View Online / Journal Homepage / Table of Contents for this issue

REVIEW

The Lycopodium alkaloids

www.rsc.org/npr
NPR
Xiaoqiang Ma and David R. Gang*
Department of Plant Sciences and Bio5 Institute, University of Arizona, 303 Forbes Building,
Tucson, AZ 85721-0036, USA. E-mail: gang@ag.arizona.edu; Fax: (520)621-7186;
Tel: (520)621-7154
Received (in Cambridge, UK) 6th September 2004
First published as an Advance Article on the web 21st October 2004

Covering: 1993–2004

Lycopodium alkaloids are quinolizine, or pyridine and a-pyridone type alkaloids. Some Lycopodium alkaloids are
potent inhibitors of acetylcholinesterase (AChE). Huperzine A (HupA) is reported to increase efficiency for learning
and memory in animals, and it shows promise in the treatment of Alzheimer’s disease (AD). 201 Lycopodium
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N

alkaloids from 54 species of Lycopodium (sensu lato) have been reported so far. This review is intended to cover the
chemical, pharmacological and clinical research on Lycopodium alkaloids reported in the literature from the spring
of 1993 to August 2004. Structures of 81 new Lycopodium alkaloids are presented, classified and analyzed. The
structural characters and biogenetic relationships of the four major Lycopodium alkaloid groups (lycopodine,
lycodine, fawcettimine and miscellaneous) are discussed. Bioactivities of Lycopodium alkaloids, especially HupA, are
Downloaded by University of Sussex on 22 July 2012

summarized. In particular, the effect of HupA and other cholinesterase inhibitors (anti-AD drugs) on acetylcholine
esterase (AChE) activity in the rat cortex and butylcholine esterase activity are compared. Structure–activity
relationships and structure modifications of HupA and its analogs are described. Information on clinical trials with
HupA and its derivative ZT-1 is presented. The state of HupA availability and recent advances in in vitro propagation
of HupA producing plants are outlined. Finally, hypotheses about Lycopodium alkaloid biosynthetic pathways are
discussed.

1 Introduction 2.2 Lycodine class

2 Structure and nomenclature of Lycopodium 2.3 Fawcettimine class
alkaloids 2.3.1 Carbinolamine form
2.1 Lycopodine class 2.3.2 Keto-amine form

Xiaoqiang Ma received his BS and MS degrees from Xinjiang Agricultural University, and PhD
degree from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, where he studied
the isolation, structure elucidation and structure–activity relationships of bioactive natural products
under the guidance of Professor Dayuan Zhu. Following that, he obtained funding for a project,
“Chemical Studies on the Natural Resources of Huperzia and Its Related Genera”, supported by
the National Natural Science Foundation of China. He then had visiting scholar and postdoctoral
appointments in plant molecular biology, biochemistry and tissue culture at: the Plant Biotechnology
Center, Baylor University, USA; Biotechnology Research Institute and Department of Biology, The
Hong Kong University of Science and Technology; and the Biology Institute II (Botany), University
of Freiburg, Germany. He now works with David Gang in the Department of Plant Sciences and the
Bio5 Institute at the University of Arizona, USA.

Xiaoqiang Ma

David Gang received a PhD in Plant Physiology from Washington State University in 1999, working
on lignan biosynthesis in the lab of Norman G. Lewis. He then did post-doctoral work in the lab of
Eran Pichersky at the University of Michigan, Ann Arbor, investigating evolution in natural product
biosynthetic pathways and in the production of flavor, aroma and scent compounds in plants. He
also has a BS in Botany-Molecular Biology and a BA in German, both of which he received from
Brigham Young University. At the University of Arizona, he teaches plant biochemistry. His research
seeks to elucidate the biosynthetic pathways that produce novel and important plant natural products
(specialized metabolites), to uncover the mechanisms responsible for the evolution of these pathways
in the plant kingdom and to understand the function of a given natural product in the biology and
physiology of a given plant species. Dr Gang has received several awards and recognition for his work
DOI: 10.1039/b409720n

including: a Young Investigator Award in Plant Genome Research, USA National Science Foundation,
2002; the Arthur Neish Young Investigator Award, Phytochemical Society of North America, 2001;
and the Margaret and Herman Sokol Postdoctoral Fellowship in the Sciences, University of Michigan,
1999.
David R. Gang

752 Nat. Prod. Rep., 2004, 21, 752–772 This journal is © The Royal Society of Chemistry 2004

2.4 Miscellaneous group
3 Bioactivities of Lycopodium alkaloids
3.1 Treatment of Alzheimer’s disease
3.2 Acetylcholinesterase inhibition
3.3 Effect on learning and memory function
3.4 Neural cell protection
3.5 Structure–activity relationships
3.6 Structure modification and new drug candidates
against AD
4 Clinical trials
4.1 Clinical trials to evaluate the efficacy and safety of
huperzine A
4.2 Clinical Trials with ZT-1
5 Sources of HupA
5.1 Natural sources
5.2 In vitro propagation as a source of HupA
6 Taxonomic history and chemotaxonomy
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
7 Biosynthesis
7.1 Proposed pathways
8 Acknowledgements
9 References
Downloaded by University of Sussex on 22 July 2012
1 Introduction
The Lycopodium alkaloids are a structurally related, yet di-
verse group of compounds, originally identified in Lycopodium
(sensu lato). To date, 201 Lycopodium alkaloids have been
identified from 54 species of Lycopodium (s. l.) (see Table 1).
These alkaloids usually contain a skeleton comprised of 16
carbons, although they sometimes have as many as 32 carbons
(apparent dimers) or less than 16 carbons (likely resulting from
bond cleavage). The Lycopodium alkaloids are quinolizine, or
pyridine and a-pyridone type alkaloids. The typical Lycopodium
alkaloid is lycopodine, which was also the first identified. Since
1993, the date of the last comprehensive review on this subject by
W. A. Ayer,1 81 new Lycopodium alkaloids have been reported.
A more recent review on Lycopodium alkaloids, which
was published last year,2 was written in Chinese and covered
several areas of research progress on the Lycopodium alkaloids.
Another recent review3 focused on huperzine A (HupA), es-
pecially on the synthesis and structural modification of HupA
and analogs, and on structure–activity relationships of these
compounds. This current review covers advances in research on
the Lycopodium alkaloids from the Spring of 1993 until August
2004.
The first investigations on Lycopodium alkaloids can be
traced back to 1881. Bödeker separated lycopodine, the first
identified lycopodium alkaloid, from Lycopodium complana-
tum.4 In 1938, Achmatowicz and Uzieblo isolated lycopodine,
giving the correct molecular formula, and two new alkaloids.5
Since the 1940s, several Canadian scientists (originating out
of W. A. Ayer’s group) have studied the isolation, structural
elucidation, biogenesis, and chemical synthesis of Lycopodium
alkaloids. W. A. Ayer was an outstanding chemist who spent the
majority of his professional career investigating Lycopodium
alkaloids and published many important articles and reviews
on this topic. Through the mid 1980s, investigators studied the
chemical constituents of various Lycopodium (s. l.) species, de-
veloped methods for chemical synthesis of several Lycopodium
alkaloids, and proposed biochemical pathways for the produc-
tion of these compounds in the plant. During the early 1980s,
Chinese investigators screened Lycopodium (s. l.) species for new
drugs for myasthenia gravis treatment.6 The period of 1986–1990
was a highlight in Lycopodium alkaloid research. During this
time, some Lycopodium alkaloids were found to possess potent
acetylcholinesterase inhibition activity.7,8 Of these, huperzine A
(HupA), which was isolated from the Chinese folk medicinal
herb Qian Ceng Ta (whole plant of Huperzia serrata (Thunb.
ex Murray) Trev.) by Chinese scientist Liu and co-workers,9,10 is
the most well known, and appears to be the most potent. HupA
View Online
is reported to increase efficiency for learning and memory in
animals,9,10 and it shows promise in the treatment of Alzheimer’s
disease and myasthenia gravis.6,11 Because of these discoveries,
a new tide of Lycopodium alkaloid research has been rising.
As described in this review, recent years have brought a large
number of reports on HupA and related compounds.
Lycopodium (s. l.) is a large group of species that are commonly
known as club mosses. These plants are characterized by low,
evergreen, coarsely moss-like and club-shaped strobili at the
tips of mosslike branches. They and the related Selaginella are
the oldest extant terrestrial vascular plants. They originated
sometime during the late Silurian to early Devonian.12–16 The
taxonomy of the genus is still not fixed. The key systems recently
accepted in Lycopodium (s. l.) are that of Ching,17–19 Holub,20,21
and Ollgaard.22–24 Common to these recent investigations is
the insistence that the genus Huperzia be separated from
Lycopodium (s. l.). In fact, Huperzia has been proposed to belong
to a family separate from Lycopodium, the Huperziaceae (=
Urostachyaceae) taxonomically separated from Lycopodium (s.
l.) by Rothmaler in 1944,25 comprised of two genera, Huperzia
and Phlegmariurus, with a total of about 150 species. This
taxonomic system is used in some countries, for example in
China, and it is supported by chemotaxonomic analysis.26 There
are more than 500 species in Lycopodium (s. l.) (families Ly-
copodiaceae and Huperziaceae). However, only 53 species have
been studied so far. Nevertheless, these plants are not abundant,
grow very slowly and are only found in very specialized habitats.
No successful cultivation of these plants has been performed or
reported. Tissue culture also seems to be very difficult.27–29 A few
investigations have been reported that have attempted to identify
the biosynthetic pathway of Lycopodium alkaloids, particularly
of lycopodine. However, these experiments have been limited to
feeding experiments with radiolabeled precursors.
2 Structure and nomenclature of Lycopodium alkaloids
A. W. Ayer178 separated the Lycopodium alkaloids into four
structural classes: the lycopodine class, the lycodine class, the
fawcettimine class and the miscellaneous group. Representative
compounds for these structural classes are lycopodine, lycodine,
fawcettimine and phlegmarine, respectively. Skeletons of these
compounds are shown in Fig. 1. The carbon numbering system
for the Lycopodium alkaloids is based on Conroy’s biogenetic
hypothesis.179 In this hypothesis the alkaloids are made up of
two 2-propylpiperidine units. These are joined as shown in 1
to give phlegmarine 2. Bond formation between C-4 and C-13
then gives the lycodane skeleton. Most examples of this class
have ring A oxidized to a pyridine ring, as in lycodine 3, or a
pyridone. Detachment of C-1 from N a and reattachment to N b
then gives the lycopodine skeleton 4. Finally migration of C-4
from C-13 to C-12 gives the fawcettimine skeleton 5. Details of
the biosynthesis of the alkaloids, as far as they are known, are
covered in Section 7.
2.1 Lycopodine class
Out of the 201 known Lycopodium alkaloids, 70 belong to the
lycopodine class. Twenty of these have been identified since 1993
(Figs. 2 and 3). This is the largest group of known Lycopodium
alkaloids, and appears to be the most widely distributed.
However, because relatively few species of Lycopodium have
been investigated in detail, this may change in the future. The first
Lycopodium alkaloid to be identified (lycopodine, 4) belongs
to this group. This class is characterized by four connected
six-membered rings, with rings A and C being a quinolizidine
ring system. The carbonyl group in ring B is generally at C-5,
although it may be found at C-6, such as in huperzines E 7, F
874,75 and O 677 which were isolated from H. serrata. According
to a biogenetic hypothesis,180 these compounds may be derived
directly from lycopodine via sequential oxidations (Fig. 2).
Nat. Prod. Rep., 2004, 21, 752–772 753
 View Online

Table 1 Reported Lycopodium alkaloids (through August 2004)

Popular name (chemical name) Source

I. Lycopodine class
Acetylacrifoline Lycopodium annottinum30,31
Acetylannofoline L. ophioglossoides32
Acetyldebenzoylalopecurine (2a-OAc-lycopecurine) L. alopecuroides31,33–35
Acetyldihydrolycopodine L. clavatum var. borbonicum,36 L. contiguum,37 L. magellanicum,38
L. paniculatum,39 L. thyoides,37 L. tristachyum31
Acetylfawcettiine L. annotinum,40 L. clavatum,41 L. contiguum,37 L. fawcettii,42 L. magellanicum,38
L. saururus,31 L. thyoides37
Acetyllofoline L. annottinum31,43
Acetyllycoclavine L. clavatum var. megastachyon31,44
Acetyllycofawcine (8-OAc-lycofawcine) L. fawcettii31,45
Acetyllycofoline (5-OAc-lycofoline) L. fawcettii31,45
Acrifoline [D11,12 ,8-oxo-dihydrolycopodine (5b-OH)] L. annottinum,46 L. annotinum var. acrifolium,31 L. selago47
Acrifolinol L. annotinum,30 L. obscurum48
Alopecurine (2a-benzoyloxylycopecurine) L. alopecuroides31,33,34
Anhydrodeacetyl-paniculine (deacetoxy-D4,5 -paniculine) L. paniculatum31,39,49
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N

Anhydrodihydrolycopodine L. tristachyum32
Anhydrolycodoline (D11,12 -lycopodine) L. alopecuroides,33 L. carolinum,31 L. clavatum var. borbonicum,36 L. gnidioides,50
L. inundatum,31 L. phlegmaria,31 L. saururus31
Annofoline (8-oxo-5b-OH dihydrolycopodine) L. annottinum30,31,40
Annopodine (27) L. annottinum51
Annotine (29) L. annottinum31,52
Downloaded by University of Sussex on 22 July 2012

Annotinine (28) L. annotinum,53 L. annotinum var. acrifolium,54 L. flabelliforme,31 L. obscurum,31

L. selago55
Clavolonine (8b-hydroxylycopodine) Huperzia miyoshiana,56 L. alopecuroides,31 L. alpinum,41,57 L. clavatum,42,58–60
L. clavatum var. megastachyon,44 L. contiguum,37 L. densum,31 L. deuterodensum,36
L. flabelliforme,31 L. inundatum,31 L. magellanicum,61 L. obscurum,48,62
L. saururus,41 L. serratum,31 L. serratum var. serratum f. serratum,63 L. serratum
var. serratum f. intermedium,63 L. sieboldii,31 L. stichense,41 L. thyoides31
Deacetylfawcettiine L. clavatum,64 L. contiguum,31 L. fawcettii,42,65 L. magellanicum,61 L. thyoides31
Deacetyllycoclavine (O-des-Ac-lycoclavine) L. clavatum var. megastachyon,44 L. paniculatum,39 L. serratum31,66
Deacetylpaniculine L. paniculatum,49 L. confertum31
Debenzoylalopecurine (2a-hydroxylycopecurine) L. alopecuroides31,33–35
Dehydrolycopecurine (5-ketone-lycopecurine) L. alopecuroides,31,35 L. inundatum67
Diacetyllycofoline L. fawcettii31,45
dihydrolycopodine (5a-OH) L. carolinum,41 L. clavatum,59,64 L. clavatum var. borbonicum,36 L. clavatum var.
inflexum,31 L. clavatum var. megastachyon,31 L. contiguum,31 L. flabelliforme,31
L. magellanicum,31 L. obscurum,48 L. paniculatum,39 L. saururus,31 L. thyoides,31
L. volubile68
4a,6a-Dihydroxyserratidine (11) H. serrata69
12-Epilycodoline (isolycodoline or pseudoselagine) H. miyoshiana,56 L. lucidulum,31,70 L. selago55,71
Fawcettiine L. annottinum,72 L. clavatum,58 L. contiguum,37 L. fawcettii,45,65 L. magellanicum61
(b-lofoline,5b-O-acetyl-8b-hydroxydihydro-lycopodine) L. saururus,31,41 L. thyoides37
Flabelliformine (4a-hydroxylycopodine) H. miyoshiana,56 L. clavatum,5,46 L. clavatum var. megastachyon,44
L. flabelliforme,73 L. lucidulum31,70
Flabelline (5-NHAc-anhydrolycopodine) (21) L. flabelliforme,31 L. deuterodensum36
Gnidioidine (D11,12 ,8b-hydroxylycopodine) L. gnidioides,50 L. phlegmaria31
Huperzine E (7) H. serrata74,75
Huperzine F (8) H. serrata74,75
Huperzine G (22) H. serrata76
Huperzine O (6) H. serrata77
6a-Hydroxylycopodine H. serrata,78 L. lucidulum79
Inundatine (5-ketone,2b-hydroxylycopecurine) L. inundatum31,67
4a-Hydroxyserratidine (10) H. serrata69
6a-Hydroxyserratidine (9) H. serrata69
Isoinundatine (2-oxolycopecurine 5-ketone) L. inundatum31,67
a-Lofoline (8a-hydroxydihydrolycopodine) L. annottinum31,72
Lucidioline (34) H. serrata,26,80 L. gnidioides,50 L. lucidulum,70 L. ophioglossoides,31 L. serratum66
(D11,12 -5b,6a-dihydroxydihydro-lycopodine)
Lycoclavine L. alpinum,57 L. clavatum var. megastachyon,44 L. gnidioides,50 L. paniculatum,49
(5b-O-acetyl-6a-hydroxy-dihydrolycopodine) L. serratum81,82
Lycodoline (33) H. miyoshiana,56 H. serrata,78 L. alopecuroides,31 L. annottinum,31 L. annotinum
var. acrifolium,31 L. clavatum,46,59 L. clavatum var. borbonicum,36 L. clavatum var.
inflexum,31 L. carolinum,31 L. fawcettii,31 L. inundatum,67 L. lucidulum,70
L. phlegmaria,31 L. saururus,31 L. selago,47 L. serratum,66,81,82 L. serratum var.
serratum,83 L. serratum var. serratum f. serratum,63 L. serratum var. serratum f.
intermedium,63 Phlegmariurus fordii63,84,85
Lycofawcine L. fawcettii31,86
(5b-O-acetyl-8-endo,12b-dihydroxy-dihydrolycopodine)
Lycofoline (D11,12 ,5b,8b-di-hydroxydihydrolycopodine) L. annottinum,72,87 L. fawcettii,31,45 L. obscurum48
Lycognidine (5b-O-[3-(3,4-dimethoxyphenyl)propionyl]- L. gnidioides31,50
6a-hydroxydihydro-lycopodine)
Lyconesidine C (25) L. chinense88
Lyconnotine (30) L. annottinum26,31,48,89
Lyconnotinol (31) L. ophioglossoides48

