Syntheses of dihydroartemisinic acid and dihydro-epi-deoxyarteannuin B incorporating a stable isotope label at the 15-position for studies into the biosynthesis of artemisinin

Article abstract of DOI:10.1016/S0040-4020(01)00711-6

[15-¹³C²H₃]-Dihydroartemisinic acid (3a) and [15-¹³CH₃]-dihydro-epi-deoxyarteannuin B (7b), intended for evaluation in vivo as biosynthetic precursors to artemisinin, have been obtained from a reconstructive synthesis. The decalenone acid 8 from acid degradation of artemisinin (1) serves as a common intermediate: following addition of labeled methyl Grignard reagent to 8, either labeled precursor can be prepared in good yield by varying the work-up conditions employed. It is shown that both compounds are prone to autoxidation on storage and that the products of such oxidation and subsequent rearrangement reactions might be confused with bona fide metabolites when using these labeled precursors in feeding experiments designed to determine the biosynthetic route to artemisinin in Artemisia annua.

Full text of DOI:10.1016/S0040-4020(01)00711-6

TETRAHEDRON
Pergamon
Tetrahedron 57 /2001) 8495±8510
Syntheses of dihydroartemisinic acid and
dihydro-epi-deoxyarteannuin B incorporating a stable
isotope label at the 15-position for studies into the biosynthesis
of artemisinin
Lai-King Sy, Nian-Yong Zhu and Geoffrey D. Brownp
Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, People's Republic of China
Received 25 April 2001; revised 11 June 2001; accepted 5 July 2001
AbstractÐ[15-13C2H3]-Dihydroartemisinic acid /3a) and [15-13CH3]-dihydro-epi-deoxyarteannuin B /7b), intended for evaluation in vivo
as biosynthetic precursors to artemisinin, have been obtained from a reconstructive synthesis. The decalenone acid 8 from acid degradation of
artemisinin /1) serves as a common intermediate: following addition of labeled methyl Grignard reagent to 8, either labeled precursor can be
prepared in good yield by varying the work-up conditions employed. It is shown that both compounds are prone to autoxidation on storage
and that the products of such oxidation and subsequent rearrangement reactions might be confused with bona ®de metabolites when using
these labeled precursors in feeding experiments designed to determine the biosynthetic route to artemisinin in Artemisia annua. q 2001
Elsevier Science Ltd. All rights reserved.
1. Introduction
other hand, Wang et al. found that arteannuin B was not
transformed into artemisinin,8 and went on to provide
evidence that two alternative oxidation products from
compounds 2/3, epi-deoxyarteannuin B /6) and its 11,13-
dihydro analogue, dihydro-epi-deoxyarteannuin B /7), are
the actual metabolic intermediates derived from 2/3 en
route to 1 /Fig. 1).9 The results of Wang et al. were
based on leaf homogenate studies, using precursors which
were radio-labeled at the 15-position, while Nair et al.
reached their conclusions without the bene®t of isotopic
labeling.
Artemisinin /qinghaosu) /1), ®rst reported as a constituent
of Artemisia annua by Chinese scientists in 19721 as the
result of a screening programme for natural products with
anti-malarial activity, is now one of the most promising new
drugs for treating malaria and appears to be particularly
useful in the treatment of multi-drug resistant forms of
Falciparum malaria in both Asia and Africa. The 1,2,4-
trioxane ring in artemisinin, which is responsible for its
anti-malarial activity, has attracted the attention of many
natural product chemists and a number of attempts to deter-
mine the biosynthetic origins of this unique system have
been described. Although most investigators agree that
artemisinic acid /2)2 or its 11,13-dihydro analogue, dihydro-
artemisinic acid /3),3 both of which are natural products
present in substantial amounts in A. annua,4 are late pre-
cursors in the biogenesis of artemisinin, the precise
mechanism by which this remarkable transformation of
2/3 to 1 occurs in vivo has remained elusive. On the one
hand, experimental evidence has been presented by Nair5
for the natural product arteannuin B /4) /which is a
metabolite of artemisinic acid)6 and by Jain7 for its 11,13-
We reasoned that the best way to resolve the apparent
controversy, regarding the mechanism for the conversion
of 3 into 1 in vivo, would be to synthesize both dihydro-
artemisinic acid /3) and dihydro-epi-deoxyarteannuin B /7)
incorporating stable isotope labels, for use as /postulated)
biogenetic precursors which could then be fed to whole
plants of A. annua. The advantage of using stable-isotope
labeled precursors is that their metabolism can be monitored
directly by NMR, yielding more extensive and more
immediate information on the chemical nature of trans-
formations occurring in vivo than is possible for radio-
labeling studies. We herein describe a single synthetic
route by which both [15-13C2H3]-dihydroartemisinic acid
/3a) and [15-13CH3]-dihydro-epi-deoxyarteannuin B /7b)
were synthesized from artemisinin /1) via a common inter-
mediate 8. The use of these labeled precursors in feeding
experiments with whole plants of A. annua will be described
in a future communication.
dihydro analogue, dihydroarteannuin
converted into 1 by cell free extracts of A. annua. On the
B
/5), being
Keywords: Artemisia annua; artemisinin; dihydroartemisinic acid; dihydro-
epi-deoxyarteanuin B; isotopic labeling.
Corresponding author. Tel.: 1852-2859-7915; fax: 1852-2857-1586;
p
e-mail: gdbrown@hkucc.hku.hk.
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020/01)00711-6

8496
L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
Figure 1. Natural products from A. annua which have been proposed as late biosynthetic precursors of artemisinin /1), on the basis of experimental evidence.
2. Results and discussion
1010,11 which can then be converted into the decalenone
acid 8 by known reactions.10,13 Whilst optimizing the acid
degradation procedure for converting 1 into 10, we also
occasionally obtained the novel compound 11 as a very
minor product /Scheme 1, Tables 1 and 2). Based on both
this ®nding and a detailed recent study of the acid degrada-
tion of the closely related compound peroxofabianane,14 we
have proposed a mechanism for the acid degradation of 1 to
10, involving the 1,2-shift of an aldehyde groupto the
internal oxygen atom of a tertiary hydroperoxide inter-
The acid degradation of artemisinin /1) and its derivatives
has been extensively studied and found to yield a wide
variety of products such as compounds 9 and 10, depending
upon the precise conditions employed.10±12 Epimerization at
the 1- and 7-positions of the acid degradation products may
also become a problem if the reaction conditions are not
carefully controlled.10,12 In the context of this study, we
sought to maximize the yield of the nor-sesquiterpene
Scheme 1. Proposed mechanism for the acid degradation of 1 to 10 in H2SO4/MeOH and its application in the reconstructive synthesis of decalenone
intermediate 8, which is common to the syntheses of both dihydroartemisinic acid and dihydro-epi-deoxyarteannuin B.

