Transformations of manool. tri- and tetracyclic norditerpenoids with in vitro activity against Plasmodium falciparum

Article abstract of DOI:10.1021/np0701071

The known 17-norisopimar-15-ene-8β,13β-diol (5) and five new semisynthetic norditerpenoids, ethyl 17-norabiet-13-(15)-E-en-8β-ol-16-oate (6), ethyl 17-norabiet-13(15)-Z-en-8β-ol-16-oate (7), 17-norpimaran- 13α-ethoxy-8,16-olactone (8), 17-norisopimarane-8β,15-diol (9), and 17-norarabiet-13(15)-ene-8β,16-diol (10), were prepared from manool (11). Standard spectroscopic data including X-ray crystal analysis were used to determine the structures of 5-10. All five compounds exhibited in vitro antiplasmodial activity against the malarial parasite Plasmodium falciparum at varying μg mL^-1 concentrations.

Full text of DOI:10.1021/np0701071

J. Nat. Prod. 2007, 70, 1253-1258
1253
Transformations of Manool. Tri- and Tetracyclic Norditerpenoids with in Vitro Activity against
Plasmodium falciparum
†
†
‡
§
,†
Albert W. W. van Wyk, Kevin A. Lobb, Mino R. Caira, Heinrich C. Hoppe, and Michael T. Davies-Coleman*
Department of Chemistry, Rhodes UniVersity, Grahamstown, South Africa, Department of Chemistry, UniVersity of Cape Town, Rondebosch,
South Africa, and DiVision of Pharmacology, Faculty of Health Sciences, UniVersity of Cape Town, ObserVatory, South Africa
ReceiVed March 12, 2007
The known 17-norisopimar-15-ene-8â,13â-diol (5) and five new semisynthetic norditerpenoids, ethyl 17-norabiet-13-
(
(
15)-E-en-8â-ol-16-oate (6), ethyl 17-norabiet-13(15)-Z-en-8â-ol-16-oate (7), 17-norpimaran-13R-ethoxy-8,16-olactone
8), 17-norisopimarane-8â,15-diol (9), and 17-norarabiet-13(15)-ene-8â,16-diol (10), were prepared from manool (11).
Standard spectroscopic data including X-ray crystal analysis were used to determine the structures of 5-10. All five
compounds exhibited in Vitro antiplasmodial activity against the malarial parasite Plasmodium falciparum at varying
-
1
µg mL concentrations.
Malaria affects 300-660 million individuals per annum world-
wide and results in an estimated 1-2 million deaths, mostly in
1
children under the age of 5. More than 70% of the malaria cases
and deaths occur in sub-Saharan Africa, where the disease has a
crippling impact on the already fragile socioeconomic and public
2
health structures of some of the world’s poorest nations. The
erythrocytic stages of the malarial protozoan parasites of the genus
Plasmodium, predominantly P. falciparum, are readily cultured in
Vitro and continue to provide a target for the renewed and urgent
antimalarial drug discovery efforts prompted by the alarming and
widespread occurrence of parasite resistance to the foremost
antimalarial drugs, notably chloroquine and sulfadoxine-py-
rimethamine.3
Terrestrial plants have historically been a vital source of
antimalarials, with quinine from the South American Cinchona tree,
from which chloroquine and related quinolines were derived, and
artemesinin and its derivatives from the Chinese herb Artemisia
annua, currently among the most promising antimalarials.4 The
disappointing rate at which synthetic chemical libraries and defined
molecular target-based drug discovery programs are perceived to
be delivering new drugs has renewed interest in natural products
as a potential source of novel antimalarial compounds.6 Recently
a series of isopimarane diterpenoids (1-4) with antiplasmodial
,5
,7
-
1
activity (IC50 7-25 µg mL ) and reportedly hemolytic properties
were isolated from the Iranian tree Platycladus orientalis (L.) Franco
8
(
Cupressaceae). In order to explore the possibility of improved
antiplasmodial activity and reduced hemolytic properties in com-
pounds possessing either norisopimarene, norabietene, or norpi-
marane as opposed to an isopimarane skeleton, we have prepared
a cohort of six 17-norditerpenoids (5-10), related to 1-4, from
the naturally occurring labdane diterpenoid (+)-manool (11). The
semisyntheses and antiplasmodial and hemolytic properties of 5-10
are reported here.
Results and Discussion
(
+)-Manool is regularly used as a semisynthetic precursor in
9-11
the syntheses of both marine and terrestrial natural products,
and our initial target, 5, was also originally synthesized from this
compound.12 Wenkert and co-workers’ semisynthesis of 5 incor-
porated an initial intramolecular aldol condensation of the diketone
(12) followed by nucleophilic addition of vinyl magnesium bromide
to the resultant 8-hydroxy-13-podocarpanone (13).12 The apparent
si-facial selectivity of the Grignard reaction was corroborated by
absorbances consistent with a cis 1,3-diol in the IR spectrum of
*
To whom correspondence should be addressed. Tel: +27 46 603 8294.
Fax: +27 46 6225109. E-mail: M.Davies-Coleman@ru.ac.za.
12
5.
Diketone 12 has been previously accessed in 72% and 36%
overall yield, respectively, from 11 via oxidation of this compound
with either a combination of OsO /HIO at pH 6 or KMnO and
†
Rhodes University.
Department of Chemistry, University of Cape Town.
Division of Pharmacology, University of Cape Town.
‡
§
4
5
4
1
0.1021/np0701071 CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/11/2007

1
254 Journal of Natural Products, 2007, Vol. 70, No. 8
Van Wyk et al.
