-

reochemistry of H-3 of the tropanol moiety was de-

duced as b based on the one-proton triplet at d 5.31

(1 H, J = 5.2 Hz). Further conﬁrmation of the struc-

ture was achieved by synthesis of 2 and comparison

of the spectral properties. We propose the trivial name

merresectine A for this novel nortropane alkaloid

which has been isolated also from the roots of

M. quinquefolia.

Compound 3, named merresectine B, showed a

molecular ion peak at m/z 559 in the EIMS and its

molecular formula was determined to be C₃₁H₄₅NO₈

by HRMS. The base peak at m/z 124, and character-

istic fragments at m/z 140, 94, 83, and 82 in the EIMS

spectrum were indicative of a tropan-3-ol ester. The

(ꢀ)-FABMS of 3 exhibited a quasi-molecular ion peak

at m/z 558 [MꢀH]^ꢀwhereas the ion at m/z 396

[(MꢀH)-162]^ꢀsuggested the loss of one hexosyl resi-

due. The occurrence of a tropan-3-ol moiety was fur-

ther conﬁrmed by the ¹H NMR spectrum. A one-

proton triplet at d 5.18 (1 H, J = 5.2 Hz) indicated a

b-orientation of H-3. In addition, the acyl moiety dis-

played two singlets at d 1.73 and 1.76 for six protons

each, a broad doublet at d 3.62 (4 H, J = 7.4 Hz) cou-

pled to a broad triplet at d 5.32 (2 H, J = 7.4 Hz).

These characteristic signals indicated the presence of

two prenyl groups. Moreover, a singlet for two aro-

matic protons at d 7.71 was observed, indicative of

a symmetric tetrasubstituted benzoyl moiety. A dou-

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K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

blet at d 4.82 (J = 7.4 Hz) was assigned to the ano-

meric proton of the hexose residue (H-1^{0 0 0 0}), thus

revealing a b-linkage to the aglycone. In addition,

irradiation of this anomeric proton signal caused a

NOE enhancement of the signal at d 3.62, indicating

that the prenyl groups were attached to C-3⁰and C-

5⁰, respectively. Thus, the structure of the acyl moiety

was identiﬁed as kurameric acid which had been dis-

covered as acyl component of ester alkaloids of cho-

line or a necine base, isolated from Liparis kurameri

F^RENCHand SAV. and L. kumokiri F. MAEKAWA

(Orchidaceae), respectively (Nishikawa et al., 1967).

Kurameric acid was then established to be the 4-b-^D-

glucoside of 3,5-bis-(3-methyl-but-2-enyl)-4-hydroxy-

benzoic acid (nervogenic acid). Merresectine B (3)

has been isolated also from the roots of M. cissoides

and M. quinquefolia, respectively.

The molecular formula of 4 was determined to be

C₂₅H₃₅NO₃by HRMS. In the EIMS spectrum, it

was characterized by a molecular ion peak at m/z

397 and signiﬁcant fragments hinted again to a deriv-

ative of tropan-3-ol. This was conﬁrmed by the ¹H

NMR and ¹³C NMR spectra. The additional signals

were similar to those of the kurameric acid moiety

of merresectine B (3) but no signals of a glucosyl res-

idue could be detected. Thus, compound 4 had to be

the aglycone of merresectine B and was named merre-

sectine C.

aﬀorded merresectine B (3) and one further compound

6. Its EI mass spectrum showed a molecular ion peak

at m/z 521 corresponding to a molecular formula of

C₂₇H₃₉NO₉determined by HRMS. The fragment at

m/z 359 (base peak) again indicated the loss of a hexosyl

moiety. The ¹H NMR data of 6 were very similar to that

of merresectine B, but showed only one prenyl residue.

Instead, a signal for an aromatic methoxy group was ob-

served. Thus, 6 which we named merresectine D b-^D-glu-

coside, has to be an ester of tropan-3a-ol with 4-(b-^D-

glucopyranosyloxy)-3-methoxy-5-(3-methyl-but-2-enyl)

benzoic acid.

The 4-b-^D-glucoside (72a) of Merresectine E (72) has

been identiﬁed in six Merremia spp. and was structurally

elucidated based on the isolation from the roots of Con-

volvulus sabatius Viv. ssp. sabatius. The compound

exhibited a quasi molecular ion peak at m/z 492

[M+H]⁺in the (+)-FABMS spectrum, corresponding

to a molecular formula of C₂₆H₃₇NO₈. The base peak

at m/z 124, and further characteristic fragments in the

EIMS spectrum were again indicative of a tropan-3-ol

ester. The structural features deduced from the ¹H

NMR spectrum resembled those of merresectine D b-

D-glucoside with the exception that three aromatic pro-

tons at d 7.03, 7.82, and 7.84, but no methoxy group

were observed. Thus the aglycone of merresectine E b-

D-glucoside (72a) has to be 3a-[4-hydroxy-3-(3-methyl-

but-2-enyl)benzoyloxy]tropane which is glucosylated

via the 4⁰-hydroxy group.

Compound 5 exhibited a molecular ion peak at m/z

425, corresponding to the molecular formula C₂₄

H₂₇NO₆determined by HRMS. Other diagnostic peaks

could be observed at m/z 274, 138, 135, 122, 107, 95

and 94 (base peak). This pattern hinted to a derivative

of 6-hydroxytropan-3-ol. The ¹H NMR (Table 5) exhib-

ited signals for two p-substituted benzoyl moieties and a

singlet for six protons at d 3.87, hinting to two aromatic

methoxy groups. The orientation of the hydroxyl groups

of the tropane moiety was deduced from the coupling

constants. H-3 exhibited a triplet (J = 5.0 Hz) at d 5.33,

characteristic for a b-orientation of this proton. H-6

was observed as a dd (J = 3.0 Hz, 7.5 Hz) at d 5.87. This

is in agreement with an angle of 90ꢁ between H-6 and H-5

corresponding to a coupling constant of more or less

0 Hz, thus proposing an a-orientation of H-6. According

to MMX-calculations¹for the epimer with a b-orienta-

tion of H-6 a dihedral angle of about 30ꢁ (coupling con-

stant about 8.0 Hz) should be expected. These results

were conﬁrmed by NOE experiments. In conclusion

compound 5 has to be 3a,6b-bis-(4-methoxybenzoyl-

oxy)tropane which we named merredissine.

2.1.2. Occurrence of aromatic 3a-acyloxytropanes

detected by GC–MS

Aromatic 3a-acyloxytropanes have been discovered

in Convolvulus spp. from Middle Asia, e.g. convolamine

(3a-veratroyloxytropane; Orechoﬀ and Konowalowa,

1934) or phyllalbine (3a-vanilloyloxytropane, 65; Shar-

ova et al., 1980). 65 could be detected also in the genus

Merremia (M. guerichii, M. kentrocaulos), but its verat-

royl congener has not been found in any of its species.

Instead, 3a-(4-hydroxybenzoyloxy)tropane (62) turned

out to be a constituent of M. dissecta, M. guerichii,

and M. kentrocaulos. Furthermore, its 4-O-methyl con-

gener datumetine (1) and also the corresponding N-

demethylated (nor) derivative merresectine A (2) have

been present together in M. dissecta as well as in M.

quinquefolia.

The merresectines C–E (4, 75, 72) have been detected

in 3 species (75), 6 spp. (72), and 8 spp. (4), respectively.

The b-^D-glucoside B (3) is not detectable by GC–MS di-

rectly and therefore not included in the analysis; how-

ever, it is detectable as its aglycone C (4) after

decomposition in the instrument. Thus, D (75) as well

as E (72) may also be present as glycosides in the plants,

as has been proven by isolation from M. guerichii and C.

sabatius, respectively. All these compounds diﬀer from

the simple aromatic 3a-acyloxytropanes mentioned

The crude alkaloid fraction of the roots of M. gueri-

chii showed two main spots giving a positive reaction

with DragendorﬀÕs reagent on TLC. Preparative HPLC

1

PC Model 4.0, Serena Software.

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

1451

above in that their aromatic acyl moieties are prenylated

in one (72, 75) or even both (4) ortho positions to the hy-

droxyl at C-4⁰. Finally, merresectine F (79) should be

mentioned; it is unequivocally a dehydro derivative of

merresectine C (4), i.e. 4 minus two hydrogen atoms.

Since there is no other plausible possibility 79 should

long-range correlations to a quaternary oleﬁnic carbon

at d 145.4, another oleﬁnic carbon at d 123.5 corre-

sponding to a singlet at d 5.42 in the proton NMR spec-

trum, and an oxygen bearing carbon at d 67.5. The ¹³C

NMR spectrum showed four further carbon atoms: two

methylene carbons at d 31.2 and 28.9 which had to be in

neighbourhood due to the H-H-COSY, one quaternary

oxygen bearing carbon at d 72.1, and one carbonyl

carbon at d 175.3 which had to be esteriﬁed to the tro-

pan-3-ol residue. These structural features very much

resembled those of consabatine (68), the molecular mass

of which is two units lower and which bears a carbonyl

function in position 4⁰instead of the secondary hydroxy

group in 4⁰-dihydroconsabatine (70).

Since 68 and 70 are accompanied by the prenylated

merresectines, especially E (72) in C. sabatius as well

as in M. quinata it may be concluded that 72 has to be

the precursor of 68 and 70 rather than vice versa. Other-

wise it would not be plausible that merresectines are

present in seven additional Merremia spp. which did

not even show traces of 68 and/or 70.

be

3a-[4-hydroxy-3-(3-methyl-buta-1,3-dienyl)-5-(3-

methyl-but-2-enyl)benzoyloxy]tropane. 79 is present in

all eight species containing 4 (Table 1). Again the 3b epi-

mer of 79 (80) could be detected in four of the six species

containing 4 as well as 83 and in addition even in one

species, M. aegyptia in which 4 has been found but

not 83. Thus, merresectine G (81) lacking four hydrogen

atoms, only present in M. dissecta, should be a congener

of 4 with an additional double bond each in both prenyl

residues. Furthermore, merresectine H (69) represents

merresectine E (72) minus 2 H.

