In vivo transformations of artemisinic acid in Artemisia annua plants

Article abstract of DOI:10.1016/j.tet.2007.06.062

Artemisinic acid labeled with both ¹³C and ²H at the 15-position has been fed to intact plants of Artemisia annua via the cut stem, and its in vivo transformations studied by 1D- and 2D-NMR spectroscopy. Seven labeled metabolites have been isolated, all of which are known as natural products from this species. The transformations of artemisinic acid-as observed both for a group of plants, which was kept alive by hydroponic administration of water and for a group, which was allowed to die by desiccation-closely paralleled those, which have been recently described for its 11,13-dihydro analog, dihydroartemisinic acid. It seems likely therefore that similar mechanisms, involving spontaneous autoxidation of the Δ^4,5 double bond in both artemisinic acid and dihydroartemisinic acid and subsequent rearrangements of the resultant allylic hydroperoxides, may be involved in the biological transformations, which are undergone by both compounds. All of the sesquiterpene metabolites, which were obtained from in vivo transformations of artemisinic acid retained their unsaturation at the 11,13-position, and there was no evidence for conversion into any 11,13-dihydro metabolite, including artemisinin, the antimalarial drug, which is produced by A. annua. This observation led to the proposal of a unified biosynthetic scheme, which accounts for the biogenesis of many of the amorphane and cadinane sesquiterpenes that have been isolated as natural products from A. annua. In this scheme, there is a bifurcation in the biosynthetic pathway starting from amorpha-4,11-diene leading to either artemisinic acid or dihydroartemisinic acid; these two committed precursors are then, respectively, the parents for the two large families of highly oxygenated 11,13-dehydro and 11,13-dihydro sesquiterpene metabolites, which are known from this species.

Full text of DOI:10.1016/j.tet.2007.06.062

Tetrahedron 63 (2007) 9548–9566
In vivo transformations of artemisinic acid in Artemisia annua
plants
a,* b
Geoffrey D. Brown and Lai-King Sy
a
School of Chemistry, The University of Reading, Whiteknights Road, Reading RG6 6AD, UK
Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and
b
Synthesis, Area of Excellence Scheme of University Grant Committee (Hong Kong), The University of Hong Kong, Pokfulam Road,
Hong Kong, People’s Republic of China
Received 29 January 2007; revised 1 June 2007; accepted 21 June 2007
Available online 27 June 2007
1
3
2
Abstract—Artemisinic acid labeled with both C and H at the 15-position has been fed to intact plants of Artemisia annua via the cut stem,
and its in vivo transformations studied by 1D- and 2D-NMR spectroscopy. Seven labeled metabolites have been isolated, all of which are
known as natural products from this species. The transformations of artemisinic acid—as observed both for a group of plants, which was
kept alive by hydroponic administration of water and for a group, which was allowed to die by desiccation—closely paralleled those, which
have been recently described for its 11,13-dihydro analog, dihydroartemisinic acid. It seems likely therefore that similar mechanisms, involv-
4
,5
ing spontaneous autoxidation of the D double bond in both artemisinic acid and dihydroartemisinic acid and subsequent rearrangements of
the resultant allylic hydroperoxides, may be involved in the biological transformations, which are undergone by both compounds. All of the
sesquiterpene metabolites, which were obtained from in vivo transformations of artemisinic acid retained their unsaturation at the 11,13-
position, and there was no evidence for conversion into any 11,13-dihydro metabolite, including artemisinin, the antimalarial drug, which
is produced by A. annua. This observation led to the proposal of a unified biosynthetic scheme, which accounts for the biogenesis of
many of the amorphane and cadinane sesquiterpenes that have been isolated as natural products from A. annua. In this scheme, there is a bi-
furcation in the biosynthetic pathway starting from amorpha-4,11-diene leading to either artemisinic acid or dihydroartemisinic acid; these
two committed precursors are then, respectively, the parents for the two large families of highly oxygenated 11,13-dehydro and 11,13-dihydro
sesquiterpene metabolites, which are known from this species.
Ó 2007 Elsevier Ltd. All rights reserved.
2
1
. Introduction
artemisinin (1)), arteannuin E (qinghaosu V) (5), arteannuin
F (artemisilactone) (6), annulide (7),
1
6
6,17
isoannulide
1
6,17
6,18,19
7,20
The Chinese plant Artemisia annua L. (sweet wormwood;
Compositae) has been the subject of extensive phytochemi-
cal investigation, following the discovery more than two
(8),
epi-deoxyarteannuin B (9),
deoxyarteannuin
6,7-dehydroarte-
a seco-cadinane (13), and the methyl
6
B (10), a-epoxyartemisinic acid (11),
misinic acid (12),
ester of artemisinic acid (14).
1
5,19
21
5
decades ago of the amorphane sesquiterpene, artemisinin
(
2
qinghaosu (1)), which has now become the lead compound
for an important new class of antimalarial drugs. Of the 47
amorphane and cadinane sesquiterpenes, which are currently
3
The first ever experimental study of the biosynthesis of arte-
misinin (1), using a radioisotopically-labeled precursor,
which was supplied to a plant homogenate assay system,
seemed to indicate that artemisinic acid (2) was converted
to both artemisinin (1) and arteannuin B (3) via separate path-
2
,4–8
known from this species,
13 incorporate a terminal dou-
ble bond at the 11,13-position (Fig. 1). The most abundant
sesquiterpenes from A. annua, which present this structural
feature are artemisinic acid (qinghao acid, arteannuic acid)
2
2
ways. Subsequent investigations, using similar cell-free
systems appeared to confirm this result (arrows in Scheme
2,9–11
2,11,12
(
2)
and arteannuin B (qinghaosu II) (3),
both of
2
3–25
which have been suggested as biogenetic precursors to arte-
misinin (1) (see below). Other 11,13-dehydro natural prod-
ucts, which have been isolated in smaller amounts include:
1 labeled ‘Refs. 22–25’).
The most detail of these studies
2
5
by Wang et al. presented evidence that the conversion of ar-
temisinic acid (2) to artemisinin (1) might proceed by a path-
way, which involved artemisitene (4) as an intermediate; and
that its conversion to arteannuin B (3) may have proceeded
via a-epoxy-artemisinic acid (11). However, in an accompa-
1
3–15
artemisitene (4)
(the 11,13-dehydro analog of
Keywords: Terpenes and terpenoids; Biogenesis; Autoxidation; NMR.
*
2
6
nying communication, these same authors also discussed
the alternative possibility that epi-deoxyarteannuin B (9)
Corresponding author. Tel.: +44 118 378 7418; fax: +44 118 931 6331;
e-mail: g.d.brown@reading.ac.uk
0
040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.06.062

G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
9549
1
4
H
O
2
5
H
H
H
H
1
0
3
O
9
1
6
5
O
4
7
O
8
H
H
15
O
H
H
H
R2
13
O
HO
O
R
O
R O
11
1
11
1
3
R
1
2
O
O
O
1
Artemisinin, R=CH3
Artemisitene, R=CH2
2 Artemisinic acid, R = H; R =CH
3 Arteanuin B, R=CH
2
22 Dihydroarteannuin B, R=CH
3
5 Arteannuin E, 5-O-β
6 Arteannuin F, 5-O-α
1
2
2
4
14 Artemisinic acid methyl ester, R =Me; R =CH
1
2
2
1
6 Dihydroartemisinic acid, R =H; R =CH
1
2
3
H
H
H
H
3
4
6
15
6
7
H
H
R
O
H
O
H
R
O
HO
HO
O
O
O
O
4
,15
3
7
8
2
2
Annulide, R=CH ; Δ
9 epi-Deoxyarteannuin B, 6-O-β; R=CH
11 α-Epoxyartemisinic acid
12 6,7-Dehydroartemisinic acid
2
2
,4
4
Isoannulide, R=CH ; Δ
10 Deoxyarteannuin B, 6-O-α; R=CH
2
2
,15
3 Arteannuin I, R=CH ; Δ
15 Dihydro-epi-deoxyarteannuin B, 6-O-β; R=CH
3
3
3
3
,4
4 Arteannuin J, R=CH ; Δ
27 Dihydro-deoxyarteannuin B, 6-O-α; R=CH
3
O
H
O
H
H
H
R
HO
O
1
3 seco-Cadinane, R=CH
5 seco-Cadinane, R=CH
2
3
2
1
1,13
Figure 1. Amorphane and cadinane sesquiterpenes incorporating a terminal D
double bond that have been reported in the literature for A. anuua.
H
O
H
H
H
?
(Ref. 21)
via
?
(Ref. 25)
via
O
H
O
H
HO
1
O
HO
O
O
1
O 13
Refs. 22-25
O
Refs. 28 and 29
but not Ref. 25
3
H
O
H
H
Refs. 22-25
O
O
H
H
O
H
11
HO
via
? (Ref. 25)
O
11
1
3
O
O
13
H
O
2
O
O
1
O
4
Ref. 26
Ref. 26
H
O
O
9
Scheme 1. Various metabolic routes postulated for the biogenesis of artemisinin (1) from artemisinic acid (2) in the early literature (pre-1993), describing the
biosynthesis of A. annua.
might be the biogenetic precursor of 1 (arrows in Scheme 1
labeled ‘Ref. 26’). Confusingly, experimental studies by
artemisinin (1) (indeed, a hypothetical mechanism for this
transformation, involving the natural product 13, has since
been proposed). Although the results of Nair and Basile
y
8,29
2
21
Nair and Basile,
published at around the same time, sug-
30
gested that arteannuin B (3) could itself be a precursor to
supported a previous suggestion by Akhila et al., it was
contradicted by Wang et al. who had found that arteannuin
B was not a precursor to artemisinin.
2
5
y
The 11,13-dihydro analog of compound 9, dihydro-epi-deoxyarteannuin
B (15) (see Fig. 1), was also suggested to be a biogenetic precursor to 1
in this reference—the companion paper to this manuscript describes
biosynthetic experiments, employing compound 15 as a labeled precursor,
which failed to find any evidence for this proposal.
27
The various possibilities, which are presented in the early lit-
erature (pre-1993) describing the biogenesis of artemisinin
are summarized in Scheme 1. Although a singular emphasis

9550
G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
has been given to artemisinic acid (2) as the biosynthetic pre-
cursor of artemisinin (1) in this Scheme, it should be noted
that additional possibilities were also considered by the orig-
inal investigators. For example, the paper by Wang et al.,
which had described artemisinic acid (2) as a precursor to
artemisinin (1), had indicated that dihydroartemisinic acid
are described in Sections 2.2 and 2.3 to be performed. This
successful synthesis encouraged further investigations in
order to determine whether this same oxidative selenation
strategy might also prove useful for producing some of the
other 11,13-unsaturated natural products, which have been
postulated as biosynthetic precursors in Scheme 1. Thus,
in view of a recent synthesis of isotopically-labeled dihy-
2
5
(
16), the 11,13-dihydro analog of artemisinic acid (2) (see
2
7,32
Fig. 1), was also a biogenetic precursor to artemisinin (1).
The conversion of 16 to 1 obviously requires extensive oxi-
dation to the amorphane ring system in order to generate the
dro-epi-deoxyarteannuin B (15a),
the preparation of
epi-deoxyarteannuin B (9) from dihydro-epi-deoxyartean-
nuin B (15) was also investigated, as is described in Section
2.1.2. An attempt to produce artemisitene (4) from artemisi-
nin (1) by this same methodology is discussed briefly in
Section 2.1.3.
1
,2,4-trioxane ring of artemisinin. It should be noted that the
corresponding conversion of any of the compounds 2, 3 or
—all of which are proposed as biosynthetic intermediates
9
to 1 in Scheme 1—would require a similar complex series
of oxidative transformations and, in addition, the reduction
1
3 2
2.1.1. Preparation of [15- C H ]-artemisinic acid (2a)
3
1
1,13
13
2
of the D
double bond at some late stage in the biogenetic
from [15- C H ]-dihydroartemisinic acid (16a).
3
[15- C H ]-Artemisinic acid (2a) of both high chemical
3
and isotopic purity was prepared from [15- C H ]-di-
3
hydroartemisinic acid (16a) in three steps, as is shown in
Scheme 2 (the labeled compound 16a was itself prepared
in four to five steps from commercially available artemisi-
1
3 2
route. However, there is still no experimental evidence in the
literature to support such a reductive step for any of the com-
pounds 2, 3 or 9.
1
3 2
The current study sets out to resolve the apparent confusion
in the earlier literature relating to the status of artemisinic
acid (2) as a biogenetic precursor, and to establish the true
nature of its transformations in vivo in A. annua. In order
to achieve this, use has been made of an assay system con-
sisting of A. annua plants, which were fed with a labeled
version of compound 2 via the cut stem, rather than plant
homogenates or cell-free systems, as in all previous biosyn-
thetic studies with artemisinic acid. The guiding philosophy
has been that this more ‘natural’ approach should produce
more reliable results to indicate the true fate of this precursor
in vivo, as well as reducing the possibility for the introduc-
tion of experimental artifacts. Similarly, the use of a precur-
3
1,33
nin, as has been described in the literature).
Both the ini-
tial protection of labeled dihydroartemisinic acid (16a) as its
methyl ester, compound 17a, and the deprotection of the un-
saturated methyl ester, compound 14a, in the final step, were
clean reactions, which proceeded in high yield. In contrast,
3
4,35
the second step—an oxidative selenation reaction,
which the D
in
double bond required in the target com-
1
1,13
pound was introduced—produced the methyl ester-protected
analog of artemisinic acid 14a in only moderate yield, to-
gether with appreciable quantities of two other isolable
side-products, compounds 18a and 19a, which were sepa-
rated chromatographically. The E configuration of the
D
1
3
2
7,11
sor labeled with a stable isotope (both C and H, in this
case), has allowed information regarding the transforma-
tions undergone by labeled artemisinic acid (2a) to be ob-
-double bond in the minor product 18a was established
by correlations observed in NOESY spectroscopy between
H-13 and H-5/H-6.
z
1
3
2
tained directly via the C and H chemical shifts of the
metabolites of 2a, which were recorded in their NMR spec-
tra. The ability to identify metabolites in situ within crude
plant extracts, without the need for the extensive sample
manipulation, which is required during a chromatographic
separation, further serves to reduce the possibilities for intro-
ducing experimental artifacts, in our opinion.
1
3
2
Therewas no depletion of either C or H label in the product
2a obtained from starting material 16a, as judged by the re-
1
2
13
sults of mass spectrometry and H, H, and C NMR spec-
troscopies, which are shown in Figure 2 (patterns for the
peaks, associated with the label at the 15-position of 2a in
these spectra, present strong similarities to those, which
have been previously published for the starting material,
compound 16a, in Ref. 31). The labeled product 2a obtained
from this synthesis was thus eminently suitable for use as
a biosynthetic precursor in the feeding studies with A. annua,
which are described in Sections 2.2 and 2.3. Neither labeled
2
. Results and discussion
2
.1. Preparation of postulated biogenetic precursors
artemisinic acid (2a) nor its methyl ester derivative (14a)
when
3
1
36,37
Having recently reported a synthesis of doubly-labeled
[
were found to undergo any detectable autoxidation
1
3
2
x
15- C H ]-dihydroartemisinic acid (16a), it was resolved
3
stored in the freezer for a period of up to one year. Therefore
no purification of 2a was necessary prior to performing the
feeding experiments, which are described in Sections 2.2
and 2.3. This was slightly surprising in view of the author’s
previous experiences with both dihydroartemisinic acid
to attempt to introduce the requisite 11,13-double bond in la-
beled artemisinic acid (2a) by making use of the oxidative
selenation procedure, which is described in Section 2.1.1.
The conversion of 16a to 2a proceeded in reasonable yield,
producing sufficient quantities of labeled artemisinic acid to
allow the feeding experiments with A. annua plants, which
3
1
27,32
(16) and dihdro-epi-deoxyarteannuin B (15),
which
were found to have undergone appreciable autoxidation on
storage, and therefore required chromatographic purification
immediately prior to their use in feeding experiments.
z
x
13 2
Throughout this paper, the suffix ‘a’ indicates the presence of a [ C H
label at the 15-position.
It is worth stressing that each of these compounds had also been isolated in
labeled form as their 11,13-dehydro analogs (compounds 2a, 3a, 7a, 8a,
9a, and 13a, respectively), as discussed above.
3
]
2.1.2. Preparation of epi-deoxyarteannuin B (9) from di-
hydro-epi-deoxyarteannuin B (15). The efficiency of the
3
4,35
oxidative selenation procedure
was lessened when