Figure 1. View of a molecule of ethyl 17-norabiet-13(15)-E-en-
8
â-ol-16-oate (6) from the crystal structure. Thermal ellipsoids for
the non-hydrogen atoms are shown at the 50% probability level.
subsequent ozonolysis.12-14 We, however, elected to modify our
preliminary synthesis of 12 from 11 using an initial high-yielding
pyridinium chlorochromate mediated oxidative rearrangement of
15
1
1 to give a 2:1 mixture of the E and Z isomers (as determined
Figure 2. Perspective view of the two molecules of 17-norpimaran-
from NMR analysis) of the R,â-unsaturated aldehyde (14). Reduc-
tive ozonolysis of E/Z-14 afforded 12 in an overall yield (71%)
from 11 comparable with reported yields.1
1
3R-ethoxy-8,16-olactone (8) comprising the crystallographic asym-
metric unit. Thermal ellipsoids for the non-hydrogen atoms are
shown at the 50% probability level.
2-14
With 12 in hand we performed an NaH-mediated intramolecular
aldol condensation to access 13 in quantitative yield, which was a
significant improvement in the yields reported for the intramolecular
method afforded 6 and 7, there was both a reduction in yield (32%)
and a change in the E:Z ratio (2:1 as opposed to 4:3). On one
occasion the lactone ether 17-norpimaran-13R-ethoxy-8,16-olactone
(8) was isolated as a minor product (10%) from our one-pot
12
aldol condensation of 12 induced by methanolic KOH (68%) and
ethanolic NaOEt (80%).14 The apparent instability of 13 required
prompt reaction of this aldol condensation product with vinyl
magnesium bromide under standard Grignard conditions. Silica
chromatography of the reaction mixture gave the desired 17-
norisopimarene-8,13-diol product 5 in 24% overall yield from 11.
Regrettably, Wenkert and co-workers12 did not report the specific
2
synthesis. Crystallization of 8 from Et O/hexane and subsequent
crystallographic analysis (Figure 2) provided both its structure and
absolute configuration.20 The two independent molecules A and B
in the asymmetric unit adopt essentially the same conformation,
except for the slightly different orientations of their ethyl groups.
13
1
13
rotation of 5 and provided only a melting point and C NMR data
for this compound. Comparison of melting point and 1 C NMR
data obtained for our synthetic product with published data for 512
confirmed the structure of our compound.
The fully assigned H and C NMR data for 8 are provided in
Table 1, and a putative mechanism for the formation of 8 from 12
is presented in Scheme 1.
3
To increase the number of semisynthetic norditerpenoids avail-
able for antiplasmodial studies, we attempted to reduce the ester
functionality in 6 with LAH to give the corresponding allylic
alcohol. Surprisingly, LAH reduction of 6 gave exclusively the 1,4-
Michael hydride addition product 9, as evidenced by the disap-
The synthesis of 5 highlighted the suitability of the C-13 ketone
moiety in 13 as a key to further structural elaboration. Accordingly,
the ketone functionality in 13 was converted into a trisubstituted
olefin using a Horner-Wadsworth-Emmons (HWE) modification
of the Wittig reaction.16 The stereoselectivity of the HWE reaction
is diminished when ketones are used to prepare trisubstituted
alkenes,17 and reaction of 13 with triethylphosphonoacetate under
standard HWE conditions (NaH and anhydrous THF) gave a 4:3
ratio of ethyl 17-norabiet-13(15)-E-en-8â-ol-16-oate (6) and ethyl
13
1
pearance of the olefinic resonances in both the C and H NMR
spectra of 9 and the appearance of additional methylene (δ 40.4)
and oxymethylene (δ 60.3) resonances in the DEPT135 NMR
C
C
spectrum of this compound. The absence of duplicated resonances
in the 13C NMR spectrum of 9 suggested that the addition product
was not epimeric at C-13. However, extensive overlap within the
methylene envelope in the 1H NMR spectrum of 9 in both
deuterated chloroform and benzene prevented the assignment of
the configuration at C-13. Interestingly, reduction of the Z isomer
7 yielded both the expected allylic alchol 10 and the hydride
1
7-norabiet-13(15)-Z-en-8â-ol-16-oate (7) in 70% yield. These two
compounds were assigned to the norabietane diterpenoid series as
opposed to the norpimarane or norisopimarane series because the
3(15)
C
2
substitutent at C-13 (i.e., the ∆1
olefin) in 6 and 7 has neither
a syn nor an anti relationship with the angular methyl group at
C-10 required for membership to either the pimarane or isopimarane
series, respectively.18 The two isomers were readily separated by
normal-phase HPLC (9:1 hexane/EtOAc). Crystals of 6, obtained
addition product 9. The retention of the vinylic resonances [δ
(d) and 142.1 (s)], loss of the ester carbonyl resonance (δ
C
123.5
167.2),
C
and the emergence of an additional oxymethylene resonance (δ
C
57.5) in the 13C NMR spectrum of 10 unequivocally confirmed its
2
by slow crystallization from Et O/hexane, were deemed suitable
for X-ray analysis, and a perspective view of the molecular structure
of this compound is presented in Figure 1.
structure.