754 Nat. Prod. Rep., 2004, 21, 752–772

 View Online

Table 1 (Contd.)

Popular name (chemical name) Source

Lycopecurine (4,10-cyclo-dihydrolycopodine) (32) L. alopecuroides31,35

Lycophlegmine (10b-hydroxyanhydrolycodoline) L. phlegmaria31,90
Lycopodine (4) H. miyoshiana,56 H. serrata,78 L. lopecuroides,31 L. alpinum,41,57 L. annottinum,52,91
L. annotinum var. acrifolium,54 L. carolinum,41 L. cernuum,92 L. clavatum,5,93
L. clavatum var. borbonicum,36 L. clavatum var. inflexum,31 L. clavatum var.
megastachyon,44 L. complanatumi,41,94 , L. contiguum31 , L. densum,95
L. deuterodensum,36,96 L. erythraeum,31 L. fastigiatum,36 L. flabelliforme,31
L. inundatum,67 L. issleri,41 L. lucidulum,70,97 L. magellanicum,38,61 L. obscurum,48,98
L. obscurum var. dendroideum,31 L. paniculatum,39 L. phlegmaria,99
L. sabinaefolium,41,100 L. saururus,31 L. selago,47,71 L. serratum,66 L. serratum var.
serratum f. serratum,63 L. serratum var serratum f. intermedium,63 L. stichense,41
L. thyoides,37 L. tristachyum,41,93 L. verticillatum,31 L. volubile68
Lycoposerramine G (12) L. serratum66
Lycoposerramine H (13) L. serratum66
Lycoposerramine I (14) L. serratum66
Lycoposerramine K (15) L. serratum66
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N

Lycoposerramine L (16) L. serratum66

Lycoposerramine M (20) L. serratum66
Lycoposerramine N (17) L. serratum66
Lycoposerramine O (26) L. serratum66
Lycoverticine (12-hydroxy flabelline) L. verticillatum31,50
Miyoshianine A (= lycoposerramine F) (18) H. miyoshiana,56 L. serratum66
Downloaded by University of Sussex on 22 July 2012

Miyoshianine B (= lycoposerramine J) (23) H. miyoshiana,56 L. serratum66

Paniculine (10a-hydroxy-5b-O-acetyldihydrolycopodine) L. paniculatum,39 L. confertum31
Selagoline (24) H. selago101
Serratezomine C (6a,12b-dihydrolycopodine) (19) L. serratum var. longipetiolatum83
Serratidine (7a-hydroxyanhydrolycodoline) H. serrata,69 H. selago,101 L. serratum,66 L. serratum var. serratum f. serratum,63,102
L. serratum var. serratum f. intermedium63,102
II. Lycodine class
Complanadine A (38) L. complanatum103
N-Demethylhuperzinine (43) L. casuarinoids116
Des-N-methyl-a-obscurine L. alpinum,57 L. clavatum,59 L. fawcettii,31 L. flabelliforme,104 L. obscurum48
Des-N-methyl-b-obscurine H. serrata,78 L. obscurum48
Des-N-methyl-fastigiatine (46) L. fastigiatum31,105
N,N-Dimethylhuperzine A H. serrata118
Fastigiatine (45) L. fastigiatum31,105
Flabellidine (1,2,3-trihydro-N a -Ac-lycodine) L. complanatum,31 L. flabelliforme,104 L. obscurum,48 L. paniculatum,49
L. thyoides41
Himeradine A (47) L. chinense106
Huperzine A (35) H. serrata,9 H. selago,101 L. selago,107 L. varium108
Huperzine B (36) H. serrata9
Huperzine C (40) H. serrata109
Huperzine D (41) H. serrata109
Huperzine U (2,3-dihydro-12-hydroxyhuperzine B) (48) H. serrata110
Huperzinine (39) H. serrata111
Hydroxy-des-N-methyl-a-obscurine (12-OH) L. flabelliforme31,104
6b-Hydroxyhuperzine A (42) H. serrata,112 L. selago107
11-Hydroxylycodine (49) L. complanatum113
Hydroxypropyllycodine L. ophioglossoides48
Lycodine (3) H. serrata,114 L. australianum,36 L. annottinum,115 L. clavatum,31 L. clavatum var.
borbonicum,36 L. deuterodensum,36 L. fastigiatum,36 L. fawcettii,31
L. flabelliforme,31 L. lucidulum,31 L. magellanicum,38 L. obscurum,48
L. phlegmaria,99 L. serratum var. serratum f. serratum,63 L. serratum var. serratum
f. intermedium,63 L. serratum var. thunbergii,31 L. tristachyum41
N-Methyl-huperzine B (37) H. serrata31,111
N-Methyl-lycodine L. complanatum,41 L. erythraeum,31 L. magellanicum,38 L. obscurum117
a-Obscurine (2,3-dihydro-b-obscurine) (50) L. annottinum,31,119 L. clavatum,59 L. fastigiatum,36 L. flabelliforme,104
L. magellanicum,61 L. obscurum,120 L. obscurum var. dendroideum,31 L. selago,47
L. sitchense41
b-Obscurine (51) L. annottinum,31,119 L. flabelliforme,104 L. magellanicum,61 L. obscurum,120
L. obscurum var. dendroideum,31 L. selago47
Phlegmariurine M (44) Ph. fordii121
Sauroxine (12-epi-a-obscurine) (52) L. saururus31,86,122
III. Fawcettimine class
Acetyllobscurinol L. ophioglossoides31,48
Alolycopine (D3,4 ,8a-hydroxyfawcettidine) L. alopecuroides31,34,123
Alopecuridine (4a-hydroxyfawcettimine) L. alopecuroides31,33,124
Anhydroaposerratinine (8a-hydroxyfawcettidine) L. verticillatum31,50
8-Deoxy-13-dehydro-serratinine L. phlegmaria,90 L. serratum31
8-Deoxyserratinidine L. phlegmaria31,90
8-Deoxyserratinine (98) L. serratum var. serratum f. serratum,63,125 L. serratum var. serratum f.
intermedium,63 H. serrata31,126
7a,11a-Dihydroxy-phlegmariurine B (72) H. serrata127
Epidihydrofawcettidine (5a-OH) L. phlegmaria31,90,128
Epilobscurinol (87) L. ophioglossoides48

Nat. Prod. Rep., 2004, 21, 752–772 755

 View Online

Table 1 (Contd.)

Popular name (chemical name) Source

Fawcettidine (53) L. fawcettii,65,125 L. alopecuroides,34 L. phlegmaria31

Fawcettimine (5) L. annottinum,31 L. clavatum,129 L. fawcettii,125 H. serrata130
Huperserratinine (100) H. serrata131
Huperzine H (101) H. serrata132
Huperzine I (2a-hydroxyfawcettidine) (59) H. serrata133
Huperzine P (84) H. serrata130
Huperzine Q (67) H. serrata134
Huperzine R (85) H. serrata135
Huperzine S (2b,13b-epoxyalopecuridine) (69) H. serrata110
Huperzine T (5a-hydroxy-6-oxodihydrophlegmariurine A) (82) H. serrata110
Huperzine W (102) H. serrata136
7-Hydroperoxy-phlegmariurine B (77) H. serrata137
11a-Hydroperoxy-phlegmariurine B (73) H. serrata137
2a-Hydroxyphlegmariurine B (74) H. serrata138
7a-Hydroxyphlegmariurine B (75) H. serrata127
8a-Hydroxyphlegmariurine B (78) H. serrata139
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N

8b-Hydroxyphlegmariurine B (79) H. serrata140

11a-Hydroxyphlegmariurine B (76) H. serrata127
Isoobscurinine (89) L. ophioglossoides48
Lobscurinol (86) L. ophioglossoides48
Lycoflexine (104) L. clavatum var. borbonicum,36 L. clavatum var. inflexum,31 L. carolinum,129
L. deuterodensum,36 L. fastigiatum,36 L. inundatum,31 L. phlegmaria,90
Downloaded by University of Sussex on 22 July 2012

L. serratum,141 L. serratum var. serratum f. serratum,63 L. serratum var.

serratum f. intermedium63
Lyconesidine A (57) L. chinense88
Lyconesidine B (58) L. chinense88
Lycopaniculatine (or paniculatine) (55) L. paniculatum31,142
Lycophlegmarine L. phlegmaria31,90,143
Lycopodium base R (65) L. fawcettii31,144
Lycoposerramine A (90) L. serratum145
Lycoposerramine C (60) L. serratum141
Lycoposerramine D (105) L. serratum141
Lycoposerramine E (83) L. serratum141
Lycoposerramine P (61) L. serratum141
Lycoposerramine Q (62) L. serratum141
Lycoposerramine S (91) L. serratum141
Lycoposerramine U (106) L. serratum141
Lycothunine (D11,12 -fawcettimine) L. serratum var. longipetiolatum31,143
Macleanine (66) L. serratum146
Magellanine (93) L. magellanicum31,61
Magellaninone (94) L. magellanicum31,38
Megastachine L. magastachyum31,147
Neohuperzinine (63) H. serrata148
Obscurinine (88) L. obscurum,48,62 L. ophioglossoides48
2-Oxophlegmariurine B (80) H. serrata138
11-Oxophlegmariurine B (81) H. serrata138
N-Oxyhuperzine Q (68) H. serrata134
Phlegmariurine A (70) H. serrata,139 Ph. fordii,84,149,150 L. serratum141
Phlegmariurine B (54) H. serrata,114,130,138,139 Ph. fordii84,150
Phlegmariurine C (71) Ph. fordii150
Serratanidine (8a-hydroxy serratine) (97) L. serratum,31,151 L. serratum var. serratum f. serratum,63 L. serratum var.
serratum f. intermedium63
Serratezomine A (103) L. serratum var. longipetiolatum83
Serratezomine B (serratinine N-oxide) (99) L. serratum var. longipetiolatum83
Serratine (96) H. serrata,152 L. serratum var. thunbergii,153 L. serratum var. serratum f.
serratum,63 L. serratum var. serratum f. intermedium63
Serratinidine (5a-NHAc,8a-OH-fawcettidine) (64) L. serratum,31,154 L. serratum var. serratum f. serratum,63 L. serratum var.
serratum f. intermedium63
Serratinine (95) H. serrata,26,80 L. serratum,31,82 L. serratum var. serratum,83 L. serratum var.
serratum f. serratum,63 L. serratum var. serratum f. intermedium,63
L. serratum var. thunbergii155,156
Sieboldine A (92) L. sieboldii157
IV. Miscellaneous group
Anhydrolycocernuine (deoxy-D12,13 -lycocernuine) L. cernuum,31,158 L. inundatum,92 L. carolinianum var. affine41,159
Carolinianine (14,15-didehydrolycocernuine) L. carolinianum var. affine31,159
Cermizine A (132) L. cernuum160
Cermizine B (133) L. cernuum160
Cermizine C (129) L. cernuum160
Cermizine D (125) L. cernuum160
Cernuine (deoxylycocernuine) (109) L. australianum,36 L. carolinianum,31 L. cernuum,158,161,162 L. chinense163
Cernuine N-oxide (123) L. cernuum160
Dihydrodeoxycernuine (deoxy-deoxolycocernuine) L. carolinianum var. affine31,41
Dihydrodesoxy lycocernuine (deoxolycocernuine) L. carolinianum var. affine31,41
Dihydroluciduline (5a-OH) (126) L. lucidulum31,164
Dihydrolycolucine (14,15,16, 17-tetradehydrolucidine B) L. lucidulum31,165
N,N-Dimethylphlegmarine L. phlegmaria31,90,171

756 Nat. Prod. Rep., 2004, 21, 752–772

 View Online

Table 1 (Contd.)

Popular name (chemical name) Source

Huperzine J (134) H. serrata166

Huperzine K (135) H. serrata166
Huperzine L (136) H. serrata166
Huperzine V (113) H. serrata167
Huperzinine B (127) H. serrata168
Lucidine A (114) L. lucidulum165,169
Lucidine B (serratanine A) (116) L. lucidulum,165,169 L. serratum var. serratum f. serratum,63 L. serratum var.
serratum f. intermedium63
Luciduline (107) L. lucidulum31,164
Lucidulinone (9-ketoluciduline) (108) L. lucidulum164,169
Lycocernuine (12a-hydroxycernuine) (110) L. carolinianum var. affine,41,159 L. cernuum,31,92,158 L. inundatum,92 Palhinhea
cernua170
Lycocernuine N-oxide (124) L. cernuum160
Lycolucine (10,11,14,15,16, 17-hexadehydro-lucidine B) L. lucidulum31,165
Lyconadine A (137) L. complanatum113
N a -Methyl-N b -acetyl-phlegmarine (111) L. clavatum var. megastachyon36,171,172
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N

N a -Methylphlegmarine (112) L. phlegmaria31,171

N b -Methylphlegmarine L. phlegmaria31,171
Oxolucidine A (115) L. lucidulum165,169,173
Oxolucidine B (or serratanine B,14b-hydroxy-lucidine B) (117) L. serratum31,165,174,175
Phlegmarine (2) L. phlegmaria171
Phlegmariurine N (128) H. serrata112,176
Downloaded by University of Sussex on 22 July 2012

Senepodine A (118) L. chinense163

Senepodine B (122) L. chinense177
Senepodine C (119) L. chinense177
Senepodine D (120) L. chinense177
Senepodine E (121) L. chinense177
Senepodine G (130) L. chinense160
Senepodine H (131) L. chinense160

Lycopodium serratum = Huperzia serrata; L. selago = H. selago; L. serratum var. serratum f. serratum = L. serratum var. thunbergii; L. cernuum =
Palhinhea cernua

The most typical compound in this class is lycopodine 4.
Positions C-4 and C-6, a to the C-5 carbonyl of 4, and the
tertiary carbons at C-7 and C-12 are commonly oxygenated
in this class. New examples are 6a-hydroxyserratidine 9,69 4a-
hydroxyserratidine 10,69 4a,6a-dihydroxyserratidine 11,69 lyco-
poserramines G 12, H 13, I 14, K 15, L 16 and N 1766
(all isolated from H. serrata and L. serratum [= H. serrata]),
miyoshianine A 1856 (from H. miyoshiana), and serratezomine
C 1983 (from L. serratum var. longipetiolatum). Compound 14
and lycoposerramine M 20,66 isolated from L. serratum, have
hydroxyl functional groups at C-11. In addition, the nitrogen
of 4 can be oxygenated, as in N-oxide 18.56 Flabelline 21
(from L. flabelliforme and L. deuterodensum) and huperzine
G 2276 (from H. serrata) are unusual derivatives of 4 with
the C-5 carbonyl being converted to an enamide (see Fig. 3).
Furthermore, the C-5 carbonyl of 4 can be reduced to a hydroxyl
group, as in miyoshianine B 2356 (from H. miyoshiana), selagoline
24101 (from H. selago), lyconesidine C 2588 (from L. chinense)
and lycoposerramine O 2666 (from L. serratum). These latter
two compounds are esterified by a feruloyl-related moieties.
Two new compounds, lycoposerramines F and J66 isolated
from L. serratum by Takayama et al., were found to be the same
compounds as miyoshianines A 18 and B 23,56 respectively. They
were published in the same years, 2003. We retain miyoshianines
A and B as the names of the two new compounds based on
their publication date (June) being earlier than that (October) of
lycoposerramines F and J.
The A, B and C rings of the lycopodine class of compounds
are stable. Skeletal variations are found mainly in the D ring.
For example, the D ring is broken between C-8 and C-15 in

Fig. 1 Representative compounds of the four major classes of Lycopodium alkaloids.