G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
9551
1
. LDA
2. PhSeBr
3. H O
H
H
H
H
H
H
CH N
2
2
2
2
6
7
+
+D 313C
D313C
D313C
D313C
D313C
H
5
H
H
O
H
H
O
H
H
O
HO
MeO
MeO
MeO
CO2Me
1
3
11
O
OH
16a
17a (94%)
14a (64%)
18a (2%)
19a (4%)
KOH
H
D313C
H
HO
O
2a (86%)
1
2
3
. LDA
H
O
H
H
. PhSeBr
. H O
2
2
+
O
H
O
O
O
O
15
9 (21%)
20 (39%)
1
. LDA
H
O
H
2. PhSeBr
H
O
O
3. H O
2 2
O
H
O
+
O
H
O
O
O
O
O
H
H
H
O
O
O
1
O
4 (5%)
21 (73%)
Scheme 2. Synthesis of labeled artemisinic acid (2a) from labeled dihydroartemisinic acid (16a) by an oxidative selenation procedure; and the preparation of
epi-deoxyarteannuin B (9) and artemisitene (4) from their respective 11,13-dihydro precursors, dihydro-epi-deoxyarteannuin B (15) and artemisinin (1), by the
same means.
1
4
1
introducing unsaturation to the 11,13-position of dihydro-
epi-deoxyarteannuin B (15), as compared with the methyl
ester of dihydroartemisinic acid (17), which is described in
the previous section. The major product isolated from the
oxidative selenation of 15 was, in fact, compound 20, the
et al., involving the ene-type reaction of O with an
2
enol ether derivative of 1. This procedure has previously
been used as part of a total synthesis of 6,7-dehydroartemi-
sinic acid (12), and it was observed that 4 can be obtained
from 1 in an overall yield of 45% by this route.
1
5
7
,11
D
2
-isomer of 9 (cf. formation of 18a from 17a in Section
.1.1); while the yield of the desired product, epi-deoxyar-
2.2. Determination of the optimum method for feeding
[15- C H ]-artemisinic acid (2a) to A. annua plants
1
3 2
teannuin B (9), was quite low of about 21% (Scheme 2). Al-
though oxidative selenation could be used as a strategy for
the synthesis of labeled 9, which would be required for use
in feeding experiments designed to establish whether or
not 9 is a biosynthetic precursor to 1 in A. annua (see
Scheme 1), it would seem worthwhile to first investigate al-
ternative synthetic strategies that might give a higher yield.
3
The presence of a carboxylic acid group in artemisinic acid
allowed the aqueous solubility of this compound to be ma-
nipulated to some extent by controlling the pH of the feeding
solution. As for the previous biosynthetic study with di-
hydroartemisinic acid, it was found that the inclusion of
TRIS buffer at pH 8.1 allowed sufficient [15- C H ]-
3
3
13 2
3
2
(
.1.3. Preparation of artemisistene (4) from artemisinin
1). Although oxidative selenation of artemisinin (1) did pro-
artemisinic acid (2a) to be incorporated reproducibly in
the feeding medium (as its more soluble conjugate base
form), in order that meaningful biosynthetic experiments
could be performed, when using H NMR spectroscopy as
the primary detection method.
duce the desired unsaturated compound, artemisitene (4),
again in low yield, the major product of the reaction was
the saturated sesquiterpene 21, in which the 11-methyl group
of artemisinin has undergone epimerization. Oxidative sele-
nation is not therefore the preferred procedure for the
synthesis of labeled 4, which would be required for use
in feeding experiments designed to establish whether or
not artemisitene (4) is a biosynthetic precursor to 1 in A. an-
nua (see Scheme 1). Rather, it would be better to convert 1
into 4 via a route, which was first reported by El-Feraly
2
3
8
However, the uptake of labeled artemisinic acid (2a) via the
root of intact plants of A. annua was found to be very limited
by comparison with the previous studies with labeled dihy-
3
3
droartemisinic acid (16a), as was shown by a preliminary
experiment involving two groups of plants. In this trial, one
group of plants was cut just above the root and immersed by

9552
G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
1
{
2
13
13
2
13 2
3 3
]-artemisinic acid (2a) obtained by synthesis from [15- C H ]-
Figure 2. (a) H NMR, (b) H NMR, (c) C NMR, and expansion (d) spectra of [15- C H
dihydroartemisinic acid (16a).
{
The region from d
1.58–1.20 ppm (comprising H-2b, H-1, H-10, H-8a, and H-8b—see Table 2) integrates as approximately [5H]. These integrations confirm the absence of
signal for H-15, which appears as an intense three proton singlet at d 1.58 ppm in the H NMR spectrum of isotopically-normal artemisinic acid.
H
H H
2.00–1.58 ppm (comprising H-2a, H-3b, H-3a, and H-9b—see Table 2) integrates as approximately [4H]; while the area from d
1

G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
9553
Figure 2. (continued)
the cut stem in a buffered feeding solution containing 2a,
while the second group, consisting of intact plants, which
had been washed clean of soil, was immersed by the root.
The feeding solution was taken up within 24 h by both
groups (when administered at a rate of roughly 1 ml—con-
taining ca. 1 mg of 2a—to every 1 g of fresh plant material).
Following compete uptake of the feeding solution, each
group of plants was then separated into root, stem, and
leaf compartments and extracted by CH Cl . The resulting
from the leaf and stem compartments of the ‘cut’ stem-fed
plants, less than 2% of the label was found in the stem com-
partment of the ‘intact’ root-fed plants, and none in the
leaves. (The majority of label recovered from the ‘intact’
plants remained associated with the root compartment, and
it is believed that this represents physical adsorption of
labeled artemisinic acid on to the root surface, rather than
assimilation of 2a by the root.)
2
2
2
crude CH Cl extracts were immediately analyzed by H
2
It was therefore concluded that there is a physiological bar-
rier to the uptake of artemisinic acid by the roots of A. annua.
This, in turn, results in a degree of uptake of artemisinic acid,
2
NMR spectroscopy, which showed no significant metabo-
lism over the 24-hour period of the feeding experiment
2
2
(
predominantly by a doublet centered at dD 1.59 ppm,
the H NMR signal in each crude extract was represented
which is too low to allow studies employing H NMR spec-
troscopy as the detection method, when artemisinic acid is
fed to intact plants via the root. In consequence, the feeding
experiments, which are described in the next section were
performed using ‘stem-feeding’ to cut plants. Whilst this is
clearly less satisfactory than direct administration of the
labeled artemisinic acid precursor to intact A. annua plants
1
JCD¼19.1 Hz, corresponding to the feeding precursor, com-
2
pound 2a). However, H NMR spectroscopy also indicated
very significant quantitative differences in the extent of up-
take of label by the two groups. Thus, while between 15 and
2
2
5% of the H label was recoverable in the CH Cl extracts
2
2

9554
G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
via the root—as was practiced in previous experiments with
dihydroartemisinic acid (for which no such physiological
feeding vials; while the second group was deprived of water,
and allowed to die by desiccation. Three plants in each group
were then harvested on a daily basis and extracted by
CH Cl . The experiment was completed after 7 days when
3
3
barrier to uptake was observed) —it is still a more ‘natural’
feeding route than the use of homogenates or cell-free
extracts, which were employed in the very first biosynthetic
experiments. (The term ‘natural’ is used in the sense that
the technique involves considerably less disturbance to the
biological system, and is therefore more likely to produce
results, which more closely mimic the true metabolic fate
for artemisinic acid that is naturally present in A. annua
plants.)
2
2
the last two sets of triplicate plant extracts had been made.
After 3 days the weight of the group of plants, which had
been deprived of water was found to have dropped to about
one-third of that of the group, which had been kept alive
hydroponically (the weight of the live group remained rela-
tively constant over the study). This is considered to be
good evidence that this group of plants was dead well before
the half-way point of the experiment was reached (or, equiv-
alently, for the discussion that follows, that enzymatic activ-
ity would have ceased in this group of plants).
2
The recovery of H label, which was achieved when ‘stem-
feeding’ labeled artemisinic acid to ‘cut’ plants in the defin-
itive experiments, which are described in the next section
varied between 10 and 40%. Although this is quite respect-
able for a biosynthetic experiment, it is significantly less
than the incorporations, which had been achieved in the pre-
2
13
2
2.4. H NMR and C– H COSY NMR analysis of the
crude CH Cl extracts of A. annua, which had been fed
2
13 2
2
3
3
vious experiments with dihydroartemisinic acid, for which
quite remarkable recoveries of up to 80% of the total admin-
istered label had been reported.
with [15- C H ]-artemisinic acid (2a)
3
2
Each CH Cl plant extract was analyzed immediately by H
2
2
NMR spectroscopy. The extracts made after 1 day showed
1
2
.3. Feeding and extraction of A. annua plants
only a doublet peak at d 1.59 ppm ( J ¼19.1 Hz), corre-
D
CD
sponding to the labeled precursor 2a. However, this doublet
peak was observed to decline over the course of the experi-
ment, and to be replaced by several other doublets in the
Forty two A. annua plants were cut just above the root and
fed individually via the cut stem with 2 mg each of
1
3 2
[
15- C H ]-artemisinic acid. These plants were then di-
3
range d 1.0–2.5 ppm from the second day onwards (weak
D
vided into two groups. One group of 21 plants was kept alive
hydroponically, by periodic replacement of water in their
peaks were also occasionally resolved in the range d 4.8–
D
5.3 ppm). For example, Figure 3 shows a H NMR spectrum
2
2
Figure 3. H NMR spectrum of a crude CH
acid.
13 2
extract from the ‘dead’ group of A. annua plants, made 4 days after feeding with [15- C H
2
Cl
2
3
]-artemisinic

G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
9555
1
3
2
from a sample, which was extracted 4 days after feeding, in
which new doublet peaks at dD 1.33, 1.66, and 2.12 ppm
have appeared, in addition to that of the labeled precursor
for labeled 2a in this C– H COSYexperiment—according
to such an analysis). The deuterium and carbon chemical
shifts recorded at the 15-position for the four metabolites,
which can be identified in Figure 4 would then be entirely
2
a (at d 1.59 ppm).
D
2
,9–11
consistent with their assignments as compounds 2a,
3a,
1
3
2
2,11,12
6,18,19
21
Although the largish one-bond C– H coupling constant
9a,
and 13a, respectively, based on the
1
(
JCDz19 Hz) of these and other doubly-labeled metabo-
lites of 2a made a detailed visual analysis of the one-dimen-
fully-assigned NMR data, which has been reported in the
literature for each of these compounds (see references).
No signals were observed in the 2D- C– H COSY spectra
of any of the crude extracts that would suggest the presence
of either of the labeled metabolites [15- C H ]-artemisinin
3
(1) or [15- C H ]-artemisitene (4), both of which have been
3
2
13
2
sional H NMR spectra from the crude extracts of the aerial
parts more difficult, it did allow for the direct analysis of
these crude plant extracts by two-dimensional C– H corre-
1
3
2
13 2
3
3,39,40 13
2
13 2
lation NMR spectroscopy. C– H COSY analysis of
the crude plant extracts (Fig. 4) indicated that the most abun-
dant deuterium resonances at dD 1.59, 1.33, 1.66, and
proposed previously as metabolites of 2 in A. annua (see
Section 1).
2
2–25
2.12 ppm were connected by a single bond to carbon reso-
nances at d 22.8, 21.9, 22.3, and 29.0 ppm, respectively.
2.5. Isolation and identification of labeled metabolites
from the CH Cl extracts of the aerial parts of A. annua
that had been fed with [15- C H ]-artemisinic acid (2a)
3
C
2
2
2
13 2
When allowance is made for the effects of H isotopic
enrichment on C chemical shift (resulting in a ca.
1
3
1
3
0
.3 ppm upfield shift in the C resonance for each deute-
In order to confirm the structures of the metabolites of 2a
whose identities had been suggested in the previous section
rium atom, which is directly attached to the carbon), the iso-
topically-normal values for each of these four metabolites
can then be inferred as: d 23.7, 22.7, 23.2, and 29.9 ppm,
1
3
2
from the C and H NMR chemical shifts observed at their
15-position, the crude CH Cl extracts obtained from feed-
ing [15- C H ]-artemisinic acid (2a) to both the ‘live’ and
C
2
2
1
3 2
respectively, assuming that there has been complete reten-
tion of the H label in the 15-methyl group. (Note that the
3
2
‘dead’ groups of A. annua plants were subjected to extensive
HPLC chromatographic separations (see Section 4.5). This
procedure resulted in the isolation of seven doubly-labeled
amorphane and cadinane sesquiterpenes (Scheme 3), in
carbon in the 15-position of isotopically-normal artemisinic
acid resonates at d 23.8 ppm, which is in good agreement
C
with the carbon chemical shift—observed at d 22.8 ppm
C
1
3
2
Figure 4. Expansion of the C– H COSY spectrum from three combined CH
13 2
2 2 3
Cl plant extracts of A. annua, which had been fed with [15- C H ]-artemisinic
acid (2a), then allowed to die by desiccation, and extracted after 4 days.