19
8
Asili et al. provided evidence to show that 1-4 and a number
The instability of 13 coupled with the use of NaH in anhydrous
THF for both the intramolecular aldol condensation and the HWE
reaction encouraged us to investigate a one-pot synthesis of 6 and
of unrelated labdane diterpenoids also caused echinocytic or
stomatocytic changes to the erythrocyte membrane. Accordingly,
they hypothesized that the weak or moderate antiplasmodial activity
exhibited by nonpolar secondary metabolites was therefore not a
sign of inhibitory activity against P. falciparum but rather an indirect
effect arising from host cell membrane damage that occurs at
7
from the diketone 12. Thus, 12 was first stirred with NaH (2.6
equiv) in dry THF (4 h) before dropwise addition of a solution of
deprotonated triethylphosphonacetate HWE reagent. Although this

Transformations of Manool
Journal of Natural Products, 2007, Vol. 70, No. 8 1255
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data of 8
position
1
δC, mult
δH, mult (J)
0.85, m
.62, m
1.39, m
.60, m
1.12, m
.40, m
HMBC
COSY
39.5
2, 10, 17
2
1, 3, 4, 10
1b, 2a
1
1a, 2a, 2b
1a, 2b, 3a
1a, 2a, 2b, 3b
2a, 2b, 3b
2b, 3a
2
3
18.4
41.9
1
2, 3, 18
1, 2, 4, 10
1
4
5
6
33.2
55.8
17.7
0.87, dd (12.2, 1.9)
1.54, m
6, 10
7
6
5, 7b
1
.70, m
1.52, m
.96, m
7
40.0
6, 8, 9, 13, 16
5, 6, 8, 13
7b
6, 7a
1
8
9
1
1
83.0
56.1
37.1
18.6
1.06, dd (12.5, 3.9)
1.20, m
11a, 11b
0
1
10, 12
8, 9, 13
13, 15
11b, 12a, 12b
11a, 12a
11a, 11b, 12b, 15a, 15b
11a, 12b
1
.80, m
1.57, m
.94, m
1
2
35.6
1
9, 13, 14
1
1
3
4
71.6
44.4
1.67, dd (12.8, 2.4)
.75, dd (12.8, 3.0)
2.58, dd (18.0, 1.8)
.66, dd (18.0, 2.3)
8, 9, 13, 15
8, 9, 13, 15
12, 13, 14, 16
12, 13, 14, 16
14b, 15a, 15b
14a, 15a, 15b
12a, 15b
1
1
5
41.1
2
14a, 15a
1
1
1
1
2
1
6
7
8
9
0
′
170.7
33.6
21.7
14.8
56.2
0.87, s
0.84, s
0.94, s
3.44, q (8.5)
3, 4, 5, 19
3, 4, 5, 19
9, 10, 18
2′, 13
2′, 13
1′
2′
2′
1′
3.45, q (7.0)
2
′
16.0
1.15, t (7.0)
Scheme 1 Putative mechanism for the Formation of 8 during the
HWE Reaction
compounds (6, 7, 10) show hemolytic activity in the 100-200 µg
-
1
mL range, measured as hemoglobin release into the culture
medium (Table 2), suggesting that these compounds may ac-
8
-1
cumulate in the erythrocyte membrane bilayer. At 100 µg mL
,
the most hemolytic compound (10) caused the majority of the
erythrocytes to assume stomatocyte type III shapes and occasional
erythrocyte ghosts were also present in the culture (Table 2, Figure
3
G, H).21 However, at lower concentrations erythrocytes retained
their normal discocyte shape. The second most hemolytic compound
(
1
7) caused a mixture of knizocytes and type III stomatocytes in
7% of the erythrocytes at 100 µg mL (Table 2, Figure 3D),
-
1
while a small percentage of type II stomatocytes were formed by
incubation with 6 (Figure 3C). Except for a modest increase in
type I echinocytes found with 8 (Figure 3E; control cultures contain
3
-4% type I echinocytes), the other compounds did not signifi-
-
1
cantly affect erythrocyte shape at 100 µg mL (Table 2, Figure
). Compounds 6, 7, and 10, which are the most hemolytic and
stomatocyte inducing”, have the lowest IC50’s, while 5, 8, and 9,
3
“
which have little or no hemolytic activity and do not cause
stomatocyte formation in the concentration range tested, display
the highest IC50’s (Table 2). From the disparity between the IC50’s
and the concentrations at which shape changes and hemolysis occur
we argue that erythrocyte shape changes per se may not be
accountable for the parasite inhibitory activity of 5, 8, and 9 and
allows for the possibility that these three compounds affect a
parasite-specific intracellular target. However, the possibility does
remain that the binding of 5, 8, and 9 to the erythrocyte membrane,
at concentrations too low to cause erythrocyte shape changes or
lysis, may remain sufficient to compromise erythrocyte function
and hence parasite viability.
Experimental Section
General Experimental Procedures. Melting points were determined
using a Reichert hot-stage microscope and are uncorrected. Optical
rotations were measured using a Perkin-Elmer 141 polarimeter cali-
brated at the sodium D line (598 nm). IR spectra were recorded on a
subhemolytic concentrations and is manifested by changes in the
shapes of the erythrocytes. At least three of our semisynthetic

1
256 Journal of Natural Products, 2007, Vol. 70, No. 8
Van Wyk et al.
Table 2. Parasite-Inhibitory and Hemolytic Properties of 5-10
5
6
7
8
9
10
CQ
IC50 (µg mL 1) ( SDa
-
10.7 ( 5.3
5.7 ( 4.6
3.9 ( 1.2
19.6 ( 6.6
13.3 ( 5.1
10.6 ( 3.3
0.0103 ( 0.0023
b
%
hemolysis
0.2
17
34
0
5
102
ND
-
1
2
%
00 µg mL
hemolysis
0.6
1
3
8
0
2
2
9
0
0
9
1
2
0
3
2
1
3
2
ND
ND
ND
ND
ND
-
1
1
%
00 µg mL
c
17d
0
stomatocytes
64
0
-
1
1
%
00 µg mL
echinocytes
2
-
1
1
%
00 µg mL
stomatocytes
1
11
0
5
-
1
5
%
0 µg mL
echinocytes
3
1
-
1
50 µg mL
a
Mean IC50 values and standard deviations (SD) were derived from IC50 determinations carried out on four separate occasions for 5-10 and on
b
c
three occasions for chloroquine (CQ). Percentage hemolysis was calculated relative to that obtained with 0.2% saponin. The percentages of
malformed erythrocytes (stomatocytes and echinocytes) present in erythrocyte samples incubated with the various compounds for 48 h are also
indicated. Erythrocyte shapes were defined according to Lim et al.21 ND ) not determined. d Mixture of knizocytes and stomatocytes.