3a-(4-Hydroxybenzoyloxy)tropane (62) might be a

precursor of a biogenetic sequence merresectine E

(72) ! C (4) ! B (3) and on the other hand of a se-

quence phyllalbine (65) ! merresectine D (75), respec-

tively. The co-presence of 3a- as well as 3b-epimers of

C (3a: 4; 3b: 83) in six species, of D (3a: 75; 3b: 77),

and of 3-(4-hydroxybenzoyloxy)tropane (3a: 62; 3b:

63) in two species each may indicate that the corre-

sponding acyl moieties have been synthesized ﬁrst before

they are transmitted to both 3-hydroxytropane epimers

in a last step of the alkaloid biosynthesis. Anyhow,

datumetine (1) and its N-demethylated congener merre-

sectine A (2) both only formed in the same two species

lead – apparently not lying on the principal pathway –

to a dead end from the biogenetical point of view due

to their 4-methoxy substituent.

2.1.4. Occurrence of 3a-benzoyloxytropane (56) and 3a-

nicotinoyloxytropane (57) detected by GC–MS

Two other, rare tropane alkaloids, 3a-benzoyloxytro-

pane (56) and 3a-nicotinoyloxytropane (57), were

discovered previously in the brassicaceous genus Coch-

learia and structurally conﬁrmed by synthesis (Bach-

mann, personal communication). Both have been

detected unequivocally in M. vitifolia though in very

low concentrations. Nicotinic acid is an unusual aro-

matic acyl component for tropanols because all known

aromatic or arylaliphatic acids esteriﬁed with this skele-

ton are biogenetic derivatives of phenylalanine.

2.1.3. Occurrence of consabatine (68) and 4⁰-dihydro-

consabatine (70) detected by GC–MS; isolation and

structure elucidation of 70

2.1.5. Occurrence of aliphatic 3a-acyloxytropanes

detected by GC–MS

Consabatine (68), an ester of tropan-3a-ol with 1-hy-

droxy-3-(3-methyl-but-2-enyl)-cyclohex-2-en-4-one car-

boxylic acid (2-deoxyconsiculic acid) discovered in

Convolvulus sabatius Viv. ssp. mauritanicus (Jenett-Siems

et al., 1998) turned out to be also a metabolite of M. qui-

nata. This is also true for its 4⁰-dihydro congener (70),

3a-[1,4-dihydroxy-3-(3-methyl-but-2-enyl)-cyclohex-2-

enecarboxy]tropane which has been structurally eluci-

dated based on the isolation from the roots of Convolvu-

lus sabatius Viv. ssp. sabatius. 4⁰-Dihydroconsabatine

(70) again showed characteristic fragments for a tro-

pan-3-ol ester in the EIMS. Its molecular formula was

deduced as C₂₀H₃₁NO₄from the molecular ion peak

Esters of tropan-3a-ol (tropine, 16) with aliphatic

acids seem to be rather rare in the genus: 3a-acetoxy-

tropane (20) could be detected in three Merremia spp.

(M. dissecta, M. kentrocaulos, M. vitifolia); further ali-

phatic 3a-acyloxytropanes are conﬁned to M. guerichii

and M. vitifolia, respectively. Thus, such aliphatic es-

ters have been detected only in altogether four out

of 18 species. In addition a few corresponding N-

demethylated (nor) derivatives (25, 34, 39) occur in

M. guerichii.

2.1.6. Occurrence of 6-substituted tropanes detected by

GC–MS

1

at m/z 349 in the HRMS. The H NMR spectrum also

Remarkably only three species, M. aegyptia, M. dis-

secta, and M. guerichii have shown the additional

occurrence of 6-substituted tropanes. 6b-Hydroxytro-

pane-3-one (23) has been detected in every three species

suggesting that a hydroxylation at C-6 might take place

already at an early stage of the biosynthesis. Further-

displayed typical tropan-3a-ol resonances. The acyl res-

idue was characterized by a prenyl moiety as in the mer-

resectines but in contrast to these, no aromatic proton

resonances were observed. In the HMBC spectrum,

the methylene protons of the prenyl moiety showed

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K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

more, in the samples of M. dissecta 6-acetoxytropan-3-

ol (32) and 6-(methoxybenzoyloxy)tropan-3-ol (67)

have been identiﬁed. However, M. guerichii turned

out to possess an even more complex pattern concern-

ing 6-substituted tropanes: 6-acyloxytropan-3-ols (41,

50, 52, 53, 55), 6-acyloxynortropan-3-ols (40, 48), 6-hy-

droxy-3-acyloxytropanes (49, 54), and one 3,6-diacyl-

oxytropane (59). The stereochemistry at C-6 and also

at C-3 could not be determined by GC–MS without

corresponding references (Table 1). However, there

are two facts supporting at least for some compounds

6b-conﬁguration: (1) The co-occurrence of the unequiv-

ocal 6b-hydroxytropan-3-one (23); (2) the isolation and

structure elucidation of 3a,6b-di-(4-methoxybenzoyl-

oxy)tropane (merredissine, 5) from M. dissecta leading

to the assumption that the monoacylated congener (67)

mentioned above might be its precursor, i.e. 67 should

be 6b-(4-methoxybenzoyloxy)tropan-3a-ol. Moreover,

this is additionally supported by the fact that the tro-

pan-3a-ol derivative datumetine (1), the alternative po-

tential precursor of 5 is also a component of the

alkaloid pattern of this species. However, 5 and 67

are unique in the genus. Whether or not the 6-substi-

tuted tropanes of M. guerichii are also – in total or

in part – 3a,6b-conﬁgurated remains to be elucidated

though 23 is also present in this species, which could

be in favour of 6b-conﬁguration. Moreover, conspicu-

ously neither any aromatic 6-acyloxy derivative nor

any 6-hydroxy derivative of an aromatic 3-acyloxytro-

pane and nortropane, respectively, was detected though

M. guerichii is a rich source of, e.g. merresectines. Nev-

ertheless, this might be at least in part due to method-

ological problems, since detection of authentic

merredissine (5) was not possible with our GC–MS sys-

tem. This is the ﬁrst report on 6-substituted tropanes in

the Convolvulaceae.

species these metabolites are accompanied by their

corresponding 3a-isomers. Five 3b-acyloxytropanes

bearing aromatic acyl residues (63, 66, 77, 80, 83)

turned out to be constituents of M. guerichii and M.

kentrocaulos, respectively. The 3b-epimer 83 of merre-

sectine C (4) has also been present in M. cissoides, M.

dissecta, M. quinquefolia, and M. vitifolia, whereas the

3b-epimer 80 of merresectine F (79) is also a constit-

uent of M. aegyptia, M. cissoides, and M. dissecta.

All these aromatic 3b-acyloxytropanes again are

accompanied by their 3a-congeners (see also Section

2.1.2). 3b-Vanilloyloxytropane, named concneorine

(66) has been originally isolated from the epigeal veg-

etative parts of the Mediterranean silver bush, Convol-

vulus cneorum L. (Mann, 1997). The structure

elucidation by spectral data is documented now for

the ﬁrst time. The compound displayed a molecular

ion peak at m/z 291 in the EIMS spectrum corre-

sponding to a molecular formula of C₁₆H₂₁NO₄

(HRMS) and thus should be an isomer of phyllalbine

(=3a-vanilloyloxytropane, 65). Indeed, the proton

NMR spectrum showed characteristic signals for a

vanilloyl residue and resonances of a tropanol moiety

with the typical multiplet of H-3a at d 5.24. Thus,

concneorine is the 3b-isomer of phyllalbine. This is

the ﬁrst report on 3b-acyloxytropanes in the Convol-

vulaceae.

2.1.8. Occurrence of methylpseudoecgonine (30) detected

by GC–MS

Methylpseudoecgonine (2a-methoxycarbonyltropan-

3b-ol, 30) has been discovered in Datura stramonium

(Solanaceae) by comparison of all four synthesized

isomers (Bachmann and Witte, personal communica-

tion). Interestingly, it has been detected also in the genus

Merremia, but only in M. aegyptia and M. gemella,

respectively. This is the ﬁrst report on 30 in the

Convolvulaceae.

2.1.7. Occurrence of 3b-acyloxytropanes detected by GC–

MS; isolation and structure elucidation of concneorine

(66)

2.2. Pyrrolidine alkaloids including nicotine

The broad occurrence and distribution of calyste-

gines (polyhydroxynortropan-3b-ols) as well as their

lipophilic precursors tropan-3b-ol (pseudotropine, 17)

and nortropan-3b-ol (15), respectively, in the family

Convolvulaceae in general and also within the genus

Merremia has been documented recently (Schimming

et al., 2005). In the present study 17 could be detected

in 10 species. However, the occurrence of acylated

derivatives is conﬁned to altogether seven species

suggesting that not all Merremia spp. which are syn-

thesizers of 3a-acyloxytropanes seem to possess pseu-

dotropine acyl transferase activities (Robins et al.,

1991, 1994, Rabot et al., 1995). Only four aliphatic

3b-esters have been detected: the acetyl derivative 22

in M. dissecta and the n-butyryl derivative 26 as well

as its congeners 36 and 47 in M. guerichii. In both

2.2.1. Structure elucidation of two N-

methylpyrrolidinylhygrines (7, 8) by synthesis

GC–MS analysis revealed the occurrence of a large

number of pyrrolidine and tropane alkaloids. The de-

tected N-methylpyrrolidinylhygrines, isomers of cus-

cohygrine (43), were, like most other aliphatic tropane

esters, previously known from the Solanaceae (Witte

et al., 1987), where their structures were established

on the basis of MS-data alone. They were proposed

to be isomeric 2⁰,4-N-methylpyrrolidinylhygrines. We

now synthesized both compounds according to a meth-

od proposed by Galinovsky et al. (1951) for the prepa-

ration of cuscohygrine and elucidated their structures

by NMR spectrometry. The ¹³C NMR was interpreted

in analogy to that of 43. For compound 7 C-2/C-2⁰

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

1453

Table 1

Pyrrolidine and tropane alkaloids in Merremia species detected by GC–MS (relative abundance in %); for mass fragmentation patterns see Table 3

Alkaloid

RI

M⁺

Pyrrolidine alkaloids

Hygrine (9)

1060 141 t/-

1075 143 -/-

1088 143 -/-

1310 162 t/t

1442 197 -/-

t/-

-/-

t/t

-/-

t/-

-/-

t/t

t/-

60/-

-/-

t

7/50

-/-

13/1

-/-

t

-

t

8/-

-/-

t/t

t

-

t

9/5

-/-

t/t

t/-

-/-

-/t

t/-

2/-

-/-

-/t

t/-

6/25

-/-

2

-

10/1

-/-

5/-

5/t

8/t

t/t

10/-

-/-

Hygroline A (10)

Hygroline B (11)

Nicotine (21)

-

-/-

-

-/-

t/t

-

-/-

8

-

-/-

t/-

5-(2-Oxopropyl)-

-/-

5/5

1/1

-/-

-

-/-

1/t

-/-

t/-

hygrine (27)

5-(2-Hydroxypropyl)- 1452 199 -/-

hygrine (28)

-/-

t/-

-/-

5/-

4/-

t

-

t/5

t/-

-

5/-

20/-

t/-

-

3/t

-/-

2/-

10/t

t/-

1/t

-

-/-

2/-

3/-

-/-

1/-

4/-

3/-

2/-

5-(2-Hydroxypropyl)- 1478 201 -/-

hygroline (31)

t/-

1/-

35/80 3/-

2’,4-N-Methyl-

pyrrolidinylhygrine (8)

1567 224 -/-

1577 224 -/-

5/1

10/3

55/20

23/60

40/-

5/t

-/-

2’,3-N-Methyl-

t/-

t/?

pyrrolidinyhygrine (7)

Cuscohygrine (43)

Phygrine (58)

1650 224 -/-

2043 280 -/-

-/-

-/-^b

-/-

25/-

-/-

-

65/15 2/10

-

48/-

-/-

t

-

3/t

-/-

-/?

