The synthesis and antibacterial activity of totarol derivatives. Part 3: modification of ring-B

Article abstract of DOI:10.1016/S0968-0896(00)00096-1

Ring-B derivatization of totarol (1) afforded the series of compounds 2-22 which were screened in vitro against: β-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and multiresistant Klebsiella pneumoniae. Several of the derivatives retained much of the antibacterial activity of totatol against the first three of these organisms (all Gram-positive), but none was more active. The Gram-negative Klebsiella was resistant to all compounds examined. Totarol (1) was shown to uncouple oxidative phosphorylation in isolated mitochondria at 50 μM. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00096-1

Bioorganic & Medicinal Chemistry 8 (2000) 1663±1675
The Synthesis and Antibacterial Activity of Totarol Derivatives.
Part 3: Modi®cation of Ring-By
Gary B. Evans,a,* Richard H. Furneaux,a Graeme J. Gainsforda
and Michael P. Murphyb
aIndustrial Research Limited, PO Box 31-310, Lower Hutt, New Zealand
bBiochemistry Department, University of Otago, PO Box 56, Dunedin, New Zealand
Received 9 December 1999; accepted 9 March 2000
AbstractÐRing-B derivatization of totarol (1) a€orded the series of compounds 2±22 which were screened in vitro against: b-lac-
tamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, methicillin-
resistant Staphylococcus aureus (MRSA), and multiresistant Klebsiella pneumoniae. Several of the derivatives retained much of the
antibacterial activity of totatol against the ®rst three of these organisms (all Gram-positive), but none was more active. The Gram-
negative Klebsiella was resistant to all compounds examined. Totarol (1) was shown to uncouple oxidative phosphorylation in
isolated mitochondria at 50 mM. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
antibacterial activity,3,4 we now report a study of the
relative antibacterial properties of 1 and of its deriva-
tives 2±22 which were obtained by synthetic manipula-
tion of ring-B. Particular interest was taken in the e€ect
of relative lipophilicity on the antibacterial activities.
Lien et al.1 have shown that lipophilic compounds are
trapped in the cell wall lipids of Gram-negative bacteria
(lipid content of up to 25% of the dry weight) but are
able to pass through Gram-positive bacterial cell walls
(0±2.5% lipid content), which results in antibacterial
lipophilic alcohols and phenols showing selective activ-
ity against Gram-positive bacteria. In keeping with this,
Kobayashi et al.2 have shown that the magnitude and
selectivity of the inhibition of Gram-positive bacterial
growth by pisiferic acid and its derivatives are a func-
tion of the lipophilicity of these compounds.
Results and Discussion
Syntheses of compounds
Firstly, totarol's lipophilicity was modi®ed by hydro-
xylation of ring-B. Treatment of O-methyltotarol with
lead tetraacetate in acetic acid by a previously described
procedure3 a€orded the 7a-acetate of the starting mate-
rial and the product formed by replacement of the iso-
propyl by an acetoxy group in 42 and 16% yield,
respectively. Lithium aluminum hydride reduction of
the former yielded the 7a-hydroxy derivative 2 (88%)
which proved to be unstable to a variety of demethyla-
tion protocols (e.g., BBr3, pyridine hydrochloride).
Synthesis of the corresponding diol 3 was achieved via
totaryl acetate,5 treatment of which with lead tetra-
acetate in acetic acid gave the previously reported 7a,13-
diacetoxy derivative6 in 44% yield as well as a mixture
of other products which ran together on TLC plates.
Lithium aluminum hydride reduction of the diacetate
provided the desired 7a-hydroxytotarol (3),7 and similar
treatment of the mixed fraction a€orded triol 7 in 12%
In a continuation of our investigation into the relation-
ship between the structures of diterpenoid compounds
based on the natural product totarol (1) and their
yFor part II, see preceding paper.
*Corresponding author. Tel.: +64-45690040; fax:+64-45690055; e-
mail: g.evans@irl.cri.nz
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00096-1

1664
G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
yield (based on totaryl acetate). The stereochemistry of
this compound was assigned on the basis of H-6 show-
ing 11.6 Hz coupling with H-5 and small coupling with
H-7. The former was therefore axial and b and the latter
equatorial and also b. In order to prepare the alkene 117
by eliminating acetic acid from the 7a,13-diacetate, the
latter was heated in ethanolic HCl under re¯ux. This
process, however, gave three readily separable products:
the 7a-ethoxy analogue of the starting material (39%)
formed by displacement at the benzylic center and
which provided a convenient source of the ethyl ether 4,
the desired alkene 11 (30%) and the product of its de-
acetylation 12 (26%).
Increase in the size of the substituent groups at C-7
should impact on the spatial arrangement of the C-14
isopropyl group and consequently on the phenolic
hydroxy group at C-13 since non-bonding peri-interac-
tions occur between groups (even hydrogen atoms) at
C-7 and the C-14 isopropyl residue.9 Conceivably bio-
logical activity might also be sensitive to such change.1
Consequently, the 7-oxo-13-O-acetyl compound was
treated with MeLi in THF and yielded the tertiary
alcohol (69%), which on reductive removal of the O-13
acetyl group a€orded the phenolic derivative 6. The
presence of the methyl group was evident from the
NMR characteristics [dH 1.69, 3H (s); dC 29.5 (q), 74.9
(s)], and the 7-a-con®guration was assigned to the
methyl group by analogy, nucleophilic attack at 7-oxo
groups of such compounds occurring from the less hin-
dered a-face of the molecules.10
In an attempt to ascertain whether the stereochemistry
of the hydroxyl group at C-7 has a bearing on anti-
bacterial activity, the alcohol 5, the C-7 epimer of 3, was
synthesized in 91% yield by the lithium aluminum
hydride reduction of the previously reported 13-O-ace-
tyl-7-oxo derivative.8 The b-stereochemistry at C-7 of
compound 5 was con®rmed by the characteristic split-
ting pattern of the C-7-carbinol proton [dH 5.15 (t,
Hydrogenolysis of the epoxyacetate 17, as previously
described,11 gave 13-acetoxytotara-8,11,13-trien-6a-ol
(42%), and lithium aluminum hydride reduction of this
product a€orded 6a-hydroxytotarol (8) (79%), the 6-
epimer (9) being obtained (69%) by lithium aluminum
1
J=8.4 Hz)] in the H NMR spectrum.9

G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
1665
hydride reduction of 6-oxototarol (14) (see below). The
b-stereochemistry at C-6 in 9 was assigned on the basis
of the deshielding of the C-10 methyl group resonance
[dH 1.60 (3H, s)] relative to the corresponding signals in
14 [dH 1.33 (3H, s)] and 8 [dH 1.17 (3H, s)].
Sugiol, a diterpene closely related to totarol (1), has an
sp2 center at C-7 in the form of a carbonyl group and is
inactive against Gram-positive bacteria up to 400 mg/
mL.12 In contrast, ferruginol, which is structurally
identical to sugiol except that it has a methylene group
instead of the carbonyl group at C-7, has been reported
as having good antibacterial activity against a variety of
Gram-positive bacteria (active at about 8 mg/mL).3
Figure 1.
CH2Cl2 to a€ord 14 in 42% overall yield based on the
acetoxy-methoxy starting material.
The 6a-bromo derivative 15, in which ring-B exists in an
approximate boat conformation,13 was synthesized by
bromination of the 13-acetoxy-7-one to give the 6a-
bromide (74%), which on saponi®cation a€orded 15 in
88% yield. Dehydrobromination of the acetylated bro-
mide using lithium bromide and sodium bicarbonate in
DMF gave the corresponding enone (91%) from which
the hydroxy-enone 16 was obtained in 89% yield by
deacetylation.
The generality of this loss in antibacterial activity, in
consequence of the introduction of an sp2 center in ring-
B, was then studied by way of a range of compounds
incorporating one or more sp2 centers in ring-B of
totarol. Such incorporation should lead to: (1) further
¯attening of the ring-B, (2) the alteration of steric com-
pression between the C-7 methylene group and the C-14
isopropyl group which may have a subsequent e€ect on
the relative spatial arrangement of the isopropyl group
and the phenolic group, (3) electronic changes in the
aromatic ring, and one or more of these e€ects could
impact on biological properties.
The incorporation of fused and bridging heterocyclic
ring systems onto the totarol skeleton was also investi-
gated. Treatment of the acetylated alkene 11 with
mCPBA gave the previously described epoxide 1711
(92%) which, on saponi®cation, a€orded the depro-
tected derivative 18 (82%).
The introduction of fused oxolane rings, by remote
functionalization of either of the b-methyl groups at C-4
or C-10 in similar diterpenoid systems, has been descri-
bed previously.11,14 Treatment of 6a-hydroxytotaryl
acetate, obtained by the hydrogenation of epoxide 17,
with lead tetraacetate and iodine in benzene gave the
6a,18-epoxy derivative (96%)10 from which compound
19 was derived (96%) by reductive deacetylation. The
intermediate acetate appears to have been incorrectly
assigned as the 6b,19-epoxy derivative on the basis of
1H NMR data.11 The absence, in the 13C NMR spec-
trum, of the characteristic resonance of C-18 (dC 33.0)
and the presence of that of C-19 [dC 19.1 (q)] indicates
the 6a,18-epoxy derivative had been formed.
Compound 10 [dH 4.97 (1H, d, J=3.7 Hz, H-6), 3.72
(3H, s); dC 154.1 (s, C-7), 98.8 (d, C-6), 54.4 (q, CH3)]
was obtained (72%) by lithium aluminum hydride
reduction of its O-acetate which, in turn, was produced
(76%) from the 7-one by treatment with trimethyl
orthoformate in the presence of Amberlyst 15 (H+)
resin. Steric impedence apparently inhibited the pro-
duction of the expected dimethyl acetal.
The Á6,7 derivatives, compounds 11 and 12, were syn-
thesized as described above.
7-Oxototarol (13), obtained by saponi®cation of its
acetate (83%), was shown by X-ray crystallographic
analysis (Fig. 1) to have its B-ring in a half-boat con-
formation as had been proposed earlier by Cut®eld et
al.13
Similar oxidative treatment of the 13-methyl ether of the
6b-alcohol 9, produced by lithium aluminum hydride
reduction of the methyl ether of ketone 14, a€orded the
6b,20-epoxy derivative 20 in 90% yield. The structure of
1
20 was established by the presence in its H and 13C
The isomeric 6-oxototarol (14) was synthesized by acid-
catalyzed elimination of acetic acid from the acetate of
2, epoxidation of the resulting alkene, and isomerization
of the product by treatment with p-TsOH in benzene
under re¯ux.14 16 The O-methyl-protected 6-oxo deri-
vative was then demethylated using boron tribromide in
NMR spectra of resonances characteristic of a cyclic
ether [dH 4.57 (1H, brs, H-6), 4.01, 3.73 (2H, 2d,
J=7.2 Hz, H-20ab); dC 78.4 (t, C-20), 77.6 (d, C-6)] and
by the absence of the signal due to the 10b-methyl group
and the presence of those characteristic of the C-18 and
C-19 methyl groups.