Figure 3. Phase-contrast micrograph of erythrocytes incubated with medium alone (A) or 100 µg mL-1 of compound 5 (B), 6 (C), 7 (D),
8
(E), 9 (F), and 10 (G, H). The erythocytes were mounted under coverslips and viewed directly by phase-contrast light microscopy. Small
arrows indicate type II stomatocytes in C and knizocytes in D. Large arrow denotes a type I echinocyte in E, and arrowheads indicate
erythrocyte ghosts in H.
Perkin-Elmer Spectrum 2000 FT-IR spectrometer with the compounds
as films (neat) on NaCl discs. NMR spectra were acquired on Bruker
with literature values;22 HRFABMS m/z 289.2531 [M + H]+ (calcd
for C20 33O 289.2531).
14,15,17-Trinorlabda-8,13-dione (12). The isomeric mixture of 14
(100 mg, 0.35 mmol) was dissolved in anhydrous CH Cl (5 mL) and
cooled to -78 °C. A steady stream of O was bubbled through this
solution (10 min) until the solution had turned a pale blue color,
whereupon N was bubbled through the solution to remove excess O
H
4
00 MHz Avance and 600 MHz Avance II spectrometers using standard
pulse sequences. Chemical shifts are reported in ppm, referenced to
residual solvent resonances (CDCl 7.25, δ 77.2, C 7.15, δ
28.02), and coupling constants are reported in Hz. LREIMS (70 eV)
2
2
3
δ
H
C
D
6 6
δ
H
C
3
1
2
3
.
and HRFABMS data were obtained on a Finnegan-Matt GCQ and a
JEOL SX102 spectrometer, respectively. Kieselgel 60 (230-400 mesh)
was used for initial flash chromatographic separations. Semipreparative
HPLC was performed using a Whatman’s Magnum 9 Partisil 10 column
After the addition of triphenyl phosphine (548 mg, 2.09 mmol, 6 equiv)
the solution was stirred at ambient temperature (4 h). The solution was
cooled to 0 °C, H
0.5 h). The solution was finally washed with H
the combined organic extracts were dried (anhydrous MgSO
2
O
2
(30%, 1 mL) was added, and stirring continued
O (3 × 5 mL), and
) and
(
2
-
1
(
10 mm i.d., length 50 cm) with an eluent flow rate of 4 mL min.
Reactions where exclusion of water was necessary were performed in
flame-dried glassware under Ar. Immediately prior to their use Et
and THF were distilled from sodium metal/benzophenone ketyl and
CH Cl was distilled from CaH . General laboratory solvents were
4
concentrated. Subsequent purification of the concentrated organic
2
O
material on a silica column (EtOAc/hexane, 1:1) afforded 12 (72.1 mg,
1
13
0
.273 mmol, 78.4%): colorless oil; H and C NMR data consistent
2
2
2
14,23,24
20
14
with literature values;
[R]
D
-28.3 (c 1.3, CHCl ), lit. -11; IR
max 2940, 1709, 1698, 1355, 1164 cm ; HRFABMS m/z 265.2167
3
distilled before use. Reactions were monitored by thin-layer chroma-
tography (DC-Plastikfolien Kieselgel 60 F254 plates) and visualized
under UV light and developed by spraying with either 10% concentrated
-1
ν
+
[
M + H] (calcd for C17
28 2
H O 265.2168).
8-Hydroxy-13-podocarpanone (13). NaH (60%, 18.3 mg, 1 equiv)
H
2
SO
E)- and (Z)-(5S,9R,10S)-Labda-8(17),13-dienal (14). A solution
of manool (11) (170 mg, 0.586 mmol) and pyridinium chlorochromate
417 mg, 1.93 mmol, 3.3 equiv) in CH Cl (8 mL) was stirred (26 h)
at room temperature. Et O (8 mL) was added, and the formation of an
4 2
in MeOH or I .
was added to a solution of 12 (241.2 mg, 0.913 mmol) in anhydrous
THF (60 mL) and the resulting mixture stirred under Ar at ambient
temperature (6 h). The yellow solution was concentrated to dryness,
(
(
2
2
2 2
taken up in Et O (5 mL), and washed with H O acidified with a few
drops of 1 M HCl (3 × 5 mL). The organic fraction was concentrated
2
orange precipitate noted. The solution was then filtered through a Celite
in Vacuo to yield 13 (228 mg, 95%) as a pale yellow, amorphous
2
8
1
13
plug and the filtrate concentrated in Vacuo to yield a dark brown oil
mass: [R]
D
3
+11.6 (c 2.2, CHCl ); H and C NMR data consistent
12
(195 mg). The oil was purified on a silica column (EtOAc/hexane, 15:
with literature values.