Nat. Prod. Rep., 2004, 21, 752–772 757


Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
Downloaded by University of Sussex on 22 July 2012
Fig. 2 Possible biogenesis of carbonyl at C6 in lycopodine group
compounds (structures of 3 new members included); * indicates a new
compound.
758
View Online
annopodine 27 and annotinine 28 and between C-7 and C-8
in annotine 29, lyconnotine 30 and lyconnotinol 31, see Fig. 3.
C-15 of 27 and 28 connects with C-10 and C-12, respectively,
whereas in 29 it connects directly to C-7. In another twist, C-4
and C-10 in lycopecurine 32 are connected to form a fifth ring.
2.2 Lycodine class
Twenty six of the 201 Lycopodium alkaloids belong to the
lycodine class. Seven of these compounds have been identified
since 1993 (see Fig. 4).
So far, all of the Lycopodium alkaloids with acetyl-
cholinesterase inhibition activity belong to this class, most
notably: HupA 35, huperzine B 36, N-methyl-huperzine B 37
and huperzinine 39. Due to the structural similarity between
HupA 35 and selagine, isolated from Lycopodium selago L.,
reinvestigation of the selagine structure was performed, reveal-
ing that the earlier structure assignment was incorrect and that
selagine was identical to HupA.107
This class also has four rings in general, with the B, C and
D rings being the same as in the lycopodine class. However,
the A ring is opened and rearranged to form a pyridine or
pyridone ring. The representative compound of this group
is lycodine 3 (Fig. 1). Complanadine A 38,103 isolated from
L. complanatum, is the second dimeric Lycopodium alkaloid
reported so far, with a C-1 to C-2 linkage between two lycodine
moieties. HupA 35, huperzinine 39, huperzines C 40 and D
41,109 6b-hydroxyhuperzine A 42 (35, 39–42 all isolated from
H. serrata), N-demethylhuperzinine 43116 (from L. casuarinoids)
and phlegmariurine M 44 (from Ph. fordii) are the products
of C ring cleavage and elimination of C-9 (Fig. 4), giving a
Fig. 3 Seventeen new members and selected compounds of the lycopodine class; * indicates a new compound.
Nat. Prod. Rep., 2004, 21, 752–772

C15 N2 skeleton. Compound 41 is a very unusual compound
in the Lycopodium alkaloids, being the only compound with
its C-16 methyl group oxygenated. Five-ring compounds, such
as fastigiatine 45, des-N-methyl-fastigiatine 46, and himeradine
A 47106 (consisting of a fastigiatine-type skeleton C16 N2 and
a quinolizidine moiety C11 N) with a C4 –C10 bond are also
uncommon. Huperzine U 48110 and 11-hydroxylycodine 49,113
isolated from H. serrata and L. complanatum, respectively, have
hydroxyl substituents at C-12 and C-11, respectively (Fig. 4).
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
Downloaded by University of Sussex on 22 July 2012
Fig. 4 Seven new members and selected compounds of lycodine class;
* indicates a new compound.
Except for at C-12, the lycodine class of compounds is rarely
hydroxylated. Variations in structure are mainly due to dehydro-
genation, skeletal additions (C-methylations), N b -methylation,
N a-acetylation and other substitutions. Conformational isomers
are found in this class. The compounds in this class usually exist
with the C/D rings in the trans configuration (i.e. a-obscurine 50
View Online
and b-obscurine 51). However, the C/D rings have been found
in the cis configuration as well, as in sauroxine 52.
2.3 Fawcettimine class
This group contains 65 of the 201 known Lycopodium alkaloids.
Of these, 35 have been discovered since 1993 (Figs. 5, 6 and 7).
This class of compounds can be regarded as the products
of C4 –C13 to C4 –C12 bond migration from lycopodine group
precursor(s). The C13 –C14 double bond of fawcettidine 53, being
an enamine, is easily hydrated to give a hydroxyl group on C-13
(i.e. fawcettimine 5), which can then lead to C13 –N bond-cleavage
to form a carbonyl group (Fig. 5). This been confirmed by
demonstration of equilibrium between the carbinolamine form
(5a) and the keto-amine form (5b).1,180,181
2.3.1 Carbinolamine form. Compounds like fawcettimine
5 in which the N is connected to C-13 are the most common in
the fawcettimine group and are referred to as the carbinolamine
form (even though many of them are not in fact carbinolamines).
This form can be divided into two sub-forms, depending on
whether the C12 –C13 bond is broken or not, with phlegmariurine
B 54 and fawcettidine 53, respectively, representing these sub-
forms (Fig. 5).
Active positions on fawcettidine 53 are at C-2, C-6, C-8
and C-13. Lyconesidines A 57 and B 5888 (from L. chinense),
huperzine I 59133 , lycoposerramines C 60, P 61 and Q 62,141
and neohuperzinine 63148 [59–63 being new compounds from
H. serrata and L. serratum (= H. serrata)], belong to the
fawcettidine sub-form (Fig. 6). A second nitrogen atom is
introduced in compounds such as serratinidine 64. Five-ring
compounds of this sub-form can be formed by intramolecular
reaction of the C-13 hydroxyl group (or its amino form)
with other positions on the skeleton, such as is observed in
lycopodium base R 65, macleanine 66,146 huperzine Q 67,134 N-
oxyhuperzine Q 68,134 and huperzine S 69110 which were isolated
from L. serratum (= H. serrata) and H. serrata.
The phlegmariurine B sub-form consists of three-ring
alkaloids of the carbinolamine form with the C4 –C13 bond
being broken, e.g. phlegmariurines A 70 and B 54. With two
phlegmariurine A moieties coupled at C-6, phlegmariurine C
71 was the first dimeric lycopodium alkaloid to be reported
(in 1988).150 7a,11a-Dihydroxyphlegmariurine B 72,127 11a-
hydroperoxyphlegmariurine B 73,137 2a-hydroxyphlegmariurine
B 74,138 7a-hydroxyphlegmariurine B 75,127 11a-
hydroxyphlegmariurine B 76,127 7-hydroperoxyphlegmariurine
B 77,137 8a- and 8b-hydroxyphlegmariurine B 78139 and 79,140 and
2-oxo- and 11-oxophlegmariurines B 80 and 81, all isolated from
H. serrata,138 are a series of derivatives of the phlegmariurine B
core structure. Huperzine T 82110 and lycoposerramine E 83,141
isolated from H. serrata and L. serratum (= H. serrata), have
structures similar to phlegmariurine A 70. Huperzine P 84130
and R 85,135 isolated from H. serrata, are compounds with new
skeletal structures and are possibly derived from this sub-form.
In 84, the five-membered ring contains an inserted oxygen
atom, and in 85, C-5 is replaced by an oxygen atom.
2.3.2 Keto-amine form. Alkaloids of this form have the N–
C13 bond of the carbinolamine form cleaved to give initially
a keto-amine 5b, although in many members of the form the
ketone has subsequently been reduced or otherwise modified.
This form also has two sub-forms having either three-ring or
four-ring skeletons, depending on whether a N–C4 (or N–C17 )
bond has been made or not (Fig. 7).
Lobscurinol 86 (5a-OH) and epilobscurinol 87 (5b-OH)
possess the common three-ring sub-form. They are possible
precursors of obscurinine 88 and isoobscurinine 89 in which
an extra ring is formed by a nitrogen atom bridging between
C-3 and C-13. Lycoposerramines A 90 and S 91,141 isolated
from L. serratum, also have an additional ring, formed in this
case by a nitrogen atom linking C-13 and C-5. Sieboldine A
Nat. Prod. Rep., 2004, 21, 752–772 759
 View Online

Fig. 5 Potential biogenesis of some fawcettimine group compounds. Structures 57–85 can be found in Fig. 6 and 86–106 in Fig. 7.
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
Downloaded by University of Sussex on 22 July 2012

Fig. 6 Twenty five new members and selected compounds with carbinolamine form of the fawcettimine class; * indicates a new compound.

Fig. 7 Ten new members and selected compounds with keto-amine form of the fawcettimine class; * indicates a new compound.

760 Nat. Prod. Rep., 2004, 21, 752–772

 View Online

92, isolated from L. sieboldii,157 has a unique structure with

an additional tetrahydrofuran ring formed by insertion of an
oxygen atom between C-1 and C-4. Lycopaniculatine 55 (Fig. 5)
is a possible precursor of magellanine 93 and magellaninone 94,
unusual compounds with a C3 –C10 bond.
Serratinine 95 and its relatives, serratine 96, serratanidine 97
and 8-deoxyserratinine 98, are representatives of the four-ring
keto-amine form with an N–C4 bond. Serratinine N-oxide has
been isolated from L. serratum var. longipetiolatum and named
serratezomine B 99.83 Huperserratinine 100, isolated from H.
serrata, 131 is a p-methoxyphenylacetyl ester of serratinine.
Huperzine H 101,132 huperzine W 102 and serratezomine A
103,83 isolated from H. serrata and its variety L. serratum var.
longipetiolatum (L. serratum = H. serrata), are special examples
of the four-ring sub-form. Huperzine H 101 has a cleaved C4 –C12
bond, leaving only three rings, while 102136 only has 14 carbons
and two rings and is possibly derived from serratinine 95 with
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N

elimination of C-5 and C-6 and cleavage of the C4 –C12 bond.

Based on the skeleton of serratinine 86, serratezomine A 103 has
a lactone ring formed by cleavage of the C4 –C5 bond and then
attachment of the C-5 carbonyl group to the C-13 hydroxyl. This
gives a spiro structure, which is very unusual in the Lycopodium
alkaloids.
Downloaded by University of Sussex on 22 July 2012

C17 N alkaloids such as lycoflexine 104 could be the product

of N-methylation followed by C–C bond formation between the
N-methyl and C-4. On this skeleton C-2 and C-8 are reactive
positions and are often substituted by hydroxyl groups, as in
lycoposerramines D 105 and U 106, which were also isolated
from L. serratum.141

2.4 Miscellaneous group

Forty of the 201 Lycopodium alkaloids belong to the miscella-
neous group. Nineteen of these have been identified since 1993.
This group includes all of the Lycopodium alkaloids that
do not belong in one of the first three classes, and represent
quite a diversity of structural motifs. A representative and
key compound belonging to this group is phlegmarine 2, see
Figs. 1 and 8. All of the compounds in this group can be
viewed as being derived from phlegmarine 2 (e.g. Fig. 8).
Furthermore, as discussed in Section 7, phlegmarine 2 appears
to play a key role in the biosynthesis of the other Lycopodium
alkaloids, perhaps being an intermediate in the formation of all
Lycopodium alkaloids. The configuration of phlegmarine was
not determined in the original paper. Relative configurations of
an acetylated derivative (N a-methyl-N b -acetylphlegmarine 111)
and three isomers of N a-methylphlegmarine 112 were confirmed
by Leniewski et al.172,182 using synthetic methods and 13 C-NMR
spectroscopy.
In all of the miscellaneous group compounds, C-4 remains
unconnected to C-12 or C-13. In the three other classes, C-
4 of phlegmarine 2 invariably forms a C–C bond with either
C-13 or C-12 during the C–C coupling reaction that leads to
the formation of these other classes. C-13 is tertiary in the
miscellaneous group and the compounds in this group generally
have three rings in their structural backbones, instead of four or
more as is common in the other classes. Although compounds in
the other classes may contain only three rings, these are believed
to be formed after the basic four ring backbone structure of the
class is formed, as discussed above. In the miscellaneous group,
the compounds begin with only three rings. Huperzine V 113167
(from H. serrata), lucidine A 114, oxolucidine A 115, lucidine B
116, oxolucidine B 117 (Fig. 9) are exceptions and have six rings
and a C27 N3 skeleton. However, these structures appear to be the
result of coupling of one additional pelletierine (or derivative
thereof) moiety and a C3 unit to the phlegmarine 2 skeleton.
Senepodines A–E (118–122),163,177 isolated from L. chinense,
have a C22 N2 skeleton. However, they still possess the core
phlegmarine structure. Phlegmarine is an obvious candidate as
precursor for these compounds.
Fig. 8 Possible biogenesis of some miscellaneous group compounds.
Cernuine 109, lycocernuine 110 (Fig. 8) and their N-oxides
123 and 124 (Fig. 9), recently isolated from L. cernuum,160 have
a large structural difference compared to phlegmarine 2. It is
likely that they are formed by a 4+2 cycloaddition reaction with
a derivative of phlegmarine 2 that has undergone opening of
ring D via cleavage of the C7 –C12 bond as shown in Fig. 8. In
contrast, luciduline 107 and lucidulinone 108 are the products of
elimination of Na and the C-1 to C-4 carbons of phlegmarine 2
and C–C bond formation between C5 and C10 . Dihydroluciduline
126 (Fig. 9) has also been isolated from the same source.1,180,181
Five two-ring alkaloids, huperzinine B 127168 (from H. serrata),
phlegmariurine N 128, cermizine C 129160 (from L. cernuum),
and senepodines G 130 and H 131160 (from L. chinense) have
the simplest lycopodium alkaloid structures. The quinolizidine
rings of 129, 130 and 131 might be related to the biogenesis of
the quinolizidine ring system (rings A and C) of the lycopodine
class. Cermizine D 125160 (from L. cernuum) is a new C16 N2 type
alkaloid consisting of a quinolizidine and a piperidine ring, and
is a possible precursor of cernuine 109 and lycocernuine 110.
Cermizines A 132 and B 133160 (from L. cernuum) possess a
very similar structure to phlegmarine 2, with differences only
in subsitituents at N a and C9 . huperzines J 134, K 135 and L
136,166 isolated from H. serrata, also are very similar in structure
to phlegmarine 2, but they possess an N-oxide functional group
at the N a position and other simple modifications. Lyconadine
A 137,113 isolated from L. complanatum, does not have the C-4
to C-13 bond of the lycodine class and instead is a phlegmarine-
type compound in which C-4 has become bonded to C-10 and
N b to C-6 (Fig. 9).