1666
G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
Attempts to deprotect compound 20 with BBr3 and
leave the ether ring intact were unsuccessful, and two
new compounds, diol 21 (39%) and triol 22 (44%), were
isolated. The structure of 13,20-dihydroxytotara-
6,8,11,13-tetraene (21) was con®rmed by the presence in
pneumoniae, up to the highest concentration tested (32
mg mL 1). The key conclusions are:
1. a trend was observed between antibacterial activity
and lipophilicity since the 7a-ethoxy derivative 4
exhibited antibacterial activity comparable with
that of 1, as did 6, whereas the diol analogues 3
and 5 showed only weak inhibitory activity, and
the least active compound was the trihydroxy
derivative 7;
1
its H and 13C NMR spectra of signals characteristic of
Á
6,7-alkene group [dH 6.89 (1H, dd, J=10.1, 3.1 Hz, H-
7), 5.96 (1H, dd, J=10.1, 3.1 Hz, H-6); dC 130.1 (d, C-
6), 124.3 (d, C-7)] and a hydroxylmethyl group at C-10
[dH 3.62, 3.38 (2H, 2t, J=6.4 Hz, H-20ab); dC 59.8 (t)].
The structure of 6b,13,20-trihydroxytotara-8,11,13-tri-
2. the introduction, into ring-B, of a double bond,
i.e., two new sp2 centers as in 10 and 12 had little
or no e€ect on antibacterial activity relative to
totarol (1) whereas, as in the case of ferruginol and
sugiol, the 7-oxo derivative of totarol, compound
13, was inactive despite the fact that the other 7-
oxo derivatives 15 and 16 and the 6-oxo derivative
14 exhibited persisting degrees of activity;
3. the addition of a fused and bridging heterocyclic
ring system into totarol (1) was of no assistance;
however, compounds 21 and 22 were both active,
the more lipophilic, 21, being slightly the more
potent;
ene (22) was con®rmed by the presence in its 1H and 13
C
NMR spectra of signals characteristic of a 6b-hydroxyl
group [dH 5.29 (1H, d, J=2.1 Hz, H-6a); dC 66.9 (t)]
and a hydroxylmethyl group at C-10 [dH 4.27 (1H, d,
J=8.2 Hz, H-20a), 2.80 (1H, dd, J=8.2, 1.6 Hz, H-20b);
dC 67.6 (t)]. The large di€erence in the NMR shifts of
the C-20 protons is likely to be due to restricted rotation
about the C-10,C-20 bond caused by intramolecular
hydrogen bonding between the cis-related hydroxyl
groups at C-6 and C-20, the former being axial and the
latter being part of the axial hydroxylmethyl substituent
at C-10.
4. again,3 O-methylation or acetylation of the phe-
nolic hydroxyl group (compounds 2, 11, 17 and
20) virtually eliminated activity;
In vitro antibacterial screening
Listed in Table 1 are the minimum inhibitory con-
centrations of compounds 2±22 together with those of
the reference compound totarol (1) when screened
against the Gram-positive b-lactamase-positive and
high level gentamycin-resistant Enterococcus faecalis,
penicillin-resistant Streptococcus pneumoniae, methi-
cillin-resistant Staphylococcus aureus (MRSA), and the
5. also in keeping with earlier ®ndings,3 activity
against Streptococcus pneumoniae was more easily
retained than against the other two Gram-positive
strains.
The interaction of totarol (1) and 7-oxototarol (13) with
mitochondria
Gram-negative multi-resistant Klebsiella pneumoniae at
1
the concentrations 2, 8 and 32 mg mL
.
Some studies on the mechanism of the antibacterial
activity of lipophilic phenolic compounds such as
totarol have implicated cell wall biosynthesis as the
likely target. There is another possible mechanism,
As with totarol itself, none of the derivatives displayed
activity against the Gram-negative organism Klebsiella
Table 1. In vitro antibacterial activity MICa, mg/mL (values in brackets are mM)
Compound
Enterococcus faecalis
Streptococcus pneumoniae
Staphylococcus aureus
Klebsiella pneumoniae
1
2
3
4
5
6
7
8
2 (7)
>32
2 (7)
>32
8 (26)
2 (6)
2 (7)
2 (6)
32 (100)
2 (7)
8 (27)
2 (6)
>32
2 (7)
>32
8 (27)
2 (5)
8 (27)
32 (93)
32 (11)
8 (27)
>32
2 (7)
>32
32 (106)
2 (6)
>32
8 (25)
32 (100)
>32
8 (27)
8 (25)
>32
2 (7)
>32
8 (27)
8 (21)
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
32 (106)
8 (24)
32 (106)
8 (25)
>32
32 (106)
32 (106)
8 (25)
>32
8 (28)
>32
8 (27)
8 (21)
>32
32 (93)
>32
>32
>32
32 (107)
>32
9
10
11
12
13
14
15
16
17
18
19
20
21
22
>32
32 (11)
>32
>32
32 (107)
8 (25)
8 (27)
8 (25)
aMinimum inhibitory concentrations.

G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
1667
however, that has yet to be examined: totarol and its
analogues could be acting by disrupting bacterial energy
metabolism. This could be by three possible means:
either by acting as speci®c proton ionophores, thus
uncoupling oxidative phosphorylation, by non-speci®-
cally disrupting bacterial membrane structure or by
speci®cally inhibiting an enzyme in energy metabolism,
in much the same way as cyanide does.
Oxidative phosphorylation, which is the major process
by which cells synthesize ATP, requires proton translo-
cation across the lipid bilayer membrane which sur-
rounds the cell. In this way cells maintain a proton
electrochemical gradient across their membrane which is
then used as an energy source to drive ATP synthesis. A
large number of lipophilic and weakly basic phenolate
compounds, called uncouplers, prevent the formation of
this proton electrochemical potential gradient by cross-
ing the lipid bilayer, bringing protons from regions of
high potential to regions of low potential. As a result,
the cell speeds up respiration in an attempt to maintain
the proton electrochemical potential across its mem-
brane. Uncoupling prevents the cells from synthesizing
ATP and thus leads to cells dying due to energy deple-
tion. General membrane disruption also has a similar
result. This can occur when large amounts of hydro-
phobic/amphipathic compounds partition into the
hydrophobic lipid bilayer and thus disrupt its structure,
making it ``leaky'' to protons and thus impossible for
the cell to maintain a proton electrochemical potential.
In addition, this disruption of membrane structure may
also directly inhibit the membrane-embedded enzymes
necessary for energy metabolism. Neither of these
mechanisms is particularly appealing for an antibiotic if
it is to have therapeutic applications as the energy fac-
tories in mammalian cells are mitochondria, small
intracellular vesicles surrounded by a lipid bilayer. Any
compound that uncoupled or disrupted this bilayer
would be potentially toxic. For example, the potent
uncoupler 2,4-dinitrophenol was once used as a slim-
ming agent but was later withdrawn because of its
toxicity.
Figure 2.
respiration rate, but at 50 mM both accelerated respira-
tion about 2-fold, and at higher concentrations both
shut down respiration. These results are consistent with
both diterpenoids being able to increase the proton
permeability of the membrane, either by acting as clas-
sical uncouplers or by disrupting the lipid bilayer struc-
ture. This increase in respiration occurs as the
respiratory chain speeds up in an e€ort to compensate
for the increased proton leakage through the mem-
brane. However, this increase in proton leakage only
occurred at concentrations 10- to 100-fold higher than
required for complete antibacterial activity and is
therefore unlikely to contribute to the antibacterial
e€ect.
A second experiment (data not shown) con®rmed that
the initial stimulation of respiration caused by these
compounds was due to increasing the proton leakage
through the mitochondrial membrane, and not to a
speci®c interaction with the respiratory chain. To do
this, combinations of glutamate and malate, or ascor-
bate and TMPD, were used as respiratory substrates.
These substrates were chosen because they utilize dif-
ferent sections of the respiratory chain from succinate.
The results were very similar to those found with succi-
nate as substrate, with stimulation of respiration occur-
ring in the presence of 50±100 mM of the diterpenoid
compounds. This again is consistent with both com-
pounds stimulating respiration non-speci®cally by
increasing the proton permeability of the membrane.
For these reasons, we chose to speci®cally examine these
possible mechanisms of action, using isolated rat liver
mitochondria. Mitochondrial respiration is a good
model for that in Gram-positive bacteria, and at the
same time results might provide an indication of likely
mammalian sensitivity to the new class of antibiotics
under study. As test compounds we chose totarol (1),
which is antibacterial at 2 mg mL 1, and 7-oxototarol
(13), which is inactive against the three strains of Gram-
positive bacteria at 32 mg mL 1. We considered that
these compounds are likely to be not too dissimilar with
regard to lipophilicity and basicity and, therefore,
would disrupt biological membranes and act as classical
uncouplers to a similar extent.
A third set of experiments examined respiration in the
presence of a known potent uncoupler, FCCP, together
with each of the various respiratory substrates men-
tioned above, separately. The concentration of FCCP
(333 nM) was selected to be sucient to completely
uncouple the mitochondria. This was done so that
any stimulation of respiration by uncoupling by the
The ®rst experiment looked at the ability of the two
diterpenoid compounds to interfere with coupled mito-
chondrial respiration. Using succinate as the respiratory
substrate, the results shown in Figure 2 were obtained.
At 10 mM, neither compound 1 nor 13 had an e€ect on

1668
G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
diterpenoid compounds would be eliminated and only
their inhibitory e€ects would remain. Under these con-
ditions, one would expect that, if the diterpenoid com-
pounds were acting as membrane disrupters (or indeed
as speci®c inhibitors of any other part of the respiratory
chain), a substantially enhanced inhibition of respira-
tion would be seen. Results using succinate as a sub-
strate are shown in Figure 3. Indeed, both totarol (1)
and 7-oxototarol (13) were able to progressively inhibit
respiration at 50 mM and above, but not at 10 mM or
below. Similar results were obtained with glutamate/
malate as respiratory substrates, but with ascorbate/
TMPD the inhibition of respiration brought about with
either of the diterpenoids was less (data not shown).
This inhibition is probably due to non-speci®c disrup-
tion of the lipid bilayer interfering with the activity of
the oxidizing enzymes in the mitochondrial membrane.
respiration towards totarol (1) at concentrations above
its in vitro antibacterial MIC removes one possible
impediment to the use of this class of compounds as
therapeutics.
X-ray single crystal analysis
The ORTEP diagram of 7-oxototarol (13) is shown in
Figure 1. The independent molecules are hydrogen
bonded through the hydroxyl hydrogen atom on O-13
and the carbonyl oxygen atom (O-7) of an adjacent
molecule forming chains along the z axis: O-7Á Á ÁH(O-
13)=1.96 (4) A; O-7Á Á ÁO-13=2.731(4) A; O-13Á Á ÁH(O-
13)Á Á ÁO-7=171(4)ꢀ (O-7 translated by: 1/2 x, 1 y,
z 1/2). Ring A adopts a slightly distorted chair con-
formation with best mean plane through C-1, C-10, C-3,
and C-4 (Æ 0.008 A) with C-2, C-5, respectively,
0.688(6) and +0.577(5) A out of plane; the slight dis-
tortion is shown in the Q(2),Å17 values of 0.069(4) A,
7.4(4)ꢀ compared with a pure chair 0 A, 0ꢀ. Ring-B
adopts a twist boat [Å,>73.4(4), 333.6(4)ꢀ compared
with theoretical 90, 330ꢀ];17 the twist results in the car-
bonyl atoms C-7, O-7 being 0.316(5), 1.043(6) A
respectively from the plane of ring-C, in the direction of
the W-methyl group C-20. Molecular modeling studies
(vapor phase)18 indicated that the alternative con-
formation in which the carbonyl group is twisted
towards the a-face of the molecule is less stable by 0.7
kcal mol 1. This suggests that the twist direction is a
phenomenon associated with the molecule itself and not
determined by intermolecular hydrogen bonding in the
crystal.
Finally, the e€ect of the diterpenoid compounds upon
phosphorylating respiration through a speci®c inhibi-
tion of ATP-synthase was probed, using each of the
various respiratory substrates mentioned above, sepa-
rately. No evidence for speci®c inhibition of this enzyme
was found.
In conclusion, it is apparent that both diterpenoids 1
and 13 interfere modestly with mitochondrial respira-
tion, by increasing the proton permeability of the mem-
brane, either by acting as classic uncouplers, by general
membrane disruption, or both. There are two strong
arguments against these mechanisms being the cause of
the antibacterial activity of totarol and its analogues,
however. Firstly, e€ects upon respiration only became
evident at concentrations 10- to 100-fold higher than
required for complete antibacterial activity. Secondly,
and most persuasively, 7-oxototarol (13) was slightly
more potent than totarol (1) in a€ecting respiration and
yet it is considerably less potent as an antibacterial
agent. The relative insensitivity of mitochondrial
The aromatic ring-C is essentially planar with average
deviations of 0.024(3) A, and O-13 and C-10 only
0.036(6) and 0.049(6) A from the mean plane respec-
tively. Atom C-15 is out of plane by 0.164(6) A anti to
O-7. The aromatic ring plane does not exactly bisect the
isopropyl carbon atoms (C-16, C-15, C-17) angle, C-16
and C-17 being +1.445(7), 1.064(7) A, respectively,
from the plane. The overall geometry of the molecule is
very similar to that of methyl 6a-bromo-13-isopropyl-7-
oxopodocarpa-8,11,13-trien-15-oate,13 with a slightly
¯atter ring-B (dihedral angle C-6±C-7±C-8±C-9=
33.7(5)ꢀ) being the only concession to the bulky bromine
atom.
Experimental
Syntheses
1
General methods. H and 13C NMR data were recorded
on a Bruker AC300 spectrometer and assignments were
made with the assistance of DEPT and COSY experi-
ments, the infra-red spectra were measured on a Perkin
Elmer 1600 series FTIR spectrometer, elemental ana-
lyses were obtained on a Carlo Erba EA 1108 elemental
analyzer, low and high resolution (HRMS) mass spectra
were obtained on a VG-70-250S double focusing
magnetic sector mass spectrometer (VG Analytical)
equipped with a standard VG-70S EI/CI ion source.
Melting points were determined on a Reichert hot stage
Figure 3.