-/-

t/-

75/?

2/-

50/60

3/1

t

70/95 5/-

70/-

1/-

-/-

-/3

-/-

-

-/-

N-Methylpyrrolidinyl- 2165 307 -/-

cuscohygrine A (60)

-/-

1/-

-

1/-

N-Methylpyrrolidinyl- 2173 307 -/-

cuscohygrine B (61)

-/-

-

-/-

-

-/-

-

-/-

t/-

-/-

1/-

-

-/-

1/-

Simple tropanes and

aliphatic 3-

acyloxytropanes

Nortropan-3-one (12)

Cyclotropine^c(13)

Tropan-3-one (14)

1133 125 -/-

1137 139 -/-

1150 139 -/-

1160 127 -/-

1167 141 -/-

1187 141 -/-

1292 169 -/-

-/-

t/-

-/-

1/t

-/-

-

t

-/-

t/t

-/-

t

-

t

-/-

-

t

t/8

-/-

t/1

-/-

1/-

-/-

2/-

1/-

t/-

1/t

-/-

t/-

3/t

t/-

-/-

t/1

-/-

t/-

-/-

-

-/-

t/-

-/-

1/-

-/-

1/t

1/-

-/-

t/-

-/-

1/-

-/-

t/-

-/-

t/-

1/-

-/-

3/15

-/-

-

t

40/-

5/-

-

t

-

2/-

12/-

-/-

Nort

-ol (15)

-ol (16)

-ol (17)

-Acetoxynortropane

-/-

30/-

5/-

-/-

t

t/-

-

-/-

-

t/-

-/-

(18)

1296 169 -/-

1308 183 -/-

1315 183 -/-

1405 197 -/-

1435 197 -/-

1440 197 -/-

1455 211 -/-

-/-

-

-/-

1/1

-/-

-

-/-

-

-/-

t/-

-/-

1/-

-/-

t/-

-/-

-/t

-/-

t/-

-/-

t/-

3/t

-/-

t/-

2/t

-

-/-

t/-

-/-

-Acetoxynortropane

(19)

-Acetoxytropane

(20)

-Acetoxytropane

(22)

-Propionyloxy-

tropane (24)

-n-Butyroxy-

nortropane (25)

-n-Butyroxy-

nortropane (26)

-Isobutyroxytropane

(29)

Methylpseudoecgonine 1473 199 -/-

(30)

3-(Hydroxybutyroxy)- 1524 227 -/-

tropane (33)

3-(2-Methylbutyroxy)- 1532 211 -/-

nortropane (34)

1547 225 -/-

-(2-Methylbutyr-

oxy)tropane (35)

(continued on next page)

1454

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

Table 1 (continued)

Alkaloid

RI

1552 225 -/-

M⁺

-/-

-

-/-

-

-/-

-

-/-

1/t

t/-

-

-/-

-(2-Methylbutyr-

oxy)tropane (36)

3-(Hydroxypentanoyl- 1603 227 -/-

oxy)nortropane (37)

3-(Hydroxypentanoyl- 1616 241 -/-

oxy)tropane (38)

-/-

-

-/-

-

-/-

-

-/-

t/-

-/t

t/-

-

-/-

1630 209 -/-

1646 224 -/-

1669 241 -/-

-

-Tigloyloxy-

nortropane (39)

-Tigloyloxytropane

(42)

3-(Hydroxyisovaler-

-/- -/-

oxy)tropane (44)

3-(Hydroxybutyroxy)- 1672 227 -/-

tropane (45)

-

-/-

1698 241 -/-

1705 241 -/-

-(Hydroxy-2-

methyl-

butyroxy)tropane (46)

-(Hydroxy-2-methyl-

butyroxy)tropane (47)

-/-

-

-/-

-

-/-

-

-/-

t/-

-/-

t/-

-

-/-

3-Hexanoyloxytropane 1755 255 -/-

(51)

6-Substituted

tropanes

1325 155 -/-

1500 199 -/-

-/-

-

-/-

-

-/-

-

1/t

-/-

5/-

t/-

-/-

t/1

2/-

t/-

-

-/-

6 -Hydroxytropan-3-

one (23)

6-Acetoxytropan-3-ol

(32)

6-Butyroxynortropan- 1632 213 -/-

3-ol (40)

6-Butyroxytropan-3-ol 1634 227 -/-

(41)

6-Pentanoyloxy-

nortropan-3-ol (48)

1719 227 -/-

1728 241 -/-

6-Hydroxy-3-(2-

methylbutyroxy)-

tropane (49)

6-(2-Methylbutyroxy)- 1730 241 -/-

tropan-3-ol (50)

-/-

-

-/-

-

-/-

-

-/-

8/-

t/-

-

-/-

6-(2-Hydroxy-2-

methylbutyroxy)-

tropan-3-ol (52)

1809 257 -/-

1811 257 -/-

1823 239 -/-

6-(2-Hydroxy-2-

methylbutyroxy)-

tropan-3-ol (53)

-/-

-

-/-

-

-/-

-

-/-

t/-

-

-/-

6-Hydroxy-3-

-/-

-

-/-

-

-/-

-

-/-

t/-

-

-/-

tigloyloxytropane (54)

6-Tigloyloxytropan-3- 1835 239 -/-

ol (55)

6-(Hydroxybutyroxy)- 2150 327 -/-

3-pentanoyloxytropane

(59)

6-(Methoxybenzoyl-

oxy)tropan-3-ol (67)

Aromatic 3-acyl-

oxytropanes and

derivatives

2412 291 -/-

-/-

-

-/-

-

-/-

-

-/-

t/-

-/-

-

-/-

1907 245 -/-

1953 246 -/-

-/-

-

-/-

-

-/-

-

-/-

t/-

-/-

-

-/-

-Benzoyloxytropane

(56)

-Nicotinoyloxy-

tropane (57)

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

1455

Table 1 (continued)

Alkaloid

RI

M⁺

Merresectine A (2)

Datumetine (1)

2195 261 -/-

-/-

-

-/-

-

-/-

-

-/-

1/-

-/-

-

-/-

t/-

-/-

2207 275 -/-

2275 261 -/-

-/-

-

-/-

-

-/-

-

-/-

1/-

-/-

t/-

-

-/-

t/-

-/-

t/-

-(4-

Hydroxybenzoyl-

oxy)tropane (62)

2282 261 -/-

2290 247 -/-

-/-

-

-/-

-

-/-

-

-/-

t/-

-/-

-/t

-/-

-

-/-

t/-

-/-

-(4-

Hydroxybenzoyl-

oxy)tropane (63)

3-(Hydroxybenzoyl-

-

oxy)nortropane (64)

Phyllalbine (65)

2340 291 -/-

2365 291 -/-

2482 347 -/-

2522 327 -/-

-/-

-

-/-

-

-/-

-

-/-

1/-

t/-

-/-

t/-

-/-

-

-/-

t/-

-/-

Concneorine (66)

Consabatine (68)

Merresectine H (69)

-

10 -/-

-/- t/-

10 -/-

4'-Dihydroconsabatine 2540 349 -/-

(70)

3-Acyloxytropane (71)

2598

?

-/-

-

-/-

-

-/-

-

t/-

-/-

-

-/-

1/-

-/-

t/-

-/-

t/-

-/-

Merresectine E (72)

2685 329 -/-

2737 353 -/-

2738 317 -/-

-

-/-

1/-

5/1

5

-

3-Acyloxytropane (73)

-

-/-

t/-

-/-

3β-Feruloyloxytropane

(74)

t/-

-

Merresectine D (75)

3-Acyloxytropane (76)

3β-Merresectine D (77)

3-Acyloxytropane (78)

Merresectine F (79)

2785 359 -/-

-/-

-

-/-

-

-/-

-

-/-

t/-

-/-

t/-

1/-

-/-

t/-

-/-

t/-

-/-

2/-

1/-

t/-

-/-

t/-

-/-

t/-

-/-

1/-

t/-

-/-

8/1

-/-

t/-

t

-/-

t/-

-/-

t/-

-/-

t/-

-/-

t/-

-/-

t/-

-/-

t/-

-/-

2790

2813 359 -/-

2890 -/-

?

-/-

-

t

?