9556
G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
H
O
H
H
H
H
3
+
6
+
4
2H 13C
3
2H 13C
15
2H 13C
2H
13
3
H
O
3
O
C
2 or 3
O
HO
O
O
O
3a
O
7a
4
,15
(2H2)
9a 6-O-
2
a
1
0a 6-O-
3
,4 2
8
a
( H )
3
O
H
H
2H 13C
4
3
1
+
5
6
+
OHC
HO
2
H313C
H
H
O
MeO
O
1
3a
14a
Scheme 3. Labeled metabolites 3a, 7a, 8a, 9a, 10a, 13a, and 14a, which were isolated following chromatographic separation of the crude plant extracts from the
13 2
3
in vivo transformations of [15- C H ]-artemisinic acid (2a) in A. annua.
addition to the recovery of the untransformed labeled feed-
ing precursor 2a. The structure and relative stereochemistry
of the seven labeled metabolites: 3a, 7a, 8a, 9a, 10a, 13a,
and 14a were all then unambiguously confirmed by compar-
ison with physical data (in particular H and C NMR spec-
tra), which has been reported in the literature.
was recovered from chromatography represents the endoge-
nous natural product, which would have been present in A.
annua in any case, even if labeled artemisinic acid had not
been administered to the plant. Hence, it can be concluded
that there has been no depletion of isotopic label as a result
of metabolism. This interpretation was further confirmed
1
13
1
13
by H NMR, C NMR, and MS analyses of the seven puri-
fied labeled metabolites: 3a, 7a, 8a, 9a, 10a, 13a, and 14a,
which were also isolated by chromatography (see Sections
4.5.2–4.5.8). Each of these samples exhibited a comparable
ratio of the endogenous isotopically-normal form to the
labeled form of the natural product (the labeled secondary
metabolite consistently accounted for between 10 and 30%
of the total), with no significant quantities of partially
labeled forms being observed.
The most abundant labeled compound to be recovered from
HPLC separation was the untransformed feeding precursor
1
3
2
1
[
15- C H ]-artemisinic acid (2a). Analysis by H NMR
3
and MS of the purified sample of artemisinic acid, which
was recovered after feeding suggested that it consisted of
approximately 15% of the doubly-labeled form of di-
hydroartemisinic acid (2a) and ca. 85% the intensity of
isotopically-normal dihydroartemisinic acid (2). Thus, the
integral for the 15-position at d 1.59 in H NMR was ca.
1
H
8
5% of that expected for [3H]; and molecular ions were ob-
The most abundant metabolite of 2a to be isolated from
HPLC separation was [15- C H ]-arteannuin B (3a). Ar-
+
13 2
served in an approximately 6:1 ratio for M : M+4 in mass
spectrometry. Significantly, there was no evidence in either
C NMR or MS for the accumulation of any partially la-
3
teannuin B has been implicated previously as a metabolite
of artemisinic acid
1
3
22–25
and was also one of the first natural
products ever to have been isolated from A. annua. The next
most significant metabolites were epi-deoxyarteannuin B
2
beled forms of artemisinic acid, beyond those, which were
already present in the sample of synthetic 2a that had been
used as the feeding precursor. Thus, while the mass spectrum
of artemisinic acid, which was recovered from the feeding
experiment contained both a molecular ion for isotopi-
cally-normal artemisinic acid (at m/z¼234) as well as a sec-
2
5
21
(9a) and the seco-cadinane (13a), both of which have
also been suggested previously as metabolites of artemisinic
acid (Scheme 1), although this is the first experimental evi-
dence that 2 can indeed be transformed into 13. Doublet
peaks for each of 3a, 9a, and 13a, are clearly visible in the
1
3 2
ond less abundant peak for the C H -labeled form (at
3
2
m/z¼238), only very low abundance peaks were observed
H NMR spectrum of the crude CH Cl extract, which is
2
2
at m/z 237 and 236, which might correspond to the partially
labeled [15- C H H] and [15- C HH ] forms, respec-
shown in Figure 3 (at d 1.33, 1.66, and 2.12 ppm, respec-
D
1
3
2
13
2
13
2
tively), as well as in the C– H COSY spectrum of Figure 4
(at dD 1.33/dC 21.9, dD 1.66/dC 22.3, and dD 2.12/dC
29.0 ppm, respectively), confirming that the isolation of
these compounds is unlikely to have been an artifact of the
chromatographic procedures. The remaining four metabo-
2
2
1
3
tively. Similarly, the C NMR spectrum of this sample con-
sisted predominantly of a singlet at d 23.7 ppm (for C-15 of
isotopically-normal artemisinic acid) as well as a 1:3:6:7:
6:3:1 septet at d 22.8 ppm (ca. 0.9 ppm upfield of the isoto-
C
pically-normal value, due to the presence of a C H label at
the 15-position of 2a). The difficulties encountered in defin-
itively identifying either a 1:2:3:2:1 quintet at ca. 0.6 ppm
upfield of the isotopically-normal value, which would corre-
spond to the partially labeled [15- C H H] form of 2a, or
2
a 1:1:1 triplet at ca. 0.3 ppm upfield of the isotopically-nor-
mal value, which would correspond to the partially labeled
C
1
3
2
13 2
lites of labeled artemisinic acid: [15- C H ]-annulide
2
3
1
3
2
13 2
(7a), [15- C H ]-isoannulide (8a), [15- C H ]-deoxyar-
3 3
1
3 2
teannuin B (10a), and [15- C H ]-artemisinic acid methyl
3
ester (14a) were all isolated in trace amounts. All four
metabolites have also been reported previously as bona
fide natural products from A. annua (see Section 1).
1
3 2
1
3 2
[
15- C HH ] form of 2a, confirmed the low abundance of
2
Although a large number of other natural products were also
isolated from this chromatographic separation (including
several non-sesquiterpenoid metabolites that are typical of
these partially deuteriated forms. It seems therefore that
the isotopically-normal form of artemisinic acid, which

G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
9557
2
A. annua), none contained a detectable amount of H label. It
was of particular interest to establish the presence or absence
of label in all of those 11,13-dihydro sesquiterpenes, which
were also isolated in pure form by this chromatographic sep-
aration. These included dihydroartemisinic acid (16), dihy-
droarteannuin B (22), arteannuin I (23), arteannuin J (24),
therefore that artemisinic acid (2) is not being converted di-
rectly into artemisinin (1) to any significant extent by the ‘di-
rect’ route, which is indicated by the arrow labeled ‘Refs.
22–25’ in Scheme 1. Two suggestions can be made to ex-
plain the apparent anomaly between earlier studies—con-
2
2–26,28–30
ducted with cell-free systems and radioisotopes
dihydro-epi-deoxyarteannuin B (15), and the seco-cadinane
5 (Fig. 1). In addition, pure samples of the 11,13-dihydro
—which appear to have observed the direct conversion of ar-
temisinic acid (2) to artemisinin (1), and the current study—
conducted in vivo with a stable isotope label—which does
not confirm this. Firstly, it may be that the cell-free systems,
x
2
4
metabolites: arteannuins H, K, L, and M, deoxyartemisinin,
and artemisinin (1) were also isolated (however, the 11,13-
dehydro analogs of these compounds were not obtained
2
2–
which were employed in the earlier biosynthetic studies
2
26,28–30
from chromatography). H NMR spectra of each of these pu-
rified 11,13-dihydro metabolites showed no detectable H
were inappropriate, permitting the appearance of ar-
tifacts, which have not been observed in the current experi-
ment, when stem-feeding the A. annua plants (indeed,
several recent papers have made reference to the possibility
of artifacts arising from the autoxidation of various natural
2
signal, even when acquired for extended periods of time.
In particular, the H NMR spectrum of a ca. 100 mg sample
2
of pure artemisinin, which was recovered from the feeding
experiment contained no detectable deuterium signal, even
when recorded with 15,000 repetitions of the pulse sequence
3
1–33,36,37,39,40
products from A. anuua).
Secondly, it is well
known that the use of a radioisotopic label can permit the de-
tection of a metabolite at a level, which is typically several
orders of magnitude lower than that for a stable isotope label.
Hence, if a bona fide minor biosynthetic pathway from arte-
misinic acid to artemisinin does exist, then it is quite possible
that this transformation would have been observed when us-
ing radioisotopes, but would have escaped detection with
stable isotopes. (Or, alternatively, this result could also be
explained if artemisinic acid was to be degraded to simpler
metabolites, which were then capable of re-assembly to arte-
misinin by the cell-free assay system, if even to only a very
limited extent).
(
1
for comparison, experiments employing between 10 and
00 transients were generally more than sufficient to identify
2
a H-labeled metabolite in a chromatographic fraction, when
purifying a labeled metabolite, which was present in that
fraction at a level of 0.1 mg or more). The apparent absence
of any H label from all of the 11,13-dihydro metabolites,
which were isolated in this study is discussed further in the
next section.
2
3. Conclusion
The in vivo transformations of artemisinic acid, which have
been observed in A. annua in this study are summarized in
Scheme 3. The first and most obvious conclusion to be drawn
from this feeding experiment is that arteannuin B (3) is the
major metabolite from the in vivo transformations of artemi-
sinic acid (2) in A. annua plants (Scheme 3). This is in agree-
Although these results appear to rule out any significant role
for the ‘direct’ pathway from artemisinic acid (2) to artemi-
sinin (1), which is shown in Scheme 1, it is still possible that
the ‘indirect’ route, which involves arteannuin B (3) as an in-
termediate (upper arrow from 2 to 3, labeled ‘Refs. 22–25’;
then arrow from 3 to 1, labeled by ‘Refs. 28 and 29 but not
Ref. 25’), may play a role in the biogenesis of 1. The current
feeding experiment was terminated after 7 days, and it is
conceivable that—if the rate of conversion of arteannuin B
(3) to artemisinin (1) was very much slower than the exper-
imentally-observed rate of conversion of artemisinic acid (2)
to arteannuin B (3)—there would have been insufficient time
for a detectable amount of labeled artemisinin to be accumu-
lated by this route. This issue could most easily be resolved
by performing a future feeding experiment using stable iso-
tope-labeled arteannuin B (3a) as a hypothetical biogenetic
2
2–25
ment with studies in the earlier literature,
performed with radioisotopic labels and plant homogenates
see arrow labeled ‘Refs. 22–25’ in Scheme 1). A further
which were
(
six stable isotope-labeled metabolites were also recorded
from the current study (Scheme 3), in addition to labeled
arteannuin B (3a). One of these compounds, epi-
deoxyarteannuin B (9), has previously been suggested to be
an intermediate in the metabolism of artemisinic acid en
2
6
route to artemisinin (arrow labeled ‘Ref. 26’ in Scheme 1);
but the remaining five metabolites (7, 8, 10, 13, and 14) have
not been reported previously as metabolites of artemisininic
acid (2), although all are known as natural products from
A. annua (Fig. 1).
2
2–25
precursor.
deoxyarteannuin B (9) or the seco-cadinane 13 (both of
Similarly, it is also possible that either epi-
2
6
21
which were isolated in significantly smaller amounts than
3
), might be undergoing conversion to artemisinin at
Perhaps the most intriguing result from this study is the
absence of any detectable incorporation of label into artemi-
sinin (1) itself. (Indeed, artemisitene (4), the 11,13-dihydro
derivative of artemisinin, which might also have been ex-
a rate, which is much slower than the rate of their formation
from artemisinic acid (2). For this reason, it might also be
worthwhile performing future feeding experiments using
either stable isotope-labeled epi-deoxyarteannuin B (9a) or
labeled seco-cadinane (13a) as the hypothetical biogenetic
precursor.
2
pected to incorporate H isotopic label, based on the earlier
biosynthetic studies that are summarized in Scheme 1, was
not even detected in these experiments.) It can be concluded
2
5
k
Although the absence for any detectable transformation of 2
into 1 in this study is at variance with the earlier literature
k
However, this is not too surprising, since artemisitene is a ‘scarce’ natural
product, which has been reported only very infrequently from nature.
22–26,28–30
(pre-1993)
it is consistent with the more recent literature (post-
for the biosynthesis of artemisinin,
1
3
The author has never isolated artemisitene from A. annua, although he
has reported some of the most extensive and detailed phytochemical
3
3,41–44
1999), in which there is now a gathering consensus
that dihydroartemisinic acid (16), rather than artemisinic
4–7
investigations of this species to date.