L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
8497
Table 1. 13C NMR data /d, ppm) for novel compounds described in Schemes 1±4 /rigorous assignments made by the 2D-NMR techniques HSQC, HMBC,
1H±1H COSY in all cases). Splittings observed for labeled compounds /suf®x `a' or `b') are given in Section 3
Position
11
14a
15a
17
18
19
20
21
22
23a
24a
25a
26a
1
2
3
4
5
6
7
8
59.8
22.4
41.5
106.0
200.4
94.2
49.1
25.2
35.5
32.0
37.9
176.2
18.0
20.5
24.1
51.4
43.2
27.8
31.2
137.9
119.8
131.9
127.2
25.3
31.5
34.2
40.4
180.1
15.1
20.4
24.2b
±
134.6
27.8
28.7
44.9
23.0
34.9
68.6
124.4
142.9
47.5
32.7
35.6
38.3
41.0
177.1
16.3
20.0
29.3
51.4
44.7
24.3
35.6
68.4
124.9
142.8
46.9
31.7
35.5
39.7
41.1
176.8
15.7
20.2
29.8
51.4
43.1
27.8
31.2
137.4
119.9
131.1
127.8
25.4
31.5
34.2
40.5
175.5
15.3
20.3
24.1
51.6
45.4
26.2
27.7
143.7
120.4
145.1
47.8
32.9
35.6
38.6
44.1
177.0
15.9
20.0
133.7
27.9
28.9
132.1
124.0
128.2
40.3
23.4
27.6
33.2
43.6
176.9
15.7
19.0
23.0
51.3
45.0
26.0
42.2
27.8
119.1
131.8
26.3
33.4
43.7
25.8
35.5
27.8
42.4
183.4
15.1
20.1
23.6b
±
46.8
28.5
32.3
29.4
35.8
133.3
128.5
24.5
30.6
35.8
41.0
179.6
15.2
20.7
17.8b
±
45.0
24.5
27.7
30.3
123.3
140.0
47.4
33.4
35.9
38.3
41.1
182.1
16.1
20.0
21.9b
±
40.0
28.8
30.5
31.1
116.7
130.1
118.2
136.6
47.5
33.7
35.5
37.7
41.1
177.0
15.9
19.9
21.6
51.4
a
±
123.5
127.9
40.1
23.3
27.8
33.2
43.1
179.5
15.5
19.0
23.1b
±
130.1
±
a
47.0
26.9
29.9
40.2
41.0
180.4
15.1
20.0
22.0b
±
9
10
11
12
13
14
15
12-OMe
109.2
51.4
a
Not resolved.
b
1
13C chemical shift given is for the isotopically-normal compound Ð see experimental for the effects of substitution of H by 2H.
1
Table 2. H NMR assignments /d, ppm) of novel compounds reported in Schemes 1±4 /rigorous assignments made by the 2D-NMR techniques HSQC,
HMBC, 1H±1H COSY and NOESY in all cases). Splittings for labeled compounds /suf®x `a' or `b') are given in Section 3
Position
11
14a
15a
17
18
19
20
1.62
1.82
1.64
2.16
2.27
±
21
22
23a
24a
25a
26a
1
1.15
1.80
1.32
1.93
2.20
±
9.90
±
±
1.66
1.69
1.25
0.95
1.73
2.34
3.19
1.23
0.89
1.28
3.64
1.62
2.09
1.18
2.03
2.13
±
6.11
±
±
±
1.53
1.89
1.55
1.56
1.64
±
5.26
±
±
2.02
1.69
1.16
1.18
1.74
1.28
2.73
1.22
0.92
1.26
3.67
1.49
1.88
1.51
1.47
1.71
±
5.29
±
±
2.04
1.68
1.25
1.20
1.74
1.26
2.74
1.22
0.95
1.29
3.67
1.61
2.09
1.19
2.03
2.13
±
6.11
±
±
±
1.66
2.36
2.16
5.26
±
±
5.37
±
1.21
2.15
2.05
5.29
±
1.52
1.80
1.19
1.50
1.65
2.05
2.43
1.87
±
1.48
1.74
1.61
1.13
1.56
2.17
5.11
±
1.80
2.05
1.00
0.93
2.05
1.70
5.27
±
2a
2b
3a
3b
4
5a
5b
6
2.19
1.96
1.96
2.06
±
5.55
±
±
2.35
1.78
1.50
1.27
1.88
2.22
2.68
1.18
0.98
1.78b
±
2.17
1.93
1.94
2.06
±
5.51
±
±
2.32
1.81
1.42
1.26
1.84
2.18
2.63
1.13
0.97
1.78
3.64
±
5.82
±
1.57
1.92
2.09
1.69
1.70
1.48
1.00
1.67
1.40
2.30
1.19
0.82
1.63b
±
±
2.15
±
±
±
7
±
±
2.25
1.73
1.21
1.17
1.74
1.47
2.74
1.24
0.87
1.69
3.68
±
2.02
1.79
1.20
1.21
1.76
1.37
2.70
1.26
0.90
0.92b
±
2.21
1.86
1.52
1.20
1.48
1.07
2.74
1.14
0.92
0.85b
±
a
8a
8b
9a
9b
10
11
13
14
15
12-OMe
2.10
2.07
1.23
1.71
1.26
3.82
1.21
0.99
1.78b
±
2.09
1.89
1.20
1.70
1.25
3.77
1.20
0.99
1.78
3.65
±
2.00
1.94
1.13
1.63
1.28
3.67
1.15
0.95
0.91b
±
a
±
1.24
1.76
1.40
2.75
1.12
0.93
4.72, 4.69
3.66
a
Not resolved.
Often absent or seen as low intensity doublet /ca. 126 Hz) in spectra of labeled compound.
b
mediate, with accompanying loss of the second oxygen
atom of the hydroperoxide group as water, which is
shown in Scheme 1. In this mechanism, the 5-aldehyde
groupis ultimately eliminated as formic acid.
Compound 8 then serves as a common intermediate for the
synthesis of both of the natural products dihydroartemisinic
acid and dihydro-epi-deoxyarteannuin B. Treatment of 8
with the Grignard reagent from methyl iodide is expected
to form the tertiary allylic alcohol addition product 13
/Fig. 2). Although this putative intermediate was never
actually isolated, its formation can be inferred from the
differing products of reaction obtained from two
alternative work-up procedures /Scheme 2). Thus, at pH
1±2, the predominant product from the Grignard addition
of [13C2H3]-methyl iodide with 8 was [15-13C2H3]-6,7-dehy-
Optimal conditions for the preparation of 10 from 1 were
found to involve short reaction times and a high concentra-
tion of acid. Treatment of 10 with barium hydroxide octa-
hydrate then resulted in the decalenone acid 8, which has a
pronounced tendency to undergo lactonization to compound
12 under the acidic work-upconditions emlopyed.
However, subsequent conversion of the lactonized side-
product 12 back to the corresponding free acid 8, by treat-
ment of either puri®ed 12 or a mixture of compounds 8
and 12 with alkali, was found to be a simple and reliable
procedure, which could be routinely employed to ensure a
high overall yield of 8 from 10.
²
dro-11,13-dihydroartemisinic acid /14a) /Tables 1 and 2)
²
The suf®x `a' indicates the replacement of the 15-[CH3] groupby 15-
[13C2H3]; the suf®x `b' indicates replacement by 15-[13CH3]; the suf®x `p'
indicates an unspeci®ed isotopic enrichment at the 15-position.

8498
L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
Figure 2. Participation of the 12-carboxylic group in determining the product formed from Grignard addition of methyl iodide to 8.
which is presumed to be formed by elimination of the H-7
proton and of the allylic hydroxyl group in 13a. At pH 4±5,
by contrast, [15-13CH3]-dihydro-epi-deoxyarteannuin B
gate base form, the carboxylate anion instead participates in
SN20 intramolecular addition at the 6-position of the allylic
hydroxyl groupof intermediate 13b forming a lactone /Fig.
2).
²
/7b) was formed as the sole product in good yield from
reaction of 8 with [13CH3]-methyl iodide /a better yield than
for our recently reported procedure for the preparation of
dihydro-epi-deoxyarteannuin B by a more direct route).15
On the basis of these experiments, we propose that when
protonated /i.e. under acidic work-up conditions), the
carboxylic acid proton in intermediate 13a undergoes an
intramolecular transfer to the 4-hydroxyl group, as shown
in Fig. 2, thereby catalyzing the dehydration of 13a to a
diene. Alternatively, at pH 4±5, when the carboxylic acid
group exists predominantly in its more nucleophilic conju-
When the decalenone methyl ester 16 /readily available by
treatment of 8 with diazomethane, Scheme 1) is used in
place of compound 8 in the Grignard reaction, both of the
expected tertiary allylic alcohol intermediates 17 and 18 can
be isolated /Tables 1 and 2) following work-upat pH 4±5.
Compounds 17 and 18 are the methyl ester derivatives of the
addition product 13, whose existence could only be inferred
for the Grignard reaction of the free acid 8; isolation of these
two epimers from the Grignard reaction of methyl ester 16
Scheme 2. Conversion of key intermediate 8 into either labeled diene 14a or biosynthetic precursor 7b by treatment with labeled methyl magnesium iodide
followed by different work-upprocedures.

L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
8499
Scheme 3. Products from treatment of 16 with isotopically-normal methyl magnesium iodide.
provides support for the mechanism shown in Fig. 2. When
17/18 were treated with acid or when acidic conditions /pH
1±2) were used in the work-upof the Grignard reaction of
16, compound 19, which is the methyl ester derivative of 14,
was the predominant dehydration product. Three alternative
dehydration products 20±22 were also isolated as very
minor components /Scheme 3): all four products presum-
ably arise from the E1 elimination of the tertiary hydroxyl
groupin 17/18 followed by elimination of either the H-7/
H-1 allylic proton /forming 19/21) or the H-15/H-3 proton
/forming 20/22) from the resulting carbocation. /It is also
possible that these diene isomers may be able to interconvert
with one another under the conditions of the reaction.)16
Alternative hydrogenation products 23a±26a were also
formed in small amounts /Scheme 4) and could be separated
by HPLC. A previous report of the synthesis of dihydro-
artemisinic acid labeled at the 15-position13 employed
dehydration of an intermediate, which is the 5,6-dihydro
analogue of 13a/b /see Fig. 2), resulting in both labeled
dihydroartemisinic acid /3p; D4) and its unwanted
D3-isomer, compound 23p, as a roughly 1:1 mixture,
which was dif®cult to separate. The current procedure, in
which the desired D4-isomer, compound 3a, is obtained by
hydrogenation of diene 14a in 58% yield after chromato-
graphy, is an improvement on the previously reported
procedure.
Hydrogenation of the D4,5D6,7-diene in compound 14a,
proceeded both regiospeci®cally, at the more highly
substituted D6,7-double bond, and stereospeci®cally, from
the a-face, to give the desired D4,5-alkene, [15-13C2H3]-
dihydroartemisinic acid 3a, as the predominant product.
It should be noted that one disadvantage of our strategy is
that there is some unavoidable loss of H label from the
15-postion of 14a during the hydrogenation step. Thus,
while compound 14a was formed largely as the 15-
[13C2H3]-isotopomer from Grignard reaction with labeled
2
Scheme 4. Conversion of diene intermediate 14a into [15-13C2H3]-dihydroartemisinic acid 3a /and smaller quantities of other alkenes) by hydrogenation.