-/-

t/t

2895 395 -/-

2955 395 -/-

2983 393 -/-

3022 379 -/-

3042 397 -/-

3070 397 -/-

3080 469 -/-

3090 397 -/-

3095 411 -/-

3215 487 -/-

3β-Merresectine F (80)

Merresectine G (81)

3-Acyloxytropane (82)

Merresectine C (4)

t/-

-

-/-

5/5

1/-

-/-

60 8/-

55/- 5/-

15/- t/-

3β-Merresectine C (83)

3-Acyloxytropane (84)

3-Acyloxytropane (85)

3-Acyloxytropane (86)

3-Acyloxytropane (87)

-

t/-

-/-

t/-

-/-

RI = retention index; t = traces; – = not detected; symbol before the oblique: roots; symbol behind the oblique: epigeal vegetative parts. ^aEpigeal

c

vegetative parts. ^bDetected by TLC. Cyclotropine might be an artefact due to GC–MS analysis.

were located at d_C65.0 and 66.0, whereas C-5/C-5⁰

exhibited two signals at 56.6 and 57.4. C-6 appeared

as a triplet at 47.3, the N-methyl groups and C-8 as

quartets at 40.5, 40.6 and 31.2, respectively. In contrast

to cuscohygrine there are only three signals above

30.0 ppm. Those at 22.6 and 23.6 were assigned to C-

4/C-4⁰and the one at 26.3 to C-3⁰. Compared to 43

there was an additional doublet at 45.7 corresponding

to C-3, thus proposing a linkage between C-2⁰and C-

3. The other isomer (8) exhibited a very similar ¹³C

NMR. The main diﬀerence was that there were two

tripletts at 26.6 and 27.0 corresponding to C-3/C-3⁰

but only one triplets at 23.0 which was assigned to C-

4⁰. In this case the linkage has to be between C-4

(45.0) and C-2⁰. The absolute conﬁgurations remain

to be determined. In GC–MS 2⁰,3-N-methylpyrro-

lidinylhygrine eluted before its 2⁰,4-isomer.

2.2.2. Occurence of pyrrolidine alkaloids including

nicotine (21) detected by GC–MS

Tropane alkaloids in solanaceous and convolvula-

ceous species are always accompanied by simple pyrrol-

idine alkaloids like hygrine (9) or cuscohygrine (43).

This is not surprising since both groups of alkaloids

share basal parts of the biogenetic pathway (Huang

et al., 1996 and literature therein). 9 has been detected

in all 18 Merremia species of the present study (Table

1), followed by nicotine (21; 13 spp.), 43 (12 spp.), and

1456

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

two N-methylpyrrolidinylhygrines (7, 8) as well as

5-(2-hydroxypropyl)hygroline (31) (9 spp. each). More-

over, seven further structurally known pyrrolidines

(10, 11, 27, 28, 58, 60, 61) have been found as metabo-

lites of 1–8 species, respectively. Phygrine (58), an alka-

loid discovered in Physalis spp. (Solanaceae) and

apparently not occurring in other solanaceous genera

(Basey et al., 1992) turned out to be also a constituent

of M. guerichii, M. hederacea, and M. kentrocaulos.

Two species (M. medium, M. tridentata) have shown

only hygrine (9) and nicotine (21) out of altogether 13

pyrrolidines, M. pterygocaulos only 9 and hygroline B

(11), respectively. M. kentrocaulos (11 pyrrolidines)

and M. guerichii (10) apparently comprise the most

complex pattern. However, it should be taken into ac-

count in general that especially 43 could lead at least

in part to an artiﬁcial formation of 9 due to degradation

by the GC–MS procedure (Moore et al., 1995). Thus, we

could detect 43 by TLC control but not in the corre-

sponding GC–MS proﬁle in the samples of M. aurea.

This may explain why only 12 out of 18 species have

been found 43-positive. Similarly, 58 turned out to be

rather unstable (degradation already by air) yielding at

least in part 27 and 43, respectively, as degradation

products (Basey et al., 1992).

Anyhow, cuscohygrine (43) has been the principal

alkaloid of the common pathway with tropanes in six

species, especially in the samples from the roots.

Nicotine (21) was dominating in M. peltata, the propyl-

hygrines 27 and 28 in M. aurea, the N-meth-

ylpyrrolidinylhygrines 7 and 8 in M. hederacea, hygrine

(9) in M. tuberosa, and 5-(2-hydroxypropyl)hygroline

(31) in M. aegyptia. Tropanes as major metabolites have

only been characterized for three species: the simple alka-

loids tropan-3-one (14) and tropan-3a-ol (16) in M. dis-

secta as well as merresectine C (4) in the epigeal

vegetative parts of M. quinata and the roots of M.

quinquefolia.

2.3. Chemotaxonomic conclusions

The results of this study of altogether 18 Merremia

spp. led to three distinguishable groups of species from

the phytochemical point of view: (1) species synthesizing

only two simple pyrrolidines, i.e. hygrine (9) and nicotine

(21) but neither any other pyrrolidine nor any tropane

Table 2

Merremia species of this study listed according to the occurrence of tropane alkaloids and assignment to sections; for details see Table 1

Occurrence of metabolites in species

of the genus Merremia

Austin (1998a,b)

OÕDonell (1941)

Van Ooststroom

a

(1939,1953)

Origin

No tropanes

M. medium (L.) Hallier f.

M. tridentata (L.) Hallier f.

Transfered to Xenostegia^b

n.i.^c

Halliera

Streptandra^d

Streptandra

South Madagascar

Phuket/Thailand

Simple tropanes like 12–17

M. aurea (Kell.) OÕ Donell

M. tuberosa (L.) Rendle

Tuberosa allies

Merremia

n.i.

Schizips

n.i.

Streptandra

Eu-Merremia

Xanthips^d

Baja California/Mex.

Gran Canaria

Java/Indonesia

M. emarginata (Burm. f.) Hall. f.

M. gemella (Burm. f.) Hallier f.^e

M. hederacea (Burm. f.) Hallier f.

M. pterygocaulos (Steud. ex Choisy) Hallier f.

M. umbellata (L.) Hallier f.

n.i.

Java/Indonesia

Eu-Merremia

n.i.

Queensld/Australia

East Madagascar

Guayaquil/Ecuador

Java/Indonesia

n.i.

Xanthips

Hailale

M. peltata (L.) Merr.

n.i.

Simple tropanes like 12–17 plus merresectines

M. aegyptia (L.) Urban

M. dissecta (Jacq.) Hallier f.^f,g

M. vitifolia (Burm. f.) Hallier f.^g

M. guerichii A. Meeuse^f,g

Vitifolia allies

n.i.

Schizips

n.i.

Streptandra

Streptandra^d

Streptandra

n.i.; nested in

Operculina^h

West Madagascar

Zanzibar/Tanzania

Java/Indonesia

n.i.

Brandberg/Namibia

North.Terr./Australia

West Madagascar

Guayaquil/Ecuador

Southeast Zimbabwe

M. quinata (R.Br.) Ooststr.

n.i.

M. cissoides (Lam.) Hallier f.^g

M. quinquefolia (L.) Hallier f.^g

M. kentrocaulos (C.B.Clarke) Rendle^g

Cissoides

n.i.

Cissoides

n.i.

a

Based on Hallier (1893).

b

Novel genus: X. medium D.F.Austin & G.W.Staples; X. tridentata D.F.Austin & G.W.Staples.

n.i. = not included in the corresponding classiﬁcation of the genus.

Not included by van Ooststroom but classiﬁed by Hallier (1893).

ssp. gemella.

c

d

e

f

In addition 6-acyloxytropanes.

In addition 3b -acyloxytropanes.

By Hallier (1894) as Operculina kentrocaulos (Steud.) Hallier.

g

h

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

1457

alkaloid; (2) species synthesizing up to 11 pyrrolidines

and unacylated tropanes like 12–17; (3) species synthesiz-

ing diﬀerent pyrrolidines, unacylated tropanes, and in

addition aromatic 3-acyloxytropanes, particularly mer-

resectines (Table 1). Especially the lack or presence of

certain tropane alkaloids in general turned out to be of

chemotaxonomic value. The two species segregated from

the genus and transferred to a novel one, Xenostegia

(Austin and Staples, 1980) are unique in this study in

lacking tropanes, thus supporting this transfer chemo-

taxonomically. Furthermore, there are four taxa with

species synthesizing simple, unacylated tropanes like

12–17: the sections Eu-Merremia van Oostroom for

which Austin claims the name Merremia, Xanthips (Gri-

seb.) Hallier f., and Hailale Hallier f. as well as the Tube-

rosa allies (Table 2). From the chemotaxonomic point of

view it is obvious that each species included in this study

and assigned to one of these four taxa is capable in pro-

ducing simple tropanes. Therefore it may be assumed

that such metabolites are a common trait in the genus

since this is also true for the remaining taxa to be dis-

cussed just below. However, it has to be taken into ac-

count that the very small section Wavula van

Ooststroom (2 spp.) is not represented in this study.

The simple tropanes 12–17 can be considered as

plesiomorphic characters since they are also frequent

metabolites in the sister family Solanaceae (e.g. Hart-

mann et al., 1986; Witte et al., 1987). In contrast, the

merresectines B–H (3, 4, 75, 72, 79, 69), their deriva-

tives 68/70, and the 3b-epimers 83, 77, 80 represent

apomorphic characters of certain Convolvulaceae.

Such apomorphic characters, especially the merresec-

tines are metabolites in members (a) of the section

Cissoides (House) OÕDonell, (b) of the Vitifolia allies

(Austin, 1998a), (c) of two species originally assigned

to section Streptandra by Hallier (M. guerichii) and

van Ooststroom (M. quinata) like many other species

but in contrast to the latter have never been classiﬁed

elsewhere and ﬁnally (d) M. kentrocaulos which has

been nested within the genus Operculina again by Hal-

lier but has never been assigned to a certain section of

Merremia. Thus, the segregation of the section Cisso-

ides OÕDonell from the section Streptandra Hallier is

supported by the phytochemical results. Furthermore,

this is true for the segregation of another group from

the section Schizips OÕDonell, Vitifolia allies by Aus-

tin. In this connection it should be remembered that

the three Vitifolia allies of this study form also a mod-

erately supported subclade in the molecular cladistic

analysis.

two species (M. dissecta, M. guerichii). In addition

previous results on the occurrence and structure eluci-

dation of cyanogenic glycosides in two of the three

species of Vitifolia allies, M. dissecta (Nahrstedt

et al., 1989, 1990) and M. vitifolia (Jenett-Siems,

1996) are also in favour of the segregation of Vitifolia

allies and Tuberosa allies from Schizips. Cyanogenic

glycosides are extremely rare in the Convolvulaceae;

there are no other reports on structurally elucidated

compounds of this type in the family.

Both species of the section Cissoides integrated in the

present study (M. cissoides, M. quinquefolia) have al-

ready been characterized previously by the discovery

of lycopsamine type pyrrolizidine alkaloids (Mann,

1997). These metabolites well-known, e.g. from the

Boraginaceae turned out to be unique in the Convolvul-

aceae (>120 species checked). The additional detection

of merresectines and their 3b-epimers in these species

represents an extraordinary support also from the che-

motaxonomic point of view for the segregation of the

section Cissoides OÕDonell from the original section

Streptandra Hallier f.