1
) to yield a 2:1 inseparable mixture of the E and Z isomers of 14 (154
17-Norisopimar-15-ene-8â,13â-diol (5). A THF solution of vinyl
magnesium bromide (0.920 mmol, 1.5 equiv) was added to a cooled
1
13
mg, 90% yield) as a pale yellow oil: H and C NMR data consistent

Transformations of Manool
Journal of Natural Products, 2007, Vol. 70, No. 8 1257
(
0 °C) solution of 13 (162.4 mg, 0.614 mol) in anhydrous THF under
an Ar atmosphere. The solution was gradually warmed to ambient
temperature and stirred (1.5 h). Et O (10 mL) and a saturated aqueous
solution of NH Cl (6 mL) were added, and the resulting mixture was
stirred for 10 min, after which the organic and aqueous phases were
separated and the aqueous fraction was extracted with Et
O (3 × 5
mL). The organic fractions were pooled, dried (anhydrous MgSO ),
(CH
3
+
, C-20), 14.3 (CH
3
, -OCH
2 3
CH ) ppm; LREIMS m/z (rel int) 334
[M ] (35), 319 (100), 273 (36), 255 (27), 207 (33); HRFABMS m/z
+
2
335.2582 [M + H] (calcd for C21
35 3
H O 335.2588).
4
Compound 8: needle-like crystals (from hexane); mp 143-144 °C;
25
-1
[R]
D
+11.5 (c 0.7, CHCl ); IR νmax 2922, 2952, 1716, 1086, 986 cm ;
3
1
13
+
2
H and C data, see Table 1; LREIMS m/z (rel int) 334 [M ] (18),
4
319 (35), 288 (100), 276 (70), 228 (31); HRFABMS m/z 334.2509
+
and concentrated to yield a yellow oil (362 mg). This oil was
subsequently purified by normal-phase HPLC (EtOAc/hexane, 3:1) to
yield 5 as white crystals (from hexane): mp 157.5-159.5 (lit. 154-
1
[M] (calcd for C21
H
34
O
3
334.2508).
General Procedure for the Preparation of 17-Norisopimarane-
8â,15-diol (9) and 17-Norarabiet-13(15)-ene-8â,15-diol (10). A
solution of either the ethyl ester 6 (90.4 mg, 0.272 mmol) or the ethyl
ester 7 (59.1 mg, 0.180 mmol) in anhydrous THF (5 mL) was added
to a cooled (0 °C) solution of LAH (2.2 equiv) in THF (5 mL). The
solution was stirred (2.5 h) under Ar while gradually warming to
ambient temperature. The remaining LAH was quenched with H
mL), concentrated in Vacuo, and taken up in Et O (5 mL), which was
washed with H
O (3 × 5 mL). The combined organic partition fractions
were dried (anhydrous MgSO ) and concentrated under reduced pressure
to yield a yellow oil. The oil was purified by normal-phase HPLC
(100% EtOAc). The reduction of the Z isomer 7 yielded saturated diol
9 (7.9 mg, 14.9%) and the allylic alcohol (10, 24.3 mg, 46.2%). The
reduction of the E isomer 6 yielded exclusively 9 (34.7 mg, 43.3%).
Compound 9: yellow oil, [R]
2921, 1451, 1060, 735 cm ; H NMR (C
12
2
8
55 °C); [R]
D
-5.6 (c 2.6, CHCl
3
); IR νmax 3299, 2945, 1436, 1196,
-
1 1
7
61 cm ; H NMR (C , 600 MHz) δ 5.71 (1H, dd, J ) 17.3, 10.7
6
D
6
Hz, H-15), 5.25 (1H, d, J ) 13.7 Hz, H-16b), 4.96 (1H, d, 10.7 Hz,
H-16a), 1.76 (1H, dq, J ) 13.1, 3.2 Hz, H-11b), 1.63 (1H, m, H-7b),
1
.60 (3H, m, H-1b, H-2b, H-12b), 1.59 (1H, m, H-6b), 1.52 (1H, dd,
2
O (2
J ) 14.0, 2.6 Hz, H-14b), 1.39 (1H, m, H-3b), 1.40 (1H, m, H-6a),
1
1
d, J ) 14.0 Hz, H-14a), 1.04 (1H, ddd, J ) 13.4, 12.9, 4.0 Hz, H-7a),
0
0
2
.37 (2H, m, H-2a, H-11a), 1.21 (1H, dt, J ) 13.7, 4.1 Hz, H-12a),
.16 (1H, dd, J ) 13.3, 3.9 Hz, H-3a), 1.13 (3H, s, H -20), 1.11 (1H,
2
3
4
.90 (3H, s, H
3 3
-18), 0.89 (3H, s, H -19), 0.71 (2H, m, H-1a, H-5),
1
3
.56 (1H, d, J ) 12.6, 2.8 Hz, H-9) ppm; C NMR data consistent
1
2
+
with literature values; LREIMS m/z (rel int) 293 [M ] (1), 274 (72),
+
34
2
59 (100), 149 (39), 136 (45); HRFABMS m/z 293.2481 [M + H]
calcd for C19 293.2480).