9558
G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
acid (2), is the true late-stage precursor to artemisinin. In this
regard, it is intriguing to note that there is a very high degree
of structural homology between the various highly oxygen-
ated 11,13-dehydro sesquiterpenes, which have been iso-
lated as metabolites of artemisinic acid (2) in the current
study, and those 11,13-dihydro metabolites, which were
obtained in the previous study with dihydroartemisinic acid
product distribution observed between the two studies (the
11,13-dihydro analogs of each of the six labeled compounds
3, 7, 8, 9, 10, and 13 were all isolated in the previous stud-
3
3
y). In support of the mechanisms for the transformations
of 2, which are proposed in the top right hand box of Scheme
4, it should be noted that the allylic hydroperoxide 28 can be
produced in the laboratory by chemical reaction of 2 with
3
3
1
45–47
(
bolism observed for 16 was concisely explained in terms
16). In the previous study, the complex pattern of meta-
O ,
2
and that it has then been shown in vitro to undergo
several of the transformations, which are depicted in this
box. The proposal that the ‘metabolism’ of artemisinic
acid (2) in A. annua might proceed via an allylic hydroper-
oxide intermediate such as compound 28, perhaps created
spontaneously by a pigment-photosensitized autoxidation
reaction occurring in vivo, was further supported by the
identical pattern of transformations, which were observed
when feeding artemisinic acid to both the ‘live’ and the
‘dead’ groups of plants in this experiment.
4
,5
of the ‘ene’-type addition of singlet oxygen to the D -dou-
ble bond of dihydroartemisinic acid. This then led to a detect-
able tertiary allylic hydroperoxide intermediate (26), which
underwent reactions characteristic of such allylic hydroper-
oxides to produce a variety of compounds, including 15, 22–
2
5, and 27—as well as artemisinin (1) itself—as is shown in
the bottom right hand box of Scheme 4. Although the puta-
tive tertiary allylic hydroperoxide of artemisinic acid, com-
pound 28 (i.e., the 11,13-dehydro analog of intermediate
2
6), was not isolated as a metabolite in the current study
The third important observation is that all of the seven me-
tabolites isolated from this feeding experiment (Scheme 3)
retained the terminal double bond, which was present at
the 11,13-position of artemisinic acid (2). In fact, about
of the metabolism of artemisinic acid (2), its presence has
been inferred in the top right hand box of Scheme 4 (where
it appears in parentheses), based on the near identical
H
H
H
H
H
H
Oxn.
1O
Oxn.
Oxn.
2
-H2O
Mevalonic
acid
HOO
H
H
H
O
O
H
HO
1O
H
O
O
2
HO
OH
30
31
1O
2
O 2
O 28
O
H
3
2
9
Redn.?
Redn.?
H
-H2O2
H
+
H O?
2
H
O
HOO
HO
HOO
HO
H
O
H
O 9/10
H
O
H
H
O
H
O
O
OHC
HO
H
O
1
1,13
7
to
11,12?
11,13
11,12
O 8
to
13
;
1
1,13-Dehydro sesquiterpenes
+
H O?
2
H
H
H
H
H
Oxn.?
Oxn.?
1O
2
2
-H O
HOO
H
H
H
H
Hock
O
H
H
HO
H
H
H
cleavage;
HO
O
3
O ;
2
OH32
O 33
O
2
1O
-H2O2
O 26
O 22
16
-H2O
1O
2
H
H
H
H
O
O
O
H
H
O
H
H
O
H
H
H
H
HOO
HO
HOO
HO
O
O
O
1
O 15/27
O
H
H
H
OHC
HO
H
H
H
H
H
O
O
O
2
O
24
O
25
3
1
1,13-Dihydro sesquiterpenes
Scheme 4. Unified biosynthetic scheme to account for the biogenesis of the two families of 11,13-unsaturated and 11,13-saturated metabolites from
A. annua.

G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
9559
two thirds of all the 11,13-dehydro sesquiterpenes, which are
currently known from A. annua (see Fig. 1) have been iso-
lated in this study. In contrast, none of the 11,13-saturated
sesquiterpenes, which were also recovered from the crude
extracts showed any detectable incorporation of label, in-
cluding artemisinin (1) itself, as already noted. These con-
clusions are the exact reverse of those from the previous
experiment, when feeding dihydroartemisinic acid (16)
to A. annua, in which about half** of all the 11,13-saturated
sesquiterpene metabolites ever described from this species
were isolated in labeled form, but none of the 11,13-dehydro
sesquiterpenes.
(16) and the 11,13-dihydro series of sesquiterpenes remains
unclear (possible branching points are indicated by arrows
bearing question marks in the left hand box of Scheme 4).
Finally, it should be noted that the unified biosynthetic
pathway shown in Scheme 4 could neatly explain the oft-
5
3
reported incidence of two chemotypes of A. annua:
viz.—a low-yielding-artemisinin chemotype, which is rich
in artemisinic acid (i.e., biosynthesis is proceeding largely
along the upper route), and a high-yielding-artemisinin che-
motype, which is low in artemisinic acid (i.e., biosynthesis is
proceeding predominantly by the lower route).
3
3
4. Experimental
The inescapable conclusion seems to be therefore that nei-
ther oxidation nor reduction at the 11,13-position can occur
to any significant extent for ‘advanced’ metabolites such as
artemisinic acid (2) or dihydroartemisinic acid (16). Artemi-
sinic acid (2) is thus the exclusive precursor to the 11,13-de-
hydro metabolites from A. annua, just as dihydroartemisinic
acid (16) is the exclusive precursor to the large family of
highly oxygenated 11,13-dihydro sesquiterpenes from this
species. This conclusion is summarized diagrammatically
in the top and bottom right hand boxes of Scheme 4 in which
artemisinic acid and dihydroartemisinic acid are portrayed
as the committed intermediates on two separate pathways
to the two large families of oxygenated 11,13-dehydro and
4.1. General information
1
13
All H and C NMR experiments were recorded on either
a Bruker AMX 400, DRX 500 or AV 600 instrument. Chem-
ical shifts are expressed in parts per million (d) relative to
TMS as internal reference. Proton chemical shifts, multiplic-
ities, coupling constants, and integrals reported in this sec-
1
tion are those, which were clearly resolved in 1D- H NMR
without recourse to 2D-NMR analysis (see Table 1 in this
section for full assignments, which were made by 2D-
2
NMR). H NMR spectra were recorded in CHCl
solution
(10 ml/100 ml), which was used both as
an internal reference (d 7.43 ppm) and as an internal cali-
3
containing C D
6 6
11,13-dihydro sesquiterpene natural products, which are
known from A. annua.
D
2
bration standard for estimating the amounts of H-labeled
metabolites in solution (the ratio of the H integral for the
2
2
Consequently, the introduction of saturation/unsaturation at
the 11,13-position must be occurring at an earlier stage in the
biosynthetic pathway to the sesquiterpene metabolites found
in A. annua. In this regard, it is now well established that the
very first steps in the biogenesis of sesquiterpenes from
A. annua proceed by the normal pathways of terpenoid bio-
synthesis, generating amorpha-4,11-diene (29) as the first
committed intermediate on the pathway to artemisinin
benzene-d
other metabolites, which were resolved in the crude plant ex-
6
internal standard to the H integral for 2a and
2
tract, was routinely used to estimate the amount of H label
3
3
present in such extracts). The chemical shift of this refer-
ence compound did not interfere with the products of meta-
bolism of artemisinic acid, for which H signal was normally
2
observed only in the aliphatic region (d
occasionally, in the alkene region (d
spectrum—see Section 2.4.
1.0–2.5 ppm) or,
4.5–5.5 ppm) of the
D
D
4
8–50
(
1).
Therefore, the oxidative/reductive interconver-
sions, which link the separate routes to the two large families
of oxygenated 11,13-dehydro and 11,13-dihydro sesquiter-
penes, must be occurring at some point ‘downstream’ of
amorpha-4,11-diene (29), but ‘upstream’ of artemisinic
acid (2)/dihydroartemisinic acid (16). The left hand box in
Scheme 4 summarizes recent experimental work on the
1
3
2
33,39,40
C– H COSY NMR
spectra were acquired with
a Bruker AV 600 MHz NMR spectrometer, employing
some modifications to the procedures, which have been de-
scribed previously for a Bruker DRX 500 spectrometer.
3
3,39
2
ꢀ
The H ‘hard’ 90 pulses (70 ms/ꢁ6 dB) and composite de-
4
3,44,51,52
metabolism of A. annua,
which has suggested arte-
coupling pulses (Waltz 16; 230 ms/8 dB), which are required
1
3
2
misinic alcohol (30), artemisinic aldehyde (31), dihydroarte-
misinic alcohol (32) and dihydroartemisinic aldehyde (33)
as possible metabolites of amorpha-4,11-diene (29). Intrigu-
for the C– H COSYexperiment were created by a ‘2H-TX’
board (essentially a gated 20 W amplifier and interface),
which was incorporated as standard on the AV 600 spectro-
meter. This board converted the lock channel circuitry from
its normal function to a transmit/receivermode during pulses,
but allowed the normal operation of the lock at all other
times—therefore samples for C– H COSY spectroscopy
could be prepared in CDCl , rather than CHCl solution, as
previously,
auto-shim the sample over long periods of acquisition time.
In addition, since the BBO probe for the 600 MHz spectro-
meter had been designed for H excitation, it was possible
to use conventional 5 mm NMR tubes to prepare the C– H
COSY sample (2.5 mm microtubes had been employed previ-
ously for use with a dual probe at 500 MHz).
4
4,52
ingly, a single cytochrome P450
which catalyses each of the three oxidations on the route
9/30/31/2, although the point at which the bifurca-
tion to the lower path, leading to dihydroartemisinic acid
has now been found,
2
1
3
2
3
3
3
3,39
and it then became possible to lock and
*
*
However, note that fewer actual metabolites were detected with confi-
dence in this experiment, as compared with the previous experiment
33
with dihydroartemisinic acid (i.e., 7 metabolites from artemisinic
acid, compared with 15 from dihydroartemisinic acid). The three most
critical factors in explaining this are probably: (i) the slightly smaller
scale of this experiment; (ii) the lessened aqueous solubility of artemi-
sinic acid in the feeding solution, as compared with dihydroartemisinic
acid (a maximum of 1.3 mg/ml, vs 2.4 mg/ml); and (iii) the lower effi-
ciency for the recovery of label, which was observed when feeding arte-
misinic acid, as compared to dihydroartemisinic acid (10–40%, as
compared with 60–80%).
2
1
3
2
3
3,39,40
1
1
HSQC, HMBC, H– H COSY, and NOESY spectra were re-
corded with 2048 data points in F and 256 data points in F .
2
1