Nat. Prod. Rep., 2004, 21, 752–772 761


Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
Downloaded by University of Sussex on 22 July 2012
Fig. 9 Nineteen new members and selected compounds of miscella-
neous group; * indicates a new compound.
3 Bioactivities of Lycopodium alkaloids
Qian Ceng Ta (the whole plant of Huperzia serrata (Thunb)
Trev.)9,10 and other species of Huperziaceae and Lycopodiaceae
(Lycopodium s. l., club mosses), have a long history of use in
Chinese folk medicine for the treatment of contusions, strains,
swellings, schizophrenia, myasthenia gravis and organophos-
phate poisoning.183 In addition, in vitro and in vivo pharma-
cological studies have demonstrated that Lycopodium alkaloids
762 Nat. Prod. Rep., 2004, 21, 752–772
View Online
Fig. 10 Some drugs used to treat Alzheimer’s disease.
produce definite effects in the treatment of diseases that affect the
cardiovascular or neuromuscular systems, or that are related to
cholinesterase activity. These alkaloids have been shown to have
positive effects on learning and memory.7,10,184 The most potent
of these is huperzine A (HupA) 35. Since HupA was discovered
in the 1980s, it has been extensively evaluated by the Chinese for
bioactivity, especially for activity toward cholinesterases and
for treatment of Alzheimer’s disease (AD). HupA has been
found to be a potent, reversible and selective acetylcholinesterase
inhibitor (AChEI).7,8,185 HupA crosses the blood–brain barrier
smoothly, and shows high specificity for acetylcholinesterase
(AChE) with a prolonged biological half-life.3 In fact, HupA is
able to penetrate the blood–brain barrier better, has higher oral
bioavailability, and possesses longer duration of AChEI action
than galantamine, tacrine, rivastigmine or donepezil, all drugs
approved for use in treating AD (see below). However, all of
the other Lycopodium alkaloids identified so far have either
not shown any AChE inhibition activity or possessed activity
that is significantly lower than that of HupA.2,169 HupA has
been approved as the drug for treatment of AD in China, and
is marketed in USA as a dietary supplement (as powdered H.
serrata in tablet or capsule format).
3.1 Treatment of Alzheimer’s disease
There is considerable evidence that the memory deficits as-
sociated with AD are due to impairment of cholinergic neu-
rotransmission in the central nervous system.186 Therefore,
cholinergic enhancement strategies have been the focus of many
treatment strategies. As a result, four drugs that target the
cholinergic system have been approved by the FDA for the
treatment of AD: tacrine, donepezil, rivastigmine, and galan-
tamine (Fig. 10). These compounds act as acetylcholinesterase
inhibitors (AChEIs), and have shown some success in alleviating
some of the symptoms associated with mild to moderate AD,
and in perhaps slowing the progression of AD.187
In 1993, tacrine (trade name: Cognex) was marketed in the
US for treatment of AD. Annual sales of tacrine reached $500
million by 1999. However, it produces some serious side effects
(liver toxicity) among >29% of patients, and has ceased to
be marketed by the producer (Warner-Lambert, now Pfizer).
Donepezil (trade name: Acricept), cleared by the FDA in late
1996, is produced by Eisai Inc. and is now marketed by Pfizer.
It appears to have better properties, both greater effect and
lower toxicity, than tacrine. Rivastigmine (trade name: Exelon),
produced by Novartis Pharmaceuticals and approved by the
FDA in 2000, also appears to have better properties than tacrine,
in that it does not appear to cause liver damage. However, it
can produce stomach-related side-effects such as nausea and
vomiting. Galantamine (also sometimes spelled galanthamine;
trade name: Reminyl), marketed by Belgium drug company
Janssen Pharmaceutical, was approved by the FDA in 2001.
Unlike the other three FDA approved AChEIs, galantamine is

Table 2 Effects of HupA and other cholinesterase inhibitors on AChE
activity in the rat cortex and BuChE activity in rat serum in vitro
IC50 a /lM
Ratio of IC50
ChEI BuChE AChE BuChE/AChE
Huperzine A 74.43 0.082 907.7
Tacrine 0.074 0.093 0.8
Physostigmine 1.26 0.251 5.0
Donepezil 5.01 0.010 501.0
Galanthamine 12.59 1.995 6.3
a
The cortex homogenate was preincubated for 5 min with iso-OMPA
0.1 mM. The rate of color production was measured spectrophotomet-
rically at 440 nm. Data from Cheng et al.195 and Wang et al.,189 and
organized by Tang and Han.190
a natural plant alkaloid, produced by Galanthus nivalis L. and
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
related plants (Liliaceae family). It is currently isolated from
daffodil bulbs by Janssen Pharmaceuticals. Galantamine has
also been used to treat symptoms of other forms of dementia,
such as vascular dementia.188
A number of other potential AChEIs have been identified
Downloaded by University of Sussex on 22 July 2012
and are being evaluated for efficacy in treating AD. Of these,
HupA, especially as its 5-Cl-O-vanillin derivative called ZT-1
and discussed in more detail below, shows particularly great
promise.
3.2 Acetylcholinesterase inhibition
As a natural product, HupA is very effective in alleviating many
of the symptoms and in potentially slowing the progression of
AD. HupA and other cholinesterase inhibitors can produce
a marked effect in inhibiting AChE, delaying hydrolysis of
acetylcholine, and enhancing the level of acetylcholine in the
synaptic cleft. These effects are thought to be the reason why
these compounds are beneficial in treating AD symptoms. HupA
causes a distinct concentration dependent inhibition in vitro of
AChE and BuChE (butyrylcholinestrase).189 The AChEI activity
(IC50 ) of HupA relative to other AChEIs is: donepezil > HupA >
tacrine > physostigmine > galantamine. However, the inhibiting
concentration of HupA to BuChE is much higher than that
to AChE (Table 2),190 i.e. it is much more selective than these
other compounds in its action. All of these AChEIs display a
dose dependent inhibition of cerebral AChE when administered
orally, but HupA demonstrates the highest in vivo activity when
administered in this manner.190 Moreover, when the effects of
HupA, donepezil and rivastigmine on cortical acetylcholine
levels and acetylcholinesterase activity in rats were compared, it
was found that HupA was 8- and 2-fold more potent in molar
terms, respectively, than donepezil and rivastigmine in increasing
cortical acetylcholine levels; and the effect of HupA was longer-
lasting.191 Furthermore, the inhibition of BuChE by tacrine is
significantly higher than that by donepezil, HupA, or the other
AChEIs (Table 2). Tacrine has the most obvious and severe side-
effect among these drugs, suggesting that BuChEI activity may
contribute to these side-effects. Thus, a high BuChE/AChE IC50
ratio is very desirable in AD drugs that target the cholinergic
system.
Structural biology investigations (particularly by X-ray crys-
tallography and computational modeling) have found that
HupA acts against AChE by directly binding to the opening
of the active site in this enzyme, thus preventing access to
the active site by the normal substrate.192 Kozikowski and co-
workers hypothesized that the three-carbon bridge substructure
of HupA was the prerequisite structure for the AChEI activity,
and that the activity would be lowered if the double bond was
eliminated from this portion of the molecule.193 The X-ray crystal
structure of the (−)-huperzine A-AChE complex showed that
this bridge in HupA was inserted into the hydrophobic area of
AChE surrounded by aromatic residues.192 The other AChEIs
View Online
function in a similar manner, but the HupA–AChE complex
has a longer half-life than these and other prophylactic agents.
As a result, and because of its very low incidence of only mild
side-effects in humans, HupA has now also been proposed as
a pretreatment drug for potential exposure to organophosphate
nerve agents194 in addition to its role in treatment of AD.
3.3 Effect on learning and memory function
Based on investigations with animal models (particularly with
mice), HupA has been shown to enhance learning and mem-
ory. This was found to be true for adult mice, aged mice,
and mice with cognition damage. Injecting HupA into the
abdominal cavity inhibits memory damage in mice treated
with cycloheximide, erinitrit, and scopolamine.196 This treatment
also promotes memory retention in aged mice. When mice
treated with HupA were evaluated by the escaping reflection
test, learning and memory were both enhanced by abdominal
cavity injection and by oral administration.196,197 HupA can also
improve performance speed in the reaction of moving avoidance
test.196
HupA has also been evaluated in recent years with other
animal models for effects on cognitive function. HupA improved
the spatial working memory in aged monkeys and in young adult
monkeys that had experimental cognitive impairment via an
adrenergic mechanism.198,199 HupA attenuated cognitive deficits
and brain injury in neonatal rats after hypoxia-ischemia and
cognitive dysfunction and neuronal degeneration in rat caused
by b-amyloid protein-(1-40).200–202 HupA reverses scopolamine-
and muscimol-induced memory deficits in chicks.203 It also
showed a memory enhancing effect, limited to the first day
in guinea pigs with certain dosages of HupA, and induced no
deleterious effects on spatial memory.204 Furthermore, Wang
and Tang,205 after comparing the effects of HupA to E2020
(donepezil) and tacrine on scopolamine-induced working and
reference memory errors in rat, concluded that HupA more
closely fit the established criteria for an AChE inhibitor to be
further evaluated in clinical studies.
3.4 Neural cell protection
The degree of cholinergic cell loss in the central nervous
system relates to the level of neuropathologic alteration, memory
impairment and cognitive lesion.206–209 AChE and choline levels
in patients’ cerebrospinal fluid have a close relationship to
the severity of dementia.210–213 Recent studies have shown that
HupA possesses additional pharmacological actions other than
affecting the hydrolysis of synaptic ACh described directly
above.198,214–218 These non-cholinergic roles include, for instance,
an antagonist effect on the NMDA (N-methyl-D-aspartate)
receptor and the protection of neuronal cells against Ab (b-
amyloid peptide), free radicals and hypoxia-ischemia induced
injury.201,218–222 These roles could also be important in AD
treatment.
3.5 Structure–activity relationships
Because it possesses properties of high anti-AChE activity
and high selectivity, HupA has become a focus of research in
recent years. To elucidate the structure–activity relationships
and search for new analogs or derivatives of HupA with
higher activity and selectivity has been a major goal recently
in AChEI research. Effort by several groups has been directed
towards preparing structurally simplified analogs of HupA and
derivatives of HupA, which may possess higher activity, longer
duration of action, less toxicity, and could be prepared by
simpler and more efficient methods when compared to HupA
itself.
The stereoselectivities of the inhibition of rat cortical AChE by
two enantiomers of HupA were first determined by McKinney
et al.223 (−)-Huperzine A was the more potent enantiomer with a
Nat. Prod. Rep., 2004, 21, 752–772 763
 View Online

K i value of 8 nM. (+)-Huperzine A inhibited the enzyme 38-fold

less potently with a K i value of 300 nM. Racemic huperzine A
was about two-fold less potent than the more active isomer, (−)-
huperzine A. Another comparison of the inhibitory effects of
two enantiomers and racemate of HupA on rat brain cholinergic
function in vitro and in vivo was made by Tang et al.224 The
synthetic racemic mixture (±)-huperzine-A was 3 times less
potent than (−)-huperzine-A in vitro (IC50 s of 3 × 10−7 M
and 1 × 10−7 M, respectively) because the former consisted
of a racemic mixture of the compound in which the (+)-
huperzine component was considerably less potent (IC50 = 7 ×
10−6 M).224 The computer-generated superposition of HupA and
acetylcholine (ACh) suggested that HupA possessed the basic
structural features of ACh.186 It is apparent that a reasonable
structural similarity can be found between the nitrogen, oxygen
and carbonyl in ACh with the corresponding amino nitrogen,
nitrogen and carbonyl in HupA. Therefore, the 5-aminomethyl-
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N

2(1H)-pyridone part of HupA may constitute a pharmacophoric

moiety.186,225,226
Because of the rigidity of the bridged ring structure, structure
modification investigations first focused on more readily altered
portions of HupA. Alterations that used a benzyl ring,227
pyramine ketone ring,228 hydroxybenzene, benzcatechin,229 or
Downloaded by University of Sussex on 22 July 2012

similar structures instead of the pyridone ring (ring A) of

HupA significantly lowered the bioactivity. Other investigations
showed that the three-carbon bridge ring and its double
bond,193 the methyl of the bridge,192,230 and the exocyclic double
bond are required for high anti-AChE activities. Elimination Fig. 11 Structures of simplified analogs of huperzine A.
or substitution of these structural features by other groups
causes the activity to drop dramatically. Activity was almost
completely lost when aminomethyl, hydroxymethyl, azide or
similar groups were introduced to the amido position of
HupA.2
Synthesis of analogs with the entire basic skeleton of HupA
is not easier than the total synthesis of HupA, which is in
itself a difficult task.2 This is a major limitation for application
of HupA or analogs as drugs. To get around this limitation,
simpler analogs were designed and prepared. All of these
possess the supposed pharmacophoric moiety of HupA: substi-
tuted 5-aminomethyl-2(1H)-pyridones 138,225,231 substituted 5-
amino-5,6,7,8-tetrahydroquinolinones 139a–d, 140,186,193,232 and
5-substituted aminoquinolinones 141.186 The single ring analogs
142a–d186 and several bridged ring analogs 143a–c186,233 derived
from HupA by the removal of the bridge and the opening of
pyridone ring were also prepared (see Fig. 11).
However, all of these structurally simplified analogs were
found to be inactive or less active in the inhibition of AChE. This
means that the comformational constraints, hydrophobic bind-
ing, and steric and electrostatic fields provided by the unsatu-
rated bridge and fused pyridone ring in HupA must be involved
in its AChE inhibitory activity.186
These experiments also demonstrated that HupA is a very
compact structure. Apparently, none of the structural ele-
ments of the HupA molecule can be removed if full activity
is to remain. Kozikowski and co-workers also modified C10 Fig. 12 Structures of some effective analogs of huperzine A.
(i.e. C6 according to the carbon numbering system for the
Lycopodium alkaloids) of HupA, as described in the next of HupA increased the potency for AChE inhibition by a factor
section.234–236 of 8; the corresponding equatorial isomer was about 1.5-fold
less active than HupA. The introduction of substituents larger
than methyl resulted in a drop in activity. For example, the
3.6 Structure modification and new drug candidates against AD
ethyl analog was found to be about 100-fold less active than
In the interest of looking for drugs against AD that are even HupA. Clear evidence that the C-10 axial methyl group points
more effective than HupA, many analogs of HupA have been into a hydrophobic region of the enzyme, while the equatorial
prepared. Among these, only a very few compounds have methyl group is directed to a less favorable hydrophilic region,
obvious AChEI activity. Those that do have this activity include was obtained through the use of molecular modeling methods
H-1, H-2, H-3, huprine X & Y, isovanihuperzine A and ZT-1 involving the docking of these analogs to the reported X-ray
(Fig. 12). crystal structure of Torpedo AChE. This enzyme has a small
H-1 [(±)-10,10-dimethylhuperzine A] is a substituted analog cavity that can just fit a methyl group.192 Substituents larger
with two methyl groups in the C-10 position of HupA.234 than methyl were found to result in a conformational energy
Introduction of an axial methyl group into the C-10 position penalty.236 Compound H-2 [(−)-10-spiro-cyclopropyl-huperzine