G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
1669
microscope and are uncorrected. All reactions were
monitored by thin layer chromatography which was
carried out on 60 PF254 silica gel-coated aluminum
sheets. All puri®cations were by column chromato-
graphy which was performed using Merck Kieselgel S
silica gel. Solvents were dried and puri®ed before use
according to standard procedures.19 ``Petrol'' refers to
the fraction of petroleum ether boiling between 60 and
80 ꢀC.
0.52 mmol) in THF (10 mL) under argon at room tem-
perature and stirring was continued for 30 min. The
reaction was quenched with water (0.1 mL), NaOH
(15%, 0.1 mL), and water (0.3 mL), ®ltered, and the ®l-
trate was concentrated in vacuo. Chromatography on
silica gel using petrol:ethyl acetate (4:1) as the eluent
gave diol 3 (142 mg, 90%); mp 216±218 ꢀC, lit.7 215±
217.5 ꢀC; 1H NMR (CDCl3) d 7.03, 6.64 (2H, 2d,
J=8.6 Hz, H-11, H-12), 5.03 (1H, br s, H-7b), 4.89 (1H,
s, OH), 3.58 (1H, sept, J=7.1 Hz, H-15), 1.44, 1.40
(2Â3H, 2d, J=7.1 Hz, H-16, H-17), 1.13 (3H, s, H-20),
1.00 (3H, s, H-18), 0.94 (3H, s, H-19); 13C NMR
(CDCl3) d 153.0, 142.9, 134.6, 133.1, 123.5, 117.2 (Ar),
65.7 (C-7), 44.2 (C-5), 41.5 (C-3), 39.2 (C-1), 38.4 (C-
10), 33.1 (C-18), 32.9 (C-4), 29.1 (C-6), 27.7 (C-15), 24.6
(C-20), 21.7 (C-19), 21.0, 20.8 (C-16, C-17), 19.5 (C-2).
13-Methoxytotara-8,11,13-trien-7ꢀ-ol (2). Lithium alu-
minum hydride (20 mg) was added to a stirred solution
of 7a-acetoxy-13-methoxytotara-8,11,13-triene3 (40 mg,
0.11 mmol) in THF (10 mL) under argon at room tem-
perature. The reaction mixture was stirred for 30 min,
quenched with water (0.04 mL), NaOH (15% aqueous,
0.04 mL), and water (0.12 mL), ®ltered and the ®ltrate
was concentrated in vacuo. Chromatography on silica
gel using petrol:ethyl acetate (19:1) as the eluent gave 2
Ethanolysis of 7ꢀ,13-diacetoxytotara-8,11,13-triene.
Dilute hydrochloric acid (1 mL, 1.2 M) was added
dropwise to a stirred solution of the diacetate (330 mg,
0.92 mmol) in ethanol (20 mL) and the reaction mixture
was heated under re¯ux for 1.5 h. The volatiles were
removed under reduced pressure and the residue was
resuspended in ethyl acetate (100 mL), washed with
brine (3Â20 mL), dried (MgSO4) and concentrated in
vacuo. Chromatography on silica gel using petrol:
CH2Cl2 (1:1) as the eluent gave the following in order of
elution: 13-acetoxy-7a-ethoxytotara-8,11,13-triene as an
(31 mg, 88%); mp 83 ꢀC; nmax 3448 (OH), 1640 (CˆC)
1
cm
;
1H NMR (CDCl3) d 7.12, 6.83 (2H, 2d,
J=8.8 Hz, H-11, H-12), 5.01 (1H, br s, H-7b), 3.78 (3H,
s, OCH3), 3.53 (1H, sept, J=6.9 Hz, H-15), 1.37, 1.33
(2Â3H, 2d, J=6.9 Hz, H-16, H-17), 1.13 (3H, s, H-20),
1.02 (3H, s, H-18), 0.93 (3H, s, H-19); 13C NMR
(CDCl3) d 157.3, 142.8, 135.8, 134.5, 128.7, 112.5 (Ar),
65.6 (C-7), 55.1 (OCH3), 44.1 (C-5), 41.5 (C-3), 39.1 (C-
1), 38.0 (C-10), 33.5 (C-18), 33.1 (C-4), 28.9 (C-6), 28.0
(C15), 24.8 (C-20), 21.7 (C-19), 21.2, 21.2 (C-16, C-17),
19.4 (C-2); m/z 316 (M+, 25%), 298 (100), 241 (82), 213
(61), 171 (45), 115 (13), 69 (14); HRMS calcd for
C21H32O2 316.2402, found 316.2399.
1
oil (133 mg, 39%); H NMR (CDCl3) d 7.17, 6.92 (2H,
2d J=8.8 Hz, H-11, H-12), 4.51 (1H, br s, H-7b), 3.79,
3.47 (2Â1H, 2dt, J=8.6, 7.0 Hz, OCH2), 3.20 (1H, sept,
J=7.0 Hz, H-15), 2.33 (3H, s, COCH3), 1.37, 1.28
(2Â3H, 2d, J=7.0 Hz, H-16, H-17), 1.27 (3H, t,
J=7.0 Hz, OCH2CH3), 1.19 (3H, s, H-20), 1.00 (3H, s,
H-18), 0.96 (3H, s, H-19); 13C NMR (CDCl3) d 169.5
(COCH3), 148.0, 147.6, 138.7, 133.6, 123.6, 123.4 (Ar),
73.4 (C-7), 63.3 (OCH2CH3), 43.6 (C-5), 41.0 (C-3), 38.7
(C-1), 38.7 (C-10), 32.9 (C-4), 32.8 (C-18), 27.5 (C-15),
24.2 (C-20), 22.8 (C-6), 21.7 (C-19), 21.6, 21.5 (C-16, C-
17), 21.1 (COCH3), 19.4 (C-2), 15.7 (CH2CH3); m/z 372
(M+, 3%), 326 (72), 284 (87), 269 (100), 227 (31), 199
(32), 185 (14), 173 (16), 157 (17), 69 (17), 55 (18), 43
(77); HRMS m/z calcd for C24H36O3 (M+) 372.2664,
found 372.2661.
Lead tetraacetate oxidation of 13-acetoxytotara-8,11,13-
triene. Lead tetraacetate (15 g, 34 mmol) was added to
a stirred solution of the acetate (7.5 g, 23 mmol) in acetic
acid (100 mL) and the solution was heated to 100 ꢀC
under argon. The mixture was stirred for 2 h at 100 ꢀC,
another portion of lead tetraacetate (7.5 g, 17 mmol)
was added and stirring was continued for a further 2 h
at this temperature. Water (1 L) was added and the
mixture was extracted with ethyl acetate (3Â150 mL)
and the combined organic extracts were washed with
water (until the washings were neutral), dried (MgSO4),
and concentrated in vacuo. Chromatography on silica
gel using petrol:ethyl acetate (50:1) as the eluent gave
7a,13-diacetoxy-totara-8,11,13-triene6 (3.9 g, 44%); mp
155 ꢀC; 1H NMR (CDCl3) d 7.13, 6.90 (2H, 2d,
J=8.8 Hz, H-11, H-12), 6.01 (1H, br s, H-7b), 2.86 (1H,
sept, J=7.0 Hz, H-15), 2.24, 1.97 (2Â3H, 2s, COCH3),
1.19, 1.15 (2Â3H, 2d, J=7.0 Hz, H-16, H-17), 1.09 (3H,
s, H-20), 0.83 (3H, s, H-18), 0.82 (3H, s, H-19); 13C
NMR (CDCl3) d 170.5, 169.9 (COCH3), 69.3 (C-7), 44.6
(C-5), 41.5 (C-3), 39.2 (C-1), 39.0 (C-10), 33.3 (C-18),
33.3 (C-4), 28.0 (C-15), 26.5 (C-6), 24.4 (C-20), 21.7,
21.6 (C-16, C-17), 21.3 (C-19), 20.9, 20.9 (COCH3), 19.7
(C-2). This was followed by an inseparable mixture of
compounds from which compound 7 was obtained (see
below).
This was followed by 13-acetoxytotara-6,8,11,13-tetra-
ene (11)7 as a solid (91 mg, 30%); mp 137±139 ꢀC, lit.7
138±140 ꢀC; 1H NMR (CDCl3) d 7.04, 6.83 (2H,
J=8.4 Hz, H-11, H-12), 6.88 (1H, dd, J=10.1, 3.0 Hz,
H-7), 6.07 (1H, dd, J=10.1, 3.0 Hz, H-6), 3.37 (1H,
sept, J=7.2 Hz, H-15), 2.32 (3H, s, COCH3), 1.32, 1.29
(2Â3H, 2d, J=7.2 Hz, H-16, H-17), 1.06 (3H, s, H-20),
1.06 (3H, s, H-19), 0.98 (3H, s, H-18); 13C NMR
(CDCl3) d 169.9 (COCH3), 147.3, 146.8, 134.5, 132.1
(Ar), 131.0 (C-6), 124.0 (C-7), 121.9, 120.7 (Ar), 49.8 (C-
5), 41.0 (C-3), 38.6 (C-10), 36.6 (C-1), 32.8 (C-4), 32.6
(C-18), 27.4 (C-15), 22.5 (C-20), 21.8 (C-19), 21.6, 21.4
(C-16, C-17), 20.1 (COCH3), 19.1 (C-2).
This was followed by totara-6,8,11,13-tetraen-13-ol
1
Totara-8,11,13-triene 7ꢀ,13-diol (3).7 Lithium aluminum
hydride (100 mg) was added cautiously to a stirred
solution of 7a,13-diacetoxytotara-8,11,13-triene (200mg,
(12)7 as an oil (67 mg, 26%); H NMR (CDCl3) d 6.89
(1H, dd, J=10.1, 3.0 Hz, H-7), 6.90, 6.56 (2H, 2d,
J=8.3 Hz, H-11, H-12), 6.06 (1H, dd, J=10.1, 3.0 Hz,