9560
G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
1
Table 1. C NMR assignments (in CDCl
3
3
) for some of the compounds obtained by synthesis in Section 2.1
a
Position (mult.)
2a
41.4
14a
41.4
25.5
26.3
134.6
120.3
37.9
42.3
25.9
18a
41.1
25.1
25.6
135.0
122.8
40.4
150.5 (C)
28.7
35.7
19a
42.3
25.9
26.2
134.5
122.0
37.0
48.0
23.6
20
47.0
19.3
31.6
143.0
121.4
87.3 (C)
167.8 (C)
21.5
35.6
26.3
122.7
173.9
8.6 (CH
17.3
21
50.5
24.7
35.9
105.3
94.1
80.7 (C)
45.5
31.1
34.0
37.6
39.7 (CH)
172.6
20.5 (CH
19.9
25.5
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
(CH)
(CH
(CH
(C)
2
)
)
25.7
26.4
134.9
120.2
38.0
42.1
26.0
35.3
27.6
142.7
172.7
126.6
19.7b
22.9
—
2
(CH)
(CH)
(CH)
(CH
(CH
2
)
)
2
35.2
27.5
35.5
27.7
77.6
0 (CH)
1 (C)
2 (C)
3 (CH
4 (CH
5 (CH
28.2
143.3
167.8
124.1
19.7b
22.8
120.3
171.3
15.0 (CH
19.4
178.7
23.9 (CH
19.7
2
3
3
)
)
)
3
)
3
)
3
)
3
)
b
b
22.6
51.4
23.0
52.9
23.2
—
5-OMe (CH
3
)
51.7
—
a
Multiplicity determined from DEPT.
Chemical shifts shown are for the 15- C H
b
13
2
13
isotopomer, which is expected to be ca. 0.9 ppm upfield of the isotopically-normal value in C NMR.
1
3
High-resolution MS were recorded in EI mode at 70 eV on
a Finnigan-MAT 95 MS spectrometer. IR spectra were re-
corded in CHCl on a Shimadzu FTIR-8201 PC instrument.
177.7 C (C-12), 135.6 C (d, J¼43.1 Hz, JCC, C-4), 119.4
CH (C-5), 51.2 CH (15-OMe), 43.9 CH (C-7), 42.0 CH
3
3
(C-11), 41.7 CH (C-1), 36.3 CH (d, J¼4.0 Hz, JCC, C-6),
3
HPLC separations were performed using a Varian chromato-
gram equipped with RI star 9040 and UV 9050 detectors and
either a YMC diol or a PREP-SIL column (both 20 mmꢂ
35.2 CH (C-9), 27.6 CH (C-10), 27.4 CH (C-8), 26.5
2
2
2
CH (d, J¼3.5 Hz, J , C-3), 25.7 CH (d, J¼2.9 Hz,
2
CC
2
3
1
J
, C-2), 22.8 ((1:3:6:7:6:3:1) sept., J¼19.2 Hz, J
,
CD
CC
1
3
2
2
2
5 cm), flow rate 8 mL/min.
15- C H ), 19.6 CH (C-14), 15.0 CH (C-13); H NMR
3 3 3
1
(d, CHCl ) ppm: 1.62 (d, J¼19.2 Hz, JCD, 15-D );
HREIMS m/z (rel int.): 254.2156 [M , C
quires 254.2154] (1.6), 253.2084 [M , C
3
3
+
13
13
2
4
.1.1. Synthesis of labeled artemisinic acid (2a). Labeled
C H H O re-
1 23 3 2
C H H O re-
1 24 2 2
1
1
5
5
+
2
artemisinic acid (2a, 102 mg) was obtained from labeled
dihydroartemisinic acid (16a, 400 mg) in three steps, as de-
scribed below. Ref. 31 contains details of the preparation of
the starting material, compound 16a, from artemisinin (1) by
a reconstructive synthesis requiring four to five steps.
quires 253.2092] (0.1), 207 (4), 205 (4), 166 (100), 153 (25),
136 (18).
1
1,13
4.1.1.2. Introduction of the D double bond in 14a
by oxidative selenation of 17a. Dry THF (3 ml) was cooled
1
3
2
ꢀ
pylamine (0.16 ml, 1.14 mmol) and n-butyllithium (0.7 ml,
4
.1.1.1. Methylation of [15- C H ]-dihydroartemi-
3
to ꢁ78 C under an atmosphere of N and distilled diisopro-
2
sinic acid (16a). DiazaldÒ (N-methyl-N-nitrosotoluene-
p-sulphonamide, 0.5 g, 2.33 mmol) was dissolved in Et O
(
1.6 M in hexane, 1.12 mmol) were added with stirring over
a period of 1 h. A solution of [15- C H ]-dihydroartemi-
2
1
3 2
10 ml) and cooled in ice. A solution of KOH (0.14 g) in
5% EtOH (3 ml) was added, and the mixture was stirred
3
9
sinic acid methyl ester (17a) (0.20 g, 0.79 mmol) in THF
(1 ml) was added and stirring continued for 30 min, then dis-
tilled hexamethylphosphoramide (0.3 ml) was added and
stirring continued for a further 30 min. The temperature
ꢀ
at 0 C for 10 min. The ethereal diazomethane solution was
distilled from a water bath and added dropwise to a solution
of dihydroartemisinic acid (400 mg, 2.27 mmol) (16a) in
ꢀ
Et O (20 ml). When the evolutionof bubbleshad ceased, ace-
2
was raised to ꢁ10 C and the mixture stirred for another 2 h,
ꢀ
tic acid (10%; 1 ml) was added to remove excess CH N . The
2
then the reaction mixture was cooled to ꢁ78 C prior to the
2
reaction mixture was extracted by Et O (3ꢂ30 ml) and the
transfer of a solution of phenylselenyl bromide (0.189 g,
0.80 mmol) in THF (2 ml). The reaction mixture was stirred
2
combined organic extracts were washed successively with
saturated NaHCO (3ꢂ5 ml) and brine (3ꢂ10 ml), then dried
ꢀ
for 15 min before the temperature was increased to ꢁ10 C
3
(
MgSO ), and the organic solvent was removed under
4
and stirring was then continued for another 1 h. Finally, ace-
tic acid (1 ml; 25%) and H O (0.5 ml; 30% in H O) were
added and the mixture was stirred at room temperature for
reduced pressure to yield the methyl ester 17a (399 mg,
.58 mmol; 94%) without the need for further purification.
2
2
2
1
3
0 min. The product was extracted with Et O (3ꢂ50 ml) and
2
1
3 2
4.1.1.1.1. [15- C H ]-Dihydroartemisinic acid methyl
ester (17a). Oil (see also Ref. 31 for physical properties).
the combined organic layers were washed by saturated
NaHCO (3ꢂ10 ml), followed bybrine(3ꢂ10 ml), thendried
3
3
ꢁ
1
IR n (cm ): 2924 (m), 2870 (m), 2849 (m), 1726 (s),
456 (m), 1435 (m), 1215 (s). H NMR (d, CDCl ) ppm:
3
(MgSO ), and solvent was removed in vacuo to yield a crude
4
1
1
5
2
2
product (0.31 g) consisting of compounds 14a, 18a, and 19a,
which were separated by HPLC (diol column, n-hexane).
3
.12 (1H, d, J¼6.7 Hz, JCH, H-5), 3.67 (3H, s, 15-OMe),
.50 (1H, m, H-6), 2.50 (1H, m, H-11), 1.94 (1H, m, H-
b), 1.91 (1H, m, H-3a), 1.80 (1H, m, H-3b), 1.62 (1H, m,
1
3 2
4.1.1.2.1. [15- C H ]-Artemisinic acid methyl ester
3
H-7), 1.59 (1H, m, H-9b), 1.55 (1H, m, H-2a), 1.41 (1H,
m, H-10), 1.25 (2H, m, H-1 and H-8a), 1.13 (3H, d,
J¼6.9 Hz, H-13), 1.08 (1H, m, H-8b), 0.94 (1H, m, H-9a),
(14a). Oil (126 mg, 64%; t 12.3 min) (see also Ref. 5 and
R
ꢁ1
Section 4.5.8 for physical properties). IR n (cm ): 3030
(w), 2951 (s), 2924 (s), 2870 (s), 1713 (s), 1624 (m), 1434
1
3
1
0
.86 (3H, d, J¼6.5 Hz, H-14); C NMR (d, CDCl ) ppm:
(m), 1271 (m), 1159 (s). H NMR (d, CDCl ) ppm: 6.28
3
3

G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
9561
1
Table 2. H NMR assignments (in CDCl
synthesis in Section 2.1
1
3
) for compounds obtained by
(m), 1228 (s). H NMR (d, CDCl ) ppm: 5.76 (1H, d,
3
3
J¼6.8 Hz, JCH, H-5), 3.78 (3H, s, 15-OMe), 3.21 (1H, s,
Position
1
2a
14a
18a
19a
20
21
–OH), 2.72 (1H, br s, H-6), 1.43 (3H, s, H-13), 1.08 (1H,
br d, J¼12.9 Hz, H-8), 0.94 (1H, dddd, J¼12.9, 12.7, 12.7,
1.43
1.92
1.53
1.74
1.88
4.99
2.61
2.67
1.38
1.32
1.04
1.70
1.42
—
1.43
1.93
1.53
1.76
1.88
4.98
2.54
2.72
1.40
1.31
1.07
1.71
1.44
—
1.37
1.97
1.54
1.81
1.94
4.96
3.34
—
2.79
1.91
1.00
1.68
1.62
—
1.22
1.93
1.59
1.79
1.89
5.76
2.72
1.77
1.42
1.08
0.94
1.66
1.49
—
1.83
1.82
1.82
2.16
2.28
5.40
—
1.47
1.94
1.49
2.08
2.39
5.93
—
1.66
1.80
1.44
1.12
1.71
1.42
2.28
1.47
1.00
1.46
—
3
.4 Hz, H-9), 0.86 (3H, d, J¼6.6 Hz, H-14)—see also Table
2
2
3
3
5
6
7
8
8
9
9
1
1
1
1
1
1
a
b
a
b
1
3
2; C NMR (d, CDCl ) ppm: 178.7 C (C-12), 134.5 C (d,
3
1
J¼43.2 Hz, JCC, C-4), 122.0 CH (C-5), 77.6 C (C-11),
5
2.9 CH (15-OMe), 48.0 CH (C-7), 42.3 CH (C-1), 37.0
3
3
CH (d, J¼4.2 Hz, J , C-6), 35.5 CH (C-9), 27.7 CH (C-
CC
2
2
1
0), 26.2 CH (d, J¼3.1 Hz, JCC, C-3), 25.9 CH (d,
—
2
2
3
a
b
a
b
0
1
3
4
2.63
2.25
1.41
1.86
1.14
—
1.82
0.84
1.69
—
J¼2.6 Hz, J , C-2), 23.9 CH (C-13), 23.6 CH (C-8),
CC 3 2
1
13 2
2
3.0 ((1:3:6:7:6:3:1) sept., J¼18.9 Hz, J , 15- C H ),
CD
3
2
3 3
1
9.7 CH (C-14)—see also Table 1; H NMR (d, CHCl )
1
ppm: 1.63 (d, J¼18.9 Hz, J , 15-D ); HREIMS m/z (rel
CD
3
+
13
2
int.): 270.2104 [M , C
C H H O requires 270.2104]
5 1 23 3 3
1
6.45, 5.55
0.89
1.58
6.28, 5.43
0.90a
1.59
3.74
1.88
0.89a
1.62
3.37
1.43
0.86a
1.60
3.78
(3), 269 (0.2), 252 (5), 236 (2), 220 (9), 193 (5), 167 (100).
a
5
5-OMe
13 2
4.1.1.3. Deprotection of [15- C H ]-artemisinic acid
methyl ester (14a) yielding [15- C H ]-artemisinic acid
2a). To a solution of 15-[ C H ]-artemisinic acid methyl
3
3
—
1
3 2
3
a
1
Not observed in the H NMR spectrum of the [15- C H
13
2
13 2
3
]-isotopomer.
(
ester (14a) (126 mg, 0.50 mmol) in MeOH (10 ml) was
added an aqueous solution of KOH (180 mg, 3.2 mmol;
10 ml) and the reaction mixture was heated to reflux over-
night. After cooling and acidification to pH 4 (HCl, 10%),
(
1H, dd, J¼1.2, 1.0 Hz, H-13a), 5.43 (1H, dd, J¼1.4, 1.2 Hz,
CH
3
H-13b), 4.98 (1H, d, J¼6.7 Hz, J , H-5), 3.74 (3H, s, 15-
OMe), 2.72 (1H, ddd, J¼12.2, 3.5, 3.5 Hz, H-7), 2.54 (1H,
the mixture was extracted by CHCl (3ꢂ20 ml). The com-
3
br, H-6), 0.89 (3H, d, J¼6.1 Hz, H-14)—see also Table 2;
bined organic layers were washed with brine (3ꢂ5 ml), dried
13 2
1
3
C NMR (d, CDCl ) ppm: 167.8 C (C-12), 143.3 CH (C-
3
(MgSO ), and solvent was removed under reduced pressure
4
1
1
1
1), 134.6 (d, J¼43.3 Hz, J , C-4), 124.1 CH (C-13),
to yield [15- C H ]-artemisinic acid (2a) without the need
for further purification.
CC
2
3
20.3 CH (C-5), 51.7 CH (15-OMe), 42.3 CH (C-7), 41.4
3
3
CH (C-1), 37.9 CH (d, J¼4.0 Hz, J , C-6), 35.2 CH (C-
CC
2
2
13 2
9
2
(
), 27.5 CH (C-10), 26.3 CH (d, J¼2.9 Hz, JCC, C-3),
4.1.1.3.1. [15- C H ]-Artemisinic acid (2a). Oil
3
2
3
5.9 CH (C-8), 25.5 CH (d, J¼2.6 Hz, J , C-2), 22.8
(102 mg; 86%) (see also Refs. 9 and 10 and Section 4.5.1
for physical properties). IR n (cm ): 3400–2600 (m),
2
2
CC
13
1
2
ꢁ1
(1:3:6:7:6:3:1) sept., J¼19.1 Hz, J , 15- C H ), 19.7
CD
3
2
CH (C-14)—see also Table 1; H NMR (d, CHCl ) ppm:
3
2924 (m), 2870 (m), 1692 (s), 1622 (m), 1435 (m), 1279
(s), 1221 (m). H NMR (d, CDCl ) ppm: 6.45 (1H, s, H-
3
1
1
1
2
2
1
.58 (d, J¼19.1 Hz, J , 15-D ); HREIMS m/z (rel int.):
CD
3
3
+
13
5
2
3
52.1998 [M , C
51 (5), 250 (2), 249 (0.5), 237 (7), 220 (33), 207 (12),
92 (51), 166 (36), 153 (100).
C H H O requires 252.1998] (75),
1
13), 5.55 (1H, s, H-13), 4.99 (1H, d, J¼6.5 Hz, JCH, H-5),
1
21
3 2
2.67 (1H, ddd, J¼12.4, 4.1, 4.1 Hz, H-7), 2.61 (1H, br s,
1
3
H-6), 0.89 (3H, d, J¼5.8 Hz, H-14)—see also Table 2;
C
NMR (d, CDCl ) ppm: 172.7 C (C-12), 142.7 C (C-11),
3
1
3
2
1
4.1.1.2.2. [15- C H ]-E-7,11-Dehydro-dihydroartemi-
sinic acid methyl ester (18a). Oil (4.5 mg, 2%; t
1
134.9 C (d, J¼43.0 Hz, J , C-4), 126.6 CH (C-13),
3
CC
2
120.2 CH (C-5), 42.1 CH (C-7), 41.4 CH (C-1), 38.0 CH
(d, J¼4.0 Hz, J , C-6), 35.3 CH (C-9), 27.6 CH (C-10),
R
ꢁ
1
3
3.9 min). IR n (cm ): 3028 (m), 2924 (m), 2870 (m),
709 (s), 1457 (m), 1435 (s), 1286 (m), 1248 (m), 1198 (s).
H NMR (d, CDCl ) ppm: 4.96 (1H, d, J¼6.6 Hz, JCH, H-
CC
2
2
1
26.4 CH (d, J¼3.4 Hz, J , C-3), 26.0 CH (C-8), 25.7
2
CC
2
1
3
3
CH (d, J¼3.0 Hz, J C-2), 22.9 ((1:3:6:7:6:3:1) sept.,
3
2
CC
13
CD 3 3
1
2
5
), 3.73 (3H, s, 15-OMe), 3.34 (1H, br s, H-6), 2.79 (1H,
dddd, J¼13.7, 1.6, 1.6, 1.6 Hz, H-8), 1.88 (3H, d,
J¼1.8 Hz, H-13), 0.89 (3H, d, J¼6.6 Hz, H-14)—see also
J¼19.1 Hz, J , 15- C H ), 19.7 CH (C-14)—see also
2
Table 1; H NMR (d, CHCl ) ppm: 1.58 (d, J¼19.1 Hz,
3
1
+
J
, 15-D ); HREIMS m/z (rel int.): 238.1850 [M ,
3
CD
13
1
3
2
Table 2; C NMR (d, CDCl ) ppm: 171.3 C (C-12), 150.5
C14 C H H O requires 238.1842] (94), 237 (6), 236
1 19 3 2
3
1
C (C-7), 135.0 C (d, J¼42.9 Hz, JCC, C-4), 122.8 CH (C-
(2), 235 (0.5), 220 (18), 210 (16), 209 (9), 193 (25), 192
(22), 166 (14), 153 (15), 140 (35), 125 (100), 123 (86).
5
4
), 120.3 C (C-11), 51.4 CH (15-OMe), 41.1 CH (C-1),
3
3
0.4 CH (d, J¼4.0 Hz, J , C-6), 35.7 CH (C-9), 28.7
CC
2
2
11,13
CH (C-8), 28.2 CH (C-10), 25.6 CH (d, J¼3.0 Hz, JCC,
4.1.2. Introduction of the D double bond in epi-de-
oxyarteannuin B (9) by oxidative selenation of dihydro-
2
2
3
C-3), 25.1 CH (d, J¼3.0 Hz,
J
,
CC
C-2), 22.6
2
1
13
2
3
2
(
(1:3:6:7:6:3:1) sept., J¼19.0 Hz, J , 15- C H ), 19.4
epi-deoxyarteannuin B (15). To dry THF (3 ml) cooled to
ꢀ
CD
CH (C-14), 15.0 CH (C-13)—see also Table 1; H NMR
(
m/z (rel int.): 252.1995 [M , C
2
(
ꢁ78 C under an atmosphere of N were added distilled di-
3
3
2
1
d, CHCl ) ppm: 1.62 (d, J¼19.0 Hz, J , 15-D ); HREIMS
isopropylamine (0.16 m, 1.14 mmol) and n-butyllithium
(0.7 ml, 1.6 M in hexane, 1.12 mmol). The mixture was
stirred for 1 h, then dihydro-epi-deoxyarteannuin B (15)
(0.2 g, 0.85 mmol) in THF (1 ml) was added and stirring
continued for a further 30 min. Distilled hexamethylphos-
phoramide (0.3 ml) was then added and the mixture stirred
for another 30 min before raising the temperature to
3
CD
3
+
13
2
C H H O requires
1 21 3 2
1
5
52.1998] (52), 251 (5), 250 (2), 249 (0.5), 220 (38), 193
12), 166 (50), 153 (100), 136 (72).
1
3 2
4
acid methyl ester (19a). Oil. (7.6 mg, 4%; t 55 min). IR
n (cm ): 3528 (m), 2926 (m), 2870 (m), 1724 (s), 1456
.1.1.2.3. [15- C H ]-11-Hydroxy-dihyroartemisinic
3
R
ꢁ
1
ꢀ
ꢁ10 C and stirring for a further 2 h. The reaction mixture