764 Nat. Prod. Rep., 2004, 21, 752–772


A] has a cyclopropane group at C-10. Its activity in vitro was
similar to that of (−)-huperzine A (natural HupA).236 H-3 is a
compound with the structure of tacrine and the possible effective
structural essence of HupA (A and B rings) connected by an
alkane tether. H-3 also has good AChEI activity, but much less
selectivity than HupA.237 Huprine X and Y are analogs that
combine tacrine with the bridgehead ring of HupA. They have
much better anti-AChE activities than HupA.238
The laboratory of Professor Dayuan Zhu (China) has pro-
duced a large number of HupA analogs and derivatives, and
has a large collection of other Lycopodium alkaloids. Isovani-
huperzine A, selected from their collection of Schiff bases at the
HupA amino group, was found to have activity close to that of
HupA in some indexes.239 But isovanihuperzine A has a stability
problem. ZT-1 was finally selected by Professor Zhu’s lab from
over 100 HupA derivatives as the most efficacious. ZT-1 is a
Schiff base made by a condensation reaction between HupA
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
and 5-Cl-O-vanillin. This semi-synthesis pathway only requires
two steps and the materials are readily available, inexpensive,
and easily prepared. Patents in China, Japan, the USA, Europe
and internationally, for the synthesis and application/use of ZT-
1, have been granted to and/or applied for by Professor Zhu.
An application has been made to the FDA for the use of ZT-1
Downloaded by University of Sussex on 22 July 2012
to treat AD.
Experimental data demonstrated that ZT-1 possesses AChEI
activity similar to HupA. However, it has more selective
inhibition on AChE as well as less toxicity in mice than
HupA. ZT-1 has similar properties to HupA regarding the ability
to cross the blood–brain barrier, its oral bioavailability, and its
longevity of action. Time course analysis of in vivo inhibition of
cholinesterases (ChEs) after infusion of ChEIs to mice and of
ACh levels in the cerebral cortex of mice after administration
of ChEIs also demonstrated that ZT-1 had better properties
than other ChEIs. These data demonstrate that ZT-1 is a
promising candidate for clinical development as a symptomatic
treatment for AD. This information about ZT-1 was obtained
from Professor Zhu by personal communication.
4 Clinical trials
Clinical trails with Lycopodium alkaloids have been carried
out since the 1990s, after the anti-AChE activity of HupA was
detected. Many reports on clinical trials of HupA and ZT-1 have
been published in recent years.
4.1 Clinical trials to evaluate the efficacy and safety of
huperzine A
Clinical trials performed with HupA have demonstrated that
HupA produces significant improvements in memory deficien-
cies in aged and AD patients. Most of these studies have been per-
formed in China, where an estimated 100 000 people have been
treated by HupA.240 Results of these studies indicate that HupA
is an effective and safe drug that improves cognitive function.
For example, Zhang et al. conducted a randomized, placebo-
controlled, multicenter study with 202 patients diagnosed with
possible or probable AD.241 One group of 100 patients was
administered 400 lg day−1 HupA for 12 weeks and 102 patients
received placebo. The treatment group displayed improvements
in cognition measured on the AD Assessment Scale (ADAS-
Cog) as well as an increase in the ability to do activities of
daily living (ADL) and improvement in behavior and mood
(ADAS non-Cog). Mild and transient adverse events (edema of
bilateral ankles and insomnia) were observed in 3% of HupA
treated patients. After observing about 200 cases of AD and
benign senile hypofunction patients in clinical research, it has
been found that both oral administration and muscle injection
of HupA can lessen the symptom of patients and enhance their
memory function.242
In addition, HupA may have application for younger people
as well. Sun and co-workers243 reported that HupA enhanced the
View Online
memory and learning performance of adolescent students. With
a double blind and matched-pair method, 34 pairs of junior
middle school students complaining of memory inadequacy
were divided into two groups. The memory quotient of the
students receiving HupA was higher than those of the placebo
group, and the scores on Chinese language lessons in the treated
group were also markedly elevated.
Xu and co-workers also evaluated AD patients.244 Sixty AD
patients were divided into two groups taking HupA (200 lg twice
a day orally for 60 days) in either capsules or tablets, respectively.
There were significant differences on all the psychological
evaluations between “before” and “after” the 60 days trials for
the two groups. No severe side effects except mild to moderate
nausea were observed. No difference was observed between the
two groups.
Ma et al. reported double-blind clinical trials of HupA
on cognitive deterioration in 314 cases of benign senescent
forgetfulness, vascular dementia and AD.245,246 The first trial was
conducted by the double blind method on 120 patients of age-
associated memory impairment with memory quotients (MQ)
< 100 based on the Wechsler Memory Scale (WMS).245,246 The
dose was 0.03 mg intramuscularly twice daily for 14–15 days. The
effective rates were 68.3% and 26.4%, respectively, in the treated
and control groups. No significant side effects were observed.
The second trial was conducted on 88 patients with cognitive
deterioration, with MQ (WMS) < 100. The mean values of MQ
of 44 treated and 44 controls were 82.8 ± 14.3 and 81.5 ± 14.4
(p > 0.05), respectively. The dose was 0.1 mg HupA four times
a day orally for 14–15 days. The mean values of MQ after the
treatment (the interval between pre- and post-tests of WMS with
A and B form, respectively, is 2 months) of treated and controls
were 93.5 ± 14.5 (p < 0.01), and 85.5 ± 16.5 (p < 0.01). The
effective rates for the treated and control groups were 68.18% and
34.09%, respectively. No significant side effects were observed
except for minor complaints of gastric discomfort (2 instances),
dizziness (1 instance), insomnia (1 instance) and mild excitement
(1 instance) in the treated group. None of these symptoms led to
withdrawal from the trial.
Another trial of 25 cases with vascular dementia and 55
cases with AD was conducted with a dose of 0.1 mg HupA
four times a day orally for 14–15 days.245,246 The memory quotient
increase of the treated group was significantly higher than that
of the control. The effective rate of the treated group was 60%,
significantly higher than the control (35%). No marked side
effects were observed.
In the United States the safety and efficacy of HupA were
evaluated in 26 patients meeting the DSM IV-R (Diagnostic
and Statistical Manual of Mental Disorders - Fourth Revision)
and the NINCDS-ADRDA (National Institute of Neurological
and Communicative Disorders and Stroke) criteria for uncom-
plicated AD and possible or probable AD.247 This study (office-
based) lasted 3 months and was open label. Other therapies,
including tacrine, donepezil and G. biloba were continued. An
oral dose of 50 lg HupA was given twice a day to 22 patients, and
the 4 other patients received a dose of 100 lg twice daily. A mean
dementia baseline score of 22.6 was measured with the MMSE
(Mini-Mental State Examination). The changes in this score, for
the 50 lg group and for the 100 lg group, respectively, were 0.5
and 1.5 points at 1 month; 1.2 and 1.8 points at 2 months, and 1.1
and 1.0 points at 3 months. Despite the small number of patients,
the authors observed dose-related improvements with higher
MMSE scores at higher dosage, and no serious side effects.
Thus, it appears that HupA is more effective and safer
than other drugs that affect the cholinergic system and that
are currently on the market in the USA for the treatment of
mild to moderate AD. Because HupA was discovered in China
during the years just after the Cultural Revolution, however,
no patents were filed and all of this information resides in the
public domain, rendering HupA non-viable as a commercially
developable drug.
Nat. Prod. Rep., 2004, 21, 752–772 765
 View Online

4.2 Clinical Trials with ZT-1

Clinical trials were performed with ZT-1 to determine its efficacy
and safety in treating AD, and to see if it behaved the same as
HupA in human subjects. These clinical trials were initiated
in China and in Europe. The following information was also
obtained from Professor Zhu by personal communication.
Phase I trials in China were performed from 2002 to 2003 and
were designed to evaluate tolerance to dosage level and to study
pharmacokinetics. These investigations have been completed,
with very encouraging results. At the same time, four phase I
clinical trials were carried out in Europe. The goals of these
studies were to evaluate: (1) the inhibition of AChE and BuChE
by ZT-1; (2) antagonism of cognitive impairment caused by
Fig. 13 Picture of Huperzia serrata (Thunb. ex Murray) Trev. growing
scopolamine; (3) pharmacokinetics; and (4) safety. These four in the wild. Note that these plants are over 15 years old (at least) and yet
phase I studies demonstrated that administration of ZT-1 to reach a height of less than 10 cm.
humans appears to be safe and well tolerated. The incidence
of possible drug-related adverse events, in particular nervous methods to propagate these plants. Thus far, however, as
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N

system and gastrointestinal symptoms, was similar to placebo.
An international multicenter phase II trial to determine optimal
dose and to more fully assess efficacy, in mild to moderate AD
patients, is now underway.
Downloaded by University of Sussex on 22 July 2012
mentioned above, no successful mass production method (either
via traditional cultivation practices or via tissue culture) has been
developed for any Lycopodium or related species. Cultivation
of these plants is very difficult. Wild collected plants that are
transferred to greenhouses or other growth environments grow

5 Sources of HupA very slowly and do not survive for more than a few months, with
no substantial increase in biomass. Because of this, tissue culture
Availability and acquisition of raw materials for the production
(in vitro propagation) of these plants has attracted attention in
of HupA are currently the bottleneck in new drug development
recent years. Many institutes and universities in China, Hong
and production from HupA derivatives. Availability of both
Kong, the USA, France, Germany, Japan and South Korea have
HupA and ZT-1 depend on the availability of raw plant
attempted to reproduce these species by these means, with no
material, because HupA cannot be chemically synthesized in
success. No breakthroughs in propagation of these plants have
an industrially feasible manner. Thus, natural plant tissues are
been previously reported.
the only realistic source of HupA in the foreseeable future.

5.1 Natural sources

HupA is only found in a small group of closely related plants
belonging to the Huperziaceae. These plants are a diminishing
and limited natural resource. H. serrata, a moss-like small herb,
was the source for the original identification of HupA.10 HupA
is also produced in other species of Huperziaceae, which have
close taxonomic relationship with H. serrata.26 As mentioned
in Section 1, Huperziaceae was taxonomically separated from
Lycopodium s. l. by Rothmaler in 1944. Lycopodium sensu stricto
species do not produce HupA or derivatives of it. The Huperzi-
aceae comprises two genera, Huperzia and Phlegmariurus, with a
total of about 150 species. The distribution of this plant family is
global, but these plants are found in relatively greater abundance
in tropical habitats of America. Nevertheless, these plants are not
abundant, and are only found in very specialized habitats.
Furthermore, H. serrata possesses a very low content of HupA
(ca. 0.007%) and grows very slowly. It takes at least 15 years
from spore germination through the gametophyte stage to finally Fig. 14 Developing Phlegmariurus squarrosus (Forst.) Löve et Löve
reach the mature sporophyte, the plant body that is harvested sporophyte propagated in vitro from spores. The plants are white because
for HupA collection. The sporophytes of H. serrata only reach they were grown in total darkness. Transfer to light induces chloroplast
a height of a few centimeters (see Fig. 13). Because of the low differentiation and greening in cultured plants. However, because of the
abundance of HupA in these small plants, the demand for HupA high levels of HupA produced by the plants under these conditions, we
may find that growth in the dark is most desirable.
will soon lead to the decimation of all wild populations of H.
serrata and related species. So far, no successful mass production
One of us, Ma, tried a great of variety methods to realize our
has been reported either for the cultivation or the tissue culture of
aim to propagate in vitro H. serrata and taxonomically related
these plants, although a number of laboratories around the world
species that contain HupA. These experiments were performed
have attempted to either introduce these plants into cultivation
in the laboratory of Professor Dr E. Wellmann (Germany).
or propagate them via in vitro methods. Finding a useful method
H. serrata and several closely related species containing HupA
for solving this problem will be a significant contribution to
were selected as raw materials for these experiments. Finally
the treatment of AD, because it will allow for the large scale
and fortunately (perhaps luckily), we succeeded in propagating
and renewable production of HupA, which can then serve as a
Phlegmariurus squarrosus (Forst.) Löve et Löve. Spores of
precursor for economical production of ZT-1.
Ph. squarrosus were treated by an optimized special method
(patent is being applied for in Germany by Wellmann and Ma),
5.2 In vitro propagation as a source of HupA
leading two to four months later to germination. Gametophytes
Because of the significant health benefits of HupA (and now its (prothalli) and mature sporophytes (Fig. 14) were successfully
derivative ZT-1), Huperzia and other Lycopodium s. l. species produced from in vitro cultures of Ph. squarrosus after six months
have been the targets of investigations seeking to develop to one years of culture. These cultured plants are now growing

766 Nat. Prod. Rep., 2004, 21, 752–772


well, and will provide an invaluable resource for production
of HupA, precursor for ZT-1, as they accumulate HupA at
levels that are significantly higher than found in H. serrata
(Ma and Gang, unpublished results). The potential economic
and health benefits of these plants cannot be stressed enough.
In addition, the ability to culture these plants now allows
for investigations into the biosynthesis of the Lycopodium
alkaloids.
6 Taxonomic history and chemotaxonomy
For a many years, species of Lycopodiales were put in the
sensu lato genus Lycopodium of Lycopodiaceae. Lycopodiaceae
was considered to comprise two genera: Lycopodium and
Phylloglossum. The former is distributed all over the world,
although mainly in tropical areas, while the latter is a single
species genus only occurring in Australia, Tasmania, and New
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
Zealand. Lycopodium was named by Linnaeus in 1753. In 1887,
Baker248 divided Lycopodium into 4 subgenera. Herter proposed
in the early part of the 20th century a two genera system in-
cluding Lycopodium and Urostachys.249,250 Two separate families,
Lycopodiaceae and Urostachyaceae, were set up by Rothmaler25
in 1944 and based on differences in the prothallus. Under
Downloaded by University of Sussex on 22 July 2012
Rothmaler’s system, three genera (Lycopodium, Diphasium and
Lepidotis) belonged to Lycopodiaceae. Urostachys was replaced
by Huperzia in his system. In 1964, Holub251 published two novel
genera: Lycopodiella and Phlegmariurus. A system with three
families (Huperziaceae, Lycopodiaceae and Phylloglossaceae)
was proposed by him in 1975. 252 The first family, contained 2
genera (Huperzia and Phlegmariurus), the second had 7 genera
and the third contained a single genus, Phylloglossum. A study by
Wilce253 based on an examination of lycopod spores suggested
that one family, Lycopodiaceae, and two genera, Lycopodium
and Phylloglossum, are adequate to describe the order. The genus
Lycopodium may be divided, according to Wilce, into subgenera,
Urostachys (containing three sections, Cernua, Inudata, and
Lateralia) and Lycopodium, containing seven sections. In 1978,
a taxonomic system with two families (Huperziaceae and
Lycopodiaceae) based on species of Lycopodiales in China
and founded on Holub’s system (of 1975) was proposed by
Ching.254 The former family included 2 genera (Huperzia and
Phlegmariurus) and the latter had 6 genera. Holub published
a new system20 in 1985 with two families (Huperziaceae and
Lycopodiaceae), the former with only one genus, i.e. Huperzia,
and the latter with 10 genera. Ollgaard proposed another system
with only one family Lycopodiaceae and four genera Huperzia,
Lycopodium, Lycopodiella and Phylloglossum.22
The taxonomic system of the Lycopodiales is still not fixed.
Nevertheless, the most popular systems used in recent publica-
tions are from Ching,254 Holub,20 and Ollgaard.22 Furthermore,
since 1960, several articles on chemotaxonomy in the Lycopo-
diales have been published. Some results are useful for a natural
system of Lycopodiales.
Flavones are common in ferns. Voirin255,256 carried out a
chemotaxonomic study on the Lycopodiales in 1967. His
results shown that chrysoeriol was a common constituent in
Lycopodiales. Lycopodium plants usually contained chrysoeriol,
while some also had luteolin. Diphasium contained chrysoeriol
and apigenin. Lepidotis contained chrysoeriol, luteolin and
apigenin. Instead of these three compounds, Huperzia contained
selahin and tricin. Voirin pointed out that possession of these
compounds was a primitive character of the Lycopodiales.255
In 1965, Towers and co-workers257 analyzed phenolic acids
and lignins in 21 species of Lycopodiales. They found that
Lycopodium and Diphasium had the same constituents and were
easy to distinguish from Huperzia and Lepitotis, based on these
chemical differences. Braekman and co-workers41,258 discussed
the alkaloid content of the Lycopodiales and the relationships
of chemical constituents to the botanical classification of 40
species. In these investigations, they evaluated the occurrence of
View Online
8 standard compound types: cernuane, lucidulane, phlegmarane,
fawcettidane, lycodane, lycopodane, inundatane and lucidane.
Their results suggested that Huperzia (= Urostachys) and
Lycopodiella s. l. (= Lepidotis, excl. L. deuterodensum) should be
separated from Lycopodium. In addition, Groupe fastigiatum of
Sect. Lycopodium and Sect. Complanata (= Diphasiastrum) were
different, based on chemical profiles. They drew the conclusion
that the system of Wilce253 was reasonable for the Lycopodiales.
Tsuda and co-workers153,155,259 investigated the chemo-
taxonomic relationship among 17 species and 3 vari-
eties of Lycopodium s. l. using: a-onocerin; 13 new
triterpenes (16-oxoserratenediol, 16-oxo-21-episerratediol, 16-
oxodiepiserratenediol, 16-oxoserratriol, 16-oxolycoclavanol, ly-
cocryptol, 21-epilycocryptol, diepilycocryptol, lyclaininol, ly-
clanitin, 16-oxolyclanitin, lycernuic acid-A and lycernuic acid-
B); 2 new compounds: glycosides of serratenediol and tohogenol;
and 1 bisnortriterpenoid: clavatol. They found differences in
chemical constituents between L. complanatum from Hokkaido
and Taiwan. They found that a-onocerin was present in plant
material from Hokkaido, but absent from the material from
Taiwan. They found that some species such as L. fargesii
and L. fordii had similar profiles of triterpenes. And, they
proposed that these two species should have a close taxonomic
relationship.
One of the authors, Ma, and co-workers26 studied the relation-
ships between Huperzia and related genera by chemotaxonomic
analysis. Huperzia (18 species, 1 variety and 2 formas) and its
related genus Phlegmariurus (8 species), Lycopodium (3 species),
Lycopodiella (1 species), Palhinhaea (1 species), Diphasiastrum
(2 species), and Lycopodiastrum (1 species) were examined by
thin layer chromatography for the presence of 10 reference
Lycopodium alkaloids: HupA 35, huperzine B 36 (see Fig. 4),
serratine 96, serratinine 95 (Fig. 7), lycopodine 4, lycodine 3
(Fig. 1), annotinine 28, lycodoline 33, lucidioline 34 (Fig. 3),
and cernuine 109 (Fig. 9). The results of this study indicated
that chemical characters could be used to distinguish the various
genera from one another. According to their results, Ching’s
system seems to be appropriate for the taxa of Lycopodiales (or
Lycopodiaceae) occurring in China. Ma used random ampli-
fied polymorphic DNAs (RAPDs) to evaluate the systematic
relationship of 35 species of Huperzia and related genera.260
Results from this investigation supported the chemotaxonomic
investigation.
Recently, Wikstrom and Kenrick14 estimated divergence times
in the Lycopodiaceae (Lycopsida) from rbcL gene sequences.
They used nonparametric rate-smoothing to draw conclusions
about the evolution of 64 species of different taxa, mainly
from Lycophytina, 10 species of Huperzia, 14 species from
Lycopodium, 7 species of Lycopodiella, 18 species of Selaginella,
1 species of Phylloglossum, and a few species of Gymnosperms.
Their results demonstrated that Huperzia was obviously distinct
from Lycopodium.
7 Biosynthesis
As mentioned in the introduction, very few biosynthetic studies
have been performed with Lycopodium alkaloids. The main
reason for this lack of knowledge on the biosynthesis of
these important compounds, as outlined above, is that the
club mosses have not yielded to cultivation and culture until
very recently (see section 5.2). Although no investigations have
been reported that have attempted to identify the biosynthetic
pathway leading directly to HupA, other Lycopodium alkaloids,
particularly lycopodine, have been the subject of this type of
investigation.51,164,171,179,261–274 No enzymes have been identified in
the Lycopodiaceae that might be involved in the production of
the Lycopodium alkaloids. One enzyme (lysine decarboxylase)
has been proposed as the entry point enzyme into the pathway(s)
to these compounds.275 However, work on this enzyme has only
been performed in nonrelated taxa.276 Nevertheless, the feeding
Nat. Prod. Rep., 2004, 21, 752–772 767

Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
Downloaded by University of Sussex on 22 July 2012
Fig. 15 Proposed biosynthetic pathway to pelletierine and 4PAA,
precursors of Lycopodium alkaloids, such as HupA.
experiments that have been performed have laid a very good
framework for future work that will seek to identify the enzymes
Fig. 16 Proposed biosynthetic pathway from pelletierine and 4PAA to HupA and related Lycopodium alkaloids.
768 Nat. Prod. Rep., 2004, 21, 752–772
View Online
(and corresponding genes) that catalyze key transformations in
the biosynthesis of HupA and other Lycopodium alkaloids.
7.1 Proposed pathways
Despite a lack of convincing direct biochemical evidence, a
number of potential biosynthetic pathways to several of the
Lycopodium alkaloids have been proposed over the years. These
are described in detail in reviews by Ayer,51,178,181 MacLean,277
Blumenkopf,278 and Hemscheidt.275 Originally, these hypothe-
ses were based on the identification of new members of
the respective classes of Lycopodium alkaloids that were
thought to represent putative intermediates in hypothesized
pathways.164 Later, feeding experiments that sought to identify
pathway intermediates were conducted, mostly in the laboratory
of Professor Ian Spenser, at McMaster University, Ontario,
Canada.264–266,272,274,279,280 In these experiments, 14 C- and 13 C-
labeled precursors were fed to shoots of Lycopodium species
growing in their natural habitat in the wild in Northern Ontario,
Canada. Such an approach was necessary because these plants
could not be cultivated at that time. A few days (usually one
to five days) after application of the radio- or stable isotope-
labeled putative precursor, the shoots of the plant were harvested
and these tissues were then analyzed for the incorporation
of label into end product alkaloids or into potential pathway
intermediates. Although difficult to perform, these experiments
produced some interesting results that led to several conclusions
about the biosynthetic pathway that produces the Lycopodium
alkaloids (including HupA), see Figs. 15 and 16.
First, feeding studies with lysine demonstrated that the entry
point into the pathway is indeed through the decarboxylation of

lysine (by lysine decarboxylase, enzyme A) to form cadaverine.
Second, cadaverine is then transformed via 5-aminopentanal to
D1 -piperideine,276 by the action of enzyme B (probably diamine
oxidase). In the meantime, two molecules of malonyl-CoA
are condensed by a ketosynthase type enzyme (C) to form
acetonedicarboxylic acid (or its bisCoA ester). D1 -Piperideine
is then coupled to acetonedicarboxylic acid (or its bisCoA
ester) to form 4-(2-piperidyl) acetoacetate (4PAA) (or 4-(2-
piperidyl) acetoacetyl-CoA, 4PAACoA),279 via the action of
unknown enzyme D. 4PAA/4PAACoA is then decarboxylated
(4PAACoA is perhaps hydrolyzed first) by unknown decarboxy-
lase E to form pelletierine, the first general intermediate to
Lycopodium alkaloids.275,279 Pelletierine and 4PAA/4PAACoA,
or some derivatives thereof (perhaps with the ketones reduced)
are then coupled (see Fig. 16), accompanied by requisite decar-
boxylation, by an unknown enzyme(s), to form phlegmarine, the
second general intermediate to all Lycopodium alkaloids.171,275
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
Lycopodine has been proposed to be a central intermediate in the
formation of other classes of Lycopodium alkaloids. However,
current evidence suggests that phlegmarine is likely to be this
intermediate. For the formation of HupA, the following scenario
can be envisioned. After oxidative ring closure of phlegmarine
to form lycodane (by an enzyme likely to be similar to berberine
Downloaded by University of Sussex on 22 July 2012
bridge enzyme281 ), other oxidative modifications, presumably all
catalyzed by cytochrome P450s or 2-oxoglutarate-dependent
dioxygenases, lead through a series of precursors to HupA
(Fig. 16). Interestingly, all of these proposed intermediates have
been identified in H. serrata and related species. Even more
interesting and significant, however, is that we have been able to
identify most of the compounds (huperzine A, huperzine B,
des-N-methyl-b-obscurine, a-obscurine and b-obscurine) that
occur near or at the end of the proposed pathway in extracts
from in vitro propagated callus and regenerated Huperziaceae
plants (Ma and Gang, unpublished). Fortunately, as described
above, we have now succeeded in propagation of Huperziaceae
plants in vitro. This will enable the tools of modern biochemistry
and genomics to be brought to bear on this area of research
and allow the identification of specific enzymes and (hopefully)
the elucidation of the entire pathway(s) to the Lycopodium
alkaloids.
8 Acknowledgements
We wish to thank Professor Dr E. Wellmann for the photo-
graph of the developing Phlegmariurus squarrosus sporophyte,
Professor D. Zhu for information on ZT-1 and clinical trials,
and Professor F. Leeper for critical review of the manuscript.
9 References
1 W. A. Ayer and L. S. Trifonov, Alkaloids (Academic Press), 1994,
45, 233–266.
2 C.-H. Tan and D.-Y. Zhu, Zhongguo Tianran Yaowu, 2003, 1, 1–7.
3 H. L. Jiang, X. M. Luo and D. L. Bai, Curr. Med. Chem., 2003, 10,
2231–2252.
4 K. Bödeker, Justus Liebigs Ann. Chem., 1881, 208, 363.
5 O. Achmatowicz and W. Uzieblo, Rocz. Chem., 1938, 18, 88–95 (in
English 94–95).
6 Y. S. Cheng, C. Z. Lu, Z. L. Ying, W. Y. Ni, C. L. Zhang and G. W.
Sang, New Drugs Clin. Remedies, 1986, 5, 197–199.
7 X. C. Tang, Y. F. Han, X. P. Chen and X. D. Zhu, Acta Pharmacol.
Sin., 1986, 7, 507–511.
8 X. C. Tang, P. De Sarno, K. Sugaya and E. Giacobini, J. Neurosci.
Res., 1989, 24, 276–285.
9 J. S. Liu, C. M. Yu, Y. Z. Zhou, Y. Y. Han, F. W. Wu, B. F. Qi and
Y. L. Zhu, Acta Chim. Sin. (Engl. Ed.), 1986, 44, 1035–1040.
10 J. S. Liu, Y. L. Zhu, C. M. Yu, Y. Z. Zhou, Y. Y. Han, F. W. Wu and
B. F. Qi, Can. J. Chem., 1986, 64, 837–839.
11 R. W. Zhang, X. C. Tang, Y. Y. Han, G. W. Sang, Y. D. Zhang, Y. X.
Ma, C. L. Zhang and R. M. Yang, Acta Pharmacol. Sin., 1991, 12,
250–252.
12 K. Kubitzki, The families and genera of vascular plants, ed.
K. U. Kramer and P. S. Green, Springer-Verlag, Berlin, 1990.
View Online
13 D. L. Nickrent, C. L. Parkinson, J. D. Palmer and R. J. Duff, Mol.
Biol. Evol., 2000, 17, 1885–1895.
14 N. Wikstrom and P. Kenrick, Mol. Phylogenet. Evol., 2001, 19, 177–
186.
15 Y. Tanabe, M. Uchida, M. Hasebe and M. Ito, J. Plant. Res., 2003,
116, 71–75.
16 T. Matsunaga, T. Ishii, S. Matsumoto, M. Higuchi, A. Darvill, P.
Albersheim and M. A. O’Neill, Plant Physiol., 2004, 134, 339–351.
17 R. C. Ching, Acta Bot. Yunnanica, 1981, 3, 1–9.
18 R. C. Ching, Acta Bot. Yunnanica, 1982, 4, 119–128.
19 R. C. Ching, Acta Bot. Yunnanica, 1982, 4, 213–226.
20 J. Holub, Folia Geobot. Phytotaxon., 1985, 20, 67–80.
21 J. Holub, Folia Geobot. Phytotaxon., 1991, 26, 81–94.
22 B. Ollgaard, Opera Bot., 1987, 92, 153–178.
23 B. Ollgaard, Index of the Lycopodiaceae, Det Kongelige Danske
Videnskabernes Selskab (The Royal Danish Academy of Sciences
and Letters), Copenhagen, 1989.
24 B. Ollgaard, Ann. Missouri Bot. Gard., 1992, 79, 687–717.
25 W. Rothmaler, Feddes Repertorium Specierum Novarum, 1944, 54,
55–82.
26 X.-Q. Ma, S.-H. Jiang and D.-Y. Zhu, Biochem. Syst. Ecol., 1998,
26, 723–728.
27 J. A. Freeberg and R. H. Wetmore, Phytomorphology, 1957, 7, 204–
217.
28 J. A. Freeberg and R. H. Wetmore, Phytomorphology, 1967, 17,
78–91.
29 T. Dwyer and A. S. Waegel, Acta Hortic., 2004, 631, 181–185.
30 G. S. Perry and D. B. MacLean, Can. J. Chem., 1956, 34, 1189–1199.
31 I. W. Southon and J. Buckingham, Dictionary of Alkaloids, Chap-
man Hall, London, 1989.
32 W. A. Ayer, L. M. Browne, A. W. Elgersma and P. P. Singer,
Can. J. Chem., 1990, 68, 1300–1304.
33 W. A. Ayer, B. Altenkirk, S. Valverde-Lopez, B. Douglas, R. F.
Raffauf and J. A. Weisbach, Can. J. Chem., 1968, 46, 15–20.
34 W. A. Ayer, B. Altenkirk, N. Masaki and S. Valverde-Lopez,
Can. J. Chem., 1969, 47, 2449–2455.
35 W. A. Ayer and N. Masaki, Can. J. Chem., 1971, 49, 524–527.
36 R. V. Gerard and D. B. MacLean, Phytochemistry, 1986, 25, 1143–
1150.
37 W. A. Ayer and S. Dikko, Phytochemistry, 1974, 13, 653–654.
38 L. A. Loyola, G. Morales and M. Castillo, Phytochemistry, 1979,
18, 1721–1723.
39 M. Castillo, G. Morales, L. A. Loyola, I. Singh, C. Calvo, H. L.
Holland and D. B. MacLean, Can. J. Chem., 1976, 54, 2900–2908.
40 F. A. L. Anet and N. H. Khan, Chem. Ind. (London), 1960, 1238–
1239.
41 J. C. Braekman, L. Nyembo, P. Bourdoux, N. Kahindo and C.
Hootele, Phytochemistry, 1974, 13, 2519–2528.
42 R. H. Burnell and D. R. Taylor, Chem. Ind. (London), 1960, 1239–
1240.
43 W. A. Ayer, A. N. Hogg and A. C. Soper, Can. J. Chem., 1964, 42,
949–951.
44 W. A. Ayer and D. A. Law, Can. J. Chem., 1962, 40, 2088–2100.
45 R. H. Burnell, B. S. Mootoo and D. R. Taylor, Can. J. Chem., 1960,
38, 1927–1932.
46 W. J. Rodewald and G. Grynkiewicz, Bull. Acad. Pol. Sci., Ser. Sci.
Chim., 1967, 15, 579–581.
47 W. J. Rodewald and G. Grynkiewicz, Rocz. Chem., 1968, 42, 465–
474.
48 W. A. Ayer and G. C. Kasitu, Can. J. Chem., 1989, 67, 1077–1086.
49 G. Morales, L. A. Loyola and M. Castillo, Phytochemistry, 1979,
18, 1719–1720.
50 L. Nyembo, N. Kahindo, J. C. Braekman and C. Hootele, Bull. Soc.
Chim. Belg., 1976, 85, 595–604.
51 W. A. Ayer, MTP Int. Rev. Sci.: Org. Chem., Ser. One, 1973, 9, 1–25.
52 A. Bertho and A. Stoll, Chem. Ber., 1952, 85, 663–685.
53 R. H. F. Manske and L. Marion, Can. J. Res., Sect. B, 1943, 21,
92–96.
54 R. H. F. Manske and L. Marion, J. Am. Chem. Soc., 1947, 69,
2126–2129.
55 O. Achmatowicz and W. Rodewald, Bull. Acad. Polon. Sci., Cl. 3,
1955, 3, 553–555.
56 X.-T. Tong, C.-H. Tan, X.-Q. Ma, B.-D. Wang, S.-H. Jiang and
D.-Y. Zhu, Planta Med., 2003, 69, 576–579.
57 N. Miller, F. Mees and J. C. Braekman, Phytochemistry, 1971, 10,
1931–1934.
58 R. H. Burnell and D. R. Taylor, Tetrahedron, 1961, 15, 173–182.
59 W. J. Rodewald and G. Grynkiewicz, Rocz. Chem., 1977, 51, 1271–
1275.
60 C. Yang, Zhongyao Tongbao, 1981, 6, 12–16.
Nat. Prod. Rep., 2004, 21, 752–772 769