1670
G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
H-6), 3.49 (1H, sept, J=7.1 Hz, H-15), 1.42, 1.39
(2Â3H, 2d, J=7.1 Hz, H-16, H-17), 1.06 (3H, s, H-20),
1.04 (3H, s, H-19), 0.99 (3H, s, H-18); 13C NMR
(CDCl3) d 152.3, 142.1, 131.8 (Ar), 130.9 (C-6), 129.6
(Ar), 124.3 (C-7), 120.5, 115.0 (Ar), 50.2 (C-5), 41.1 (C-
3), 38.4 (C-10), 36.8 (C-1), 32.7 (C-4), 32.6 (C-18), 27.4
(C-15), 22.4 (C-20), 21.4 (C-19), 21.1, 20.4 (C-16, C-17),
19.2 (C-2).
reaction mixture was stirred for 2 h, quenched with
water (0.05 mL), NaOH (15%, 0.05 mL), and water
(0.15 mL), ®ltered and the ®ltrate was concentrated in
vacuo. Chromatography on silica gel using petrol:ethyl
acetate (10:1) as the eluent gave 5 as an oil (74 mg,
82%); 1H NMR (CDCl3) d 6.98, 6.62 (2H, 2d
J=8.6 Hz, H-11, H-12), 5.37 (1H, s, OH), 5.15 (1H, t,
J=8.4 Hz, H-7a), 3.55 (1H, sept, J=7.1 Hz, H-15),
1.49, 1.37 (2Â3H, 2d, J=7.1 Hz, H-16, H-17), 1.29 (3H,
s, H-20), 0.96 (2Â3H, s, H-18, H-19); 13C NMR
(CDCl3) d 153.4, 144.2, 136.2, 133.4, 122.7, 116.5 (Ar),
68.4 (C-7), 48.8 (C-5), 41.5 (C-3), 39.9 (C-1), 37.9 (C-
10), 33.1 (C-4), 32.9 (C-18), 30.9 (C-6), 28.7 (C-15), 24.8
(C-20), 21.5 (C-19), 21.0, 20.9 (C-16, C-17), 19.3 (C-2).
7ꢀ-Ethoxytotara-8,11,13-trien-13-ol (4). Lithium alumi-
num hydride (60 mg) was added cautiously to a stirred
solution of 13-acetoxy-7a-ethoxytotara-8,11,13-triene
(120 mg, 0.32 mmol) in THF (10 mL) under argon at
room temperature. The reaction mixture was stirred for
30 min, quenched with water (0.06 mL), NaOH (15%,
0.06 mL), and water (0.18 mL), ®ltered and the ®ltrate
was concentrated in vacuo. Chromatography on silica
gel using petrol:ethyl acetate (19:1) as the eluent gave as
an oil 4 (92 mg, 87%); nmax 3369 (OH), 1584 (CˆC)
cm 1; 1H NMR (CDCl3) d 6.99, 6.56 (2H, 2d J=8.6 Hz,
H-11, H-12), 4.92 (1H, s, OH), 4.51 (1H, t, J=2.5 Hz,
H-7b), 3.80, 3.48 (2H, 2dq J=8.6, 7.0 Hz, OCH2CH3),
3.18 (1H, sept, J=7.0 Hz, H-15), 1.44, 1.40 (2Â3H, 2d,
J=7.0 Hz, H-16, H-17), 1.27 (3H, t, J=7.0 Hz,
OCH2CH3), 1.15 (3H, s, H-20), 0.99 (3H, s, H-18), 0.95
(3H, s, H-19); 13C NMR (CDCl3) d 152.7, 143.0, 133.3,
133.2, 123.3, 117.1 (Ar), 73.7 (C-7), 63.4 (OCH2CH3),
43.8 (C-5), 41.1 (C-3), 38.9 (C1), 38.3 (C-10), 32.9 (C-4),
32.9 (C-18), 27.8 (C-15), 24.5 (C-20), 22.9 (C-6), 21.7 (C-
19), 20.8, 20.5 (C-16, C-17), 19.5 (C-2), 15.8 (CH2CH3);
m/z 330 (M+, 11%), 284 (68), 269 (100), 227 (46), 199
(52), 157 (37), 43 (32); HRMS m/z calcd for C22H34O2
330.2559 (M+), found 330.2554.
13-Acetoxy-7ꢀ-methyltotara-8,11,13-trien-7ꢁ-ol. The
13-acetoxy-7-one (see above) (300 mg, 0.88 mmol) was
dissolved in THF (10 mL) and cooled to 78 ꢀC with
stirring. MeLi (1.5 mL, 1.0 M in diethyl ether) was
added dropwise to the resulting soultion and after
10 min the reaction mixture was allowed to warm to
ambient temperature and then quenched with aqueous
NH4Cl (10%, 100 mL). The resulting aqueous layer
was extracted with ethyl acetate (3Â50 mL) and the
combined organic phases were washed with brine
(3Â20 mL), dried (MgSO4), and concentrated in vacuo.
Chromatography on silica gel using petrol:ethyl acetate
(19:1) as the eluent gave the title tertiary alcohol
(210 mg, 69%); mp 122 ꢀC; nmax 3486 (OH), 1754 (CO),
1
1194 cm
;
1H NMR (CDCl3) d 7.12, 6.87 (2H, dd,
J=8.8 Hz, H-11, H-12), 4.30 (1H, sept, J=6.9 Hz, H-
15), 2.31 (3H, s, COCH3), 1.66 (3H, s, CH3), 1.29, 1.28
(2Â3H, 2d, J=7.0 Hz, H-16, H-17), 1.25 (3H, s, H-20),
0.98 (3H, s, H-18), 0.92 (3H, s, H-19); 13C NMR
(CDCl3) d 169.7 (COCH3), 148.9, 147.2, 140.3, 139.0,
123.6, 123.1 (Ar), 74.6 (C-7), 48.0 (C-5), 41.1 (C-3), 40.6
(C-1), 39.6 (C-6), 39.6 (C-10), 32.8 (C-4), 32.6 (C-18),
29.7 (CH3), 27.7 (C-15), 25.2 (C-20), 21.6 (COCH3),
21.6 (C-19), 21.3, 20.8 (C-16, C-17), 19.4 (C-2); m/z 358
(M+, 3%), 340 (69), 283 (100), 267 (15), 213 (42), 187
(32), 171 (19), 91 (10), 69 (23); HRMS m/z calcd for
C23H34O3 (M+) 358.2508, found 358.2506.
13-Acetoxytotara-8,11,13-trien-7-one.8 N-Bromosuccini-
mide (16.3 g, 75 mmol) was added to a stirred solution
of totaryl acetate (10 g, 30 mmol) and CaCO3 (9 g,
60 mmol) in THF (200 mL) and water (5 mL) at room
temperature, and the resulting reaction mixture was
stirred for a further 1 h while being irradiated with vis-
ible light.20 The mixture was ®ltered, the ®ltrate was
concentrated in vacuo, water (500 mL) was added and
the resulting aqueous suspension extracted into ethyl
acetate (3Â100 mL). The combined organic layer was
washed with aqueous Na2S2O3 (3Â50 mL), brine
(3Â50 mL), dried (MgSO4) and concentrated in vacuo.
Chromatography on silica gel using petrol:ethyl acetate
(19:1) as theꢀeluent gave the title ketone (7.4 g, 71%);
7ꢀ-Methyltotara-8,11,13-triene-7ꢁ,13-diol (6). Lithium
aluminum hydride (100 mg) was added portionwise to a
stirred solution of 13-acetoxy-7a-methyltotara-8,11,13-
trien-7b-ol (100 mg, 0.3 mmol) in THF (10 mL) under
argon at room temperature. The mixture was stirred for
2 h, quenched with water (0.1 mL), NaOH (15%,
0.1 mL), and water (0.3 mL), ®ltered, and the ®ltrate
was concentrated in vacuo. Chromatography on silica
gel using petrol:ethyl acetate (9:1) as the eluent gave diol
6 (85 mg, 96%); mp 160 ꢀC; nmax 3461, 3224 (OH), 1583
ꢀ
1
mp 170±171 C, lit.8 169±170 C; H NMR (CDCl3) d
7.21, 7.09 (2H, 2d, J=8.6 Hz, H-11, H-12), 3.73 (1H,
sept, J=7.0 Hz, H-15), 2.35 (3H, s, COCH3), 1.38, 1.23
(2Â3H, d, J=7.0 Hz, H-16, H-17), 1.14 (3H, s, H-20),
1.04 (3H, s, H-18), 0.94 (3H, s, H-19); 13C NMR
(CDCl3) d 202.1 (C-7), 169.5 (COCH3), 153.5, 148.2,
139.9, 133.9, 127.1, 121.3 (Ar), 47.5 (C-5), 41.4 (C-3),
38.7 (C-1), 38.4 (C-6), 38.2 (C-10), 33.2 (C-4), 32.1 (C-
18), 27.9 (C-15), 22.8 (C-20), 21.6, 21.4, 21.3, 21.3 (C-19,
C-16, C-17, COCH3), 18.8 (C-2).
1
(CˆC), 1190 cm 1; H NMR (CDCl3) d 6.99, 6.62 (2H,
2d J=8.6 Hz, H-11, H-12), 5.40 (1H, s, OH), 4.26 (1H,
sept, J=7.1 Hz, H-15), 1.69 (3H, s, CH3), 1.42 (2Â3H,
2d, J=7.1 Hz, H-16, H-17), 1.24 (3H, s, H-20), 0.99
(3H, s, H-18), 0.93 (3H, s, H-19); 13C NMR (CDCl3) d
153.9, 142.2, 139.9, 133.1, 123.5, 116.4 (Ar), 74.9 (C-7),
48.1 (C-5), 41.1 (C-3), 40.5 (C-1), 39.7 (C-6), 39.1 (C-
10), 32.6 (C-4), 32.6 (C-18), 29.5 (CH3), 28.0 (C-15),
25.3 (C-20), 21.5 (C-19), 20.5, 20.1 (C-16, C-17), 19.3
(C-2); m/z 316 (M+, 10%), 298 (85), 283 (100), 213 (44),
Totara-8,11,13-triene-7ꢁ,13-diol (5).13 Lithium aluminum
hydride (50 mg) was added portionwise to a stirred solution
of the 13-O-acetyl-7-one (see above, 100 mg, 0.3 mmol)
in THF (10 mL) under argon at room temperature. The