9562
G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
ꢀ
13
was cooled to ꢁ78 C and a solution of phenylselenyl bro-
mide (0.189 g, 0.80 mmol) in THF (2 ml) was added and
stirring continued for 15 min. The temperature was then in-
H-14)—see also Table 2; C NMR (d, CDCl ) ppm—see
3
+
Table 1; EIMS m/z (rel int.): 250 [M ꢁO ] (26), 236 (15),
2
232 (17), 209 (32), 192 (100), 180 (34), 152 (72).
ꢀ
creased to ꢁ10 C and stirring was continued for another 1 h
before acetic acid (1 ml; 25%) and H O (0.5 ml; 30% in
2
4.2. A. annua plants
2
H O) were added and the mixture allowed to warm to
2
room temperature, and then stirred for 30 min. The reaction
mixture was extracted by Et O (3ꢂ50 ml) and the organic
A. annua plants were grown outdoors from seed (seeds were
supplied by Prof. Guan-Yi Liang from Guizhou province,
Guiyang, and taxonomically verified specimens are held
by him; a voucher specimen of plant material grown from
the seeds (no. HKU2285) has also been deposited in The
University of Hong Kong herbarium collection). Plants
were used for this feeding experiment after approximately
three months growth, when mature and beginning to come
into flower.
2
layers were combined and washed by saturated NaHCO
(
3
3ꢂ10 ml), followed by brine (3ꢂ10 ml), dried (MgSO ),
4
and solvent was removed in vacuo to yield a crude product
0.29 g), consisting of compounds 20 and 9, which were sep-
arable by HPLC (silica column, 5% EtOAc/n-hexane).
(
4.1.2.1. epi-Deoxyarteannuin B (9). Oil (42 mg,
.18 mmol, 21%; t 19.5 min—see Ref. 6 for NMR proper-
0
ties, and Refs. 17 and 18 for other physical properties; see
R
1
3 2
4.2.1. Preparation of a feeding solution of [15- C H ]-
3
+
also Section 4.5.5). HREIMS m/z (rel int.): 232.1463 [M ,
C H O requires 232.1464] (100), 217 (30), 204 (11),
artemisinic acid (2a) in TRIS buffer. The maximum solu-
bility of compound 2a in TRIS buffer (50 mM, pH 8.1) was
found to be around 1.3 mg/ml after ultrasonification for
1
5 20 2
1
87 (53), 173 (35).
1
2
sition of 2a as a result of ultrasonification and previous
–3 h. H NMR analysis indicated no change in the compo-
4.1.2.2. 7,11-Dehydro-dihydro-epi-deoxyarteannuin
B (20). Oil (78 mg, 0.34 mmol, 39%; t 26.1 min). IR n
3
3
studies had determined that this concentration of TRIS
buffer caused no discernible physical injury to intact
A. annua plants. Therefore, feeding solutions were prepared,
which contained around 1 mg of 2a/1 ml buffer.
R
ꢁ
1
(
(
(
8
1
cm ): 3017 (m), 2945 (m), 2862 (m), 1732 (s), 1682
m), 1454 (m), 1261 (m). H NMR (d, CDCl ) ppm: 5.40
1
3
1H, d, J¼1.3 Hz, H-5), 2.63 (1H, dd, J¼12.7, 9.1 Hz, H-
), 2.30–2.10 (3H, m, H-3 and H-8), 1.82 (3H, s, H-13),
.69 (3H, s, H-15), 1.41 (1H, ddd, J¼12.6, 10.1, 10.1 Hz,
4.3. ‘Stem-feeding’ of 2a to cut A. annua plants
1
3
H-9), 0.84 (3H, d, J¼6.7 Hz, H-14)—see also Table 2; C
NMR (d, CDCl ) ppm—see Table 1; HREIMS m/z (rel
int.): 232.1458 [M , C H O requires 232.1464] (75),
1
Forty-two A. annua plants were cut just above the root and
weighed individually (fresh plant weights were in the range
10–30 g). Individual plants were then fed with the labeled
precursor at a rate of approximately 1 mg of 2a/10 g fresh
plant material, by immersing the cut stem in a vial contain-
ing 2 ml of the feeding solution (using a solution prepared
from 90 mg of 2a in 90 ml of TRIS buffer (50 mM, pH
8.1)). The plants were exposed to a strong light source
(500 W bulb) and a constant flow of air (from a nearby
fan) in order to promote the uptake of the feeding solution
via the transpiration stream. Under these conditions,
‘stem-cut’ plants were normally able to take up all the feed-
ing solution over a period of 4–6 h. A small amount of TRIS
buffer was then added to the feeding vial in order to rinse any
remaining labeled precursor from the walls of the vial, and
the plants were left for a short while longer to take up this
additional small volume.
3
+
5 20 2
2
17 (28), 204 (26), 177 (23), 153 (100), 136 (82).
1
1,13
4
.1.3. Introduction of the D
double bond in artemisi-
tene (4) by oxidative selenation of artemisinin (1). To dry
ꢀ
1 ml, 7.1 mmol), followed by n-butyllithium (1.4 ml, 1.6 M
THF (15 ml) cooled to ꢁ10 C was added diisopropylamine
(
in hexane, 2.24 mmol). The reaction mixture was stirred for
ꢀ
1
0 min and then cooled to ꢁ78 C, before the addition of
a solution of artemisinin (1) (0.56 g, 1.97 mmol) in THF
5 ml). Stirring was continued for another 30 min, then a so-
(
lution of phenylselenyl bromide (0.345 g, 1.46 mmol) in
THF (5 ml) was transferred to the reaction mixture, which
ꢀ
acid (2 ml; 25% in H O) and H O (1 ml; 50% in H O)
was stirred at ꢁ78 C for a further 2 h. Solutions of acetic
2
2
2
2
were added and stirring was continued at room temperature
for a further 30 min, before the mixture was extracted by
Et O (3ꢂ50 ml), washed by saturated NaHCO (3ꢂ5 ml)
Half of the plants were then kept alive outdoors for a period
of 1 day to one week by continually replenishing their feed-
ing vials with deionized water (hydroponic conditions). For
the other half, the feeding vials were not replenished with
water, once dry, and the plants in this group were therefore
allowed to die by desiccation. The ambient temperature
2
3
followed by brine (3ꢂ10 ml), dried (MgSO ), and solvent
4
removed in vacuo to yield a crude product (0.64 g).
4.1.3.1. Artemisitene (4). Yield 5% (0.03 g,
0
.11 mmol)—see Refs. 13–15 for physical properties.
ꢀ
was 21–27 C over the course of the experiment and the
skies were mostly sunny.
4.1.3.2. 11-epi-Artemisinin (21). Oil (0.41 g, 1.45 mmol,
3%). IR n (cm ): 2966 (m), 2931 (m), 2878 (m), 1732 (s),
ꢁ1
7
1
1
450 (w), 1379 (m), 1107 (s). H NMR (d, CDCl ) ppm: 5.93
3
4.3.1. Extraction procedures. Extracts of the 42 individual
plants were made at 1, 2, 3, 4, 5, 6, and 7 days after the ter-
mination of feeding. The total plant weight at extraction for
the live group was found to be between 60 and 85% of the
initial weight that had been determined before feeding;
this slight loss in weight may indicate some dehydration as
a result of the forcing conditions, which were employed in
(
(
1H, s, H-5), 2.39 (1H, ddd, J¼13.4, 13.4, 3.9 Hz, H-3), 2.28
1H, q, J¼7.4 Hz, H-11), 2.08 (1H, m, H-3), 1.94 (1H, m,
H-2), 1.80 (1H, dddd, J¼13.4, 3.6, 3.6, 3.6 Hz, H-8), 1.71
(
1H, dddd, J¼13.1, 3.6, 3.5, 3.5 Hz, H-9), 1.47 (3H, d,
J¼7.4 Hz, H-13), 1.46 (3H, s, H-15), 1.12 (1H, dddd,
J¼13.3, 13.3, 13.3, 3.6 Hz, H-9), 1.00 (3H, d, J¼5.9 Hz,