8500
L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
Figure 3. 13C NMR and 2H NMR spectra of compounds 3a and 14a showing ca. 32% overall depletion of 2H label /but not 13C) from the 15-position during the
2
2
hydrogenation step. /a) Expansion of 13C NMR spectrum of 14a; /b) H NMR spectrum of 14a; /c) expansion of 13C NMR spectrum of 3a; /d) H NMR
spectrum of 3a.
methyl iodide, compound 3a from hydrogenation of 14a
was shown to be a mixture of all four possible isotopomers
at the 15-position by 13C NMR /Fig. 3). Interpretation of the
13C NMR spectra depicted in Fig. 3 rests on two assump-
tions. Firstly, the carbon resonance for the 15-position is
expected to be shifted up®eld by approximately 0.3 ppm
normal 14 is 24.2 ppm) shown in Fig. 3, which is consistent
with approximately 88% deuteriation at the 15-position
2
2
/77% [15-13C1 H3]-14; 13% 15-[13C1 H2H]-14; 8% 15-
2
[13C1 HH2]-14; 2% 15-[13CH3]-14). However, all three
possible splitting patterns are seen superimposed on one
another for the 15-position of 3a in Fig. 3; we estimate
that 3a is approximately 56% deuteriated at the 15-position
from this 13C NMR expansion /35% [15-13C2H3]-3, 21%
2
for each H atom directly attached at C-15. Secondly, a
1
2
single replacement of H by H at C-15 is predicted to
produce a 1:1:1 triplet /1JCD,19 Hz) in the proton-
decoupled 13C NMR spectrum; two deuterium replacements
result in a 1:2:3:2:1 quintet; and complete deuteriation at the
15-position produces a 1:3:6:7:6:3:1 septet. Such a septet
pattern dominates in the expansion of the 13C spectrum of
14a at dC 23.3 ppm /which is the expected chemical shift for
the [15-13C2H3] isotopomer of 14 since dC for isotopically-
[15-13C2H2H]-3,
21%
[15-13C2HH2]-3
and
24%
[15-13CH3]-3). The hydrogenation stephas thus resulted in
the loss of approximately one deuterium atom from the 15-
position. However, these NMR analyses must be considered
as semi-quantitative only, because no account is taken of the
poorer ability of 2H to induce a nuclear Overhauser
enhancement at 13C as compared with H, which may in
1

L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
8501
Figure 4. /a) 13C NMR and /b) 1H NMR spectra of 7b showing essentially complete 13C labeling at the 15-position. /c) 13C NMR and /d) 1H NMR spectra of 7a
2
showing ca. 100% retention of both 13C and H labels at the 15-position.
turn lead to an over-estimation of the extent of deuterium
depletion.
ion then span the range 240±237 and 166±163 daltons for
the base peak. However, there are believed to also be
inaccuracies inherent in such mass spectral analysis because
additional peaks caused by hydrogen/deuterium loss in frag-
mentation reactions may also occur superimposed in these
same mass ranges, and these fragments are then of suf®-
ciently similar mass in high resolution mass spectroscopy
as to be confused with the isotopomer peaks under study.
Mass spectroscopic analysis indicated a lesser degree of
deuterium depletion for 3a as compared to 13C analysis
Analysis of the molecular ions and daughter ions formed by
fragmentation of the 7-substituent /M12C3H6O2), which
constitute the base peak in the mass spectrum of 3a, should
be a more straightforward procedure for establishing the
extent of deuterium depletion relative to 14a since each
isotopomer is expected to give only a single peak differing
by one mass unit. All possible isotopomers for the molecular

8502
L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
and no depletion at all in the 13C label. This was con®rmed
by H NMR spectroscopy /Fig. 3) in which signal for the
tion to much larger quantities of the labeled precursor itself.
The structures of 1a and 7a were con®rmed by their 1D 1H
NMR spectra, while the planar structure of novel compound
27a was determined by the 2D-NMR experiments HSQC,
HMBC and 1H±1H COSY as for all other novel compounds
reported herein /Table 3). Unfortunately, the relative stereo-
chemistry of the newly formed epoxide group in 27a was
not clearly shown by NOESY experiments. However, we
were able to synthesize much larger amounts of an isotopi-
cally normal compound with NMR spectra exactly match-
ing those of 27a /after making allowances for the effects of
isotopic enrichment) by treating dihydroartemisinic acid /3)
with m-chloroperoxybenzoic acid. The X-ray crystallo-
graphic structure of 27 from synthesis con®rmed that the
epoxy group had been introduced to the a-face of dihydro-
artemisinic acid /Fig. 5) and the oxidation product 27a
formed from 3a on storage has been assigned as a-epoxy-
dihydroartemisinic acid accordingly. We have recently
isolated a natural product from A. annua with NMR spectra
identical to those of isotopically normal a-epoxy-dihydro-
artemisinic acid /27) /unpublished results); compound 27 is
the 11,13-dihydro analogue of the natural product a-epoxy-
artemisinic acid, which has been described previously from
this species.17
2
15-position appears entirely as a doublet /1JCD ca. 19 Hz) for
both 3a and 14aÐany replacement of 13C by 12C would be
expected to lead to the appearance of a singlet peak in H
NMR.
2
13C, 2H and mass spectral analyses of the other minor
products 23a±26a from hydrogenation of 14a indicated
varying degrees of depletion in the 2H label at the
15-position, and we believe that this effect might therefore
2
be ascribed to the exchange of the allylic H atoms at the
15-position of 14a with H radicals adsorbed on the Pd
catalyst surface. The effect is particularly marked in
compound 23a which has also clearly undergone double
bond isomerization /again probably by a radical process).
1
The H and 13C NMR spectra of [15-13CH3]-dihydro-epi-
deoxyarteannuin B /7b) indicated ca. 100% incorporation
of 13C label at the 15-position as expected /Fig. 4: the
corresponding spectra for [15-13C2H3]-dihydro-epi-deoxy-
arteannuin B /7a), showing close to 100% incorporation
2
of both 13C and H labels at the 15-position is also shown
for comparison). We judged that the extent of stable isotope-
labeling determined for both compounds 3a and 7b was
suf®ciently high that both labeled precursors might be
used in feeding experiments with A. annua in order to
delineate the biosynthesis of artemisinin /1) by studying
We propose that all three products 1a, 7a and 27a arise by
the initial reaction of oxygen with the D4,5-double bond of
the labeled precursor 3a. This results in an allylic tertiary
hydroperoxide, which then undergoes spontaneous conver-
sion to these three isolated products under the conditions of
storage. There are several precedents in the literature for
the transformations of 3 suggested in Scheme 6.3,18±21 All
these references state or imply that it is singlet oxygen
/1O2), which reacts with the D4,5-double bond in dihydro-
artemisinic acid in order to form the initial tertiary allylic
hydroperoxide. Since our sample of 3a was stored in the
dark there is no obvious source of singlet oxygen which
would be required to initiate the formation of 1a, 7a and
27a, according to our understanding of current theory
/which states that both a photosensitizer and a light source
2
the 13C and/or H NMR spectra of extracts and puri®ed
compounds which would be isolated from the fed plants.
The labeled precursors 3a and 7b were stored in the freezer
at 2208C whilst awaiting the growth of A. annua plants to
be used in such feeding experiments. Surprisingly, it was
found that both samples of 3a and 7b had undergone some
changes after storage for six months /Schemes 5 and 6).
Re-chromatography of sample 3a after storage yielded
signi®cant amounts of [15-13C2H3]-artemisinin /1a),
[15-13C2H3]-dihydro-epi-deoxyarteannuin
B
/7a) and
[15-13C2H3]-a-epoxy-dihydroartemisinic acid /27a) in addi-
Scheme 5. Proposed autoxidation and subsequent rearrangement reactions of 3a occurring during storage at 2208C for six months.

L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
8503
Scheme 6. Proposed autoxidation and subsequent rearrangement reactions of 7b occurring during storage at 2208C for six months.
Table 3. NMR assignments /d, ppm) for labeled compounds formed from the slow autoxidation of 3a and 7b on storage /Schemes 5 and 6) /rigorous
assignments made by the 2D-NMR techniques HSQC, HMBC, 1H±1H COSY and NOESY in all cases). Splittings for labeled compounds /suf®x `a' or `b') are
given in Section 3
dC
dH
Position
27a
28b
29b
30b
31b
32b
33b
34b
27a
28b
29b
30b
31b
32b
33b
34b
1
40.2
22.3
39.9
27.2
38.8
27.2
47.3
27.9
41.4
25.9
47.7
37.5
46.4
26.1
38.6
37.4
1.07
1.31
1.65
1.84
1.67
±
2.63
2.04
1.74
1.64
1.10
1.05
1.69
1.27
2.60
±
1.49
2.27
1.93
5.78
1.44
2.27
2.00
5.67
1.64
2.50
2.40
6.73
1.58
2.43
1.85
4.35
1.85
2.70
2.61
±
1.33
2.05
2.30
4.43
2.04
2.60
2.19
±
2a
2b
3a
3b
4
5
6
7
8a
8b
9a
9b
10
11
12
13
14
15
24.8
129.6
126.5
145.1
81.3
194.0
83.5
203.2
57.8
58.7
38.1
42.8
28.7
127.0
84.4
84.9
38.8
24.4
131.2
70.1
85.2
38.0
24.4
132.6
195.7
84.1
37.9
24.1
137.0
128.5
82.1
42.0
23.7
139.4
141.0
81.1
42.5
24.0
140.7
126.9
82.2
42.6
23.6
61.3
61.9
83.3
41.0
24.1
±
4.17
±
±
3.70
±
±
±
±
±
5.88
±
±
6.76
±
±
5.82
±
±
3.46
±
2.93
1.80
1.15
1.13
1.67
1.36
3.21
±
2.72
1.76
1.13
1.09
1.67
1.37
3.11
±
2.75
1.85
1.16
1.02
1.70
1.56
3.25
±
2.13
1.75
1.20
1.09
1.69
1.47
3.12
±
2.25
1.84
1.23
1.11
1.75
1.55
3.16
±
2.09
1.72
1.20
1.07
1.71
1.51
3.13
±
2.54
1.85
1.21
1.10
1.69
1.37
3.24
±
34.8
32.0
32.1
31.9
32.2
32.2
32.3
31.8
29.5
42.0
181.2
15.5
19.0
23.6
31.9
39.5
179.7
9.4
19.8
20.8
31.7
39.0
180.4
9.3
19.9
21.2
31.5
40.8
179.3
9.2
19.0
15.3
29.2
39.4
179.1
9.4
19.4
21.0
30.3
39.8
178.1
9.3
18.8
15.8
29.5
39.6
178.8
9.4
20.8
19.5
30.7
39.2
177.9
9.3
19.2
14.6
1.29
0.86
1.32a
1.17
0.93
1.79
1.14
0.94
1.79
1.12
0.96
1.77
1.15
0.99
1.84
1.19
0.94
1.81
1.15
0.96
1.82
1.21
0.90
1.42
a
Low intensity and split into a doublet /125.9 Hz) in spectrum of isotopically-labeled 27a.