G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
1671
171 (16), 98 (11), 83 (85); HRMS calcd for C21H32O2
316.2402, found 316.2402.
(Ar), 69.1 (C-6), 57.3 (C-5), 43.0 (C-3), 40.0 (C-1), 38.7
(C-10), 37.5 (C-7), 35.2 (C-18), 34.0 (C-4), 27.4 (C-15),
23.6 (C-20), 21.3, 22.4 (C-19), 20.9, 20.8 (C-16, C-17),
19.2 (C-2); m/z 302 (M+, 69%), 287 (100), 269 (40), 227
(43), 199 (32), 69 (15); HRMS m/z calcd for C20H30O2
(M+) 302.2246, found 302.2238.
Totata-8,11,13-trien-6ꢀ,7ꢀ,13-triol (7). Lithium alumi-
num hydride (150 mg) was added cautiously to a stirred
solution of the mixed fraction derived by lead tetra-
acetate treatment of totaryl acetate (see above) (400 mg)
in THF (20 mL) under argon at room temperature. The
reaction mixture was stirred for 30 min, quenched with
water (0.15 mL), NaOH (15%, 0.15 mL), and water
(0.45 mL), ®ltered and the ®ltrate was concentrated in
vacuo. Chromatography on silica gel using petrol:ethyl
acetate (9:1) as the eluent gave the title triol 7 as an oil
Totara-8,11,13-triene-6ꢁ,13-diol (9). Lithium aluminum
hydride (50 mg) was added portionwise to a stirred
solution of 6-oxototarol (14) (see below) (100 mg,
0.30 mmol) in THF (10 mL) under argon at room tem-
perature. The reaction mixture was stirred for 2 h,
quenched with water (0.05 mL), NaOH (15%, 0.05 mL),
and water (0.15 mL), ®ltered and the ®ltrate was con-
centrated in vacuo. Chromatography on silica gel using
1
(131 mg); nmax 3412 (OH), 1630 (CˆC) cm 1; H NMR
(CDCl3) d 6.94, 6.62 (2H, 2d J=8.6 Hz, H-11, H-12),
5.57 (1H, s, OH), 4.93 (1H, br s, H-7b), 4.31 (1H, d,
J=11.6 Hz, H-6b), 3.47 (1H, sept, J=7.0 Hz, H-15),
1.46, 1.35 (2Â3H, 2d, J=7.0 Hz, H-16, H-17), 1.30 (3H,
s, H-20), 1.21 (3H, s, H-18), 1.16 (3H, s, H19). dC
(CDCl3) 153.7, 143.2, 134.6, 133.4, 122.3, 116.9 (Ar),
78.2, 77.2 (C6, C7), 53.3 (C5), 43.6 (C-3), 40.3 (C-1),
39.4 (C-10), 36.1 (C-18), 33.6 (C-4), 28.9 (C-15), 26.2 (C-
20), 22.0 (C-19), 21.0, 21.0 (C-16, C-17), 19.2 (C-2); m/z
318 (M+, 23%), 300 (76), 267 (30), 243 (52), 215 (70),
202 (28), 187 (22), 173 (25), 69 (42), 55 (22), 41 (28);
HRMS m/z calcd for C20H30O3 (M+) 318.2195, found
318.2190.
petrol:ethyl acetate (9:1) as the eluent gave the diol 9 as
1
an oil (79 mg, 90%); nmax 3401 (OH), 1502 (CˆC) cm
;
1H NMR (CDCl3) d 7.06, 6.58 (2H, 2d J=8.6 Hz, H-11,
H-12), 4.88 (1H, s, OH), 4.75 (1H, d, J=4.8 Hz, H-6a),
3.27 (1H, sept, J=7.1 Hz, H-15), 3.08, 3.01 (2H, dd,d,
J=17.0, 4.8; 17.7 Hz H-7a 7b), 1.60 (3H, s, H-20), 1.38
(2Â3H, 2d, J=7.1 Hz, H-16, H-17), 1.31 (3H, s, H-19),
1.08 (3H, s, H-18); 13C NMR (CDCl3) d 152.7, 142.5,
132.8, 131.6, 121.3, 114.2 (Ar), 69.1 (C-6), 57.3 (C-5),
43.0 (C-3), 40.0 (C-1), 38.7 (C-10), 37.5 (C-7), 35.2 (C-
18), 34.0 (C-4), 27.4 (C-15), 23.6 (C-20), 21.3, 22.4 (C-
19), 20.9, 20.8 (C-16, C-17), 19.2 (C-2); m/z 302 (M+,
44%), 269 (100), 227 (21), 199 (17), 149 (20), 69 (18);
HRMS m/z calcd for C20H30O2 (M+) 302.2246, found
302.2248.
13-Acetoxytotara-8,11,13-trien-6ꢀ-ol.11 A mixture of
the epoxyacetate 1711 (1.3 g, 3.8 mmol), Pd/C (250 mg)
and isopropanol (20 mL) was stirred at room tempera-
ture under an atmosphere of hydrogen for 14 h. The Pd/
C was removed by ®ltration through Celite and the
volatiles were removed in vacuo. Chromatography on
silica gel using petrol:ethyl acetate (19:1) as the eluent
gave the title alcohol (630 mg, 49%); mp 136±138 ꢀC,
13-Acetoxy-7-methoxytotara-6,8,11,13-tetraene. Trimethyl
orthoformate (1 mL) was added dropwise to a stirred
suspension of 13-acetoxytotara-8,11,13-triene-7-one (see
above, 350 mg, 1 mmol) and Amberlyst1 15 (400 mg) in
methanol (10 mL) and the resulting solution warmed to
50 ꢀC. After 1 h the reaction mixture was cooled, ®ltered
through Celite and the ®ltrate was concentrated in
vacuo. Chromatography on silica gel using petrol:ethyl
acetate (99:1) as the eluent gave the title compound
(270 mg, 76%); mp 103 ꢀC; nmax 1758 (CO), 1676
lit.11 136±139 ꢀC; H NMR (CDCl3) d 7.08, 6.80 (2H,
1
2d, J=8.6 Hz, H-11, H-12), 4.33 (1H, m, H-6), 3.30
(1H, sept, J=7.2 Hz, H-15), 3.11 (2H, m, H-7a,b), 2.30
(3H, s, COCH3),1.28, 1.28 (2Â3H, 2t, J=7.2 Hz, H-16,
H-17), 1.17 (3H, s, H-20), 1.11 (2Â3H, 2s, H-18, H-19);
13C NMR (CDCl3) d 169.8 (COCH3), 147.6, 147.1,
136.5, 133.1, 121.3, 120.8 (Ar), 68.6 (C-6), 57.0 (C-5),
42.7 (C-3), 39.6 (C-1), 38.8 (C-10), 37.0 (C-7), 34.8 (C-
18), 33.8 (C-4), 27.2 (C-15), 23.0 (C-20), 22.2 (C-19),
21.3, 21.1 (C-16, C-17), 21.3 (COCH3), 18.9 (C-2).
1
(CˆC), 1206 cm 1; H NMR (CDCl3) d 7.02, 6.90 (2H,
2d J=8.5 Hz, H-11, H-12), 4.97 (1H, d, J=3.7 Hz, H-
6), 3.72 (3H, s, OCH3), 3.66 (1H, sept, J=7.1 Hz, H-15),
2.34 (3H, s, COCH3), 1.35, 1.21 (2Â3H, 2d, J=7.1 Hz,
H-16, H-17), 1.08 (2Â3H, 2s, H-20, H-18), 0.99 (3H, s,
H-19); 13C NMR (CDCl3) d 169.9 (COCH3), 153.8 (C-
7), 149.1, 148.4, 136.3, 131.8, 123.1, 120.7 (Ar), 99.0 (C-
6), 54.5 (OCH3), 49.5 (C-5), 41.2 (C-3), 39.8 (C-10), 36.8
(C-1), 33.3 (C-4), 32.9 (C-18), 29.8 (C-15), 22.0 (C-20),
21.9 (C-19), 21.7, 21.6 (C-16, C-17), 20.0 (COCH3), 19.2
(C-2); m/z 356 (M+, 49%), 342 (50), 300 (100), 285 (37),
267 (58), 243 (18), 215 (33), 128 (10), 83 (12), 55 (28);
HRMS m/z calcd for C23H32O3 (M+) 356.2352, found
356.2364.
Totara-8,11,13-triene-6ꢀ,13-diol (8). Lithium aluminum
hydride (50 mg) was added portionwise to a stirred solu-
tion of 13-acetoxytotara-8,11,13-trien-6a-ol11 (100mg,
0.30mmol) in THF (10 mL) under argon at room tem-
perature. The reaction mixture was stirred for 2 h,
quenched with water (0.05 mL), NaOH (15%, 0.05 mL),
and water (0.15 mL), ®ltered and concentrated in vacuo.
Chromatography on silica gel using petrol:ethyl acetate
(10:1) as the eluent gave diol 8 (86 mg, 98%); mp 177 ꢀC;
n
max 3372 (OH), 1589 (CˆC) cm 1; 1H NMR (CDCl3) d
7-Methoxytotara-6,8,11,13-tetraen-13-ol (10). Lithium
aluminum hydride (100 mg) was added portionwise
to a stirred solution of 13-acetoxy-7-methoxytotara-
6,8,11,13-tetraene (300 mg, 0.8 mmol) in THF (10 mL)
under argon at room temperature. The reaction mixture
was stirred for 2 h, quenched with water (0.1 mL),
NaOH (15%, 0.1 mL), and water (0.3 mL), ®ltered and
6.92, 6.55 (2H, 2d J=8.4 Hz, H-11, H-12), 4.81 (1H, s,
OH), 4.35 (1H, m, H-6b), 3.39 (1H, sept, J=6.9 Hz, H-
15), 3.22, 3.00 (2H, 2d, J=16.6 Hz, 6.3 Hz; 16.6, 4.8 Hz,
H-7a,7b), 1.37 (2Â3H, 2d, J=6.9 Hz, H-16, H-17), 1.17
(3H, s, H-20), 1.13 (3H, s, H-18), 1.12 (3H, s, H-19); 13C
NMR (CDCl3) d 152.7, 142.5, 132.8, 131.6, 121.3, 114.2