61 M. Castillo, L. A. Loyola, G. Morales, I. Singh, C. Calvo, H. L.
Holland and D. B. MacLean, Can. J. Chem., 1976, 54, 2893–2899.
62 T. Hu, R. F. Chandler and A. W. Hanson, Tetrahedron Lett., 1987,
28, 5993–5996.
63 Y. Inubushi, H. Ishii, B. Yasui, T. Harayama, M. Hosokawa, R.
Nishino and Y. Nakahara, Yakugaku Zasshi, 1967, 87, 1394–1404.
64 R. H. Burnell and B. S. Mootoo, Can. J. Chem., 1961, 39, 1090–
1093.
65 R. H. Burnell, J. Chem. Soc., Abstr., 1959, 3091–3093.
66 H. Takayama, K. Katakawa, M. Kitajima, K. Yamaguchi and N.
Aimi, Chem. Pharm. Bull., 2003, 51, 1163–1169.
67 J. C. Braekman, C. Hootele and W. A. Ayer, Bull. Soc. Chim. Belg.,
1971, 80, 83–90.
68 S. R. Johns, J. A. Lamberton and A. A. Sioumis, Aust. J. Chem.,
1969, 22, 1317.
69 C.-H. Tan, X.-Q. Ma, S.-H. Jiang and D.-Y. Zhu, Nat. Prod. Lett.,
2002, 16, 149–153.
70 W. A. Ayer, B. Altenkirk, R. H. Burnell and M. Moinas,
Can. J. Chem., 1969, 47, 449–455.
71 O. Achmatowicz and W. Rodewald, Rocz. Chem.., 1956, 30, 233–
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
242.
72 F. A. L. Anet and N. H. Khan, Can. J. Chem., 1959, 37, 1589–1596.
73 M. Curcumelli-Rodostamo and D. B. MacLean, Can. J. Chem.,
1962, 40, 1068–1070.
74 D. Y. Zhu, M. F. Huang, B. D. Wang, X. M. Kong and Y. Q. Yang,
Chin. J. Appl. Environ. Biol., 1996, 2, 352–355.
Downloaded by University of Sussex on 22 July 2012
75 B.-D. Wang, J. Wang, H.-F. Sun and D.-Y. Zhu, Youji Huaxue, 2001,
21, 606–610.
76 B.-D. Wang, S.-H. Jiang, W.-Y. Gao, D.-Y. Zhu, X.-M. Kong and
Y.-Q. Yang, Zhiwu Xuebao, 1998, 40, 842–845.
77 B. D. Wang, N. N. Teng and D. Y. Zhu, Chin. J. Org. Chem., 2000,
20, 812–814.
78 S. Yuan, R. Feng and G. Gu, Zhongcaoyao, 1995, 26, 115–117.
79 W. A. Ayer, J. A. Berezowsky and D. A. Law, Can. J. Chem., 1963,
41, 649–657.
80 B. N. Zhou, D. Y. Zhu, M. F. Huang, L. J. Lin, L. Z. Lin, X. Y. Han
and G. A. Cordell, Phytochemistry, 1993, 34, 1425–1428.
81 C. Yu, W. Shen, J. Han, Y. Chen and Y. Zhu, Yaoxue Xuebao, 1982,
17, 795–797.
82 C. Yu, W. Shen, J. Han, Y. Chen and Y. Zhu, Zhongcaoyao, 1982,
13, 15.
83 H. Morita, M. Arisaka, N. Yoshida and J. i. Kobayashi, J. Org.
Chem., 2000, 65, 6241–6245.
84 S. Tong and G. Xiang, Zhiwu Xuebao, 1984, 26, 411–415.
85 W. A. Ayer and G. G. Iverach, Tetrahedron Lett., 1962, 87–92.
86 W. A. Ayer, T. E. Habgood, V. Deulofeu and H. R. Juliani,
Tetrahedron, 1965, 21, 2168–2172.
87 F. A. L. Anet, M. Ahmad and N. H. Khan, Can. J. Chem., 1962,
40, 236–239.
88 Y. Hirasawa, H. Morita and J. i. Kobayashi, Tetrahedron, 2002, 58,
5483–5488.
89 F. A. L. Anet and et al., Tetrahedron Lett., 1964, pp. 751–757. .
90 Y. Inubushi and T. Harayama, Yakugaku Zasshi, 1982, 102, 434–
439.
91 O. Achmatowicz and W. Rodewald, Rocz. Chem., 1955, 29, 509–530.
92 Y. Inubushi, T. Harayama, T. Hibino and M. Akatsu, Yakugaku
Zasshi, 1971, 91, 980–986.
93 L. Marion and R. H. F. Manske, Can. J. Res., 1944, 22B, 137–139.
94 R. H. F. Manske and L. Marion, Can. J. Res., 1942, 20, 87–92.
95 R. H. F. Manske, Can. J. Chem., 1953, 31, 894–895.
96 H. Fuchino, H. Nakamura, Y. Toyoshima, T. Hakamatsuka, N.
Tanaka, R. C. Cambie and J. E. Braggins, Aust. J. Chem., 1998, 51,
175–176.
97 R. H. F. Manske and L. Marion, Can. J. Res., Sect. B, 1946, 24,
57–62.
98 R. H. F. Manske and L. Marion, Can. J. Res., Sect. B, 1944, 22,
53–55.
99 R. Rouffiac, Ann. Pharm. Fr., 1963, 21, 685–698.
100 L. Marion and R. H. F. Manske, Can. J. Res., 1946, 24B, 63–65.
101 D. Staerk, J. Larsen, L. A. Larsen, E. S. Olafsdottir, M. Witt and
J. W. Jaroszewski, Nat. Prod. Res., 2004, 18, 197–203.
102 Y. Inubushi, T. Harayama, M. Akatsu and H. Ishii, Chem. Commun.,
1968, 1138–1139.
103 J. Kobayashi, Y. Hirasawa, N. Yoshida and H. Morita, Tetrahedron
Lett., 2000, 41, 9069–9073.
104 S. N. Alam, K. A. H. Adams and D. B. MacLean, Can. J. Chem.,
1964, 42, 2456–2466.
105 R. V. Gerard, D. B. MacLean, R. Fagianni and C. J. Lock,
Can. J. Chem., 1986, 64, 943–949.
106 H. Morita, Y. Hirasawa and J. Kobayashi, J. Org. Chem., 2003, 68,
4563–4566.
770 Nat. Prod. Rep., 2004, 21, 752–772
View Online
107 W. A. Ayer, L. M. Browne, H. Orszanska, Z. Valenta and J. Liu,
Can. J. Chem., 1989, 67, 1538–1540.
108 G. D. Ainge, S. D. Lorimer, P. J. Gerard and L. D. Ruf, J. Agric.
Food. Chem., 2002, 50, 491–494.
109 J. S. Liu and M. F. Huang, Phytochemistry, 1994, 37, 1759–1761.
110 C.-H. Tan, X.-Q. Ma, G.-F. Chen and D.-Y. Zhu, Can. J. Chem.,
2003, 81, 315–318.
111 S. Q. Yuan and T. T. Wei, Yaoxue Xuebao, 1988, 23, 516–520.
112 S. Q. Yuan and Y. M. Zhao, Zhongcaoyao, 2000, 31, 498–499.
113 J. i. Kobayashi, Y. Hirasawa, N. Yoshida and H. Morita, J. Org.
Chem., 2001, 66, 5901–5904.
114 S. Yuan, R. Feng and G. Gu, Zhongcaoyao, 1994, 25, 453-454, 473.
115 F. A. L. Anet and C. R. Eves, Can. J. Chem., 1958, 36, 902–909.
116 Y.-C. Shen and C.-H. Chen, J. Nat. Prod., 1994, 57, 824–826.
117 W. A. Ayer and G. G. Iverach, Can. J. Chem., 1960, 38, 1823–1826.
118 P. Hu, M. L. Gross, S. Q. Yuan, T. T. Wei and Y. Q. Lu, Org. Mass
Spectrom., 1992, 27, 99–104.
119 W. A. Ayer, J. A. Berezowsky and G. G. Iverach, Tetrahedron, 1962,
18, 567–573.
120 W. A. Ayer and G. G. Iverach, Tetrahedron Lett., 1960, 19–25.
121 Z. C. Mou, Z. S. Yang, Y. Q. Lu and X. T. Liang, Acta Chim. Sin.,
1989, 47, 702–704.
122 V. Deulofeu and J. de Langhe, J. Am. Chem. Soc., 1942, 64, 968–969.
123 W. A. Ayer and B. Altenkirk, Can. J. Chem., 1969, 47, 2457–2459.
124 W. A. Ayer, B. Altenkirk and Y. Fukazawa, Tetrahedron, 1974, 30,
4213–4214.
125 H. Ishii, B. Yasui, R. Nishino, T. Harayama and Y. Inubushi, Chem.
Pharm. Bull., 1970, 18, 1880–1888.
126 J. Li, Y. Han and J. Liu, Zhongcaoyao, 1987, 18, 50–51.
127 C.-H. Tan, B.-D. Wang, S.-H. Jiang and D.-Y. Zhu, Planta Med.,
2002, 68, 188–190.
128 T. Harayama, T. Taga, K. Osaki and K. Kuriyama, Heterocycles,
1984, 22, 1327–1330.
129 W. A. Ayer, Y. Fukazawa, P. P. Singer and B. Altenkirk, Tetrahedron
Lett., 1973, 5045–5048.
130 C. H. Tan, S. H. Jiang and D. Y. Zhu, Tetrahedron Lett., 2000, 41,
5733–5736.
131 D.-Y. Zhu, S.-H. Jiang, M.-F. Huang, L.-Z. Lin and G. A. Cordell,
Phytochemistry, 1994, 36, 1069–1072.
132 W. Y. Gao, Y. M. Li, B. D. Wang and D. Y. Zhu, Chin. Chem. Lett.,
1999, 10, 463–466.
133 W.-Y. Gao, B.-D. Wang, Y.-M. Li, S.-H. Jiang and D.-Y. Zhu,
Chin. J. Chem., 2000, 18, 614–616.
134 C. H. Tan, X. Q. Ma, G. F. Chen and D. Y. Zhu, Helv. Chim. Acta,
2002, 85, 1058–1061.
135 C. H. Tan, G. F. Chen, X. Q. Ma, S. H. Jiang and D. Y. Zhu, J. Nat.
Prod., 2002, 65, 1021–1022.
136 C. H. Tan, X. Q. Ma, G. F. Chen, S. H. Jiang and D. Y. Zhu, Chin.
Chem. Lett., 2002, 13, 331–332.
137 C. Tan, X. Ma, H. Zhou, S. Jiang and D. Zhu, Zhiwu Xuebao, 2003,
45, 118–121.
138 C.-H. Tan, G.-F. Chen, X.-Q. Ma, S.-H. Jiang and D.-Y. Zhu,
J. Asian Nat. Prod. Res., 2002, 4, 227–231.
139 X. J. Tan, H. Q. Wang, H. L. Jiang, W. L. Zhu, S. H. Jiang, D. Y.
Zhu, K. X. Chen and R. Y. Ji, Acta Chim. Sin. (Engl. Ed.), 2000,
58, 1386–1392.
140 S. Q. Yuan and Y. M. Zhao, Yaoxue Xuebao, 2003, 38, 596–598.
141 H. Takayama, K. Katakawa, M. Kitajima, K. Yamaguchi and N.
Aimi, Tetrahedron Lett., 2002, 43, 8307–8311.
142 M. Castillo, G. Morales, L. A. Loyola, I. Singh, C. Calvo, H. L.
Holland and D. B. MacLean, Can. J. Chem., 1975, 53, 2513–2514.
143 Y. Inubushi and T. Harayama, Chem. Pharm. Bull., 1981, 29, 3418–
3421.
144 R. H. Burnell, A. Chapelle, J. Fischer and L. Ricard, J. Chem. Soc.,
Chem. Commun., 1974, 391.
145 H. Takayama, K. Katakawa, M. Kitajima, H. Seki, K. Yamaguchi
and N. Aimi, Org. Lett., 2001, 3, 4165–4167.
146 W. A. Ayer, Y.-T. Ma, J.-S. Liu, M.-F. Huang, L. W. Schultz and J.
Clardy, Can. J. Chem., 1994, 72, 128–130.
147 J. C. Braekman, C. Hootele, N. Miller, J. P. Declercq, G. Germain
and M. Van Meerssche, Can. J. Chem., 1979, 57, 1691–1693.
148 S. Yuan, Y. Zhao and R. Feng, Yaoxue Xuebao, 2002, 37, 946–949.
149 Z. Wan, J. Zhang, J. Dai and D. Liang, Kexue Tongbao (Foreign
Lang. Ed.), 1986, 31, 489–492.
150 B. M. Chu and J. Li, Yaoxue Xuebao, 1988, 23, 115–121.
151 Y. Inubushi, T. Harayama, M. Akatsu, H. Ishii and Y. Nakahara,
Chem. Pharm. Bull., 1968, 16, 561–563.
152 X. Zhang, H. Wang and Y. Qi, Zhongcaoyao, 1990, 21, 146–147.
153 Y. Inubushi, Y. Tsuda, H. Ishii, T. Sano, M. Hosokawa and T.
Harayama, Yakugaku Zasshi, 1964, 84, 1108–1113.