8504
L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
Figure 5. ORTEP diagram for compound 27, which was obtained by treatment of isotopically-normal 3 with mCPBA.
are required in order to produce the singlet state of
molecular oxygen /1O2)). Based on the foregoing results,
we suggest that there may be something lacking in current
theories regarding the generation of singlet oxygen and/or
the absolute requirement for singlet oxygen in reacting with
tri-substituted double bonds, at least when it comes to the
slow spontaneous autoxidation of unsaturated organic
in Scheme 6. The formation of 29b from a-hydroperoxide
28b is thus consistent with this structure revision for
arteannuin K).15 The formation of the a,b-unsaturated
ketone 30b can be explained in an analogous fashion by
dehydration of 28b23 /cf. the proposed biogenesis of
arteannuin N in A. annua from dihydroartemisinic acid).18
The secondary allylic hydroperoxide 31b is almost certainly
the result of 3,2-rearrangement /Schenck reaction) of allylic
hydroperoxide 28b23 /several analogous rearrangement
reactions of the secondary allylic hydroperoxide from
photo-oxidation of dihydroartemisinic acid have been
reported recently),19 and 32b may be formed by dehydration
of this rearranged hydroperoxide. The well-known Smith
epimerization reaction of allylic hydroperoxides23 can then
be invoked to account for the formation of 33b from
compound 31b. Compound 34b was shown to have the
rather unusual structure of an epoxy-ketone by 2D-NMR
analysis /summarized in Table 3). A tentative suggestion
is made for the derivation of 34b from 28b in Scheme 6,
based on the reported rearrangements of allylic hydro-
peroxides of fatty acids to b-hydroxy epoxides;24 further
oxidation of the alcohol groupof such a hydroxy-epoxide
intermediate would then yield 33b, possibly by a radical
process.23
compounds, which, although
a matter of common
experience is a phenomenon which has not often been
studied or reported. In addition to the current unexpected
®nding, we would draw the reader's attention to previous
reports both by ourselves18 and others3 regarding the slow
spontaneous autoxidation of dihydroartemisinic in the
absence of photosensitizers and a recent report of a much
more rapid and apparently un-photosensitized spontaneous
autoxidation of a natural product.22
Re-chromatography of sample 7b after a period of six
months storage at 2208C resulted in the isolation of seven
compounds 28b±34b /Table 3), of which six are novel, in
addition to much larger amounts of the labeled precursor
starting material. The secondary allylic hydroperoxide 28b
is once again clearly formed from `ene-type' reaction of
oxygen with the D4,5-double bond in 7b /note that because
there is no H-6 proton in 7b, formation of a tertiary allylic
hydroperoxide is not possible). Compound 29b is the
[15-13CH3]-isotopomer of the natural product arteannuin
K,18 which might then be formed by homolysis of the hydro-
peroxide group in 28b.23 /N.B. The stereochemistry at the
5-position of this natural product, originally reported as
5b-OH,18 is believed to have been wrongly assigned and
we have recently submitted a manuscript revising the stereo-
chemistry of the 5-OH groupin arteannuin K to that shown
We have obtained evidence for some of the transformations
proposed in Scheme 6 for allylic hydroperoxide 28b by
allowing a pure sample of this compound to stand in
CDCl3 for several weeks. By recording the 1H NMR spectra
of this sample at regular intervals it was possible to observe
the appearance of peaks characteristic of all of the products
29b±32b within a few days. After 45 days, peaks of the
starting material had almost disappeared, leaving a complex

L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
8505
mixture containing ca. 10% 29b, 10% 30b, 15% 31b and
10% 32b by 1H NMR. Although no peaks due to 33b or 34b
were seen in 1H NMR under these conditions, there were a
large number of unidenti®ed signals in these spectra,
suggestive of alternative transformations occurring in
CDCl3, which are not observed in the solid state. Once
again, conventional wisdom would require a source of
singlet oxygen, in order to produce the allylic hydro-
peroxide 28b /from which all of 29b±34b are proposed to
be derived) from 7b, although there is no obvious source for
this species under the conditions of storage.
were recorded by a Perkin±Elmer differential scanning
calorimeter 7 /DSC 7). Optical rotations were measured
by a Perkin-Elmer 343 polarimeter /Na 589 nm). [a]D values
are given in 1021 deg cm2 g21 and CHCl3 was used as solvent.
dC values for the 15-position of compounds labeled by 2H at
the 15-position /suf®x `a') generally appeared as 4 sets of
superimposed signals: a singlet at the isotopically-normal
value [15-13CH3]; a 1:1:1 triplet which is 0.3 ppm up®eld of
this [15-13CH2 H]; a 1:2:3:2:1 quintet 0.6 ppm up®eld
2
[15-13CH2H2]; and a 1:3:6:7:6:3:1 septet 0.9 ppm up®eld
1
[15-13C2H3]. JCD couplings for these multiplets were in
The formation of the labeled natural products artemisinin
/1a), dihydro-epi-deoxyarteannuin B /7a) and a-epoxy-
dihydroartemisinic acid /27a) from the slow spontaneous
autoxidation of [15-13C2H3]-dihydroartemisinic acid /3a),
as well as the appearance of the labeled natural product
arteannuin K /29b) from prolonged storage of [15-13CH3]-
dihydro-epi-deoxyarteannuin B /7b), is of some concern
from the point of view of feeding experiments in which
the labeled precursors 3a and 7b are intended to be tested
as biosynthetic precursors en route to natural products from
A. annua. Clearly, great care must be taken to ensure that
allylic hydroperoxides and their rearrangement products,
which can readily be formed from autoxidation of the
double bond in these compounds in vitro even in the absence
of light and photosensitizers, are not confused with bona
®de metabolites which might then be re-isolated in vivo.
It is thus important to prepare these labeled precursors
freshly or to re-purify them after storage and prior to their
use in feeding experiments. Finally, we make the suggestion
that the unexpected ease by which both dihydroartemisinic
acid and dihydro-epi-deoxyarteannuin B undergo spon-
taneous oxidation /by non-enzymatic processes) may have
some bearing on the rather confusing results concerning the
biogenesis of artemisinin which have been reported in the
literature.
the range 19.0±19.3 Hz for all compounds reported. For
brevity, only the four chemical shifts at the labeled position
are quoted, together with an estimated percentage for each
isotopomer. Four sets of molecular ions and four sets of base
peaks were generally observed in MS for 13C/2H-labeled
compounds /suf®x `a') corresponding, in order of decreas-
ing mass, to [15-13C2H3], [15-13CH2H2], [15-13CH2 H] and
2
[15-13CH3] isotopomers, respectively. For brevity, experi-
mental and calculated high-resolution mass data is given
only for the [15-13C2H3] isotopomer. High-resolution data
for the other isotopomers are not explicitly stated, but were
universally found to be within the same range of tolerance
as that for the [15-13C2H3] isotopomer.
3.2. Acid degradation of artemisinin *1) to 10
To a cooled solution of conc. H2SO4 in MeOH /120 ml;
180 ml) in an ice bath was added artemisinin /1) /12.0 g,
42.6 mmol). The mixture was stirred for 10 min after which
iced water /250 ml) was added. The mixture was ®ltered and
extracted with CHCl3 /5£100 ml) and the combined organic
layers were washed with brine /3£50 ml), dried /MgSO4)
and solvent was removed under reduced pressure to yield a
crude product /9.05 g; 75% w/w), consisting predominantly
of 10 /90% by 1H NMR).
Compound 10: [a]Dˆ234.4 /c 1.3, CHCl3); IR nmax
/CHCl3): 3028, 2928, 1728, 1712 cm21; other physical
properties as for Ref. 10. Compound 11 was sometimes
isolated as a very minor product by CC.
3. Experimental
3.1. General
1
Compound 11: H NMR /d, CDCl3) ppm: 9.90 /1H, d,
1
All H and 13C NMR experiments were recorded on a
Jˆ1.7 Hz), 3.64 /3H, s), 3.19 /1H, m), 1.28 /3H, s), 1.23
/3H, d, Jˆ7.2 Hz), 0.89 /3H, d, Jˆ6.5 Hz)Ðsee Table 2;
13C NMR: see Table 1.
Bruker DRX 500 instrument. Chemical shifts are expressed
in ppm /d) relative to TMS as internal standard. Proton
chemical shifts, multiplicities, coupling constants and
integrals reported in this section are those which were
clearly resolved in 1D-1H NMR without recourse to
2D-NMR analysis /see tables in the main text for full assign-
3.3. Robinson annulation of 10 to 8
2
ments made by 2D-NMR). H NMR spectra were recorded
BaOH2´8H2O /8 g) in MeOH /150 ml) was added to the
crude product consisting mostly of 10 /9.05 g, 33.8 mmol,
90% purity by 1H NMR) and the reaction mixture was stir-
red at room temperature for 3 h, then ®ltered and neutralized
to pH 7 by dropwise addition of CH3COOH under ice bath
cooling. The volume of the solvent was reduced in vacuo /to
ca. 30 ml) and the mixture was gradually acidi®ed by
addition of HCl /5%) until no more solid precipitated.
/N.B. addition of excess HCl causes lactonization of product
8). The precipitate was ®ltered and dried by suction to yield
a crude product /7.08 g, 78% w/w) from which compound 8
/4.98 g, 21.1 mmol, 62%) could be obtained by recrystal-
lization from EtOAc. See Ref. 10 for physical properties of
in CDCl3 at 42.4 MHz and CDCl3 was referenced to
7.29 ppm. HSQC, HMBC, 1H±1H COSY and NOESY spec-
tra were recorded with 1024 data points in F2 and 256 data
points in F1. High-resolution MS were recorded in EI mode
at 70 eV on a Finnigan-MAT 95 MS spectrometer. IR spec-
tra were recorded in CHCl3 on a Shimadzu FT-IR-8201 PC
instrument. Column chromatography /CC) was performed
using silica gel 60±200 mm /Merck). HPLC separations
were performed using a Varian chromatograph equipped
with RI star 9040 and UV 9050 detectors and either a
normal phase Intersil PREP-SIL or
a YMC diol
20 mm£25 cm column, ¯ow rate 8 ml/min. Melting points