1672
G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
the ®ltrate was concentrated in vacuo. Chromatography
on silica gel using petrol:ethyl acetate (9:1) as the eluent
gave the methoxytotarol 10 (190 mg, 72%); mp 238 ꢀC;
(100 mL), washed with aqueous solutions of KI
(10%, 2Â20 mL), Na2S2O3 (10%, 2Â20 mL), NaHCO3
(2Â20 mL), and brine (2Â20 mL), dried (MgSO4) and
concentrated in vacuo to give the 6,7-a-epoxide. p-
Toluenesulfonic acid (0.5 g) was added to a stirred
solution of the crude epoxide (4.0 g) in benzene (50 mL)
and the solution was heated under re¯ux for 1 h before
®ltration through solid NaHCO3 and concentrated in
vacuo. Chromatography on silica gel using petrol:ethyl
acetate (19:1) as the eluent gave the title 6-one (3.2 g,
84%); mp 96±98 ꢀC; nmax 1709 (CˆO), 1630 (CˆC);
1H NMR (CDCl3) d 7.60, 7.15 (2H, 2d, J=8.8 Hz, H-
11, H-12), 3.80 (3H, s, OCH3), 3.60, 3.50 (2H, 2d,
J=21.1z H, H-7a,b), 3.10 (1H, br s, H-15), 1.33, 1.33
(2Â3H, 2d, J=7.0 Hz, H-16, H-17), 1.30 (3H, s, H-20),
1.12 (3H, s, H-18), 1.07 (3H, s, H-19); 13C NMR
(CDCl3) d 209.7 (C-6), 141.5, 133.2, 130.8, 122.7, 122.0,
110.2 (Ar), 55.3 (OCH3), 61.9 (C-5), 44.0 (C-7), 42.8
(C-3), 40.5 (C-10), 39.5 (C-1), 32.9 (C-18), 32.5 (C-4),
28.2 (C-15), 25.3 (C-20), 21.8 (C-19), 20.5, 20.4 (C-16,
C-17), 19.0 (C-2); m/z 314 (M+, 100%), 299 (92), 281
(20), 257 (32), 229 (52), 217 (23), 69 (30), 55 (18), 41
(28); HRMS m/z calcd for C21H30O2 (M+) 314.2246,
found 314.2245.
nmax 3218 (OH), 1646 (CˆC), 1570, 1486, 1342,
1
1188 cm
;
1H NMR (CDCl3) d 6.86, 6.60 (2H, 2d,
J=8.4 Hz, H-11, H-12), 4.95 (1H, d, J=3.7 Hz, H-6),
4.80 (1H, s, OH), 3.72 (3H, s, OCH3), 3.72 (1H, sept,
J=7.0 Hz, H-15), 1.44, 1.32 (2Â3H, 2d, J=7.0 Hz, H-
16, H-17), 1.06 (3H, s, H-20), 1.05 (3H, s, H-18), 0.98
(3H, s, H-19); 13C NMR (CDCl3) d 154.1 (C-7), 153.4,
144.3, 131.4, 130.9, 120.5, 116.0 (Ar), 98.8 (C-6), 54.4
(OCH3), 49.8 (C-5), 41.2 (C-3), 39.4 (C-10), 36.9 (C-1),
33.1 (C-4), 32.8 (C-18), 29.8 (C-15), 21.9 (C-20), 21.3,
21.1 (C-16, C-17), 20.2 (C-19), 19.2 (C-2); m/z 314 (M+,
17%), 300 (100), 285 (41), 267 (53), 243 (18), 215 (52),
181 (19), 131 (23), 83 (16), 69 (45), 55 (32); HRMS m/z
calcd for C21H30O2 (M+) 314.2246, found 314.2249.
13-Hydroxytotara-8,11,13-trien-7-one (13).21 Sodium
hydride (10 mg) was added to a stirred solution of 13-
acetoxytotara-8,11,13-trien-7-one (see above, 72 mg,
0.2 mmol) in methanol (10 mL) at room temperature
under argon. The reaction mixture was stirred for 14 h
and concentrated in vacuo. Chromatography on silica
gel using petrol:ethyl acetate (19:1) as the eluent gave
ketone 13 (50 mg, 83%); mp 240 ꢀC; lit.21 240±241 ꢀC;
1H NMR (CDCl3) d 6.81, 6.64 (2H, 2d, J=8.5 Hz, H-
11, H-12), 3.40 (1H, sept, J=6.9 Hz, H-15), 2.46 (2H,
m, H-6a,b), 1.17, 1.08 (2Â3H, 2d, J=6.9 Hz, H-16, H-
17), 0.87 (3H, s, H-20), 0.83 (3H, s, H-18), 0.72 (3H, s,
H-19); 13C NMR (CDCl3) d 205.9 (C-7), 156.5, 149.2,
145.8, 145.0, 122.2, 121.2 (Ar), 49.9 (C-5), 42.8 (C-3),
40.0 (C-1), 39.8 (C-6), 39.0 (C-10), 34.2 (C-4), 32.8 (C-
18), 29.8 (C-15), 23.5 (C-20), 21.7 (C-9), 21.4, 20.9 (C-
16, C-17), 20.1 (C-2).
6-Oxototara-8,11,13-trien-13-ol (14). Boron tribromide
(1.0 M, 1 mL) was added dropwise to a stirred solution of
13-methoxytotara-8,11,13-trien-6-one (45 mg, 0.14mmol)
in CH2Cl2 (5 mL) at room temperature under argon.
The solution was stirred for 30 min, diluted with CH2Cl2
(50 mL), washed with aqueous solutions of Na2S2O3 (10%,
3Â25 mL), NaHCO3 (3Â25 mL), brine (3Â25 mL), dried
(MgSO4), and concentrated in vacuo. Chromatography
on silica gel using petrol:ethyl acetate (12:1) as the elu-
ent gave the hydroxy ketone 14 (39 mg, 91%); mp 70 ꢀC;
1
nmax 3454 (OH), 1696 (CˆO), 1632 (CˆC) cm 1; H
13-Methoxytotara-6,8,11,13-tetraene.6 Dilute hydro-
chloric acid (10%, 1 mL) was added dropwise to a stir-
red solution of 7a-acetoxy-13-methoxytotara-8,11,13-
triene6 (350 mg, 0.98 mmol) in methanol (20 mL) and
the reaction heated at re¯ux for 1.5 h. The volatiles were
removed under reduced pressure and the residue resus-
pended in ethyl acetate (100 mL), washed with brine
(3Â20 mL), dried (MgSO4) and concentrated in vacuo.
Chromatography on silica gel using petrol:ethyl acetate
(99:1) as the eluent gave the title tetraene (161 mg, 55%);
mp 84 ꢀC; 1H NMR (CDCl3) d 6.97, 6.71 (2H, 2d,
J=8.5 Hz, H-11, H-12), 6.89 (1H, dd, J=10.1, 3.1 Hz,
H-7), 6.03 (1H, dd, J=10.1, 3.1 Hz, H-6), 3.77, (3H, s,
OCH3), 3.49 (1H, sept, J=7.0 Hz, H-15), 1.33, 1.33
(2Â3H, d, J=7.0 Hz, H-16, H-17), 1.04 (3H, s, H-20),
1.01 (3H, s, H-18), 0.96 (3H, s, H-19); 13C NMR
(CDCl3) d 156.5, 141.7, 131.8, 131.3 (Ar), 130.5 (C-6),
124.4 (C-7), 120.0, 110.1 (Ar), 55.2 (OCH3), 50.0 (C-5),
41.0 (C-3), 38.2 (C-10), 36.6 (C-1), 32.5 (C-4), 32.5 (C-
18), 27.3 (C-15), 24.5 (C-20), 22.3 (C-19), 21.3, 21.0 (C-
16, C-17), 19.1 (C-2).
NMR (CDCl3) d 7.04, 6.63 (2H, 2d, J=8.5 Hz, H-11,
H-12), 5.10 (1H, s, OH), 3.67, 3.49 (2H, 2d, J=21.2 Hz,
H-7a,b), 3.11 (1H, br s, H-15), 1.35, 1.33 (2Â3H, 2d,
J=7.0 Hz, H-16, H-17), 1.33 (3H, s, H-20), 1.11 (3H, s,
H-18), 1.08 (3H, s, H-19); 13C NMR (CDCl3) d 210.1
(C-6), 153.0, 141.6, 131.1, 122.5, 115.2 (Ar), 62.0 (C-5),
43.9 (C-7), 42.7 (C-3), 40.5 (C-10), 39.3 (C-1), 32.8 (C-
18), 32.4 (C-4), 27.9 (C-15), 25.2 (C-20), 21.7 (C-19),
20.4, 20.2 (C-16, C-17), 18.9 (C-2); m/z 300 (M+, 78%),
285 (100), 267 (21), 243 (31), 215 (52), 173 (15), 69 (28);
HRMS m/z calcd for C20H28O2 (M+) 300.2089, found
300.2091.
13-Acetoxy-6ꢀ-bromototara-8,11,13-trien-7-one.7,22
A
solution of bromine (250 mg, 2 mmol) in glacial acetic
acid (5 mL) was added dropwise over a period of 10 min
to a stirred solution of 13-acetoxytotara-8,11,13-trien-7-
one (500 mg, 2 mmol) in glacial acetic acid (20 mL) con-
taining 1 drop of HBr. After 30 min the mixture was
concentrated in vacuo, and the resultant residue puri®ed
by column chromatography on silica gel using petrol:
ethyl acetate (12:1) as the eluent gave the title bromo-
ketone (454 mg, 74%); mp 174±175 ꢀC, lit. 176±177 ꢀC.
1H NMR (CDCl3) d 7.14, 7.13 (2H, 2d, J=8.7 Hz, H-
11, H-12), 4.56 (1H, d, J=7.5 Hz, H-6), 3.47 (1H, sept,
J=7.0 Hz, H-15), 2.36 (3H, s, COCH3), 2.16 (1H, d,
J=7.5 Hz, H-5), 1.40, 1.26 (2Â3H, 2d, J=7.0 Hz, H-16,
13-Methoxytotara-8,11,13-trien-6-one. m-Chloroperoxy-
benzoic acid (85%, 4.0 g) was added to a stirred solution
of 13-methoxytotara-6,8,11,13-tetraene (3.8 g, 12.7 mmol)
in CH2Cl2 (40 mL) at room temperature under argon.
The solution was stirred for 2 h, diluted with CH2Cl2