Ethyl 17-Norabiet-13(15)-E-en-8â-ol-16-oate (6) and Ethyl 17-
Norabiet-13(15)-Z-en-8â-ol-16-oate (7). Triethyl phosphonoacetate
D
-9.8 (c 3.5, CHCl
, 600 MHz) δ 3.43 (2H,
-16), 1.72 (1H, m, H-12b), 1.70 (1H, m, H-13), 1.60
3
); IR νmax 3361,
-
1 1
(
H O
33 2
6 6
D
t, J ) 6.6 Hz, H
2
(1H, m, H-2b), 1.59 (2H, m, H-1b, H-2a), 1.51 (1H, m, H-7b), 1.44
(1H, m, H-11b), 1.43 (1H, m, H-6a), 1.42 (1H, m, H-11a), 1.39 (1H,
m, H-3b), 1.36 (1H, m, H-6b), 1.33 (1H, m, H-14b), 1.27 (1H, m,
H-15b), 1.25 (H-15a), 1.17 (1H, m, H-7a), 1.14 (1H, m, H-3a), 1.05
(
376 µL, 1.90 mmol, 2.5 equiv) was added to an anhydrous solution
of NaH (60%, 38 mg, 1.90 mmol, 2.5 equiv) in THF (15 mL), and the
mixture was stirred under an Ar atmosphere (0.5 h). A solution of 13
(264 mg, 0.761 mmol) in anhydrous THF (10 mL) was added via
3 3 3
(3H, s, H -20), 0.90 (3H, s, H -18), 0.89 (3H, s, H -19), 0.72 (1H, m,
cannula and the reaction mixture stirred at ambient temperature (10
h), after which HCl (1 M, 3 drops) was added and the mixture
H-5), 0.71 (1H, m, H-12a), 0.70 (1H, m, H-14a), 0.69 (1H, m, H-1a),
13
0.62 (1H, dd, J )12.4, 3.3 Hz, H-9) ppm; C NMR (CDCl
δ 71.3 (C, C-8), 60.3 (CH , C-16), 56.7 (CH, C-9), 56.5 (CH, C-5),
49.3 (CH , C-14), 42.8 (CH , C-7), 42.5 (CH , C-3), 40.4 (CH , C-15),
39.7 (CH , C-1), 37.5 (C, C-10), 33.88 (CH , C-12), 33.85 (CH , C-18),
33.5 (C, C-4), 29.4 (CH, C-13), 22.0 (CH , C-19), 20.7 (CH , C-6),
18.9 (CH , C-2), 18.5 (CH , C-11), 15.8 (CH , C-20) ppm; LREIMS
m/z (rel int) 294 [M ] (1), 279 (100), 261 (85), 243 (21), 179 (16);
3
, 150 MHz)
concentrated in Vacuo. The resulting oil was taken up in Et
and washed with H
O (3 × 5 mL). The combined organic fraction was
dried (anhydrous MgSO ) and concentrated in Vacuo to yield a yellow
2
O (5 mL)
2
2
2
2
2
2
4
2
2
3
oil, which was subsequently purified on silica (EtOAc/hexane, 1:4) to
yield a mixture of the E/Z isomers 6 and 7. This mixture was further
purified using normal-phase HPLC (EtOAc/hexane, 1:9) to yield 6 (105
mg, 41%) and 7 (76 mg, 29%). On one isolated occasion the minor
product 8 was also isolated in 10% yield.
3
2
2
2
3
+
+
HRFABMS m/z 295.2637 [M + H] (calcd for C19
H
35
O
2
295.2636).
-10.8 (c 1.4, CHCl
1
34
Compound 10: yellow oil; [R]
D
-1
3
); IR νmax
34
Compound 6: white crystals (from Et
68.1 (c 1.8, CHCl
); IR νmax 2942, 1720, 1634, 1156, 1046 cm ; 1H
NMR (CDCl , 400 MHz) δ 5.60 (1H, t, J ) 1.4 Hz, H-15), 4.13 (2H,
CH ), 3.92 (1H, m, H-12b), 2.20 (1H, ddd, J )
3.1, 1.6, 0.7 Hz, H-14b), 2.05 (1H, dd, J ) 13.1, 2.3 Hz, H-14a),
.79 (1H, m, H-11b), 1.76 (1H, m, H-12a), 1.75 (1H, m, H-7b), 1.67
2
O); mp 110-111 °C; [R]
D
3
3446, 2945, 1709, 1386, 757 cm ; H NMR (CDCl , 400 MHz) δ
-
1
-
3
5.65 (1H, dd, J ) 8.1, 6.8 Hz, H-15), 4.12 (1H, dd, J ) 11.6, 6.8 Hz,
H-16b), 3.92 (1H, dd, J ) 11.6, 8.1 Hz, H-16a), 2.49 (1H, dd, J )
13.2, 2.1 Hz, H-14b), 2.27 (1H, m, H-12b), 1.97 (1H, dt, J ) 12.8, 4.5
Hz, H-12a), 1.79 (1H, m, H-7b), 1.77 (1H, m, H-14a), 1.72 (1H, m,
H-11b), 1.64 (1H, m, H-1b), 1.58 (1H, m, H-2b), 1.54 (1H, m, H-6b),
1.45 (1H, m, H-7a), 1.40 (2H, m, H-3b, H-6a), 1.38 (1H, m, H-11a),
1.37 (1H, m, H-2a), 1.12 (1H, m, H-3a), 1.10 (1H, m, H-9), 0.94 (3H,
3
q, J ) 7.1 Hz, -OCH
2
3
1
1
(
1H, m, H-1b), 1.59 (1H, m, H-2b),1.58 (1H, m, H-6b), 1.44 (1H, m,
H-7a), 1.43 (1H, m, H-11a), 1.40 (1H, m, H-6a), 1.39 (1H, m, H-3b),
.38 (1H, m, H-2a), 1.26 (3H, t, J ) 7.1 Hz, -OCH CH ), 1.17 (1H,
m, H-9), 1.14 (m, 1H, H-3a), 0.94 (3H, s, H -20), 0.87 (3H, s, H -18),
.87 (1H, m, H-1a), 0.84 (3H, s, H -19) ppm; C NMR (CDCl , 100
MHz) δ 166.3 (C, C-16), 159.4 (C, C-13), 116.1 (CH, C-15), 73.9 (C,
C-8), 59.6 (CH , -OCH CH ), 56.4 (CH, C-9), 56.3 (CH, C-5), 53.3
, C-14), 42.1 (CH , C-3), 41.5 (CH , C-7), 39.7 (CH , C-1), 37.4
C, C-10), 33.6 (CH , C-18), 33.3 (C, C-4), 29.6 (CH , C-12), 22.4
, C-11), 21.7 (CH , C-19), 18.4 (CH , C-2), 18.3 (CH , C-6), 15.4
, C-20), 14.3 (CH , -OCH CH ) ppm; LREIMS m/z (rel int) 334
1
2
3
s, H
0.84 (3H, s, H
C-13), 123.5 (CH, C-15), 73.0 (C, C-8), 57.5 (CH
C-9), 56.4 (CH, C-5), 44.4 (CH , C-14), 42.1 (CH
C-7), 39.5 (CH , C-1), 37.4 (C, C-10), 36.7 (CH , C-12), 33.6 (CH
C-18), 33.3 (C, C-4), 23.1 (CH , C-11), 21.7 (CH , C-19), 18.4 (CH
C-6), 18.3 (CH , C-2), 15.4 (CH
292 [M ] (2), 259 (86), 179 (92), 149 (100), 91 (93); HRFABMS m/z
3
-20), 0.90 (1H, m, H-5), 0.87 (3H, s, H
3
-18), 0.85 (1H, m, H-1a),
, 100 MHz) δ 142.1 (C,
, C-16), 56.7 (CH,
-19) ppm; 13C NMR (CDCl
3
3
3
3
1
3
0
3
3
2
2
2
, C-3), 41.3 (CH
2
,
3
,
2
,
2
2
3
2
2
(
(
(
(
[
CH
2
2
2
2
2
3
3
2
2
3
, C-20) ppm; LREIMS m/z (rel int)
+
CH
2
3
2
2
+
CH
3
+
3
2
3
33 2
293.2480 [M + H] (calcd for C19H O 293.2481).