8506
L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
8. Compound 8 sometimes underwent a substantial degree
of lactonization to 12 during the acidic work-up, see Ref. 10
for physical properties of 12. The free acid 8 could be
recovered by treatment of 12 with alkali as described below.
/30), 165.1549 [M12C3H5O2; C1113C1H142H3 requires
165.1552] /100), 164 /15), 163 /10), 162 /1).
Compound 15a: oil /96 mg, 0.41 mmol, 6%, Rt 21.0 min);
IR nmax /CHCl3): 3400±2400 /br), 3026, 2940, 1715, 1603,
1458 cm21 1H NMR /d, CDCl3) ppm: 5.55 /1H, dd,
;
3.3.1. Hydrolysis of the lactone side-product 12back to
decalenone acid 8. Compound 12 /2.5 g, 10.6 mmol) was
stirred with KOH solution /15%, 30 ml) at room tempera-
ture for 15 min. The mixture was cooled in an ice bath and
HCl /10%) was added to pH 2, until no more precipitate was
formed. The precipitate was ®ltered, washed with H2O
/5 ml) and dried by suction to yield compound 8 /2.3 g,
9.8 mmol, 93%) without need for further puri®cation.
3
Jˆ6.0 Hz, JCH, 2.1 Hz), 2.68 /1H, dq, Jˆ6.9, 7.1 Hz),
2.35 /1H, m), 1.78 /H-15) is not clearly seen in the spectrum
of the isotopically enriched sample, 1.18 /3H, d, Jˆ7.1 Hz),
0.98 /3H, d, Jˆ7.0 Hz)Ðsee Table 2; 2H NMR /d, CDCl3)
1
ppm: 1.78 /d, Jˆ19.3 Hz, JCD); 13C NMR: see Table 1Ð
3
2
127.9 /d, Jˆ4.5 Hz, JCC), 28.7 /d, Jˆ4.0 Hz, JCC), 23.1/
22.8/22.5/22.2 /2:10:20:68); HREIMS m/z /rel. int.)
238.1806 [M1, C1413C1H192H3O2 requires 238.1842] /1.0),
237 /1.2), 236.1673 [M12H2; C1413C1H172H3O2 requires
236.1686] /7.5), 192 /18), 163.1392 [M12H2±C3H5O2;
C1113C1H122H3 requires 163.1396] /100), 162 /40), 161
/17), 160 /13).
3.4. Synthesis of either diene 14a or lactone 7b from keto
acid 8 by Grignard reaction with labeled methyl iodide
To small Mg chips /0.436 g, 17.9 mmol) in anhyd. Et2O
/300 ml) was added a solution of labeled MeI /either
[13C2H3] or [13CH3]) /1.32 ml; 21.0 mmol) in anhyd. Et2O
/100 ml) and the reaction mixture was re¯uxed for 1.5 h. A
solution of 8 /1.69 g, 7.16 mmol) in anhyd. Et2O /200 ml)
was added and the reaction was allowed to re¯ux for a
further 2.5 h until completion, as determined by TLC.
Work-upat pH 4±5 for [ 13CH3]-methyl iodide: the reaction
mixture was cooled in an ice bath and HCl /10%) was added
dropwise to pH 4±5. Then the reaction mixture was
extracted with Et2O /3£300 ml) and the combined organic
layers were washed by brine /3£50 ml), dried /MgSO4) and
the solvent was removed under reduced pressure to yield
lactone 7b without need for further puri®cation.
Work-upat pH 1±2 for [ 13C2H3]-methyl iodide: the mixture
was cooled in an ice bath and HCl /10%) was added until pH
1±2. Then the reaction mixture was extracted with Et2O
/3£300 ml) and the combined organic layers were washed
with brine /3£50 ml), dried /MgSO4) and the solvent was
removed under reduced pressure to yield a crude product
/1.52 g, 90% w/w) consisting mostly of 14a with
compounds 7a and 15a as side-products. The mixture was
separated either by CC /15% EtOAc/n-hexane) or by HPLC
/10% EtOAc/n-hexane/1% CH3COOH).
Compound 7b: oil /1.60 g; 6.81 mmol, 95%). See Ref. 18
for physical propertiesÐwith the following differences due
to isotopic enrichment: 1H NMR /d, CDCl3) ppm: 5.64 /1H,
3
1
d, Jˆ6.0 Hz, JCH, H-5), 1.69 /3H, d, Jˆ126.0 Hz, JCH
,
H-15); 13C NMR: 142.2 /d, Jˆ42.9 Hz, JCC, C-4), 121.7
1
/d, Jˆ2.0 Hz, 2JCC), 83.2 /d, Jˆ4.0 Hz, 3JCC, C-6), 30.8 /d,
2
Jˆ3.0 Hz, JCC, C-3), 23.5 /ca. 70 times intensity of other
13C peaks, C-15); HREIMS m/z /rel. int.) 235.1643 [M1,
C1413C1H22O2 requires 235.1654] /28), 191 /100), 162 /100).
Compound 7a: Oil /200 mg, 0.85 mmol, 12%, Rt 13.1 min).
Physical properties as for isotopically-normal 7 in Ref. 18,
with the following differences due to isotopic enrichment:
3.5. Preparation of methyl ester 16 from decalenone 8
3
1H NMR /d, CDCl3) ppm: 5.63 /1H, d, Jˆ6.2 Hz, JCH
,
H-5), 1.69 /H-15) is not clearly seen; H NMR /d, CDCl3)
Diazomethane was freshly prepared from Diazaldw
/N-methyl-N-nitrosotoluene-p-sulphonamide) /1.88 g, 88
mmol) in Et2O /30 ml) cooled in an ice bath, by adding a
solution of KOH /0.436 g) in EtOH; /96%, 10 ml) with
stirring for 5±10 min. The ethereal diazomethane solution
was distilled from a water bath and added dropwise to a
stirred solution of decalenone 8 /2.05 g, 8.69 mmol) in
Et2O /70 ml) until no more bubbles were observed. Then
CH3COOH /10%, 1 ml) was added to destroy the excess
diazomethane and the reaction mixture was washed with
brine /3£10 ml), dried /MgSO4) and concentrated to yield
the methyl ester 16 /2.0 g, 8 mmol, 98% w/w) without need
for further puri®cationÐsee Ref. 10 for physical properties.
2
1
ppm: 1.69 /d, Jˆ19.2 Hz, JCD); 13C NMR: 142.2 /d,
1
3
Jˆ42.9 Hz, JCC, C-4), 83.2 /d, Jˆ4.0 Hz, JCC, C-6), 30.8
2
/d, Jˆ3.0 Hz, JCC, C-3), 23.5/23.2/22.9/22.6 /1:6:10:83),
3
21.0 /d, Jˆ2.8 Hz, JCC, C-2); HREIMS m/z /rel. int.)
238.1826 [M1, C1413C1H192H3O2 requires 238.1842] /15),
237 /1.4), 236 /0.16), 235 /0.01), 219 /10), 194 /90),
165.1543
165.1552] /100), 164 /19), 163 /6), 162 /1).
[M12C3H5O2;
C1113C1H142H3
requires
Compound 14a: solid /1.18 g, 4.96 mmol, 69%, Rt
20.8 min), Mp119±120 8C [a]Dˆ225.3 /c 4.8, CHCl3);
IR nmax /CHCl3): 3400±2400, 2912, 2874, 2829, 1707,
1458 cm21
;
1H NMR /d, CDCl3) ppm: 6.11 /1H, d,
3.5.1. Reaction of methyl ester 16 with the Grignard
reagent from methyl iodide. To small Mg chips /89 mg,
3.66 mmol) in anhyd. Et2O /20 ml) was added a solution of
MeI /0.30 ml, 4.80 mmol) in anhyd. Et2O /5 ml) and the
reaction mixture was re¯uxed for 1.5 h. A solution of 16
/375 mg, 1.5 mmol) in anhyd. Et2O /10 ml) was added and
the reaction was allowed to re¯ux for a further 2.5 h until
completion, as determined by TLC.
3
Jˆ6.3 Hz, JCH), 3.82 /1H, q, Jˆ7.0 Hz), 1.78 /H-15) is
not clearly seen in the spectrum of the isotopically enriched
sample, 1.21 /3H, d, Jˆ7.0 Hz), 0.99 /3H, d, Jˆ6.1 Hz)Ð
see Table 2; 2H NMR /d, CDCl3) ppm: 1.78 /d, Jˆ19.2 Hz,
1JCD); 13C NMR: see Table 1Ð137.9 /d, Jˆ43.4 Hz, 1JCC),
131.9 /d, Jˆ4.9 Hz, 3JCC), 31.2 /d, Jˆ2.9 Hz, 2JCC), 27.8 /d,
Jˆ2.7 Hz, 3JCC), 24.2/23.9/23.6/23.3 /2:8:13:77); HREIMS
m/z /rel. int.) 238.1835 [M1, C1413C1H192H3O2 requires
238.1842] /4.7), 237 /0.18), 236 /0.03), 235 /0.04), 193
Work-upat pH 4±5: the reaction mixture was cooled in an