3. Experimental

3.1. General

¹H NMR (300 MHz, 400 MHz) and ¹³C NMR

(75 MHz, 100 MHz) spectra were recorded on a Bruker

AC 300 or Bruker AC 400. EIMS, HRMS, and FABMS

spectra were obtained using Varian MAT CH₇A, Finni-

gan MAT 711, and Finnigan MAT CH₅DF, respectively.

Optical rotations were measured on a Perkin Elmer 241

MC polarimeter. Preparative HPLC was carried out on

an Eurospher 100 C-18 column (10 lm, 24 · 250 mm)

or a Nucleosil 300 C-18 column (10 lm, 16 · 250 mm).

3.2. Plant material

Seeds were collected in the wild (Table 2). The plants

were grown in the greenhouse and provided roots and

shoots for alkaloid extraction. Voucher specimens are

deposited at the Institut fur Pharmazie (Pharmazeuti-

¨

sche Biologie), Freie Universita¨t Berlin.

3.3. Extraction and isolation

3.3.1. Isolation of compounds 1 and 2

Ground roots of M. dissecta (270 g) were extracted

with aq. MeOH (80%) for 72 h. The alcoholic extract

was concentrated under reduced pressure and dissolved

in aq. tartaric acid (2%). The acidic soln was extracted

successively with CHCl₃and EtOAc, its pH adjusted

to 8 (10% aq. NaHCO₃) and extracted with CH₂Cl₂-

iso-PrOH (3:1). For isolation the alkaloids were

The 3b-merresectines are also present in the section

Cissoides, the Vitifolia allies, and two African species

(M. guerichii, M. kentrocaulos) which are not classiﬁed

since 1893. Thus, they make no further contribution

from the chemotaxonomic point of view. This is also

true for 6-acyloxytropanes which are limited to only

1458

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

subjected to column chromatography on silica gel with

1

s, N-CH₃), 3.17 (2 H, br s, H-1, H-5), 3.87 (3 H, s,

OCH₃), 5.24 (1 H, t, J = 5.0 Hz, H-3), 6.95 (2 H, d,

J = 8.0 Hz, H-3⁰, H-5⁰), 7.99 (2 H, d, J = 8.0 Hz, H-2⁰,

H-6⁰); ¹³C NMR data, see Table 4; EIMS 70 eV, m/z

(rel. int.): 275 [M]⁺(19), 152 (1), 140 (17), 135 (13), 125

(9), 124 (100), 94 (16), 83 (30), 82 (27); HRMS 80 eV,

m/z: 275.1516 (C₁₆H₂₁NO^þ₃^ꢀ, calc. 275.1521), 152.0481

(C₈H₈O^þ₃^ꢀ, calc. 152.0474), 140.1067 (C₈H₁₄NO⁺, calc.

140.1075), 135.0451 (C₈H₇O^þ₂, calc. 135.0446), 124.1123

(C₈H₁₄N⁺, calc. 124.1126), 83.0735 (C₅H₉N^+., calc.

83.0735), 82.0661 (C₅H₈N⁺, calc. 82.0655).

CHCl₃-MeOH-diethylamine mixtures to yield

(23 mg) and 2 (11 mg).

3.3.2. Isolation of compounds 3, 4, and 5

Ground roots of M. dissecta (490 g) were extracted

with 70% aq. MeOH. The concentrated extract was par-

titioned between acid and base in the usual manner to

yield 531 mg of basic material. The crude alkaloids were

chromatographed on silica gel with CHCl₃–MeOH–

NH₃(20%) mixtures to yield 3 (154 mg) and 5

(27 mg). Fractions 33–43 were puriﬁed by preparative

HPLC (Eurospher 100 C-18 column, MeOH–0.5% aq.

H₃PO₄mixtures) to give 4 (6 mg).

3.5. Merresectine A (2)

Yellow solid. ¹H NMR (CDCl₃): d 1.96 (4 H, m,

2 · H-2, 2 · H-4), 2.21 (4 H, m, H₂-6, H₂-7), 2.85 (1

H, br s, –NH), 3.68 (2 H, br s, H-1, H-5), 3.87 (3

H, s, OCH₃), 5.31 (1 H, t, J = 5.2 Hz, H-3), 6.95 (2

H, d, J = 8.0 Hz, H-3⁰, H-5⁰), 7.98 (2 H, d,

J = 8.0 Hz, H-2⁰, H-6⁰); ¹³C NMR data, see Table 4;

EIMS 70 eV, m/z (rel. int.): 261 [M]⁺(11), 220 (9),

152 (40), 135 (21), 126 (5), 110 (100); HRMS 80 eV,

m/z: 261.1368 (C₁₅H₁₉NO^þ₃^ꢀ, calc. 261.1365), 220.0966

(C₁₂H₁₄NO^þ₃, calc. 220.0974), 152.0469 (C₈H₈O^þ₃^ꢀ, calc.

152.0474), 135.0446 (C₈H₇O^þ₂, calc. 135.0446),

126.0909 (C₇H₁₂NO⁺, calc. 126.0919), 110.0974

(C₇H₁₂N⁺, calc. 110.0970).

3.3.3. Isolation of compound 6

Ground roots of M. guerichii (92 g) were extracted

three times with MeOH at room temperature. The alco-

holic extract was concentrated under reduced pressure

and dissolved in aq. tartaric acid (2%). The acidic soln

was extracted successively with CHCl₃and EtOAc, its

pH adjusted to 8 (3% aq. NH₃) and extracted with

CHCl₃. For isolation the crude alkaloids were subjected

to preparative HPLC (Eurospher 100 C-18 column, 0.5

% aq. H₃PO₄–MeOH 60:40 to 30:70 after 30 min) to

yield 6 (1 mg) and again 3 (5 mg).

3.3.4. Isolation of compound 66

Ground epigeal vegetative parts of C. cneorum (500 g)

were moistened with aq. NH₃(17%) and extracted with

CH₂Cl₂at room temperature. The lipophilic extract was

concentrated under reduced pressure and then extracted

ﬁve times with aq. tartaric acid (2%). The acidic soln was

alkalinized with aq. Na₂CO₃to pH 8 and extracted with

CH₂Cl₂. For isolation the crude alkaloids were subjected

to preparative TLC on silica gel (CHCl₃–MeOH–NH₃

(25%) 80:20:1, R_f0.45) to yield 66 (8 mg).

R¹

N

3

O

1'

4'

R³

R²

3.3.5. Isolation of compounds 70 and 72a

Datumetine (1): R¹= CH₃, R²= OCH₃, R³= H

Merresectine A (2): R¹= H, R²= OCH₃, R³= H

Phyllalbine (65): R¹= CH₃, R²= OH, R³= OCH₃

Ground roots and rhizomes of C. sabatius ssp. saba-

tius (400 g) were extracted three times with MeOH at

room temperature. The alcoholic extract was concen-

trated under reduced pressure and dissolved in aq. tar-

taric acid (2%). The acidic soln was extracted

successively with petrol ether, CH₂Cl₂and EtOAc, its

pH adjusted to 8 (3% NH₃) and extracted with CHCl₃/

isopropanol 3:1. For isolation the crude alkaloids were

subjected to preparative HPLC (Nucleosil 300 C-18 col-

umn, 0.5% aq. H₃PO₄–MeOH 60:40 to 30:70 after

30 min) to yield 70 (3 mg) and 72a (3 mg).

3.6. Merresectine B (3)

20

D

1

Oil. ½aꢁ ꢀ10.2 (MeOH; c 0.1); H NMR (acetone-

d₆): d 1.72 (2 H, d, J = 15.0 Hz, H-2a, H-4a), 1.74 (6

H, s, 2 · –CH₃), 1.76 (6 H, s, 2 · –CH₃), 2.08 (4 H, m,

H₂-6, H₂-7), 2.17 (2 H, br dt, J = 15.0, 5.0 Hz, H-2b,

H-4b), 2.26 (3 H, s, N-CH₃), 3.12 (2 H, br s, H-1, H-

5), 3.25–3.50 (4 H, m, glycosidic protons), 3.62 (4 H,

br d, J = 7.4 Hz, 2 · H-1⁰⁰, 2 · H-1⁰⁰⁰), 3.67 (1 H, dd,

J = 12.0, 6.0 Hz, H-6^{000 0}a), 3.78 (1 H, dd, J = 12.0,

3.0 Hz, H-6^{000 0}b), 4.82 (1 H, d, J = 7.4 Hz, H-1^{000 0}),

3.4. Datumetine (1)

Yellow solid. ¹H NMR (CDCl₃): d 1.84 (2 H, d,

J = 14.7 Hz, H-2b, H-4b), 2.08 (4 H, br s, H₂-6, H₂-7),

2.24 (2 H, dt, J = 14.7, 5.0 Hz, H-2a, H-4a), 2.32 (3 H,

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

1459

Table 3

Mass fragmentation patterns of pyrrolidine and tropane alkaloids identiﬁed by GC–MS taken from Table 1^a

Compound

RI

M⁺(%)

m/z (%)

Cyclotropine (13)

1137

1292

1296

1435

1440

1442

1452

1473

1478

1500

1524

1532

1603

1616

1632

1634

1669

1672

1698

1705

1719

1728

1755

1809

1811

1907

1953

2150

2275

2282

2290

2412

2482

2522

2598

2685

2737

2785

2790

2813

2890

2895

2955

2983

3022

3070

3080

3090

3095

3215

139 (98)

169 (5)

197 (5)

197 (2)

197 (1)

199 (2)

199 (19)

201 (1)

199 (25)

227 (11)

211 (2)

227 (2)

241 (10)

213 (7)

227 (17)

241 (12)

227 (10)

241 (7)

241 (8)

227 (5)

241 (6)

255 (5)

257 (15)

257 (10)

245 (10)

246 (15)

327 (6)

261 (18)

261 (13)

247 (1)

291 (8)

347 (3)

327 (6)

?