G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
1673
H-17), 1.22 (3H, s, H-20), 1.15 (3H, s, H-18), 1.09 (3H,
s, H-19); 13C NMR (CDCl3) d 193.6 (C-7), 169.2
(COCH3), 149.6, 148.2, 139.2, 133.1, 127.0, 120.1 (Ar),
56.6 (C-6), 51.5 (C-5), 41.7 (C-3) 38.6 (C-10), 37.6 (C-1),
34.9 (C-4), 34.2 (C-18), 28.1 (C-15), 23.8 (C-20), 21.8 (C-
19), 21.3, 21.2 (C-16, C-17), 18.3 (C-2).
36.9 (C-4), 33.5 (C-18), 32.1 (C-20), 29.5 (C-19), 27.1 (C-
15), 20.7, 21.1 (C-16, C-17), 18.8 (C-2).
13-Acetoxy-6ꢀ,7ꢀ-epoxytotara-8,11,13-triene (17).11 m-
Chloroperoxybenzoic acid (85%, 4.0 g) was added to a
stirred solution of 13-acetoxytotara-6,8,11,13-tetraene
(11) (2.6 g, 8 mmol) in CH2Cl2 (50 mL) at room tem-
perature under argon. The solution was stirred for 2 h,
diluted with CH2Cl2 (100 mL), washed with aqueous
solutions of KI (10% w/w, 2Â20 mL), Na2S2O3 (10%,
2Â20 mL), NaHCO3 (2Â20 mL), and brine (2Â20 mL),
dried (MgSO4) and concentrated in vacuo. Chromato-
graphy on silica gel using petrol:ethyl acetate (19:1) as
the eluent gave epoxide 17 (2.5 g, 92%); mp 137±139 ꢀC,
6ꢀ-Bromo-13-hydroxytotara-8,11,13-trien-7-one (15).7,22
Sodium hydride (catalytic) was added to a stirred solu-
tion of 13-acetoxy-6-a-bromototara-8,11,13-trien-7-one
(300 mg, 0.7 mmol) in methanol (10.0 mL) at room tem-
perature under argon. The mixture was stirred for 14 h
and concentrated in vacuo. Chromatography on silica
gel using petrol:ethyl acetate (9:1) as the eluent gave 15
as a solid (223 mg, 88%); mp 219 ꢀC, lit.7 218±220 ꢀC.
1H NMR (CDCl3) d 6.98, 6.82 (2H, J=8.4 Hz, H-11, H-
12), 4.86 (1H, br s, OH), 4.55 (1H, d, J=7.5 Hz, H-6),
3.50 (1H, sept, J=7.1 Hz, H-15), 2.15 (1H, d, J=7.5 Hz,
H-5), 1.49, 1.37, (2Â3H, 2d, J=7.1 Hz, H-16, H-17),
1.22 (3H, s, H-20), 1.14 (3H, s, H-18), 1.06 (3H, s, H-
19); 13C NMR (CDCl3) d 194.3 (C-7), 153.6, 144.9,
133.9, 131.3, 120.3, 120.0 (Ar), 57.2 (C-6), 52.2 (C-5),
41.9 (C-3), 38.5 (C-10), 37.9 (C-1), 35.2 (C-4), 34.3 (C-
18), 28.1 (C-15), 24.1 (C-20), 21.9, 21.9 (C-16, C-17),
20.8 (C-19), 18.3 (C-2).
lit.11 138±139 ꢀC; H NMR (CDCl3) d 6.97, 6.86 (2H,
1
J=8.6 Hz, H-11, H-12), 3.83 (1H, d, J=4.6 Hz, H-7b),
3.48 (2H, m, H-6, H-15), 2.25 (3H, s, COCH3), 1.29,
1.27 (2Â3H, 2d, J=7.0 Hz, H-16, H-17), 1.16 (3H, s, H-
20), 1.09 (3H, s, H-18), 1.03 (3H, s, H-19); 13C NMR
(CDCl3) d 169.6 (COCH3), 149.9, 147.2, 140.0, 131.0,
123.9, 121.6 (Ar), 54.1 (C-7), 52.9 (C-6), 47.3 (C-5), 40.8
(C-3), 38.8 (C-10), 37.0 (C-1), 33.2 (C-4), 32.5 (C-18),
28.7 (C-15), 25.4 (C-20), 22.1 (C-19), 21.4, 21.4 (C-16,
C-17), 21.3 (COCH3), 18.8 (C-2).
6ꢀ,7ꢀ-Epoxytotara-8,11,13-trien-13-ol (18). Sodium
hydride (catalytic) was added to a stirred solution of the
epoxyacetate 17 (100 mg, 0.3 mmol) in methanol
(10 mL) at room temperature under argon. The mixture
was stirred for 14 h and concentrated in vacuo. Chro-
matography on silica gel using petrol:ethyl acetate
(12:1) as the eluent gave the epoxyalcohol 18 (74 mg,
13-Acetoxytotara-5,8,11,13-tetraen-7-one.7,22 13-Acetoxy-
totara-6a-bromotetraen-8,11,13-trien-7-one (see above,
260 mg, 0.6 mmol), LiBr (520 mg), and NaHCO3
(120 mg) were suspended in DMF (5 mL) with stirring
and the mixture was heated at 100 ꢀC for 48 h. The
reaction mixture was diluted with brine (100 mL),
extracted with ethyl acetate (3Â20 mL) and the com-
bined organic layers were washed with brine (3Â20 mL),
dried (MgSO4), and concentrated in vacuo. Chromato-
graphy on silica gel using petrol:ethyl acetate (9:1) as
the eluent gave the conjugated enone (190 mg, 91%);
mp 162±163 ꢀC, lit.22 164±165 ꢀC; 1H NMR (CDCl3)
7.38, 7.15 (2H, J 8.8 Hz, H-11, H-12), 6.42 (1H, s, H-6),
4.40 (1H, sept, J=7.1 Hz, H-15), 2.35 (3H, s, COCH3),
1.51 (3H, s, H-18), 1.39, 1.23 (2Â3H, 2d, J=7.1 Hz, H-
16, H-17), 1.34 (3H, s, H-19), 1.25 (3H, s, H-20); 13C
NMR (CDCl3) d 188.5 (C-7), 170.1 (C-5), 169.5
(COCH3), 151.9, 148.3, 140.0, 131.4, 127.0 (Ar), 126.3
(C-6), 123.1 (Ar), 41.6 (C-10), 39.8 (C-3), 38.0 (C-1),
36.7 (C-4), 33.2 (C-18), 31.9 (C-20), 29.4 (C-19), 26.8 (C-
15), 21.4, 21.3 (C-16, C-17), 21.2 (COCH3), 18.6 (C-2).
82%); mp 175 ꢀC; nmax 3230 (OH), 1583 (CˆC),
1
1202 cm
;
1H NMR (CDCl3) d 6.90, 6.65 (2H, J=
8.4 Hz, H-11, H-12), 4.90 (1H, s, OH), 3.95, 3.59 (2H,
2d, J=4.7 Hz, H-6, H-7), 3.62 (1H, sept, J=7.1 Hz, H-
15), 1.46 (2Â3H, 2t, J=7.1 Hz, H-16, H-17), 1.23 (3H, s,
H-20), 1.18 (3H, s, H-18), 1.12 (3H, s, H-19); 13C NMR
(CDCl3) d 152.3, 144.6, 134.8, 130.4, 121.3, 116.7 (Ar),
54.2 (C-7), 53.1 (C-6), 47.7 (C-5), 40.8 (C-3), 38.3 (C-
10), 37.1 (C-1), 33.1 (C-4), 32.4 (C-18), 28.8 (C-15), 25.5
(C-20), 22.0 (C-19), 20.8, 20.7 (C-16, C-17), 18.8 (C-2);
m/z 300 (M+, 77%), 285 (100), 243 (29), 215 (73), 203
(42), 69 (51), 55 (22), 41 (41); anal. calcd for C20H28O2:
C, 79.9; H, 9.3; found: C,79.7; H, 9.3.
13-Acetoxy-6ꢀ,18-epoxytotara-8,11,13-triene.11 Iodine
(0.6 g, 2.4 mmol) was added to a stirred solution of 13-
acetoxytotara-8,11,13-trien-6a-ol (0.5 g, 1.5 mmol) and
lead tetraacetate (1.1 g, 2.5 mmol) in benzene at room
temperature under argon. The mixture was stirred for
2 h, diluted with benzene (150 mL), and the solution was
washed with aqueous Na2S2O3 (10% w/w, 2Â50 mL),
NaHCO3 (2Â50 mL), brine (2Â50 mL), dried (MgSO4),
and concentrated in vacuo. Chromatography on silica
gel using petrol:ethyl acetate (9:1) as the eluent gave the
title tetrahydrofuranyl compound (0.35 g, 70%); mp
148±149 ꢀC, lit.11 146±148 ꢀC. 1H NMR (CDCl3) d 7.11,
6.84 (2H, 2d, J=8.6 Hz, H-11, H-12), 4.10 (1H, m, H-
6b), 3.74, 3.50 (2H, 2d, J=7.1 Hz, H-18a,b), 3.46 (1H,
m, H-7a), 3.30 (1H, sept, J=7.1 Hz, H-15), 2.66 (1H,
dd, J=15.9, 8.4 Hz, H-7b), 2.31 (3H, s, COCH3), 1.26
(2Â3H, 2t, J=7.3Hz, H-16, H-17), 1.19 (2Â3H, 2s, H-20,
13-Hydroxytotara-5,8,11,13-tetraen-7-one (16).22 Sodium
hydride (catalytic) was added to a stirred solution of 13-
acetoxytotara-5,8,11,13-tetraen-7-one (70 mg, 0.2 mmol)
in methanol (5 mL) at room temperature under argon
and the reaction mixture was stirred for 14 h and con-
centrated in vacuo. Chromatography on silica gel using
petrol:ethyl acetate (9:1) as the eluent gave the hydroxy
compound 16 (53 mg, 89%); mp 243 ꢀC, lit.22 243±
244.5 ꢀC; 1H NMR (CDCl3) d 7.23, 6.95 (2H, J=8.8 Hz,
H-11, H-12), 6.43 (1H, s, H-6), 5.75 (1H, s, OH), 4.44
(1H, sept J=7.1 Hz, H-15), 1.49, 1.36 (2Â3H, 2d,
J=7.1 Hz, H-16, H-17), 1.47 (3H, s, H-18), 1.32 (3H, s,
H-19), 1.24 (3H, s, H-20); 13C NMR (CDCl3) 189.9 (C-
7), 170.8 (C-5), 154.0, 147.2, 134.1, 131.2, (Ar), 126.5
(C-6), 123.4 120.8 (Ar), 41.5 (C-10), 40.1 (C-3), 38.4 (C-1),