G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
9563
order to stimulate the transpiration stream and to promote
uptake of the feeding solution. For the ‘dead’ group, the
plant weight had dropped to 30% of the original weight after
diol column (18% EtOAc/n-hexane) from which 20 fractions
were collected over a 50 min period. A total of 16 injec-
yy
tions were made—each injection containing approximately
150 mg of the crude plant material—and the HPLC column
was ‘washed’ with 50% EtOAc/n-hexane/1% AcOH be-
tween injections, in order to obtain a ‘polar residue’ fraction
(1.2 g), created from the combined washings. Those chro-
matographic fractions from the ‘preliminary’ separation,
3
days as a result of desiccation.
Each plant was extracted separately by grinding to a fine
powder under liquid N , followed by immersion in CH Cl
2
2
2
(
ca. 20 ml solvent/1 g plant material) in a container wrapped
2
in aluminum foil (in order to exclude light, and to prevent
any spontaneous autoxidation by oxygen), which was then
placed on a shaker for 8 h. The extraction vessels were fil-
tered free of plant particulates, and the solvent was dried
which exhibited a doublet signal in their H NMR spectra
were subjected to a second round of HPLC on the silica col-
umn, for which the polarity of the mobile phase was adjusted
to match the polarity of the fraction under study.
(
MgSO ) and removed in vacuo to yield a crude plant ex-
4
tract. This extraction procedure was repeated again over-
night (without shaking) and the two CH Cl extracts were
The ‘polar residue’ from the ‘preliminary’ HPLC was also
re-subjected to HPLC separation on a silica column (50%
EtOAc/n-hexane/1% AcOH): 13 injections were made (ca.
100 mg per injection), resulting in the collection of 20 frac-
tions over a 50-min period. Those fractions, which exhibited
2
2
combined. It was generally found that the efficiency of ex-
traction with CH Cl was between 1 and 2% (w/w). The
crude CH Cl extract from each plant was analyzed immedi-
2
2
2
2
2
ately after extraction (in order to minimize the possibility of
autoxidation) by both H and H NMR spectroscopies.
a doublet signal in their H NMR spectra, corresponding to
2
1
more polar metabolites of 2a, were then further purified in
a second round of HPLC (silica column again), with the
polarity of the mobile phase being adjusted according to
the polarity of the fraction under study.
All the plant residues were then re-extracted by MeOH
(
(
100 ml) using similar procedures to those described above
the efficiency of extraction in MeOH was between 2 and
% (w/w)). Some recovery of H label was also noted in these
2
5
MeOH extracts, although this was consistently less than that
of the corresponding CH Cl extracts. However, H NMR
2
The eight labeled metabolites, which were isolated from
these extensive preparative HPLC separations are listed in
Sections 4.5.1–4.5.8.
2
2
analysis of the MeOH extracts indicated only very broad
poorly defined resonances (unlike the CH Cl extracts,
1
3 2
4.5.1. [15- C H ]-Artemisinic acid (2a)/artemisinic acid
3
2
2
which are discussed in Section 4.4—see also Fig. 3, for an
example), and these samples were not analyzed further.
(2). Oil (3.1 mg, t 25–27 min in the preliminary separation
R
(diol HPLC column); tR 15.0 min with 17% EtOAc/
n-hexane (silica HPLC column)). (See also Section
4.1.1.3.1 for physical properties). H NMR (d, CDCl3)
2
1
4.4. H NMR analysis of the crude CH Cl extracts from
the aerial parts of A. annua, which had been fed with 2a
2
2
ppm: 6.43 (1H, s, H-13), 5.55 (1H, s, H-13), 4.99 (1H, br
s), 2.69 (1H, d, J¼12.1 Hz, H-7), 2.60 (1H, br s, H-6),
1.59 (ca. 85% of [3H], for a mixture of isotopically-normal
The initial analysis of each individual A. annua plant that
had been fed with 2a was made by H NMR spectroscopy
2
15-H3 and isotopically-labeled 15-D ), 0.90 (3H, d,
3
1
3
of the corresponding crude CH Cl extract, which was dis-
2
J¼5.8 Hz, H-14); C NMR (d, CDCl ) ppm: 170.9 (C-
2
3
solved in 0.6 ml of CHCl , containing benzene-d as both
3
12), 142.4 (C-11), 135.0 (C-4), 126.4 (C-13), 120.2 (C-5),
42.2 (C-7), 41.4 (C-1), 37.9 (C-6), 35.2 (C-9), 27.6 (C-10),
26.4 (C-3), 25.9 (C-8), 25.6 (C-2), 23.7 (s, isotopically-
6
reference and a standard for quantitative calibration (see
Section 4.1). The extent of recovery of H label in each
2
CH Cl extract was calculated by comparing the ratio of
2
normal 15-CH ), 22.8 ((1:3:6:7:6:3:1) sept., J¼19.1 Hz,
2
3
2
1
13
2
2
the total integral in the aliphatic region of each H NMR
spectrum to that of the internal calibration standard, C D
at d 7.43 ppm (Section 4.1).
J
, 15- C H ), 19.7 (C-14); H NMR (d, CHCl ) ppm:
CD 3 3
1
1.58 (d, J¼19.1 Hz, J , 15-D ); HREIMS m/z (rel int.):
6
6
CD
3
+
13
2
238.1840 [M , C
C H H O requires 238.1842] (10),
14 1 19 3 2
D
+
237 (1), 236 (0.2), 234.1612 [M , C H O requires
234.1620] (56), 206 (14), 189 (26), 121 (100), 119 (77).
15 22 2
1
3
2
4
.4.1. C– H COSY NMR analysis of the crude CH Cl
2
2
extracts from the aerial parts of A. annua, which had
been fed with 2a. The crude CH Cl extracts from all three
1
3 2
4.5.2. [15- C H ]-Arteannuin B (3a)/arteannuin B (3).
3
2
2
members of a triplicate group of plants, which had been har-
vested and extracted on the same day were combined into
a single sample (ca. 400 mg), which was re-dissolved in
ca. 0.6 ml CDCl and analyzed by C– H COSY spectros-
3
copy with the AV 600 instrument (see Section 4.1).
Oil (1.5 mg, t 22–25 min in the preliminary (diol HPLC
R
column); tR 58.3 min in 17% EtOAc/n-hexane (silica
HPLC column)). H NMR (d, CDCl ) ppm: 6.15 (1H, d,
1
3
1
3
2
J¼3.2 Hz, H-13a), 5.43 (1H, d, J¼3.2 Hz, H-13b), 2.74
(1H, dddd, J¼12.3, 3.2, 3.2, 3.2 Hz, H-7), 2.68 (1H, s, H-
5
), 2.06 (1H, dddd, J¼13.3, 3.2, 3.2, 3.2 Hz, H-8), 1.90
4.5. Separation of labeled metabolites from the CH Cl2
extracts of the aerial parts of A. annua, which had been
(2H, m, H-3 and H-8), 1.76 (1H, ddd, J¼13.3, 13.3,
6.2 Hz, H-2), 1.69 (1H, ddd, J¼13.9, 13.3, 4.6 Hz, H-3),
1.52 (1H, m, H-2), 1.49 (2H, m, H-1 and H-10), 1.41 (1H,
2
fed with 2a
All the CH Cl extracts of the 21 plants of A. annua, which
2
2
yy
2 2
The 21 CH Cl plant extracts from the ‘live’ plant experiment were also
had been fed with 2a and allowed to die by desiccation (the
dead’ group) were combined together (total weight: 2.56 g)
and subjected to a ‘preliminary’ HPLC separation using the
combined together (total weight 2.40 g) and subjected to a separate
HPLC separation, which gave similar results to those reported herein
for the ‘dead’ group.
‘

9564
G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
1
3
2
dddd, J¼13.3, 12.6, 12.3, 3.4 Hz, H-8), 1.33 (ca. 80% of
3H], for a mixture of isotopically-normal 15-H and isoto-
pically-labeled 15-D ), 1.25 (1H, m, H-9), 0.99 (3H, d,
4.5.5. [15- C H ]-epi-Deoxyarteannuin B (9a)/epi-
3
[
deoxyarteanuin B (9). Oil (0.8 mg, t 13–14 min in the pre-
R
liminary separation (diol HPLC column); t 22.2 min in 9%
R
3
3
1
3
1
J¼6.2 Hz, H-14); C NMR (d, CDCl ) ppm: 169.9 (C-
EtOAc/n-hexane (silica HPLC column)). H NMR (d,
CDCl ) ppm: 6.17 (1H, s, H-13a), 5.56 (1H, s, H-13b),
5.28 (1H, s, H-5), 2.71 (1H, dd, J¼6.6, 6.6 Hz, H-7), 2.10
(1H, dd, J¼18.1, 6.2 Hz, H-3a), 2.03 (1H, m, H-3b), 1.89
(1H, m, H-8a), 1.86 (1H, m, H-2a), 1.66 (70% of 3H, s,
3
1
5
2
2), 138.6 (C-11), 117.5 (C-13), 81.0 (C-6), 58.6 (C-5),
8.3 (C-4), 52.7 (C-7), 43.7 (C-1), 33.9 (C-9), 30.6 (C-10),
4.3 (C-3), 22.7 (s, isotopically-normal 15-CH ), 21.9
3
3
1
13 2
(
(
(1:3:6:7:6:3:1) sept., J¼19.2 Hz, J , 15- C H ), 21.7
CD
3
2
C-8), 18.5 (C-14), 16.2 (C-2); H NMR (d, CHCl ) ppm:
3
for a mixture of isotopically-normal 15-H and isotopi-
3
cally-labeled 15-D ), 1.67 (2H, m, H-2b and H-9b), 1.56
3
1
1
2
2
.33 (d, J¼19.3 Hz, J , 15-D ); HREIMS m/z (rel int.):
CD
3
+
13
2
52.1629 [M , C
48.1409 [M , C H O requires 248.1412] (17), 230
C H H O requires 252.1634] (2),
1
(1H, m, H-10), 1.38 (1H, m, H-8b), 1.25 (1H, ddd,
J¼10.5, 10.5, 2.7 Hz, H-1), 1.12 (1H, m, H-9a), 0.97 (3H,
1
4
17
3 3
+
1
5 20 3
1
3
(
49), 206 (96), 190 (100), 177 (88), 163 (70), 162 (65).
d, J¼6.9 Hz, H-14); C NMR (d, CDCl ) ppm: 169.9 (C-
3
1
2), 142.9 (C-11), 141.3 (C-4), 123.8 (C-5), 120.6 (C-13),
1
3 2
4.5.3. [15- C H ]-Annulide (7a)/annulide (7) (insepara-
ble from isoannulide (8)). Oil (0.2 mg, t 14–15 min in
83.4 (C-6), 44.9 (C-7), 44.3 (C-1), 31.1 (C-3), 30.0 (C-9),
28.5 (C-8), 28.4 (C-10), 23.2 (s, isotopically-normal 15-
2
R
1
3 2
the preliminary separation (diol HPLC column); tR
2
CH ), 22.3 ((1:3:6:7:6:3:1) sept., J¼19.2 Hz, 15- C H ),
3
3
2
2.3 min in 10% EtOAc/n-hexane (silica HPLC column)).
H NMR (d, CDCl ) ppm: 6.51 (1H, s, H-13a), 5.60 (1H, s,
21.7 (C-2), 20.0 (C-14); H NMR (d, CHCl ) ppm: 1.66
3
1
1
(d, J¼19.2 Hz, JCD, 15-D ); HREIMS m/z (rel int.):
3
3
1
3
2
H-13b), 5.20 (ca. 80% of 1H, s, for a mixture of isotopi-
cally-normal 15-H and isotopically-labeled 15-D ), 5.03
236.1677 [C
(3), 234 (0.5), 232.1462 [C H O requires 232.1464]
C H H O requires 236.1686] (25), 235
4 1 17 3 2
1
2
2
15 20 2
(
a mixture of isotopically-normal 15-H and isotopically-
1H, d, J¼11.4 Hz, H-5), 4.88 (1H, ca. 80% of 1H, s, for
(100), 217 (28), 191 (13), 188 (35), 187 (47), 173 (25).
2
1
3
2
labeled 15-D ), 2.66 (1H, ddd, J¼11.9, 5.5, 5.5 Hz, H-7),
4.5.6. [15- C H ]-Deoxyarteannuin B (10a)/deoxy-
3
2
2
.26 (1H, ddd, J¼13.9, 3.4, 3.4 Hz, H-3a), 2.17 (1H, dd,
arteannuin B (10). Oil (0.1 mg, t 13–14 min in the prelim-
R
J¼13.9, 3.0 Hz, H-3b), 2.04 (1H, m, H-2b), 2.01 (1H, m,
H-6), 1.85 (1H, m, H-9b), 1.70 (1H, m, H-10), 1.69 (2H, m,
H-8a and H-8b), 1.56 (1H, m, H-1), 1.45 (1H, m, H-2a),
inary separation (diol HPLC column); t 23.7 min in 9%
R
1
EtOAc/n-hexane (silica HPLC column)). H NMR (d,
CDCl ) ppm: 6.09 (1H, d, J¼3.2 Hz, H-13a), 5.36 (1H, d,
3
1
.17 (1H, dddd, J¼12.5, 12.5, 12.5, 3.7 Hz, H-9a), 0.92 (3H,
J¼3.2 Hz, H-13b), 5.13 (1H, s, H-5), 2.66 (1H, dddd,
J¼11.7, 2.7, 2.7, 1.5 Hz, H-7), 2.04 (1H, dddd, J¼18.1, 10.2,
6.9, 2.7 Hz, H-2a), 1.94 (2H, m, H-3a and H-3b), 1.93 (1H,
m, H-8a), 1.84 (1H, m, H-2b), 1.80 (1H, m, H-9b), 1.67
(70% of 3H, s, for a mixture of isotopically-normal 15-H3
1
3
d, J¼6.6 Hz, H-14); C NMR (d, CDCl ) ppm: 165.1 (C-12)
3
not seen due to small amount of sample, 145.3 (C-4), 139.2
C-11), 129.1 (C-13), 106.5 (s, isotopically-normal 15-
(
1
13 2
CH ), 106.0 ((1:2:3:2:1) quin., J¼24 Hz, J , 15- C H ),
2
CD
2
7
3
1
5.5 (C-5), 43.3 (C-6), 42.7 (C-1), 40.4 (C-7), 34.8 (C-9),
0.5 (C-8), 29.3 (C-3), 28.7 (C-2), 27.9 (C-10), 20.0 (C-
and isotopically-labeled 15-D ), 1.66 (1H, m, H-1), 1.56
3
(1H, m, H-10), 1.35 (1H, dddd, J¼12.6, 12.6, 12.6, 3.7 Hz,
2
13
4); H NMR (d, CHCl ) ppm: 5.24 (1H, d, J¼24.7 Hz,
H-8b), 0.99 (3H, d, J¼6.6 Hz, H-14); C NMR (d CDCl )
3
3
1
1
J
HREIMS m/z (rel int.): 235.1615 [C
, 15a-D ), 4.92 (1H, d, J¼23.5 Hz, J , 15b-D );
(ppm): 170.8 (C-12) not seen due to small amount of sam-
ple), 142.5 (C-4) not seen due to small amount of sample,
139.9 (C-11) not seen due to small amount of sample),
118.8 (C-5), 116.8 (C-13), 89.4 (C-6) not seen due to small
amount of sample, 52.5 (C-7), 44.8 (C-1), 34.3 (C-9), 30.7
CD
2
CD
2
1
3
2
C H H O requires
1 18 2 2
1
4
2
35.1622] (8), 232.1463 [C H O requires 232.1463]
15 20 2
(
100), 217 (22), 204 (11), 175 (8).
1
3
2
4.5.4. [15- C H ]-Isoannulide (8a)/isoannulide (8) (in-
separable from annulide (7)). Oil (0.1 mg, t 14–15 min
(C-10), 26.2 (C-3), 23.6 (s, isotopically-normal 15-CH ),
3
3
1
3 2
22.7 ((1:3:6:7:6:3:1) sept., J¼19.1 Hz, 15- C H ), 22.1
R
3
2
in the preliminary separation (diol HPLC column); tR
2
(C-8), 19.9 (C-2), 19.8 (C-14); H NMR (d, CHCl ) ppm:
3
1
2.3 min in 10% EtOAc/n-hexane (silica HPLC column)).
H NMR (d, CDCl ) ppm: 6.53 (1H, s, H-13a), 5.62 (1H,
1.67 (d, J¼19.1 Hz, J ,15-D ); HREIMS m/z (rel int.):
CD
2
3
1
13
236.1684 [C
C H H O requires 236.1686] (21), 235
14 1 17 3 2
3
s, H-13b), 5.47 (1H, br s, H-3), 4.84 (1H, d, J¼10.5 Hz,
H-5), 2.68 (1H, ddd, J¼11.6, 5.1, 5.1 Hz, H-7), 2.22 (2H,
m, J¼ H-2a and H-2b), 2.19 (1H, m, H-6), 1.82 (70% of
(2), 232.1460 [C H O requires 232.1464] (80), 217
15 20 2
(19), 187 (100), 173 (29), 153 (81), 136 (53).
1
3
2
3
H, s, for a mixture of isotopically-normal 15-H and isoto-
4.5.7. [15- C H ]-seco-Cadinane (13a)/seco-cadinane
3
3
pically-labeled 15-D ), 1.79 (1H, m, H-8a), 1.78 (1H, m, H-
3
(13). Oil (0.4 mg, t 18–21 min in the first HPLC of the ‘po-
R
9
1
b), 1.69 (1H, m, H-8b), 1.52 (1H, m, H-1), 1.31 (1H, m, H-
0), 1.13 (1H, dddd, J¼12.3, 12.3, 12.3, 3.9 Hz, H-9a), 0.89
lar residue’ (silica HPLC column); tR 37.2 min in 35%
EtOAc/n-hexane/0.5% AcOH (silica HPLC column)). H
NMR (d, CDCl ) ppm: 9.36 (1H, br s, H-5), 6.39 (1H, s,
3
H-13a), 5.70 (1H, s, H-13b), 2.83 (1H, br, H-7), 2.45 (1H,
m, H-3a), 2.34 (1H, m, H-3b), 2.33 (1H, m, H-6), 2.12
(80% of 3H, s, for a mixture of isotopically-normal 15-H3
1
1
3
(
(
3H, d, J¼6.1 Hz, H-14); C NMR (d, CDCl ) ppm: 166.7
3
C-12) not seen due to small amount of sample, 140.0 (C-11)
not seen due to small amount of sample, 130.5 (C-4), 129.2
C-13), 124.1 (C-3), 74.7 (C-5), 40.9 (C-1), 39.4 (C-6),
(
3
2
8.6 (C-7), 34.8 (C-9), 30.5 (C-8), 29.0 (C-10), 26.9 (C-2),
0.2 (C-14), 18.5 (s, isotopically-normal 15-CH ), 17.6
and isotopically-labeled 15-D ), 1.88 (1H, br s, H-9b),
3
1.86 (1H, m, H-2a), 1.80 (1H, d, J¼10.5 Hz, H-8), 1.51
3
1
13
2
2
(
(1:3:6:7:6:3:1) sept., J¼19.0 Hz, JCD, 15- C H );
H
(1H, m, H-2b), 1.34 (1H, m, H-9a), 1.31 (2H, m, H-1 and
H-10), 1.21 (H-8b, dddd, J¼13.2, 11.6, 11.6, 2.6 Hz, H-
3
1
NMR (d, CHCl ) ppm: 1.81 (d, J¼19.0 Hz, J , 15-D3);
3
CD
1
3
2
13
HREIMS m/z (rel int.): 236.1673 [C
quires 236.1685] (3).
C H H O re-
3
8b), 0.96 (3H, d, J¼6.2 Hz, H-14); C NMR (d, CDCl )
1
4
1
17
2
3
(ppm): 208.5 (C-5), 204.2 (C-4), 170.3 (C-12), 141.4