110 (58), 108 (40), 96 (60), 82 (20), 68 (100)

110 (100), 97 (5), 83 (5), 68 (17)

3a-Acetoxynortropane (18)

3b-Acetoxynortropane (19)

3a-n-Butyroxynortropane (25)

3b-n-Butyroxynortropane (26)

5-(2-Oxopropyl)hygrine (27)

5-(2-Hydroxypropyl)hygrine (28)

Methylpseudoecgonine (30)

5-(2-Hydroxypropyl)hygroline (31)

6-Acetoxytropan-3-ol (32)

110 (100), 80 (30), 68 (29)

110 (100), 80 (32), 68 (28)

140 (82), 124 (3), 96 (17), 82 (100)

142 (40), 140 (100), 82 (88)

182 (12), 168 (10), 155 (9), 96 (70), 82 (100)

142 (100), 84 (30)

140 (9), 113 (100), 96 (53), 94 (28), 82 (18)

140 (5), 124 (100), 96 (15), 94 (20), 82 (23), 59 (30)

110 (100), 80 (24), 68 (23), 57 (10)

3-(Hydroxybutyroxy)tropane (33)

3-(2-Methylbutyroxy)nortropane (34)

3-(Hydroxypentanoyloxy)nortropane (37)

3-(Hydroxypentanoyloxy)tropane (38)

6-Butyroxynortropan-3-ol (40)

6-Butyroxytropan-3-ol (41)

3-(Hydroxyisovaleroxy)tropane (44)

3-(Hydroxybutyroxy)tropane (45)

3a-(Hydroxy-2-methylbutyroxy)-tropane (46)

3b-(Hydroxy-2-methylbutyroxy)-tropane (47)

6-Pentanoyloxynortropan-3-ol (48)

6-Hydroxy-3-(2-methyl-butyroxy)tropane (49)

3-Hexanoyloxytropane (51)

6-(2-Hydroxy-2-methyl-butyroxy)tropan-3-ol (52)

6-(2-Hydroxy-2-methyl-butyroxy)tropan-3-ol (53)

3a-Benzoyloxytropane (56)

3a-Nicotinoyloxytropane (57)

6-(Hydroxybutyroxy)-3-pentanoyloxytropane (59)

3a-(4-Hydroxybenzoyloxy)tropane (62)

3b-(4-Hydroxybenzoyloxy)tropane (63)

3-(Hydroxybenzoyloxy)nortropane (64)

6-(Methoxybenzoyloxy)tropan-3-ol (67)

Consabatine (68)

110 (100), 80 (18), 73 (28), 68 (19)

140 (8), 124 (100), 96 (18), 94 (23), 82 (25), 73 (31)

142 (10), 110 (40), 99 (100), 80 (42), 68 (25)

140 (18), 113 (100), 96 (41), 94 (60), 82 (17)

140 (5), 124 (100), 94 (25), 82 (28), 59 (8)

140 (8), 124 (100), 96 (12), 94 (27), 82 (30), 59 (5)

140 (6), 124 (100), 96 (12), 94 (20), 82 (25), 45 (7)

140 (5), 124 (100), 96 (8), 95 (9), 94 (22), 82 (30), 45 (7)

142 (10), 99 (100), 80 (19)

197 (2), 140 (23), 94 (100), 57 (15)

140 (5), 124 (100), 96 (12), 94 (18), 82 (22)

140 (28), 113 (100), 96 (33), 82 (25), 73 (12)

140 (25), 113 (100), 96 (38), 82 (25), 73 (15)

140 (18), 124 (100), 96 (18), 94 (18), 83 (40), 82 (25)

140 (15), 124 (100), 96 (22), 94 (23), 82 (38)

242 (2), 226 (20), 138 (12), 122 (62), 95 (100), 94 (99), 57 (23)

140 (17), 124 (100), 94 (30), 83 (48), 82 (47)

140 (7), 124 (100), 94 (25), 83 (30), 82 (49)

163 (8), 138 (15), 110 (100), 91 (30), 80 (20)

135 (22), 113 (100), 96 (29)

330 (1), 179 (2), 140 (5), 124 (100), 96 (10), 94 (8)

144 (5), 140 (18), 124 (100), 94 (28), 83 (28), 82 (32)

233 (3), 140 (20), 124 (100), 95 (29), 82 (32)

Merresectine H (69)

3-Acyloxytropane (71)

Merresectine E (72)

3-Acyloxytropane (73)

329 (8)

353 (9)

359 (18)

?

312 (1), 244 (1), 189 (5), 140 (25), 124 (100), 94 (22), 83 (25), 82 (24)

140 (22), 124 (100), 94 (25), 83 (38), 82 (37)

Merresectine D (75)

3-Acyloxytropane (76)

274 (2), 236 (4), 219 (10), 140 (35), 124 (100), 94 (42), 83 (52), 82 (48)

265 (5), 140 (22), 124 (100), 94 (21), 83 (17), 82 (16)

274 (1), 236 (2), 219 (8), 140 (10), 124 (100), 94 (30), 83 (25), 82 (40)

208 (12), 140 (8), 124 (100), 94 (24), 83 (44), 82 (58)

257 (10), 212 (4), 140 (25), 124 (100), 94 (27), 83 (30), 82 (32)

257 (28), 140 (8), 124 (100), 94 (20), 83 (28), 82 (50)

255 (11), 140 (28), 124 (100), 94 (30), 83 (35), 82 (38)

140 (8), 124 (100), 94 (36), 83 (38), 82 (65)

3b-Merresectine D (77)

3-Acyloxytropane (78)

Merresectine F (79)

359 (18)

?

395 (10)

395 (12)

393 (7)

379 (12)

397 (18)

469 (3)

397 (18)

411 (7)

487 (12)

3b-Merresectine F (80)

Merresectine G (81)

3-Acyloxytropane (82)

3b-Merresectine C (83)

3-Acyloxytropane (84)

312 (1), 257 (8), 140 (10), 124 (100), 94 (30), 83 (30), 82 (42)

329 (3), 319 (22), 140 (40), 124 (100), 94 (19), 83 (34), 82 (24)

257 (4), 140 (8), 124 (100), 94 (22), 83 (22), 82 (24)

140 (28), 124 (100), 94 (26), 83 (30), 82 (26)

3-Acyloxytropane (85)

3-Acyloxytropane (86)

3-Acyloxytropane (87)

429 (10), 140 (35), 124 (100), 94 (30), 83 (34), 82 (25)

a

For mass spectral data of newly isolated compounds see Section 3, for known compounds please refer to Witte et al. (1987), Doerk et al. (1991),

Basey et al. (1992), Christen et al. (1993), and Ionkova et al. (1994).

5.18 (1 H, t, J = 5.0 Hz, H-3), 5.34 (2 H, t, J = 7.5 Hz,

H-2⁰⁰, H-2⁰⁰⁰), 7.71 (2 H, s, H-2⁰, H-6⁰); ¹³C NMR

data, see Table 4; EIMS 70 eV, m/z (rel. int.): 559

[M]⁺(3), 397 (30), 257 (5), 140 (23), 124 (100), 94

(12), 83 (24), 82 (15); (ꢀ)-FABMS m/z: 558 [MꢀH]^ꢀ,

396 [(MꢀH)-162]^ꢀ; HRMS 80 eV, m/z: 559.3147

(C₃₁H₄₅NO₈^þꢀ, calc. 559.3145), 397.2619 (C₂₅H₃₅NO^þ₃^ꢀ,

calc. 397.2617), 257.1543 (C₁₇H₂₁O^þ₂, calc. 257.1542),

140.1075 (C₈H₁₄NO⁺, calc. 140.1075), 124.1125

(C₈H₁₄N⁺, calc. 124.1126).

1460

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

(C₁₇H₂₁O^þ₂, calc. 257.1542), 140.1076 (C₈H₁₄NO⁺, calc.

140.1075), 124.1123 (C₈H₁₄N⁺, calc. 124.1126).

Table 4

¹³C NMR (75 MHz, d = ppm) data of compounds 1–4

Carbon

1 (CDCl₃) 2 (CDCl₃) 3 (DMSO-d₆) 4 (acetone-d₆)

3.8. Merredissine (5)

1

59.8

36.7

53.4

36.6

67.5

36.3

53.4

28.4

59.1

35.4

61.7

37.0

2

20

Oil. ½aꢁ ꢀ8.6 (MeOH; c 0.2); ¹H NMR data, see Ta-

3

67.7

36.7

67.0

35.4

67.4

37.0

D

4

ble 5; ¹³C NMR data, see Table 5; EIMS 70 eV, m/z (rel.

int.): 425 [M]⁺(3), 290 (2), 274 (5), 138 (12), 135 (37),

122 (31), 107 (8), 95 (91), 94 (100); HRMS 80 eV, m/z:

425.1832 (C₂₄H₂₇NO^þ₆^ꢀ, calc. 425.1838), 290.1390

(C₁₆H₂₀NO^þ₄^ꢀ, calc. 290.1392), 274.1442 (C₁₆H₂₀NO^þ₃,

calc. 274.1443), 152.0473 (C₈H₈O^þ₃, calc. 152.0474),

94.0656 (C₆H₈N^þ, calc. 94.0657).

5

59.8

25.8

59.1

25.1

61.7

26.3

6

7

25.8

40.4

25.1

39.8

26.3

40.1

N-CH₃

1⁰

123.3

131.4

113.7

163.3

165.7

55.4

122.9

131.4

113.8

163.4

165.5

55.5

126.0

128.0

132.8

156.4

164.8

122.4

129.4

129.6

158.7

167.2

2⁰/6⁰

3⁰/5⁰

4⁰

7⁰

H₃C

N

4₀⁰₀-O₀₀C₀H₃

1₀₀/1₀₀₀

2₀₀/2₀₀₀

3₀₀/3

4₀₀/4⁰⁰⁰

5 /5⁰⁰⁰

1^{000 0}

2^{000 0}

3^{000 0}

4^{000 0}

5^{000 0}

6^{000 0}

a

27.7

122.2

135.9

25.4

29.1

122.8

134.6

26.0

H₃CO

O

4''

7''

3

1''

O

7'

17.6

104.4

73.8

76.0^a

70.0

76.8^a

61.0

17.9

1'

4'

OCH₃

Merredissine (5)

Assignments interchangeable.