M ] (30), 319 (100), 273 (50), 207 (53), 149 (35); HRFABMS m/z
Malaria Parasite Viability, Hemolysis, and Erythrocyte Shape
+
3
34.2509 [M] (calcd for C21 334.2508).
Compound 7: yellow oil; [R] +66.3 (c 0.8, CHCl
1
H
34
O
3
Assays. P. falciparum (3D7 strain) was cultured under an atmosphere
3
4
D
3
); IR νmax 2941,
, 400 MHz) δ 5.75
2 2 2
of 3% CO , 4% O , and 93% N in RPMI-1640 medium supplemented
-
1
1
695, 1652, 1165, 1037 cm ; H NMR (CDCl
3
with 50 mM glucose, 0.65 mM hypoxanthine, 25 mM HEPES, 0.2%
-
1
(
1H, t, J ) 1.5 Hz, H-15), 4.12 (2H, dq, J ) 7.1, 1.0 Hz, -OCH
CH ), 3.66 (1H, dd, J ) 13.5, 0.9 Hz, H-14b), 2.34 (1H, m, H-12b),
.11 (1H, dt, J ) 12.8, 4.3 Hz, H-12a), 1.86 (1H, dd, J ) 13.5, 0.9
2
-
3
(w/v) NaHCO , 0.048 mg mL gentamicin, 0.5% (w/v) Albumax II,
+
3
and 2-4% (v/v) human O erythrocytes. Parasite-infected erythrocytes
from culture were mixed with fresh culture medium and erythrocytes
to yield a 2% parasitemia and 2% hematocrit suspension and distributed
in microtiter plates at 100 µL/well. Serial dilutions of each compound
were prepared in a duplicate plate and transferred to the parasite plate
at 100 µL/well. The plates were incubated at 37 °C for 48 h, and parasite
viability in each well was determined by measuring parasite lactate
2
Hz, H-14a), 1.78 (1H, m, H-11b), 1.77 (1H, m, H-7a), 1.67 (1H, m,
H-1b), 1.62 (2H, m, H-2b, H-11a), 1.55 (1H, m, H-6b), 1.52 (1H, m,
H-7a), 1.40 (1H, m, H-2a), 1.39 (1H, m, H-3b), 1.37 (1H, m, H-6a),
1
.25 (3H, t, J ) 7.1 Hz, -OCH
H-3a), 0.94 (3H, s, H -20), 0.88 (1H, m, H-5), 0.87 (3H, s, H
.86 (1H, m, H-1a), 0.84 (3H, s, H
MHz) δ 167.2 (C, C-16), 160.0 (C, C-13), 115.6 (CH, C-15), 74.3 (C,
C-8), 59.7 (CH , -OCH CH ), 56.7 (CH, C-9), 56.4 (CH, C-5), 45.3
, C-14), 42.4 (CH , C-7), 42.1 (CH , C-3), 39.7 (CH , C-1), 37.9
, C-12), 37.5 (C, C-10), 33.7 (CH , C-18), 33.3 (C, C-4), 23.5
, C-11), 21.7 (CH3, C-19), 18.5 (CH , C-2), 18.3 CH , C-6), 15.3
2
CH
3
), 1.16 (1H, m, H-9) 1.15 (1H, m,
-18),
, 100
3
3
1
3
25
0
3
-19) ppm; C NMR (CDCl
3
dehydrogenase activity. Parasite viabilities were determined relative
to solvent controls (wells containing 0.5% DMSO). To assess hemolytic
activity, duplicate plates containing normal, uninfected erythrocytes at
2% hematocrit were also incubated for 48 h with the various compound
dilutions. The intact erythrocytes were sedimented in the microtiter
plate wells by centrifugation at 200g for 3 min, and released hemoglobin
2
2
3
(CH
(CH
(CH
2
2
2
2
2
2
3
2
2

1
258 Journal of Natural Products, 2007, Vol. 70, No. 8
Van Wyk et al.
in the supernatants was measured by removing aliquots and measuring
absorbance at 405 nm. Wells treated with 0.2% saponin (from Quillaja
bark supplied by Sigma-Aldrich, Steinheim, Germany) were used as
(13) Wenkert, E.; Mahajan, J. R.; Nussim, M.; Schenker, F. Can. J. Chem.