G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
9565
(
(
C-11), 128.3 (C-13), 57.9 (C-6), 41.7 (C-1), 39.8 (C-7), 38.2
C-3), 34.9 (C-2), 33.4 (C-10), 32.0 (C-9), 29.9 (s, isotopi-
11. Zheng, G.-Q. Planta Med. 1994, 60, 54–57.
12. Jeremic, D.; Jokic, A.; Behbud, A.; Stefanovic, M. Tetrahedron
Lett. 1973, 3039–3042.
13. Acton, N.; Klayman, D. L. Planta Med. 1985, 51, 441–442.
14. El-Feraly, F. S.; Ayalp, A.; Al-Yahya, M. A.; McPhail, D. R.;
McPhail, A. T. J. Nat. Prod. 1990, 53, 66–71.
cally-normal 15-CH ), 29.0 ((1:3:6:7:6:3:1) sept., J¼
3
1
3
2
2
1
9.4 Hz, 15- C H ), 23.5 (C-8), 19.7 (C-14); H NMR (d,
3
1
CHCl ) ppm: 2.12 (d, J¼19.4 Hz, J , 15-D ); HREIMS
3
CD
3
1
3
2
m/z (rel int.): 252.1635 [C
C H H O requires
1 17 3 3
1
4
2
quires 248.1412] (72), 230 (85), 220 (40), 205 (100), 202
52.1634] (12), 251 (1), 250 (0.5), 248.1414 [C H O re-
15. Sy, L.-K.; Brown, G. D. J. Chem. Soc., Perkin Trans. 1 2001,
2421–2429.
1
5 20 3
(
35), 192 (77), 178 (87).
16. Zhu, D.-Y.; Deng, D.-A.; Zhang, S.-G.; Xu, R.-S. Acta Chim.
Sin. 1984, 42, 937–939.
17. Brown, G. D. Phytochemistry 1993, 32, 391–393.
18. Roth, R. J.; Acton, N. Planta Med. 1987, 53, 576.
19. El-Feraly, F. S.; Al-Meshal, I. A.; Khalifa, S. I. J. Nat. Prod.
1989, 52, 196–198.
20. Wu, Z.-H.; Wang, Y.-Y. Acta Chim. Sin. 1984, 42, 596–598.
21. Brown, G. D. Phytochemistry 1994, 36, 637–641.
22. Wang, Y.; Xia, Z.-Q.; Zhou, F.-Y.; Wu, Y.-L.; Huang, J.-J.;
Wang, Z.-Z. Acta Chim. Sin. 1988, 46, 1152–1153.
23. Sangwan, R. S.; Agrawal, K.; Luthra, R.; Thakur, R. S.; Singh-
Sangwan, N. Phytochemistry 1993, 34, 1301–1302.
24. Huang, J.-J.; Zhou, F.-Y.; Wu, L.-F.; Zhen, G.-H. Acta Chim.
Sin. 1990, 275–277.
25. Wang, Y.; Xia, Z.-Q.; Zhou, F.-Y.; Wu, Y.-L.; Huang, J.-J.;
Wang, Z.-Z. Chin. J. Chem. 1993, 11, 457–463.
26. Wang, Y.; Shen, Z.-W.; Xia, Z.-Q.; Zhou, F.-Y. Chin. J. Chem.
1993, 11, 476–478.
1
3 2
4
.5.8. [15- C H ]-Artemisinic acid methyl ester (14a)/
3
artemisinic acid methyl ester (14). Oil (0.1 mg, tR
–10 min in the preliminary separation (diol HPLC column);
t 14.3 min in 2% EtOAc/n-hexane (silica HPLC column)).
9
R
1
(See also Ref. 5 and Section 4.1.1.2.1). H NMR (d,
CDCl ) ppm: 6.30 (1H, s, H-13a), 5.46 (1H, s, H-13b), 5.01
3
(
1H, br s, H-5), 3.77 (3H, s, 15-OMe), 2.74 (1H, d,
J¼12.1 Hz, H-7), 2.57 (1H, s, H-6), 1.62 (80% of 3H, s, for
a mixture of isotopically-normal 15-H and isotopically-la-
3
1
3
beled 15-D ), 0.92 (3H, d, J¼5.9 Hz, H-14); C NMR (d,
3
CDCl ) ppm: 167.8 (C-12) not seen due to small amount of
3
sample, 143.3 (C-11) not seen due to small amount of sample,
1
34.7 (C-4) not seen due to small amount of sample, 124.1
C-13), 120.3 (C-5), 51.7 (15-OMe), 42.4 (C-7), 41.4 (C-
(
1
8
), 38.0 (C-6), 35.3 (C-9), 27.6 (C-10), 26.4 (C-3), 25.9 (C-
), 25.6 (C-2), 23.7 (s, isotopically-normal C-15), 22.8
1
13 2
(
1
1
(1:3:6:7:6:3:1) sept., J ¼19.1 Hz, 15- C H ), 19.7 (C-
27. Brown, G. D.; Sy, L.-K. Tetrahedron 2007, 63, 9536–9547 (as
a companion paper with the current manuscript).
28. Nair, M. S. R.; Basile, D. V. J. Nat. Prod. 1993, 56, 1559–1566.
29. Nair, M. S. R.; Basile, D. V. Indian J. Chem. 1992, 31B,
880–882.
CD
3
2
1
4); H NMR (d, CHCl ) ppm: 1.62 (d, J¼19.1 Hz, J
,
3
CD
1
3
2
5-D ); HREIMS m/z (rel int.): 252.1992 [C
3
C H H O
1 21 3
15
2
requires 252.1998] (7), 251 (25), 248.1773 [C H O re-
6 24 2
1
quires 248.1776] (48), 230 (7), 220 (10), 216 (25), 189
38), 153 (100).
(
30. Akhila, A.; Thakur, R. S.; Popli, S. P. Phytochemistry 1987, 26,
1
927–1930.
1. Brown, G. D.; Sy, L.-K. Tetrahedron 2004, 60, 1125–1138.
32. Sy, L.-K.; Zhu, N.-Y.; Brown, G. D. Tetrahedron 2001, 57,
495–8510.
3
Acknowledgements
8
We thank the Generic Drug Research Programme of the
Chemistry Department of The University of Hong Kong
for providing a postdoctoral fellowship to Dr. L.-K.S. The
seeds of A. annua were kindly supplied by Prof. Liang
of the Guiyang laboratory of natural products, Guizhou prov-
ince, China. This project was funded by a grant from the
Committee on Conference and Research Grants (CRCG).
33. Brown, G. D.; Sy, L.-K. Tetrahedron 2004, 60, 1139–1159.
34. Zhou, W.-S.; Huang, D.-Z.; Ping, X.-F.; Zhang, L.; Zhu, J.; Xu,
X.-X. Acta Chim. Sin. 1989, 47, 710–715.
35. Zhou, W.-S. Pure Appl. Chem. 1986, 58, 817–824.
36. Sy, L.-K.; Brown, G. D. Tetrahedron 2002, 58, 897–908.
37. Sy, L.-K.; Brown, G. D. Tetrahedron 2002, 58, 909–923.
38. Zhang, L.; Zhou, W.-S. Acta Chim. Sin. 1989, 47, 1117–1119.
3
4
9. Brown, G. D. Phytochem. Rev. 2003, 2, 45–59.
1
3
2
0. Brown, G. D. C– H Correlation Spectroscopy: Development
and Applications to Chemical and Biological Problems, 15th
International Meeting on NMR spectroscopy, University of
Durham, July 8–12, 2001.
References and notes
1
2
. Haynes, R. K.; Vonwiller, S. C. Acc. Chem. Res. 1997, 30, 73–79.
. Tu, Y.-Y.; Ni, M.-Y.; Zhong, Y.-R.; Li, L.-N.; Cui, S.-L.; Zhang,
M.-Q.; Wang, X.-Z.; Ji, Z.; Liang, X.-T. Planta Med. 1982, 44,
41. Wallaart, T. E.; van Uden, W.; Lubberink, H. G. M.;
Woerdenbag, H. J.; Pras, N.; Quax, W. J. J. Nat. Prod. 1999,
62, 430–433.
42. Wallaart, T. E.; Pras, N.; Quax, W. J. J. Nat. Prod. 1999, 62,
1160–1162.
43. Bertea, C. M.; Freije, J. R.; van der Woude, H.; Verstappen,
F. W. A.; Perk, L.; Marquez, V.; de Kraker, J.-W.;
Posthumus, M. A.; Jansen, B. J. M.; de Groot, Aa.; Franssen,
M. C. R.; Bouwmeester, H. J. Planta Med. 2005, 71, 40–47.
44. Teoh, K. H.; Polichuk, D. R.; Reed, D. W.; Nowak, G.; Covello,
P. S. FEBS Lett. 2006, 580, 1411–1416.
1
. Brown, G. D. Educ. Chem. 2006, 43, 97–99.
43–145.
3
4
. Sy, L.-K.; Brown, G. D.; Haynes, R. Tetrahedron 1998, 54,
4
345–4356.
5. Sy, L.-K.; Brown, G. D. Phytochemistry 1998, 48, 1207–1211.
6. Sy, L.-K.; Brown, G. D. Phytochemistry 2001, 58, 1159–1166.
7. Brown, G. D.; Liang, G.-Y.; Sy, L.-K. Phytochemistry 2003,
6
4, 303–323.
8
9
. Sy, L.-K.; Cheung, K.-K.; Zhu, N.-Y.; Brown, G. D.
Tetrahedron 2001, 57, 8481–8493.
. Misra, L. N.; Ahmad, A.; Thakur, R. S.; Lotter, H.; Wagner, H.
J. Nat. Prod. 1993, 56, 215–219.
0. Kim, S.-U.; Han, J.; Lim, Y.-H. Planta Med. 1996, 62, 480–481.
45. El-Feraly, F. S.; Al-Mesha, I. A.; Al-Yahya, M. A.; Hifnawy,
M. S. Phytochemistry 1986, 25, 2777–2778.
46. Hayes, R. K.; Vonwiller, S. C. J. Chem. Soc., Chem. Commun.
1990, 451–453.
1

9566
G. D. Brown, L.-K. Sy / Tetrahedron 63 (2007) 9548–9566
4
4
4
7. Acton, N.; Roth, R. J. J. Org. Chem. 1992, 57, 3610.
51. Martin, V. J. J.; Pitera, D. J.; Withers, S. T.; Newman, J. D.;
Keasling, J. D. Nat. Biotechnol. 2003, 21, 796–802.
52. Ro, D.-K.; Paradise, E. M.; Ouellet, M.; Fisher, K. J.; Newman,
K. L.; Ndungu, J. M.; Ho, K. A.; Eachus, R. A.; Ham, T. S.;
Kirby, J.; Chang, M. C. Y.; Withers, S. T.; Shiba, Y.;
Sarpong, R.; Keasling, J. D. Nature 2006, 440, 940–943.
53. Wallaart, T. E.; Pras, N.; Beekman, A. C.; Quax, W. J. Planta
Med. 2000, 66, 57–62.
8. Chen, D.-H.; Ye, H.-C.; Li, G.-F. Plant Sci. 2000, 155, 179–185.
9. Bouwmeester, H. J.; Wallart, T. E.; Janssen, M. H. A.; van
Loo, B.; Jansen, B. J. M.; Posthumus, M. A.; Schmidt,
C. O.; de Kraker, J.-W.; Konig, W. A.; Franssen, M. C. R.
Phytochemistry 1999, 52, 843–854.
5
0. Chang, Y.-J.; Seung, S.-H.; Park, S.-H.; Kim, S.-U. Arch.
Biochem. Biophys. 2000, 383, 178–184.