1674
G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
H-19); 13C NMR (CDCl3) d 169.9 (COCH3), 147.7,
146.2, 137.8, 133.2, 123.9, 121.8 (Ar), 83.8 (C-18), 71.4
(C-6), 56.3 (C-5), 40.0 (C-10), 39.3 (C-7), 37.5 (C-4),
36.7 (C-1), 35.4 (C-3), 27.6 (C-15), 23.8 (C-20), 21.6
(COCH3), 21.0, 21.0 (C-16, C-17), 20.4 (C-2), 19.1 (C-19).
and concentrated in vacuo. Chromatography on silica
gel using petrol:ethyl acetate (19:1) as the eluent gave
the epoxyether 20 (0.90 g, 90%); mp 155 ꢀC; nmax 1642
(CˆC), 1200, 1180 cm 1; 1H NMR (CDCl3) d 7.03, 6.76
(2H, 2d, J=8.6 Hz, H-11, H-12), 4.57 (1H, br s, H-6a),
4.01, 3.73 (2H, 2d, J=7.2 Hz, H-20a, H-20b), 3.83 (3H,
s, OCH3), 3.22 (1H, m, H-15), 3.13, 2.99 (2H, 2d,
J=17.2 Hz, H-7a,b), 1.37, 1.33 (2Â3H, 2d, J=7.0 Hz,
H-16, H-17), 1.11 (3H, s, H-19), 1.02 (3H, s, H-18); 13C
NMR (CDCl3) d 157.4, 142.7, 135.0, 132.2, 120.5, 109.3
(Ar), 78.4 (C-20), 77.6 (C-6), 55.2 (OCH3), 54.6 (C-5),
45.0 (C-10), 40.0 (C-1), 40.0 (C-3), 33.6 (C-18), 31.3 (C-
4), 28.3 (C-15), 28.2 (C-7), 22.7 (C-19), 20.5, 20.4 (C-16,
C-17), 18.9 (C-2); m/z 314 (M+, 96%), 269 (100), 241
(11), 185 (18); HRMS calcd for C21H30O2 (M+)
314.2246, found 314.2243.
6ꢀ,18-Epoxytotara-8,11,13-trien-13-ol (19). Lithium
aluminium hydride (100 mg) was added portionwise to
a stirred solution of 13-acetoxy-6a,18-epoxytotara-
8,11,13-triene (see above, 200 mg, 0.6 mmol) in THF
(20 mL) under argon at room temperature. The reaction
mixture was stirred for 2 h, quenched with water
(0.1 mL), NaOH (15%, 0.1 mL), and water (0.3 mL),
®ltered and the ®ltrate was concentrated in vacuo.
Chromatography on silica gel using petrol:ethyl acetate
(9:1) as the eluent gave the alcohol 19 (170 mg, 96%);
mp 209 ꢀC; nmax 3300 (OH), 1192, 1077 cm 1; H NMR
1
(CDCl3) d 6.96, 6.61 (2H, 2d, J=8.4 Hz, H-11, H-12),
5.50 (1H, br s, OH), 4.14 (1H, q, J=7.1 Hz, H-6b), 3.76,
3.52 (2H, 2d, J=7.1 Hz, H-18), 3.64 (1H, m, H-7a), 3.32
(1H, m, H-15), 2.68 (1H, dd, J=15.9, 8.4 Hz, H-7b),
1.38, 1.36 (2Â3H, 2t, J=7.0 Hz, H-16, H-17), 1.21 (3H,
s, H-20), 1.19 (3H, s, H-19); 13C NMR (CDCl3) d 152.9,
140.7, 132.5, 132.2, 123.4, 114.8 (Ar), 83.5 (C-18), 71.6
(C-6), 56.2 (C-5), 39.7 (C-10), 39.3 (C-7), 36.8 (C-4),
36.4 (C-1), 35.1 (C-3), 27.5 (C-15), 23.7 (C-20), 20.1 (C-
19), 20.0, 20.0 (C-16, C-17), 18.2 (C-2); m/z 300 (M+,
63%), 285 (35), 255 (23), 216 (100), 201 (25), 157 (16),
95 (17); anal. calcd for C20H28O2: C, 79.9; H, 9.4; found:
C, 79.7; H, 9.6.
Attempted demethylation of 6ꢁ,20-epoxy-13-methoxy-
totara-8,11,13-triene (20). Boron tribromide (10 mL,
1.0 M) was added dropwise to a stirred solution of
compound 20 (600 mg, 1.9 mmol) in CH2Cl2 (20 mL) at
room temperature under argon. The reaction mixture
was stirred for 30 min, diluted with CH2Cl2 (250 mL),
washed with aqueous Na2S2O3 (10% w/w, 3Â75 mL),
NaHCO3 (3Â75 mL), brine (3Â75 mL), dried (MgSO4),
and concentrated in vacuo. Chromatography on silica
gel using petrol:ethyl acetate (12:1) as the eluent gave in
order of elution: 13,20-dihydroxytotara-6,8,11,13-tetra-
ene (21) as an oil (222 mg, 39%); nmax 3415 (OH), 1661
1
(CˆC) cm 1; H NMR (CDCl3) d 6.85, 6.47 (2H, 2d,
J=8.3 Hz, H-11, H-12), 6.89 (1H, dd, J=10.1, 3.1 Hz,
H-7), 5.96 (1H, dd, J=10.1, 3.1 Hz, H-6), 5.06 (2H, s,
OH), 3.62, 3.38 (2H, 2t, J=6.4 Hz, H-20a,b), 3.38 (1H,
sept, J=7.1 Hz, H-15), 1.30, 1.28 (2Â3H, 2d, J=7.1 Hz,
H-16, H-17), 0.92 (3H, s, H-18), 0.90 (3H, s, H-19); 13C
NMR (CDCl3) d 153.4, 144.6, 132.4, 130.1 (C-6), 124.3
(C-7), 123.9, 114.2 (Ar), 59.8 (C-20), 50.0 (C-5), 43.4 (C-
10), 40.7 (C-3), 32.4 (C-4), 32.3 (C-18), 30.7 (C-1), 27.2
(C-15), 22.9 (C-19), 21.0, 20.8 (C-16, C-17), 18.6 (C-2);
m/z 300 (M+, 32%), 270 (100), 199 (21), 55 (10);
HRMS, calcd for C20H28O2 (M+) 300.2089, found
300.2093; 6b,13,20-trihydroxytotara-8,11,13-8,11,13-tri-
ene (22) as an oil (265 mg, 44%); nmax 3684, 3615, 3453
13-Methoxytotara-8,11,13-trien-6ꢁ-ol. Lithium alumi-
num hydride (400 mg) was added portionwise to a stir-
red solution of 13-methoxytotara-8,11,13-trien-6-one
(see above) (2.7 g, 8.6 mmol) in THF (50 mL) under
argon at room temperature. The reaction mixture was
stirred for 2 h, quenched with water (0.4 mL), NaOH
(15%, 0.4 mL), and water (1.2 mL), ®ltered and the ®l-
trate was concentrated in vacuo. Chromatography on
silica gel using petrol:ethyl acetate (19:1) as the eluent
gave the title alcohol (2.15 g, 79%); mp 110±112 ꢀC; H
1
NMR (CDCl3) d 7.18, 6.81 (2H, 2d, J=8.8 Hz, H-11,
H-12), 4.75 (1H, br s, H-6a), 3.81 (3H, s, OCH3), 3.26
(1H, m, H-15), 3.11 (1H, dd, J=17.2, 5.1 Hz, H-7a),
3.00 (1H, d, J=17.2 Hz, H-7b), 1.62 (3H, s, H-20), 1.36,
1.33 (2 3H, 2d, J=7.1 Hz, H-16, H-17), 1.32 (3H, s, H-
19), 1.08 (3H, s, H-18); 13C NMR (CDCl3) d 156.3,
141.4, 133.4, 129.7, 123.5, 110.0 (Ar), 65.8 (C-6), 54.9
(OCH3), 52.2 (C-5), 43.0 (C-3), 42.9 (C-1), 39.4 (C-7),
37.1 (C-10), 33.9 (C-4), 33.4 (C-18), 27.4 (C-15), 27.2 (C-
20), 23.6 (C-19), 20.3, 20.2 (C-16, C-17), 19.6 (C-2); m/z
316 (M+, 69%), 298 (100), 241 (24), 213 (17), 171 (11),
69 (18); anal. calcd for C21H32O2: C, 79.7; H, 10.4;
found: C, 79.9; H, 10.4.
1
(OH), 1273 cm 1; H NMR (CDCl3) d 6.76, 6.55 (2H,
2d J=8.1 Hz, H-11, H-12), 5.29 (1H, d, J=2.1 Hz, H-
6a), 4.27 (1H, d, J=8.2 Hz, H-20a), 3.37 (1H, sept,
J=7.1 Hz, H-15), 2.80 (1H, dd, J=8.2, 1.6 Hz, H-20b),
1.28, 1.27 (2Â3H, 2d, J=7.1 Hz, H-16, H-17), 1.09 (3H,
s, H-19), 0.76 (3H, s, H-18); 13C NMR (CDCl3) d 152.5,
139.2, 138.3, 128.4, 117.4, 114.1 (Ar),67.6 (C-20), 66.9
(C-6), 42.8 (C-5), 41.2 (C-3), 37.0 (C-10), 33.6 (C-4),
32.7 (C-18), 29.4 (C-1), 29.2 (C-7), 26.5 (C-15), 21.8,
21.6 (C-16, C-17), 20.8 (C-19), 18.9 (C-2); m/z 300
(M+ H2O, 22%), 270 (100), 255 (47), 202 (22), 199
(21), 171 (24), 157 (20), 145 (18), 41 (12); HRMS calcd
for C20H28O2 300.2089, found 300.2089.
6ꢁ,20-Epoxy-13-methoxytotara-8,11,13-triene (20). Iodine
(1.2 g, 4.7 mmol) was added to a stirred solution of
13-methoxytotara-8,11,13-trien-6b-ol (1 g, 3.2 mmol)
and lead tetraacetate (2.2 g, 4.8 mmol) in benzene at
room temperature under argon. The reaction mixture
was stirred for 2 h, diluted with benzene (150 mL),
washed with aqueous Na2S2O3 (10% w/w, 2Â50 mL),
NaHCO3 (2Â50 mL), brine (2Â50 mL), dried (MgSO4),
Biology
Minimum inhibitory concentrations (MIC) were
determined by the double agar dilution method with
Mueller±Hinton agar as described previously.3 Liver
mitochondria were prepared from female Wistar rats

G. B. Evans et al. / Bioorg. Med. Chem. 8 (2000) 1663±1675
1675
(200±300 g) by homogenization followed by di€erential
centrifugation at 4 ꢀC in an isolation medium containing
250 mM sucrose, 5 mM Tris±HCl and 1 mM EGTA, pH
7.4, and stored on ice at about 50 mg protein/mL.23
Protein concentrations were determined by the biuret
assay24 using bovine serum albumin as a standard. To
measure respiration rate mitochondria (1 mg protein/
mL) were incubated in medium containing 120 mM
KCl, 5 mM KH2PO4, 2 mM MgCl2, 10 mM Hepes±
KOH, 1 mM EGTA, pH 7.2, at 25 ꢀC in an oxygen
electrode chamber thermostatted at 25 ꢀC (Rank
Brothers, Bottisham, Cambridge, UK). The respiratory
substrates used were either glutamate and malate (5 mM
of each), succinate (5 mM) or ascorbate (5 mM) and
TMPD (500 mM). When succinate or ascorbate/TMPD
were used as respiratory substrates rotenone (13 mM)
was also present. Mitochondria were preincubated with
respiratory substrates for up to 1 min, then totarol (1),
7-oxototarol (13) or an equivalent volume of ethanol
carrier was added and the rate of coupled respiration
was measured; ADP (200 mM) was then added and the
rate of phosphorylating respiration measured; ®nally,
the uncoupler FCCP (333 nM) was added and the rate
of uncoupled respiration measured. The oxygen elec-
trode was calibrated assuming an oxygen concentration
of 475 nmol O atoms/mL in air-equilibrated medium at
25 ꢀC.25 Respiration rates in the presence of various
concentrations of totarol (1) or 7-oxototarol (13) were
determined in duplicate and the e€ects of them on cou-
pled, phosphorylating or uncoupled respiration for the
three di€erent respiratory substrates are expressed as a
percentage of the control incubations in the presence of
ethanol carrier. Data are the means of determinations
on at least three separate mitochondrial preparations
ÆS.E.M.
and Technology postdoctoral fellowship to GBE and the
Health Research Council of New Zealand for ®nancial
support for MPM. Carolyn Porteous and Sean Runacres
are thanked for technical support. Prof. Robin Ferrier
assisted with the preparation of the manuscript.
References and Notes
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2. Kobayashi, K.; Nishino, C.; Fukushima, M.; Shiobara, Y.;
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3. For part I, see Evans, G. B.; Furneaux, R. H.; Gravestock,
M. B.; Lynch, G. P.; Scott, G. K. Bioorg. Med. Chem. 1999, 7,
1953.
4. For part II, see Evans, G. B.; Furneaux, R. H. Bioorg. Med.
Chem. 2000, 8, 1653.
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8. Chow, Y. L.; Erdtman, H. Acta Chem. Scand. 1960, 14,
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ney, J. D.; Wenker, E. J. Org. Chem. 1964, 29, 2293.
10. Baker, K. M.; Davis, B. R. Tetrahedron 1969, 25, 2487.
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14. Matsumoto, T.; Endo, Y.; Okimoto, M. Bull. Chem. Soc.
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18. Biosym Technologies Program Discover1 2.97 using CVFF
Force®eld; Biosym Technologies Inc., San Diego, 1993.
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20. Finucane, B. W.; Thomson, J. B., J. Chem Soc., Chem
Commun. 1969, 1220.
X-ray single crystal analysis
Crystallographic data for 7-oxototarol (13). C20H28O2,
orthorhombic, space group P 212121(19),26 a=10.400(1),
b=12.002(1), c=13.852(1) A, V=1729.0(3) A3, Z=4,
DC=1.154 Mg m 3, T=173(2) K, Mo Ka radiation
(l=0.71073 A), ꢀ=0.72 cm 1. A Siemens P4 dif-
fractometer was used to measure 2985 independent
re¯ections (4ꢀꢁ2yꢁ52ꢀ) of which 1925 ``observed'' had
Inet>2 s (Inet). No absorption correction was made and
the structure was solved by direct methods27 and re®ned
on F2o using all data to give R (``observed'') 0.057, wR2
(all data, on F2o) 0.104.28 The absolute con®guration was
not determined and all hydrogen atoms were located by
di€erence Fourier maps and re®ned with individual iso-
21. Hodges, R. J. Chem. Soc. 1961, 4247.
22. Cambie, R. C.; Gallagher, R. T. Tetrahedron 1968, 24, 4631.
23. Chappell, J. B.; Hansford, R. G. In Subcellular Compo-
nents: Preparation and Fractionation; Birnie, G. D., Ed.; But-
terworths: London, 1972; pp 77±91.
24. Gornall, A. G.; Bardawill, C. J.; David, M. M. J. Biol.
Chem. 1949, 177, 751.
25. Reynafarje, B.; Costa, L. E.; Lehninger, A. L. Anal. Bio-
chem. 1985, 145, 406.
tropic thermal parameters. The ®nal maximum shift/
3
26. International Tables for Crystallography; Hahn, T., Ed.;
Kluwer Academic: Dordrecht, 1995; Vol. A.
27. Sheldrick, G. M. SHELXS-86 Program for Crystal Struc-
ture Determination. Institute for Anorganische Chemie der
Universitat: Gottingen, Germany, 1986.
error was 0.006 with Ár excursions 0.20 to 0.17 e A
.
Acknowledgements
28. Sheldrick, G. M. SHELXL-93 Program for Re®nement
of Crystal Structures. University of Gottingen: Gottingen,
Germany, 1993.
We wish to thank the Foundation for Research, Science
and Technology for a New Zealand Research, Science