154 B. Yasui, H. Ishii, T. Harayama, R. Nishino and Y. Inubushi,
Tetrahedron Lett., 1966, 3967–3973.
155 Y. Inubushi, Y. Tsuda and T. Sano, Yakugaku Zasshi, 1962, 82,
1537–1541.
156 Y. Inubushi, H. Ishii, B. Yasui, M. Hashimoto and T. Harayama,
Chem. Pharm. Bull., 1968, 16, 82–91.
157 Y. Hirasawa, H. Morita, M. Shiro and J. Kobayashi, Org. Lett.,
2003, 5, 3991–3993.
158 W. A. Ayer, J. K. Jenkins, S. Valverde-Lopez and R. H. Burnell,
Tetrahedron Lett., 1964, 2201–2209.
159 N. Miller, C. Hootele, C. Braekman-Danheux and J. C. Braekman,
Bull. Soc. Chim. Belg., 1971, 80, 629–638.
160 H. Morita, Y. Hirasawa, T. Shinzato and J. Kobayashi, Tetrahedron,
2004, 60, 7015–7023.
161 L. Marion and R. H. F. Manske, Can. J. Res., 1948, 26B, 1–2.
162 W. A. Ayer and K. Piers, Can. J. Chem., 1967, 45, 451–459.
163 H. Morita, Y. Hirasawa, N. Yoshida and J. Kobayashi, Tetrahedron
Lett., 2001, 42, 4199–4201.
164 W. A. Ayer, N. Masaki and D. S. Nkunika, Can. J. Chem., 1968, 46,
3631–3642.
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
165 W. A. Ayer, L. M. Browne, Y. Nakahara, M. Tori and L. T. J.
Delbaere, Can. J. Chem., 1979, 57, 1105–1107.
166 W. Y. Gao, Y. M. Li, S. H. Jiang and D. Y. Zhu, Planta Med., 2000,
66, 664–667.
167 H. Q. Liu, C. H. Tan, S. H. Jiang and D. Y. Zhu, Chin. Chem. Lett.,
2004, 15, 303–304.
Downloaded by University of Sussex on 22 July 2012
168 S. Yuan, Y. Zhao and R. Feng, Junshi Yixue Kexueyuan Yuankan,
2001, 25, 57–58.
169 M. Tori, T. Shimoji, E. Shimura, S. Takaoka, K. Nakashima, M.
Sono and W. A. Ayer, Phytochemistry, 2000, 53, 503–509.
170 J. Zhang, Z. Guo, D. Pan and X. Cai, Zhongcaoyao, 1997, 28, 139–
140.
171 L. Nyembo, A. Goffin, C. Hootele and J. C. Braekman,
Can. J. Chem., 1978, 56, 851–856.
172 A. Leniewski, D. B. MacLean and J. K. Saunders, Can. J. Chem.,
1981, 59, 2695–2702.
173 M. Tori, T. Shimoji, S. Takaoka, K. Nakashima, M. Sono and W. A.
Ayer, Tetrahedron Lett., 1999, 40, 323–324.
174 Y. Inubushi, H. Ishii and T. Harayama, Yakugaku Zasshi, 1980, 100,
672–674.
175 W. A. Ayer, L. F. Ball, L. M. Browne, M. Tori, L. T. J. Delbaere and
A. Silverberg, Can. J. Chem., 1984, 62, 298–302.
176 Z. C. Miao, Z. S. Yang and F. R., Yaoxue Xuebao, 1989, 24, 114–117.
177 Y. Hirasawa, H. Morita and J. i. Kobayashi, Tetrahedron, 2003, 59,
3567–3573.
178 W. A. Ayer and L. S. Trifonov, in Lycopodium Alkaloids, Academic
Press, San Diego, 1994.
179 H. Conroy, Tetrahedron Lett., 1960, 34–37.
180 C. H. Tan, Studies on the alkaloids of Huperzia serrata (Thunb.)
Trev., Chinese Academy of Sciences [D], Shanghai, 2001.
181 W. A. Ayer, Nat. Prod. Rep., 1991, 8, 455–463.
182 A. Leniewski, J. Szychowski and D. B. MacLean, Can. J. Chem.,
1981, 59, 2479–2490.
183 Jiangsu New Medical College: The Dictionary of traditional Chinese
medicine, Shanghai Sci-Tech Press, Shanghai, 1985.
184 X. D. Zhu and X. C. Tang, Yaoxue Xuebao, 1987, 22, 812–817.
185 X. C. Tang, Acta Pharmacol. Sin., 1996, 17, 481–484.
186 D. L. Bai, X. C. Tang and X. C. He, Curr. Med. Chem., 2000, 7,
355–374.
187 P. D. Sloane, S. Zimmerman, C. Suchindran, P. Reed, L. Wang, M.
Boustani and S. Sudha, Annu. Rev. Public Health, 2002, 23, 213–231.
188 M. Farlow, Clin. Pharmacokinet., 2003, 42, 1383–1392.
189 H. Wang and X. C. Tang, Acta Pharmacol. Sin., 1998, 19, 27–30.
190 X. C. Tang and Y. F. Han, CNS Drug Rev., 1999, 5, 281–300.
191 Y. Q. Liang and X. C. Tang, Neurosci. Lett., 2004, 361, 56–59.
192 M. L. Raves, M. Harel, Y. P. Pang, I. Silman, A. P. Kozikowski and
J. L. Sussman, Nat. Struct. Biol., 1997, 4, 57–63.
193 A. P. Kozikowski, C. P. Miller, F. Yamada, Y. P. Pang, J. H. Miller,
M. McKinney and R. G. Ball, J. Med. Chem., 1991, 34, 3399–3402.
194 G. Lallement, V. Baille, D. Baubichon, P. Carpentier, J. M. Collom-
bet, P. Filliat, A. Foquin, E. Four, C. Masqueliez, G. Testylier, L.
Tonduli and F. Dorandeu, Neurotoxicology, 2002, 23, 1–5.
195 D. H. Cheng, H. Ren and X. C. Tang, NeuroReport, 1996, 8, 97–101.
196 W. H. Lu, J. Shou and X. C. Tang, Zhongguo Yaoli Xuebao, 1988,
9, 11–15.
197 J. W. Ye, Y. Z. Shang, Z. M. Wang and X. C. Tang, Acta Pharmacol.
Sin., 2000, 21, 65–69.
198 J. W. Ye, J. X. Cai, L. M. Wang and X. C. Tang, J. Pharmacol. Exp.
Ther., 1999, 288, 814–819.
199 L. Y. Ou, X. C. Tang and J. X. Cai, Eur. J. Pharmacol., 2001, 433,
151–156.
View Online
200 L. S. Wang, J. Zhou, X. M. Shao and X. C. Tang, Brain Res., 2002,
949, 162–170.
201 L.-s. Wang, J. Zhou, X.-m. Shao and X.-c. Tang, Zhonghua Erke
Zazhi, 2003, 41, 42–45.
202 R. Wang, H. Y. Zhang and X. C. Tang, Eur. J. Pharmacol., 2001,
421, 149–156.
203 Y. Gao, X. C. Tang, L. C. Guan and P. Z. Kuang, Acta Pharmacol.
Sin., 2000, 21, 1169–1173.
204 P. Filliat, A. Foquin and G. Lallement, Drug Chem. Toxicol., 2002,
25, 9–24.
205 T. Wang and X. C. Tang, Eur. J. Pharmacol., 1998, 349, 137–142.
206 R. F. Butterworth, Dev. Neurosci. (Basel), 1998, 20, 478–484.
207 G. Ferreira, M. Meurisse, R. Gervais, N. Ravel and F. Levy,
Neuroscience, 2001, 106, 103–116.
208 M. Mesulam, Learn. Mem., 2004, 11, 43–49.
209 M. S. Parihar and T. Hemnani, J. Clin. Neurosci., 2004, 11, 456–467.
210 M. Ruberg, F. Rieger, A. Villageois, A. M. Bonnet and Y. Agid,
Brain Res., 1986, 362, 83–91.
211 V. Kumar, E. Giacobini and S. Markwell, Acta Neurol. Scand., 1989,
80, 461–466.
212 R. Elble, E. Giacobini and C. Higgins, Neurobiol. Aging, 1989, 10,
45–50.
213 T. Egashira, H. Goto, H. Takeda, K. Takada and T. Matsumiya,
Nippon Ronen Igakkai Zasshi, 1999, 36, 256–261.
214 R. K. Gordon, S. V. Nigam, J. A. Weitz, J. R. Dave, B. P. Doctor
and H. S. Ved, J. Appl. Toxicol., 2001, 21, S47–51.
215 X. Q. Xiao, R. Wang, Y. F. Han and X. C. Tang, Neurosci. Lett.,
2000, 286, 155–158.
216 X. Q. Xiao, R. Wang and X. C. Tang, J. Neurosci. Res., 2000, 61,
564–569.
217 H. Y. Zhang, Y. Q. Liang, X. C. Tang, X. C. He and D. L. Bai,
Neurosci. Lett., 2002, 317, 143–146.
218 J. M. Zhang and G. Y. Hu, Neuroscience, 2001, 105, 663–669.
219 X. D. Wang, J. M. Zhang, H. H. Yang and G. Y. Hu, Acta
Pharmacol. Sin., 1999, 20, 31–35.
220 X. Q. Xiao, H. Y. Zhang and X. C. Tang, J. Neurosci. Res., 2002,
67, 30–36.
221 M. Grundman and P. Delaney, Proc. Nutr. Soc., 2002, 61, 191–202.
222 H. Y. Zhang, H. Yan and X. C. Tang, Neurosci. Lett., 2004, 360,
21–24.
223 M. McKinney, J. H. Miller, F. Yamada, W. Tuckmantel and A. P.
Kozikowski, Eur. J. Pharmacol., 1991, 203, 303–305.
224 X. C. Tang, G. H. Kindel, A. P. Kozikowski and I. Hanin,
J. Ethnopharmacol., 1994, 44, 147–155.
225 A. P. Kozikowski, Y. Xia, E. R. Reddy, W. Tuckmantel, I. Hanin
and X. C. Tang, J. Org. Chem., 1991, 56, 4636–4645.
226 D. L. Bai, Pure Appl. Chem., 1993, 65, 1103–1112.
227 Y. Xia, E. R. Reddy and A. P. Kozikowski, Tetrahedron Lett., 1989,
30, 3291–3294.
228 A. P. Kozikowski, G. Campiani, V. Nacci, A. Sega, A. Saxena and
B. P. Doctor, J. Chem. Soc., Perkin Trans. 1, 1996, 1287–1297.
229 G. Campiani, A. P. Kozikowski, S. M. Wang, L. Ming, V. Nacci,
A. Saxena and B. P. Doctor, Bioorg. Med. Chem. Lett., 1998, 8,
1413–1418.
230 D. X. Liu, H. L. Jiang, Q. M. Wang, K. X. Chen and R. Y. Ji,
Bioorg. Med. Chem. Lett., 1998, 8, 419–422.
231 A. P. Kozikowski, E. Thiels, X. C. Tang and I. Hanin, Adv. Med.
Chem., 1992, 1, 175–205.
232 D. M. Fink, G. M. Bores, R. C. Effland, F. P. Huger, B. E. Kurys,
D. K. Rush and D. E. Selk, J. Med. Chem., 1995, 38, 3645–3651.
233 A. P. Kozikowski, G. Campiani and W. Tuckmantel, Heterocycles,
1994, 39, 101–116.
234 A. P. Kozikowski, Q. J. Ding, A. Saxena and B. P. Doctor, Bioorg.
Med. Chem. Lett., 1996, 6, 259–262.
235 A. P. Kozikowski, G. Campiani, L. Q. Sun, S. M. Wang, A. Saxena
and B. P. Doctor, J. Am. Chem. Soc., 1996, 118, 11357–11362.
236 A. P. Kozikowski, K. R. C. Prakash, A. Saxena and B. P. Doctor,
Chem. Commun., 1998, 1287–1288.
237 P. R. Carlier, D. M. Du, Y. F. Han, J. Liu and Y. P. Pang, Bioorg.
Med. Chem. Lett., 1999, 9, 2335–2338.
238 E. Ros, J. Aleu, I. G. de Aranda, D. Munoz-Torrero, P. Camps,
A. Badia, J. Marsal and C. Solsona, Eur. J. Pharmacol., 2001, 421,
77–84.
239 Z. Q. Xiong, X. C. Tang, J. L. Lin and D. Y. Zhu, Acta Pharmacol.
Sin., 1995, 16, 21–25.
240 H. F. Chiu and M. Zhang, Int. J. Geriatr. Psychiatry, 2000, 15,
947–953.
241 Z. Zhang, X. Wang, Q. Chen, L. Shu, J. Wang and G. Shan,
Zhonghua Yixue Zazhi, 2002, 82, 941–944.
242 C. L. Zhang and G. Z. Wang, New Drugs Clinic, 1990, 9, 339–341.
243 Q. Q. Sun, S. S. Xu, J. L. Pan, H. M. Guo and W. Q. Cao, Acta
Pharmacol. Sin., 1999, 20, 601–603.
Nat. Prod. Rep., 2004, 21, 752–772 771

244 S. S. Xu, Z. Y. Cai, Z. W. Qu, R. M. Yang, Y. L. Cai, G. Q. Wang,
X. Q. Sy, X. S. Zhong, R. Y. Cheng, W. A. Xu, J. X. Li and B. Feng,
Acta Pharmacol. Sin., 1999, 20, 486–490.
245 Y. X. Ma, Y. Zhu, Y. D. Gu, Z. Y. Yu, S. M. Yu and Y. Z. Ye,
Towards Prolongation of the Healthy Life Span, New York Academy
of Sciences, New York, 1998.
246 Y. X. Ma, Y. Zhu, Y. D. Gu, Z. Y. Yu, S. M. Yu and Y. Z. Ye, Arch.
Pharmacol. (Weinheim, Ger.), 1998, 358, R76–R76.
247 A. Mazurek, New Engl. J. Med., 2000, 342, 821.
248 J. G. Baker, Handbook of fern allies, Maggs Bros. Ltd., London,
1887, p. 8.
249 G. Herter, Bot. Jahrb., 1909, 98, 29.
250 G. Herter, Beihefte zum Botanischen Centralblatt, 1923, 39, 39.
251 J. Holub, Preslia, 1964, 36, 16–22.
252 J. Holub, Preslia, 1975, 47, 103–104.
253 J. H. Wilce, Am. Fern. J., 1972, 62, 65–79.
254 R. C. Ching, Acta Phytotax. Sin., 1978, 16, 1–9.
255 B. Voirin and P. Lebreton, Bull. Soc. Chim. Biol., 1967, 49, 1402–
1405.
256 T. S. Zhou, Wuhan Zhiwu Yanjiu, 1989, 7, 377–389.
Published on 21 October 2004 on http://pubs.rsc.org | doi:10.1039/B409720N
257 D. M. Power, G. H. Towers and A. C. Neish, Can. J. Biochem., 1965,
43, 1397–1407.
258 J. C. Braekman, l. Nyembo and J. J. Symoens, Phytochemistry, 1980,
19, 803–807.
259 Y. Inubushi, Y. Tsuda, H. Ishii, T. Sano, M. Hosokawa and T.
Harayama, Yakugaku Zasshi, 1964, 84, 1108–1113.
Downloaded by University of Sussex on 22 July 2012
260 X. Q. Ma, D. Liu, Z. B. Hu, and D. Y. Zhu, Zhongguo Yeshen Zhiwu
Ziyuan, 1998, Suppl., p. 155.
261 Y. Maki, Gifu Yakka Daigaku Kiyo, 1961, 11, 1–8.
262 R. N. Gupta, Lloydia, 1968, 31, 318–326.
263 R. N. Gupta, M. Castillo, D. B. MacLean, I. D. Spenser and J. T.
Wrobel, J. Am. Chem. Soc., 1968, 90, 1360–1361.
772 Nat. Prod. Rep., 2004, 21, 752–772
View Online
264 M. Castillo, R. N. Gupta, Y. K. Ho, D. B. MacLean and I. D.
Spenser, Can. J. Chem., 1970, 48, 2911–2912.
265 M. Castillo, R. N. Gupta, Y. K. Ho, D. B. MacLean and I. D.
Spenser, J. Am. Chem. Soc., 1970, 92, 1074–1075.
266 M. Castillo, R. N. Gupta, D. B. MacLean and I. D. Spenser,
Can. J. Chem., 1970, 48, 1893-&.
267 R. N. Gupta, Y. K. Ho, D. B. MacLean and I. D. Spenser, J. Chem.
Soc., Sect. D, 1970, 409–410.
268 R. B. Herbert, Alkaloids (London), 1971, 1, 1–30.
269 Y. K. Ho, R. N. Gupta, D. B. MacLean and I. D. Spenser,
Can. J. Chem., 1971, 49, 3352–3361.
270 J. C. Braekman, R. N. Gupta, D. B. MacLean and I. D. Spenser,
Can. J. Chem., 1972, 50, 2591–2602.
271 W. D. Marshall, Biosynthesis of lycopodium alkaloids, 1973.
272 W. Marshall, T. Nguyen, D. Maclean and I. Spenser, Can. J. Chem.,
1975, 53, 41–50.
273 T. Hemscheidt and I. D. Spenser, J. Am. Chem. Soc., 1990, 112,
6360–6363.
274 T. Hemscheidt and I. D. Spenser, J. Am. Chem. Soc., 1993, 115,
3020–3021.
275 T. Hemscheidt, Top. Curr. Chem., 2000, 209, 175–206.
276 H. J. Gerdes and E. Leistner, Phytochemistry, 1979, 18, 771–775.
277 D. B. MacLean, The alkaloids, ed. A. Brossi, Academic Press, New
York, 1985.
278 T. A. Blumenkopf and C. H. Heathcock, Alkaloids: Chemical and
Biological Perspectives, ed. S. W. Pelletier, John Wiley & Sons, New
York, 1985.
279 T. Hemscheidt and I. D. Spenser, J. Am. Chem. Soc., 1996, 118,
1799–1800.
280 J. C. Richards and I. D. Spenser, Can. J. Chem., 1982, 60, 2810–2820.
281 H. Dittrich and T. M. Kutchan, Proc. Natl. Acad. Sci., 1991, 88,
9969–9973.