L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
8507
ice bath and H2O /5 ml) was added to pH 4±5. Then, the
reaction mixture was extracted with Et2O /3£20 ml) and the
combined organic layers were washed with brine /3£10 ml),
dried /MgSO4) and solvent removed under reduced pressure
to yield a crude product /330 mg, 88% w/w) consisting of
epimers 17 and 18 which was separated by HPLC /27%
EtOAc/n-hexane).
completion, as shown by TLC. H2O /5 ml) was added and
the reaction mixture was extracted by Et2O /3£10 ml),
washed with brine /3£5 ml), dried /MgSO4) and solvent
was removed under reduced pressure to yield a crude
product /16 mg, 80% w/w), consisting mostly of compound
1
19 /95% by H NMR).
3.6. Hydrogenation of diene acid 14a
Compound 17: oil /87 mg, 0.33 mmol, 22%, Rt 27.3 min),
[a]Dˆ224.8 /c 0.33, CHCl3); IR nmax /CHCl3): 3590, 3450
To diene acid 14a /430 mg, 1.81 mmol) in EtOAc /200 ml)
was added a catalytic amount of palladium activated char-
coal and the solution was left in a hydrogenation apparatus
overnight. The reaction mixture was ®ltered and solvent was
removed under reduced pressure to yield a crude product
/414 mg, 96% w/w) which was separated by HPLC /4%
EtOAC/n-hexane/1% CH3COOH). Compound 3a: oil
/248 mg, 1.03 mmol, 58%, Rt 15.3 min). Physical properties
as for Ref. 18Ðwith the following differences due to iso-
1
/br), 3005, 2928, 2856, 1728, 1456, 1437 cm21; H NMR
/d, CDCl3) ppm: 5.26 /1H, s), 3.67 /3H, s), 2.73 /1H, dq,
Jˆ6.9, 6.8 Hz), 1.26 /3H, s), 1.22 /3H, d, Jˆ6.8 Hz), 0.92
/3H, d, Jˆ6.3 Hz)Ðsee Table 2; 13C NMR: see Table 1;
HREIMS m/z /rel. int.) 248.1780 [M12H2O, C16H24O2
requires 248.1776] /89), 214 /12), 189 /30), 161 /100).
Compound 18: oil /144 mg, 0.54 mmol, 36%, Rt 24.3 min),
[a]Dˆ270.7 /c 1.4, CHCl3); IR nmax /CHCl3): 3599, 3447
1
topic enrichment: H NMR /d, CDCl3) ppm: 5.11 /1H, d,
1
/br), 3005, 2972, 2930, 2872, 1728, 1458, 1437 cm21; H
3
2
Jˆ6.4 Hz, JCH, H-5), 1.63 /H-15) is not clearly seen; H
NMR /d, CDCl3) ppm: 1.63 /d, Jˆ19.0 Hz, JCD); 13C
1
NMR /d, CDCl3) ppm: 5.29 /1H, s), 3.67 /3H, s), 2.74 /1H,
dq, Jˆ7.1, 6.8 Hz), 1.29 /3H, s), 1.22 /3H, d, Jˆ6.8 Hz),
0.95 /3H, d, Jˆ6.1 Hz)Ðsee Table 2; 13C NMR: see Table
1; HREIMS m/z /rel. int.) 248.1774 [M12H2O, C16H24O2
requires 248.1776] /100), 216 /14), 189 /40), 161 /76).
1
NMRÐ136.0 /d, Jˆ43.2 Hz, JCC, C-4), 36.4 /d, Jˆ
3
2
3.4 Hz, JCC3, C-6), 26.6 /d, Jˆ3.5 Hz, JCC, C-3), 25.8 /d,
Jˆ3.0 Hz, JCC, C-2), 23.9/23.6/23.3/23.0 /24:21:21:35);
HREIMS m/z /rel. int.) 240.1989 [M1, C1413C1H212H3O2
requires 240.1998] /2), 239 /0.4), 238 /0.2), 237 /0.08),
192 /5), 166.1626 [M12C3H6O2; C1113C1H152H3 requires
166.1630] /100), 165 /30), 164 /14), 163 /7).
Work-upat pH 1±2: the reaction mixture was cooled in an
ice bath and HCl /10%) was added to pH 1±2. Then, the
reaction mixture was extracted with Et2O /3£50 ml) and the
combined organic layers were washed with brine /3£10 ml),
dried /MgSO4) and solvent removed under reduced pressure
to yield a crude product /328 mg, 88% w/w) consisting
predominantly of 19 with very small amounts of compounds
20±22.
Compound 23a: oil /20 mg, 0.083 mmol, 5%, Rt 19.3 min).
[a]Dˆ144.7 /c 1.34, CHCl3); IR nmax /CHCl3): 3400±2600
1
/br), 3040, 2928, 2903, 2847, 1705, 1458, 1445 cm21; H
NMR /d, CDCl3) ppm: 5.29 /1H, br), 2.30 /1H, dq, Jˆ10.7,
6.9 Hz), 2.15 /1H, dd, Jˆ17.6, 5.4 Hz), 1.63 /d,
1
Jˆ125.9 Hz, JCH)Ðca. 30% intensity expected for 3H in
Compound 19: oil. [a]Dˆ213.6 /c 0.1, CHCl3); IR nmax
the isotopically-normal spectrum, 1.19 /3H, d, Jˆ6.9 Hz),
0.82 /3H, d, Jˆ6.4 Hz)Ðsee Table 2; 2H NMR /d, CDCl3)
1
/CHCl3): 2930, 1719, 1603 cm21; H NMR /d, CDCl3)
1
ppm: 1.63 /d, Jˆ18.9 Hz, JCD); 13C NMR: see Table 1Ð
ppm: 6.11 /1H, s), 3.77 /1H, q, Jˆ7.0 Hz), 3.65 /3H, s),
1.78 /3H, s), 1.20 /3H, d, Jˆ7.0 Hz), 0.99 /3H, d,
Jˆ6.1 Hz)Ðsee Table 2; 13C NMR: see Table 1; HREIMS
m/z /rel. int.) 248.1776 [M1, C16H24O2 requires 248.1776]
/10), 211 /10), 189 /20), 149 /30), 99 /100).
1
3
131.8 /d, Jˆ48.3 Hz, JCC), 33.4 /d, Jˆ2.5 Hz, JCC), 27.8
/br, 3JCC), 26.3 /br, 2JCC), 23.6/23.3/23.0/22.7 /52:26:15:7);
HREIMS m/z /rel. int.) 240.1986 [M1, C1413C1H212H3O2
requires 240.1998] /2.0), 239 /1.2), 238 /1.2), 237 /0.8),
166.1624
166.1630] /100), 165 /63), 164 /57), 163 /40).
[M12C3H6O2;
C1113C1H152H3
requires
Compound 20: oil. 1H NMR /d, CDCl3) ppm: 5.82 /1H, s),
4.72 /1H, s), 4.69 /1H, s), 3.66 /3H, s), 2.75 /1H, dq, Jˆ7.1,
7.1 Hz), 1.12 /3H, d, Jˆ7.1 Hz), 0.93 /3H, d, Jˆ6.4 Hz)Ð
see Table 2; 13C NMR: see Table 1.
Compound 24a: oil /34 mg, 0.148 mmol, 8%, Rt 13.7 min).
[a]Dˆ280.9 /c 3.3, CHCl3); IR nmax /CHCl3): 3400±2600,
1
3030, 2924, 2872, 1705, 1458 cm21; H NMR /d, CDCl3)
Compound 21: oil. 1H NMR /d, CDCl3) ppm: 5.51 /1H, s),
3.64 /3H, s), 2.63 /1H, dq, Jˆ7.1, 7.1 Hz), 1.78 /3H, s), 1.13
/3H, d, Jˆ7.1 Hz), 0.97 /3H, d, Jˆ7.0 Hz)Ðsee Table 2;
13C NMR: see Table 1.
ppm: 3.67 /1H, q, Jˆ7.1 Hz), 2.43 /1H, d, Jˆ13.6 Hz), 1.15
/3H, d, Jˆ7.1 Hz), 0.95 /3H, d, Jˆ6.4 Hz), 0.92 is not
clearly seenÐsee Table 2; 2H NMR /d, CDCl3) ppm:
1
0.92 /d, Jˆ19.3 Hz, JCD), 13C NMR: see Table 1Ð29.4
1
/d, Jˆ35.4 Hz, JCC), 17.8/17.5/17.2/16.9 /13:17:19:50);
Compound 22: oil. 1H NMR /d, CDCl3) ppm: 5.37 /1H, s),
5.26 /1H, br s), 3.68 /3H, s), 2.74 /1H, dq, Jˆ8.8, 6.9 Hz),
1.69 /3H, d, Jˆ2.3 Hz), 1.24 /3H, d, Jˆ6.9 Hz), 0.87 /3H, d,
Jˆ6.4 Hz)Ðsee Table 2; 13C NMR: see Table 1.
HREIMS m/z /rel. int.) 240.1995 [M1, C1413C1H212H3O2
requires 240.1998] /2.9), 239 /0.4), 238 /0.1), 237 /0.02),
166.1625 [M12C3H6O2; C1113C1H152H3 requires 166.1630]
/100), 165 /24), 164 /8), 163 /3).
3.5.2. Conversion of 17/18 to 19 by treatment with
concentrated sulphuric acid. To a solution of the crude
mixture of compounds 17 and 18 /20 mg, 0.075 mmol) in
Et2O /5 ml) was added 70% H2SO4 /1 ml) and the reaction
mixture was stirred at room temperature for 5 h until
Compound 25a: oil /54 mg, 0.23 mmol, 13%, Rt 14.6 min).
[a]Dˆ245.2 /c 7.4, CHCl3); IR nmax /CHCl3): 3400±2600,
1
3032, 2924, 2864, 1705, 1458, 1447 cm21; H NMR /d,
CDCl3) ppm: 5.11 /1H, s), 2.70 /1H, dq, Jˆ8.8, 6.8 Hz),
2.17 /1H, m), 2.02 /1H, dd, Jˆ9.5, 8.8 Hz), 1.26 /3H, d,