3.9. Merresectine D b-D-glucoside (6)

H₃C

N

Oil. ¹H NMR (acetone-d₆): d 1.71 (3 H, s, H-5⁰⁰), 1.74

(3 H, s, H-4⁰⁰), 1.80 (2 H, br d, J = 15.0 Hz, H-2a, H-4a),

2.15 (4 H, m, H₂-6, H₂-7), 2.29 (2 H, m, H-2b, H-4b),

2.34 (3 H, s, N-CH₃), 3.22 (2 H, m, H-1, H-5), 3.25-

3.50 (4 H, m, H-2⁰⁰⁰-H5⁰⁰⁰), 3.51 (1 H, br d, J = 7.5 Hz,

3

O

1'

4'''

H₃C

4''

CH₃

3'

5'

3'''

3''

Table 5

1

4'

OR

1'''

1''

H (400 MHz, d = ppm, CDCl₃) and ¹³C (100.6 MHz, d = ppm,

CDCl₃) NMR data of 5

CH₃

5'''

CH₃

5''

Carbon

d_H

d_C

60.4

Merresectine B (3): R = β-D-glucose

Merresectine C (4): R = H

1

3.64 br s

1.86 br d (14.7), 2.54 m

5.33 t (5.0)

2

34.5

66.2

3

4

2.10 br d (14.7), 2.54 m

3.58 br s

33.1

66.7

5

3.7. Merresectine C (4)

6

5.87 dd (3.0, 7.5)

2.32 m, 2.84 dd (7.5, 14.2)

2.72 s

79.0

36.1

7

Oil. ¹H NMR (acetone-d₆): d 1.72 (6 H, s, 2 · –CH₃),

1.75 (2 H, d, J = 15.0 Hz, H-2a, H-4a), 1.77 (6 H, s, 2 · –

CH₃), 2.13 (4 H, br s, H₂-6, H₂-7), 2.26 (2 H, br dt,

J = 15.0, 5.0 Hz, H-2b, H-4b), 2.33 (3 H, s, N-CH₃),

3.21 (2 H, br s, H-1, H-5), 3.40 (4 H, d, J = 7.4 Hz,

2 · H-1⁰⁰, 2 · H-1⁰⁰⁰), 5.16 (1 H, t, J = 5.0 Hz, H-3),

5.37 (2 H, br t, J = 7.4 Hz, H-2⁰⁰, H-2⁰⁰⁰), 7.68 (2 H, s,

H-2⁰, H-6⁰);¹³C NMR data, see Table 4; EIMS 70 eV,

m/z (rel. int.): 397 [M]⁺(19), 257 (4), 140 (24), 124

(100), 94 (18), 83 (41), 82 (27); HRMS 80 eV, m/z:

397.2619 (C₂₅H₃₅NO^þ₃^ꢀ, calc. 397.2617), 257.1540

N-CH₃

1⁰

40.1

122.6

131.6

114.0

163.6

166.1

55.5

2⁰/6⁰

3⁰/5⁰

4⁰

8.06 d (8.0)

6.98 d (8.0)

7⁰

4₀⁰₀-OCH₃

1_{00 00}

2 /6

3⁰⁰/5⁰⁰

4⁰⁰

3.87 s

122.4

131.6

113.7

163.6

165.4

55.5

7.98 d (8.2)

6.93 d (8.2)

7⁰⁰

4⁰⁰-OCH₃

3.87 s

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

1461

H-1⁰⁰), 3.64 (1H, dd, J = 3.0, 12.0 Hz, H-6⁰⁰⁰a), 3.77 (1 H,

3.11. 4⁰-Dihydroconsabatine (70)

dd, J = 6.0, 12.0 Hz, H-6⁰⁰⁰b), 3.91 (3 H, s, OCH₃), 5.03

(1 H, d, J = 6.0 Hz, H-1⁰⁰⁰), 5.18 (1 H, t, J = 5.0 Hz, H-

3), 5.33 (1 H, t, J = 7.5 Hz, H-2⁰⁰), 7.48 (1 H, d,

J = 2.0 Hz, H-2⁰), 7.49 (1 H, d, J = 2.0 Hz, H-6⁰); EIMS

70 eV, m/z (rel. int.): 521 [M]⁺(4), 359 (100), 219 (7), 140

(20), 124 (80), 83 (17), 82 (10); (ꢀ)-FABMS m/z: 520

[MꢀH]^ꢀ, 358 [(MꢀH)-162]^ꢀ; HRMS 80 eV, m/z:

521.2621 (C₂₇H₃₉NO₉^þꢀ, calc. 521.2625), 359.2096 (C₂₁-

H₂₉NO^þ₄^ꢀ, calc. 359.2097).

1

Oil. H NMR (CDCl₃): d (ppm) 1.58 (2 H, m, H-2a,

H-4a), 1.61 (3 H, s, H-5⁰⁰), 1.70 (3 H, br s, H-4⁰⁰), 1.82 (1

H, m, H-5⁰a) 1.85–1.95 (6 H, m, H₂-6⁰, H₂-6, H₂-7), 1.97

(1 H, m, H-5⁰b), 2.04 (2 H, m, H-2b, H-4b), 2.20 (3 H, s,

N-CH₃), 2.85 (1 H, dd, J = 16.0, 8.0 Hz, H-1⁰⁰a), 2.91 (1

H, dd, J = 16.0, 8.0 Hz, H-1⁰⁰b), 3.01 (2 H, br s, H-1, H-

5), 4.03 (1 H, br t, J = 6.0 Hz, H-4⁰), 4.90 (1 H, t,

J = 5.0 Hz, H-3), 5.20 (1 H, tdq, J = 8.0, 1.0, 1.0 Hz,

H-2⁰⁰), 5.42 (1 H, br s, H-2⁰); ¹³C NMR (CDCl₃): d

(ppm) 17.8 (q, C-5⁰⁰), 25.6 (t, C-6/C-7), 25.8 (q, C-4⁰⁰),

28.9 (t, C-5⁰), 31.2 (t, C-6⁰), 32.2 (t, C-1⁰⁰), 36.4 (t, C-

2), 36.5 (t, C-4), 40.3 (q, N-CH₃), 59.6 (d, C-1/C-5),

67.5 (d, C-4⁰), 69.8 (d, C-3), 72.1 (s, C-1⁰), 120.8 (d, C-

2⁰⁰), 123.5 (d, C-2⁰), 134.2 (s, C-3⁰⁰), 145.4 (s, C-3⁰),

175.3 (s, C-7⁰); EIMS 70 eV, m/z (rel. int.): 349 [M]⁺

(14), 332 (5), 280 (1), 181 (5), 163 (6), 140 (5), 124

(100), 96 (13), 94 (13), 83 (10), 82 (15); HRMS 80 eV

H₃C

N

3

O

H₃C

R¹

4'

m/z: 349.2252 ½Mꢁ^þðC₂₀H₃₁NO^þꢀ, calc. 349.2253),

OR²

4

CH₃

332.2224 (C₂₀H₃₀NO^þ₃, calc. 332.2226), 124.1126

(C₈H₁₄N^þ, calc. 124.1126).

Merresectine D (75):

R¹= OCH₃, R²= H

Merresectine D β-D-glucoside (6): R¹= OCH₃,

R²=β -D-glucose

H₃C

N

Merresectine E (72):

Merresectine E β-D-glucoside (72a): R¹= H,

R²=β -D-glucose

R¹= H, R²= H

3

O

7'

HO

1'

4'

H

6'

5'

2'

3'

2''

3.10. Concneorine (66)

3''

5''

4''

1''

X

Oil. ¹H NMR (CDCl₃): d (ppm) 1.82 (2 H, m, H-6a, H-

7a), 2.02 (4 H, m, H₂-2, H₂-4), 2.14 (2 H, m, H-6b, H-7b),

2.45 (3 H, s, N-CH₃), 3.39 (2 H, br s, H-1, H-5), 3.94 (3 H,

s, OCH₃), 5.24 (1 H, m, H-3), 6.92 (1 H, d, J = 8.5 Hz, H-

5⁰), 7.50 (1 H, d, J = 2.0 Hz, H-2⁰), 7.61 (1 H, dd, J = 8.5,

2.0 Hz, H-6⁰); ¹³C NMR (CDCl₃): d (ppm) 26.48 (t, C-6/

C-7), 35.90 (t, C-2/C-4), 38.83 (q, N-CH₃), 55.99 (q,

OCH₃), 60.47 (d, C-1/C-5), 67.27 (d, C-3), 111.83 (d, C-

2⁰), 114.41 (d, C-5⁰), 122.30 (s, C-1⁰), 124.16 (d, C-6⁰),

146.57 (s, C-3⁰), 150.63 (s, C-4⁰), 165.95 (s, C-7⁰); EIMS

70 eV, m/z (rel. int.): 291 [M]⁺(32), 168 (3), 151 (10),

140 (9), 124 (100), 94 (19), 82 (44); HRMS 80 eV, m/z:

291.1471 (C₁₆H₂₁NO^þ₄^ꢀ, calc. 291.1471).

Consabatine (68): X = O

4'-Dihydroconsabatine (70):

X = H, OH

3.12. Merresectine E b-D-glucoside (72a)

1

Oil. H NMR (CDCl₃): d (ppm) 1.72 (3 H, s, H-5⁰⁰),

1.76 (3 H, s, H-4⁰⁰), 1.82 (2 H, br d, J = 15.0 Hz, H-2a,

H-4a), 2.07 (4 H, m, H₂-6, H₂-7), 2.20 (2 H, m, H-2b,

H-4b), 2.30 (3 H, s, N-CH₃), 3.16 (2 H, m, H-1, H-5),

3.29 (1 H, dd, J = 7.0, 16.0 Hz, H-1⁰⁰a), 3.42–3.76 (4

H, m, H-2⁰⁰⁰-H5⁰⁰⁰), 3.44 (1 H, m, H-1⁰⁰b), 3.87 (1H, dd,

J = 5.0, 12.0 Hz, H-6⁰⁰⁰a), 3.93 (1 H, dd, J = 3.0,

12.0 Hz, H-6⁰⁰⁰b), 4.98 (1 H, d, J = 7.0 Hz, H-1⁰⁰⁰), 5.08

(1 H, t, J = 5.0 Hz, H-3), 5.16 (1 H, tdq, J = 8.0, 1.0,

1.0 Hz, H-2⁰⁰), 7.03 (1 H, d, J = 8.0 Hz, H-5⁰), 7.82 (1

H, d, J = 1.0 Hz, H-2⁰), 7.84 (1 H, dd, J = 8.0, 1.0 Hz,

H-6⁰); EIMS 70 eV, m/z (rel. int.): 347 (11), 140 (5),

124 (100), 96 (17), 95 (18); (+)-FABMS, m/z: 492

[M+H]⁺.