1966, 44, 2575-2579.
(14) Do Khac Manh, D.; Fetizon, M.; Flament, J. P. Tetrahedron 1975,
3
1, 1897-1902.
1
00% hemolysis controls. Erythrocyte shape changes after 48 h
(
(
15) Sundararaman, P.; Herz, W. J. Org. Chem. 1977, 42, 813-819.
16) For reviews of the HWE reaction see: (a) Maryanoff, B. E.; Reitz,
A. B. Chem. ReV. 1989, 89, 863-927. (b) Boutagy, J.; Thomas, R.
Chem. ReV. 1974, 74, 87-99.
incubation with the various test compounds were assessed by removing
aliquots from treated wells and preparing Giemsa-stained thin blood
smears for light microscopy. In addition, aliquots were wet-mounted
under glass coverslips and viewed directly by phase-contrast micros-
copy. To assess the effects of the compounds on erythrocyte shape at
subhemolytic concentrations, 2% hematocrit suspensions of fresh
erythrocytes were incubated with parasite culture medium alone
(
17) Kelly S. E. In Alkene Synthesis. ComparatiVe Organic Synthesis;
Trost, B. M., Ed.; Pergamon: Oxford, 1991; Vol. 1, pp 761-773.
(18) Manitto, P. In Biosynthesis of Natural Products; Sammes, P. G.,
Translation Ed.; Ellis Horwood Ltd.: Chichester, 1981; pp 257-
260.
(
control) or with medium containing 100, 50, or 25 µg mL-1 of the
(
19) X-ray analysis of 6. Diffraction intensities were collected with Mo
various compounds for 48 h. After incubation, the erythrocyte suspen-
sions were mounted directly under coverslips on microscope slides and
immediately viewed with a Nikon Eclipse E600 light microscope using
a 100× Apochromat oil-immersion objective and phase contrast optics.
Images were captured with a Media Cybernetics CoolSNAP-Pro
monochrome cooled charge-coupled device camera. Discocytes, echi-
nocytes, stomatocytes, and knizocytes in the samples were defined
3
KR radiation from a cubic fragment (0.10 × 0.11 × 0.12 mm ) using
φ- and ω-scans of 1.00° on a Nonius Kappa CCD diffractometer,
with the crystal cooled to 113(2) K in a nitrogen stream. Crystal
data for 6: C21H34O3, M ) 334.48, orthorhombic, space group
P212121 (no. 19), a ) 7.3750(2) Å, b ) 9.7769(3) Å, c ) 27.077(1)
3
3
Å, V ) 1952.4(1) Å , Z ) 4, Dc ) 1.138 Mg/m , µ(Mo KR) )
-1
0
.074 mm , F(000) ) 736. A total of 4302 reflections were
2
1
according to Lim et al.
collected, of which 2405 were observed [I > 2σ(I)]. The structure
2
was solved by direct methods and refined on F using all data, with
Acknowledgment. We would like to thank Professor B. Robinson
of Westchem Industries, New Zealand, for the generous donation of
manool. Financial assistance from the South African National Research
Foundation (NRF), the Department of Environmental Affairs and
Tourism, DAAD (Ph.D. bursary A.v.W), is gratefully acknowledged.
Professor L. Fourie (North West University) is thanked for assistance
with the acquisition of HRFABMS data. M.R.C. acknowledges research
support from the University of Cape Town and the NRF.
non-hydrogen atoms treated anisotropically. All H atoms were located
and then added in idealized positions in a riding model. The final R
factors were R1 ) 0.1455 (all data), 0.0510 (observed data), wR2 )
0
.0917 (all data), 0.0742 (observed data) for 222 parameters.
Crystallographic data (excluding structure factors) have been depos-
ited at the Cambridge Crystallographic Data Centre (deposition no.
620303).
(20) X-ray analysis of 8. Diffraction intensities were collected with Mo
KR radiation from a specimen of dimensions 0.10 × 0.20 × 0.20
mm3 using φ- and ω-scans of 1.00° on a Nonius Kappa CCD
diffractometer, with the crystal cooled to 113(2) K in a nitrogen
stream. Crystal data for 8: C21H34O3, M ) 334.48, monoclinic, space
Supporting Information Available: 1H and 13C NMR spectra of
compounds 5-10. This material is available free of charge via the
Internet at http://pubs.acs.org.
group P21 (no. 4), a ) 6.9880(1) Å, b ) 22.0165(4) Å, c ) 12.6710-
3
(
3) Å, â ) 101.345(1)°, V ) 1911.36(6) Å , Z ) 4, Dc ) 1.162
3
-1
Mg/m , µ(Mo KR) ) 0.075 mm , F(000) ) 736. A total of 8635
References and Notes
reflections were collected, of which 5846 were observed [I > 2σ-
2
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(I)]. The structure was solved by direct methods and refined on F
Nature 2005, 434, 214-217.
using all data, with non-hydrogen atoms treated anisotropically. All
H atoms were located and then added in idealized positions in a riding
model. The final R factors were R1 ) 0.0828 (all data), 0.0387
(observed data), wR2 ) 0.0782 (all data), 0.0692 (observed data) for
441 parameters. Crystallographic data (excluding structure factors)
have been deposited at the Cambridge Crystallographic Data Centre
(deposition no. 620304).
(
(
(
(
(
(
(
2) Sachs, J.; Malaney, P. Nature 2002, 415, 680-685.
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(
(
10) Vik, A.; Hedner, E.; Charnock, C.; Samuelsen, O.; Larsson, R.;
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205-210.
NP0701071