8508
L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
Jˆ6.8 Hz), 0.93 is not clearly seen, 0.90 /3H, d,
tion of dihydroartemisinic acid 3 /400 mg, 1.66 mmol) in
CHCl3 /20 ml) was added m-chloroperoxybenzoic acid
/mCPBA; 440 mg, 2.55 mmol) and the mixture was stirred
overnight at room temperature. H2O /20 ml) was added and
the mixture was extracted by CHCl3 /3£100 ml), and
washed with NaHCO3 /10%, 3£50 ml). The combined
organic layers were washed with brine /3£20 ml), dried
/MgSO4) and solvent was removed under reduced pressure
to obtain a crude product from which the excess mCPBA
was removed by HPLC /12% EtOAc/n-hexane/0.5%
CH3COOH). Crystals of epoxide 27 were obtained by
recrystallization from n-hexane. Compound 27: solid
/350 mg, 1.37 mmol, 82%, Rt 29.0 min). Mp153±154 8C.
[a]Dˆ23.4 /c 1.5, CHCl3); IR nmax /CHCl3): 3028, 2928,
2
Jˆ6.4 Hz)Ðsee Table 2; H NMR /d, CDCl3) ppm: 0.93
/d, Jˆ19.1 Hz, JCD); 13C NMR: see Table 1Ð30.3 /d,
1
1
3
Jˆ35.4 Hz, JCC), 24.5 /d, Jˆ3.9 Hz, JCC), 21.9/21.6/
21.3/21.0 /25:24:29:23); HREIMS m/z /rel. int.) 240.1984
[M1, C1413C1H212H3O2 requires 240.1998] /0.08), 239
/0.03), 166.1631 [M12C3H6O2; C1113C1H152H3 requires
166.1630] /100), 165 /35), 164 /14), 163 /1).
Compound 26a: oil /10 mg, 0.042 mmol, 2%, Rt 9.9 min).
[a]Dˆ252.3 /c 0.7, CHCl3); IR nmax /CHCl3): 3400±2600,
1
3032, 2926, 2855, 1703, 1458, 1445 cm21; H NMR /d,
CDCl3) ppm: 5.27 /1H, s), 2.74 /1H, dd, Jˆ11.3, 6.7 Hz),
2.21 /1H, dd, Jˆ11.3, 4.2 Hz), 1.14 /3H, d, Jˆ6.7 Hz), 0.92
1
1
/3H, d, Jˆ6.3 Hz), 0.85 /3H, dd, Jˆ125.4 Hz, JCH
,
7.0 Hz)Ðca. 10% of peak intensity expected for 3H in
2878, 2853, 1705, 1458 cm21; H NMR /d, CDCl3) ppm:
2.65 /1H, s), 2.60 /1H, dq, Jˆ10.7, 6.9 Hz), 2.04 /1H, br m),
1.84 /1H, dd, Jˆ15.3, 6.6 Hz), 1.32 /3H, s), 1.29 /3H, d,
Jˆ6.9 Hz), 0.86 /3H, d, Jˆ6.4 Hz)Ðsee Table 3; 13C
NMR: see Table 3; HREIMS m/z /rel. int.) 252.1725 [M1,
C15H24O3 requires 252.1725] /2), 234 /60), 206 /27), 179
/100), 161 /45).
2
the isotopically normal spectrumÐsee Table 2; H NMR
1
/d, CDCl3) ppm: 0.85 /d, Jˆ18.7 Hz, JCD); 13C NMR: see
1
Table 1Ð31.1 /d, Jˆ34.5 Hz, JCC), 28.8 /d, Jˆ3.9 Hz,
3JCC), 22.0/21.7/21.4/21.1/48:29:16:7); HREIMS m/z /rel.
int.) 240.1989 [M1, C1413C1H212H3O2 requires 240.1998]
/0.24), 239 /0.12), 238 /0.10), 237 /0.05), 167.1706
[M12C3H5O2; C1113C1H162H3 requires 167.1709] /100),
166 /95), 165 /85), 164 /60).
3.8. Separation of compounds 28b±34b formed by
autoxidation of 7b on storage
Compound 7b /61 mg, 0.26 mmol) was stored at 2208C in
the freezer and found to have undergone some changes by
1H NMR after six months. The mixture was separated by
HPLC /12% EtOAc/n-hexane). Compound 7b /45 mg,
0.19 mmol, 74%, Rt 12.8 min).
3.7. Separation of compounds 1a, 7a and 27a formed by
autoxidation of 3a on storage
Compound 3a /29 mg, 0.12 mmol) stored at 2208C, was
found to have undergone some changes by 1H NMR spectro-
scopy after six months. The mixture was separated by HPLC
/8% EtOAc/n-hexane/0.5% CH3COOH).
Compound 28b: oil /1.8 mg, 0.067 mmol, 3%, Rt 39.3 min).
[a]Dˆ245.4 /c 0.18, CHCl3); IR nmax /CHCl3): 3300, 2930,
1763, 1456 cm21; 1H NMR /d, CDCl3) ppm: 8.44 /1H, br s,
OOH), 5.78 /1H, ddd, Jˆ5.9, 5.9, 2.1 Hz), 4.16 /1H, s), 3.21
/1H, dq, Jˆ6.6, 7.3 Hz), 2.93 /1H, ddd, Jˆ11.9, 6.6,
6.4 Hz), 2.27 /1H, ddd, Jˆ17.8, 5.9, 5.3 Hz), 1.93 /1H,
ddd, Jˆ17.8, 12.1, 5.9 Hz), 1.49 /1H, ddd, Jˆ12.1, 11.4,
5.3 Hz), 1.79 /d, Jˆ126.0 Hz, 1JCH), 1.17 /3H, d, Jˆ7.3 Hz),
0.93 /3H, d, Jˆ6.4 Hz)Ðsee Table 3; 13C NMR: see Table
Compound 1a: oil /6 mg, 0.021 mmol, 17%, Rt 28.6 min).
1H NMR spectrum as for artemisininÐsee Ref. 25Ðwith
the following differences due to isotopic enrichment: dH
1
1.44 /d, Jˆ129.3 Hz, JCH, H-15)Ðca. 10% of peak inten-
sity expected for 3H in the isotopically normal spectrum.
Compound 7a: oil /2 mg, 0.008 mmol, 7% Rt 14.9 min). 1H
NMR spectrum as for dihydro-epi-deoxyarteannuin BÐsee
Ref. 18Ðwith the following differences due to isotopic
2
1
3Ð129.6 /d, Jˆ2.9 Hz, JCC), 127.0 /d, Jˆ52.9 Hz, JCC),
21.5 /ca. 70 times intensity of other 13C peaks); HREIMS
m/z /rel. int.) 249.1433 [M12H2O, C1413C1H20O3 requires
249.1446] /18), 225 /15), 197 /25), 167 /65), 139 /55), 125
/85), 111 /100).
3
enrichment: dH 5.63 /1H, d, Jˆ5.9 Hz, JCH, H-5), 1.69
1
/3H, Jˆ125.4 Hz, JCH, H-15)Ðca. 10% of peak intensity
expected for 3H in the isotopically normal spectrum.
Compound 29b: oil /1 mg, 0.004 mmol, 2%, Rt 44.3 min).
Physical properties as for arteannuin KÐsee Ref. 18; H
Compound 27a: solid /1 mg, 0.004 mmol, 4%, Rt 30.9 min).
Physical properties as for isotopically normal 27Ðsee
belowÐwith the following differences due to isotopic
enrichment: 1H NMR /d, CDCl3) ppm: 1.32 /3H, d,
1
3
NMR /d, CDCl3) ppm: 5.67 /1H, dd, Jˆ5.3, JCH, 5.3 Hz),
3.70 /1H, s), 3.11 /1H, dq, Jˆ7.3, 7.1 Hz), 2.72 /1H, ddd,
Jˆ12.0, 6.4, 6.4 Hz), 2.27 /1H, m), 2.00 /1H, m), 1.79 /3H,
d, Jˆ126.2 Hz, 1JCH), 1.44 /1H, ddd, Jˆ11.0, 11.0, 5.3 Hz),
1.14 /3H, d, Jˆ7.1 Hz), 0.94 /3H, d, Jˆ6.3 Hz)Ðsee Table
1
Jˆ125.9 Hz, JCH)Ðca. 10% of peak intensity expected
for 3H in the isotopically normal spectrum; 13C NMR:
57.8
/25:22:21:32); HREIMS m/z /rel. int.) 238.1836 [M1
/C1413C1H212H3O3)±H2O; C1413C1H192H3O2
requires
/d,
Jˆ46.8 Hz,
1JCC),
23.6/23.3/23.0/22.7
3
3; 13C NMR: see Table 3Ð27.2 /d, Jˆ5.0 Hz, JCC), 21.2
/ca. 75 times intensity of other 13C peaks); HREIMS m/z
/rel. int.) 251.1601 [M1, C1413C1H22O3 requires 251.1603]
/3), 167.1048 [M12C413C1H7O for a retro-Diels Alder
fragmentation requires 167.1072] /100).
238.1841] /40), 237 /27), 236 /4), 235 /4), 183.1652
[M12C3H5O2; C1113C1H162H3O requires 183.1658] /100),
182 /35), 181 /16), 180 /12), 165.1563 [M12H2O-
C3H5O2; C1113C1H142H3 requires 165.1552] /62), 164 /44),
163 /15), 162 /5).
Compound 30b: oil /6.3 mg, 0.025 mmol, 10%, Rt
14.8 min) [a]Dˆ289.3 /c 0.1, CHCl3); IR nmax /CHCl3):
1
3.7.1. Preparation of 27 by treatment of isotopically
normal dihydroartemisinic *3) with mCPBA. To a solu-
3028, 2932, 2856, 1769, 1678, 1456 cm21; H NMR /d,
3
CDCl3) ppm: 6.73 /1H, ddd, Jˆ5.5, JCH, 5.5, 2.6 Hz),