H₃C

N

O

3

3'

OCH₃

4'

OH

Concneorine (66)

1462

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

3.13. Synthesis of 1

(d, C-2⁰), 209.5 (s, C-7); EIMS 70 eV, m/z (rel. int.):

224 [M]⁺(8), 209 (1), 167 (8), 152 (12), 140 (5), 124

(5), 110 (5), 96 (2), 84 (100); HRMS 80 eV, m/z:

224.1887 (C₁₃H₂₄N₂O, calc. 224.1889).

500 mg of tropan-3a-ol were dissolved in 3 ml pyr-

idine and 2 ml 4-methoxybenzoylchloride in 50 ml

CHCl₃added at 0 ꢁC. After further addition of cat-

alytic amounts of 4-dimethylaminopyridine the mix-

ture was stirred for 24 h at room temp. The

reaction was stopped by the addition of 20 ml ice-

water. After evaporation the residue was taken up

in saturated aq. NaHCO₃and extracted with CHCl₃.

The organic layers were puriﬁed by ﬂash CC on silica

gel (CHCl₃–MeOH–diethylamine 92:4:4) to yield

580 mg of 1.

N

2'

CH₃

4

O

3

8

CH₃

6

2

7

5

N

CH₃

2’,3-N-Methylpyrrolidinylhygrine (7)

3.14. Synthesis of 2

3.17. 2⁰,4-N-Methylpyrrolidinylhygrine (8)

200 mg of tropan-3a-yl-4⁰-methoxybenzoate and

23 mg of K₂CO₃were dissolved in 50 ml toluene. After

addition of 464 mg of 2,2,2-trichlorethylchlorformiate

the mixture was reﬂuxed for 36 h under nitrogen.

The reaction was stopped by addition of icewater

and the aq. layer extracted with CH₂Cl₂. The organic

layers were puriﬁed by ﬂash CC on silica gel (CH₂Cl₂–

MeOH 95:5) to yield 268 mg of N-(2,2,2-trichloreth-

1

Oil. H NMR (CDCl₃): d 2.21 (3 H, s), 2.27 (6 H, s),

1.52–3.12 (m); ¹³C NMR (CDCl₃): d (ppm) 23.0 (t, C-

4⁰), 26.6 (t, C-3), 27.0 (t, C-3⁰), 31.1 (q, C-8), 40.2 (q,

N-CH₃), 41.4 (q, N-CH₃), 45.0 (d, C-4), 47.3 (t, C-6),

55.5 (t, C-5), 57.6 (t, C-5⁰), 63.2 (d, C-2), 67.6 (d, C-

2⁰), 209.7 (s, C-7); EIMS 70 eV, m/z (rel. int.): 224

[M]⁺(8), 209 (1), 167 (8), 152 (12), 140 (5), 124 (5),

110 (5), 96 (2), 84 (100); HRMS 80 eV, m/z: 224.1887

(C₁₃H₂₄N₂O, calc. 224.1889).

oxycarbonyl)-tropan-3a-yl-4⁰-methoxybenzoate.

This

was dissolved in 10 ml HOAc and stirred with

550 mg Zn for 36 h at room temp. under nitrogen.

The reaction was stopped by the addition of icewater.

The aq. layer was adjusted to pH 9 (NH₃) and ex-

tracted with EtOAc. The ﬁnal puriﬁcation was

achieved by CC on silica gel (CHCl₃–MeOH–diethyl-

amine 96:2:2) to yield 65 mg of 2.

2'

O

4

N

2

CH₃

N

CH₃

2’,4-N-Methylpyrrolidinylhygrine (8)

3.15. Synthesis of 7 and 8

1.28 g of N-methyl-a-pyrrolidone dissolved in Et₂O

were reduced with LiAlH₄for 1 h. Then a mixture of

0.9 g of 3-oxoglutaric acid, phosphat buﬀer (pH 7) and

1 N NaOH was added. The Et₂O was evaporated and

the reaction mixture left for 40 h at room temp. For

working up it was acidiﬁed with HCl and extracted with

CH₂Cl₂, then alkalinised with NH₃and again extracted

with CH₂Cl₂. 550 mg of crude alkaloids were puriﬁed by

column chromatography on silica gel (CHCl₃–MeOH

95:5 to CHCl₃–MeOH–NH_{3 conc.}90:10:1) to give

120 mg cuscohygrine and 6 mg of a three to one mixture

of 7 and 8.

3.18. GC–MS analysis

Ground plant parts (50 g) were extracted three times

with 500 ml MeOH (80%). After evaporation the extract

was acidiﬁed and partitioned between water and organic

solvents. The aqueous layer was alkalinized and ex-

tracted with CHCl₃. The crude alkaloids were subjected

to GC–MS analysis. The GC–MS system consisted of a

Carlo Erba 5160/Fisons 8060 GC equipped with a

30 m · 0.32 mm fused silica capillary column coated

with the methyl silicone stationary phase DB 1 (J&W

Scientiﬁc, California). Helium was used as carrier gas.

Conditions during split injection: injector 250 ꢁC, split

1:20, temperature program 70–300 ꢁC, 6 ꢁC/min. The

capillary column was directly coupled to the quadrupole

mass spectrometer Finnigan MAT 4515/MD800. Reten-

tion indices (RI): Kovats indices (Kovats, 1958)

were calculated in respect to a set of co-injected

hydrocarbons.

3.16. 2⁰,3-N-Methylpyrrolidinylhygrine (7)

1

Oil. H NMR (CDCl₃): d 2.21 (3 H, s), 2.27 (6 H,

s), 1.52–3.12 (m); ¹³C NMR (CDCl₃): d (ppm) 22.5 (t,

C-4), 23.6 (t, C-4⁰), 26.3 (t, C-3⁰), 31.2 (q, C-8), 40.5

(q, N-CH₃), 40.6 (q, N-CH₃), 45.7 (d, C-3), 47.3 (t,

C-6), 56.5 (t, C-5), 57.4 (t, C-5⁰), 65.0 (d, C-2), 67.0

K. Jenett-Siems et al. / Phytochemistry 66 (2005) 1448–1464

1463

den x-Methylaminoaldehyden. Monatshefte fur Chemie 82, 551–

557.

¨

H₃C

O

N

Garcia-Argaez, A.N., Perez-Amador, M.C., Aguirre-Hernandez, E.,

Martinez-Vazquez, M., 1999. Two new caﬀeate esters from the

roots of Merremia tuberosa and M. dissecta. Planta Med. 65, 678–

679.

COOCH₃

OH

Hallier, H., 1893. Versuch einer naturlichen Gliederung der Convolv-

¨

ulaceen auf morphologischer und anatomischer Grundlage. Bot.

Jahrb. Syst. 16, 453–591.

Cyclotropine (13)

Methylpseudoecgonine (30)

Hallier, H., 1894. Convolvulaceae africanae. Bot. Jahrb. Syst. 18, 112–

119.

O

Hartmann, T., Witte, L., Oprach, F., Toppel, G., 1986. Reinvestiga-

tion of the alkaloid composition of Atropa belladonna plants, root

cultures, and cell suspension cultures. Planta Med. 52, 390–395.

Huang, M.N., Abraham, T.W., Kim, S.H., Leete, E., 1996. 1-

Methylpyrrolidine-2-acetic acid is not a precursor of tropane

alkaloids. Phytochemistry 41, 767–773.

N

CH₃

Phygrine (58)

Ionkova, I., Witte, L., Alfermann, A.W., 1994. Spectrum of tropane

alkaloids in transformed roots of Datura innoxia and Hyoscyamus

x gyo¨rﬀyi cultivated in vitro. Planta Med. 60, 382–384.

Jenett-Siems, K., 1996. Phytochemische Untersuchungen an Wind-

engewa¨chsen der Gattungen Calystegia, Convolvulus, Ipomoea und

Acknowledgements

Merremia unter besonderer Berucksichtigung des Alkaloidvorkom-

¨

mens. Dissertation Fachbereich Pharmazie, Freie Universita¨t

Berlin.

The authors are indebted to Professor Daniel F. Aus-

tin Ph.D., Conservation & Science Department, Ari-

zona-Sonora Desert Museum, Tucson/AZ (USA), for

helpful discussions on the assignment of the species to

sections/‘‘allies’’. In addition, the authors acknowledge

Director Clyde Dunlop (Northern Territory Herbarium,

Palmerston, N.T., Australia) as well as Mrs. Elisabeth

Ba¨umel-Eich, Berlin, for essential support in exploring

and collecting the plant material, Dr. Peter Bachmann,

Braunschweig/Bonn, for supplying some authentic com-

pounds (13, 30, 56, 57) as reference substances, as well as

Jenett-Siems, K., Eich, E., 1994. Hygrines and tropanes, alkaloids of

considerable importance for the chemotaxonomy of the Convol-

vulaceae. Eur. J. Pharm. Sci. 2, 122.

Jenett-Siems, K., Mann, P., Kaloga, M., Siems, K., Jakupovic, J.,

Witte, L., Eich, E., 1998. Tropane alkaloids with a unique type of

acyl moiety from two Convolvulus species. Phytochemistry 49,

1449–1451.

Kitagawa, I., Shibuya, H., Yokokawa, Y., Baek, N.I., Ohashi, K.,

Yoshikawa, M., Nitta, A., Wiriadinata, H., 1988. Structures of

merremosides B and D, new antiserotonic resin-glycosides from the

tuber of Merremia mammosa, an Indonesian folk medicine. Chem.

Pharm. Bull. 36, 1618–1621.

Kovats, E., 1958. Gas chromatographic characterization of organic

compounds. I. Retention indexes of aliphatic halides, alcohols,

aldehydes, and ketones. Helv. Chim. Acta 41, 1915–1932.

Mann, P., 1997. Zur Phytochemie und Chemotaxonomie tropischer

Mrs. Margrit Meyer, Institut fur Pharmazie, Freie Uni-

¨

versita¨t Berlin, for skilful technical assistance in the

preparation of extracts.

und mediterraner Convolvulaceen unter besonderer Berucksichti-

¨

gung des Alkaloid-Vorkommens. Dissertation Fachbereich Phar-

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Article Doi

Chemotaxonomy of the pantropical genus Merremia (Convolvulaceae) based on the distribution of tropane alkaloids

DOI: 10.1016/j.phytochem.2005.04.027

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Full text of DOI:10.1016/j.phytochem.2005.04.027

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