L.-K. Sy et al. / Tetrahedron 57 *2001) 8495±8510
8509
1
3.25 /1H, dq, Jˆ10.4, 6.8 Hz), 2.75 /1H, ddd, Jˆ10.4, 6.5,
6.5 Hz), 2.50 /1H, ddd, Jˆ17.5, 4.9, 4.9 Hz), 2.40 /1H, dd,
Jˆ17.5, 9.5 Hz), 1.77 /3H, d, Jˆ124.6 Hz, 1JCH), 1.12 /3H,
d, Jˆ6.8 Hz), 0.96 /3H, d, Jˆ6.0 Hz)Ðsee Table 3; 13C
70 times intensity of other 13C peaks); HREIMS m/z /rel.
int.) 249.1444 [M1, C1413C1H20O3 requires 249.1446] /100),
221 /30), 176 /28).
CDCl3 /0.6 ml) for forty ®ve days and H NMR spectra
were acquired approximately every 5 days. Over this time
characteristic peaks for compounds 29b±32b were observed
to grow in intensity, until the starting material had almost
entirely disappeared leaving a complex mixture consisting
of ca. 10% 29b, 10% 30b, 15% 31b and 10% 32b together
with a large number of unidenti®ed peaks. Characteristic
peaks used in identifying 29b in the mixture: 5.67 /1H,
m), 3.71 /1H, d, Jˆ2.7 Hz), 2.72 /1H, ddd, Jˆ12.0, 6.4,
6.4 Hz). Characteristic peaks used in identifying 30b in
the mixture: 6.73 /1H, ddd, Jˆ5.5, 5.5, 2.6 Hz), 3.25 /1H,
dq, Jˆ10.4, 6.8 Hz), 2.75 /1H, ddd, Jˆ10.4, 6.5, 6.5 Hz).
Characteristic peaks used in identifying 31b in the mixture:
5.88 /1H, d, Jˆ6.0 Hz), 4.35 /1H, br s), 0.99 /3H, d,
Jˆ6.5 Hz). Characteristic peaks used in identifying 32b in
the mixture: 6.76 /1H, d, Jˆ5.6 Hz), 2.61 /1H, dd, Jˆ17.2,
12.5 Hz), 1.20 /3H, d, Jˆ7.0 Hz).
1
NMR: see Table 3Ð132.6 /d, Jˆ45.9 Hz, JCC), 16.2 /ca.
Compound 31b: oil /4.0 mg, 0.015 mmol, 6%, Rt 53.6 min).
[a]Dˆ162.6 /c 0.4, CHCl3); IR nmax /CHCl3): 3531, 3309
1
/br), 2934, 1759, 1454 cm21; H NMR /d, CDCl3) ppm:
3
7.89 /1H, s, OOH), 5.88 /1H, d, Jˆ6.0 Hz, JCH), 4.35
/1H, br s), 3.12 /1H, dq, Jˆ6.4, 7.1 Hz), 2.43 /1H, d,
Jˆ14.6 Hz), 2.13 /1H, ddd, Jˆ10.2, 6.4, 6.1 Hz), 1.84
1
/3H, d, Jˆ127.1 Hz, JCH), 1.15 /3H, d, Jˆ7.1 Hz), 0.99
/3H, d, Jˆ6.5 Hz)Ðsee Table 3; 13C NMR: see Table
3Ð137.0 /d, Jˆ43.9 Hz, 1JCC), 21.0 /ca. 95 times the inten-
sity of other 13C peaks); HREIMS m/z /rel. int.) 249.1440
[M12H2O, C1413C1H20O3 requires 249.1446] /100), 234
/30), 221 /45), 205 /50), 179 /35), 178 /95), 160 /80).
Acknowledgements
We thank the Generic Drugs Research Programme of the
Chemistry Department of the University of Hong Kong for
providing a postdoctoral fellowship to Dr Sy. Mr Ho Kin-
Fai performed some preliminary synthetic work. This work
was funded by a grant from the CRCG.
Compound 32b: oil /6.9 mg, 0.028 mmol, 11%, Rt
22.4 min). [a]Dˆ165.6 /c 0.7, CHCl3); IR nmax /CHCl3):
1
3020, 2932, 1767, 1682, 1456 cm21; H NMR /d, CDCl3)
3
ppm: 6.76 /1H, d, Jˆ5.7 Hz, JCH), 3.16 /1H, dq, Jˆ5.9,
7.1 Hz), 2.70 /1H, dd, Jˆ17.3, 4.3 Hz), 2.61 /1H, dd,
Jˆ17.3, 12.3 Hz), 2.25 /1H, ddd, Jˆ11.5, 5.9, 5.9 Hz),
1
1.81 /3H, dd, Jˆ128.4, JCH, 1.5 Hz), 1.19 /3H, d,
References
Jˆ7.1 Hz), 0.94 /3H, d, Jˆ6.6 Hz)Ðsee Table 3; 13C
1
NMR: see Table 3Ð139.4 /d, Jˆ44.9 Hz, JCC), 15.8 /ca.
1. Liu, J.-M.; Ni, M.-Y.; Fan, Y.-F.; Tu, Y.-Y.; Wu, Z.-H.; Wu,
Y.-L.; Zhou, W.-S. Acta Chim. Sin. 1979, 37, 129±141.
2. Wang, Y.; Xia, Z.-Q.; Zhou, F.-Y.; Wu, Y.-L.; Huang, J.-J.;
Wang, Z.-Z. Acta Chim. Sin. 1988, 46, 1152±1153.
3. Kim, N.-C.; Kim, S.-U. J. Korean Agric. Chem. Soc. 1992, 35,
106±109.
70 times the intensity of other 13C peaks); HREIMS m/z /rel.
int.) 249.1442 [M1, C1413C1H20O3 requires 249.1446] /100),
221 /25), 205 /35), 176 /40).
Compound 33b: oil /1.4 mg, 0.005 mmol, 2%, Rt 24.4 min).
[a]Dˆ143.5 /c 0.14, CHCl3); IR nmax /CHCl3): 3408 /br),
4. 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.
1
2930, 1759, 1460 cm21; H NMR /d, CDCl3) ppm: 7.73
3
/1H, br s, OOH), 5.82, /1H, dd, Jˆ6.3 Hz, JCHˆ1.6 Hz),
4.43 /1H, dd, Jˆ10.1, 6.5 Hz), 3.13 /1H, dq, Jˆ6.5, 7.2 Hz),
2.30 /1H, ddd, Jˆ12.5, 6.5, 2.6 Hz), 2.05 /1H, dd, Jˆ12.5,
5. Nair, M. S. R.; Basile, D. V. J. Nat. Prod. 1993, 56, 1559±
1566.
1
10.1 Hz), 1.82 /3H, ddd, Jˆ127.4 Hz, JCH, 1.0, 1.0 Hz),
6. Sangwan, R. S.; Agarwal, K.; Luthra, R.; Thakur, R. S.; Singh-
Sangwan, N. Phytochemistry 1993, 34, 1301±1302.
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1.15 /3H, d, Jˆ7.2 Hz), 0.96 /3H, d, Jˆ6.5 Hz)Ðsee
Table 3; 13C NMR: see Table 3Ð19.5 /ca. 95 times the
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249.1444 [M12H2O, C1413C1H20O3 requires 249.1446]
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1
2860, 1774, 1713, 1458 cm21; H NMR /d, CDCl3) ppm:
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3.46 /1H, d, Jˆ1.9 Hz), 3.24 /1H, dq, Jˆ6.9, 7.1 Hz), 2.60
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1
Jˆ12.3, 5.9, 5.3 Hz), 1.42 /3H, d, Jˆ124.6 Hz, JCH), 1.21
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13C NMR: see Table 3Ð15.4 /ca. 90 times intensity of other
13C peaks); HREIMS m/z /rel. int.) 265.1409 [M1,
C1413C1H20O4 requires 265.1409] /2), 177 /85), 176 /70),
162 /100).
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