Elucidation of the topography of the thapsigargin binding site in the sarco-endoplasmic calcium ATPase

Article abstract of DOI:10.1016/j.bmc.2010.06.032

Removal of each of the acyl groups of thapsigargin at O-3, O-8 and O-10 significant reduces the affinity of the inhibitors to the SERCA1a pump. Replacement of the acyl groups at O-3 and O-10 with flexible residues could be performed with only a minor decrease of the affinity, whereas introduction of voluminous stiff residues caused dramatic reduction of the affinity. The results can be rationalized on the basis of the interactions of thapsigargin with the SERCA1a pump as revealed from 3D X-ray structural models of thapsigargin bound to the SERCA1a. In conclusion the results confirm and elaborate the previously suggested pharmocophore model of thapsigargin.

Full text of DOI:10.1016/j.bmc.2010.06.032

Bioorganic & Medicinal Chemistry 18 (2010) 5634–5646
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Elucidation of the topography of the thapsigargin binding site in the
sarco-endoplasmic calcium ATPase
Dorthe Mondrup Skytte a, Jesper Vuust Møller b, Huizhen Liu a, Helle Østergren Nielsen a,
Louise Elsa Svenningsen a, Christina Mernøe Jensen a, Carl Erik Olsen c, Søren Brøgger Christensen a,
*
a Department of Medicinal Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark
b PUMPKIN, Department of Physiology and Biophysics, University of Aarhus, Ole Worms Alle blg 1185, DK-8000 Aarhus C, Denmark
c Department of Basic Sciences and Environment, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Removal of each of the acyl groups of thapsigargin at O-3, O-8 and O-10 significant reduces the affinity of
the inhibitors to the SERCA1a pump. Replacement of the acyl groups at O-3 and O-10 with flexible res-
idues could be performed with only a minor decrease of the affinity, whereas introduction of voluminous
stiff residues caused dramatic reduction of the affinity. The results can be rationalized on the basis of the
interactions of thapsigargin with the SERCA1a pump as revealed from 3D X-ray structural models of
thapsigargin bound to the SERCA1a. In conclusion the results confirm and elaborate the previously sug-
gested pharmocophore model of thapsigargin.
Received 20 April 2010
Revised 7 June 2010
Accepted 10 June 2010
Available online 16 June 2010
Keywords:
Thapsigargin
SERCA
Ó 2010 Elsevier Ltd. All rights reserved.
Pharmacophore
Topography of binding cavity
1. Introduction
enzymes such as humane glandular kallikrein 2 (hK2) and prostate
specific antigen [PSA also known as human glandular kallikrein
The sarco- and endoplasmic Ca2+-ATPases (SEERCA) are essen-
tial for the calcium homeostasis of cells. The SERCA family includes
the products of three genes named SERCA1 (ATP2A1), SERCA2
(ATP2A2), and SERCA3 (ATP2A3), each giving rise to alternatively
spliced mRNA.1 Presently the SERCA family comprises 14 isoforms:
two SERCA1 (1a and 1b), three SERCA2 (2a–2c), and in humans six
SERCA3 (3a–3f). In some species (mice and rats) other specific iso-
forms of SERCA3 have been found.1 Thapsigargin (Tg, 1a) isolated
from the Mediterranean umbelliferous plant Thapsia garganica L.
was originately believed only to inhibit the endoplasmic Ca2+-ATP-
ases.2 Later studies, however revealed that thapsigargin is a potent
and specific inhibitor of all the subtypes of SERCA even though the
inhibition constants towards the different subtypes vary from 0.2
to 12 nM.2–7 Blockage of SERCA leads to malfunction of the calcium
homeostasis in the cells and eventually apoptosis.8–10 In contrast
to chemotherapeutics Tg provokes apoptosis at all stages of the cell
cycle.8–10 Slow proliferation of prostate cancer cells prevents treat-
ment of prostate cancer by ordinary chemotherapeutics since these
attack the cells during the mitotic phase of the cell cycle.11 Treat-
ment with Tg will overcome this problem but the ubiquitous pres-
ence of SERCA in all kind of cells precludes the use of Tg as per se.
By taking advantage of expression of characteristic proteolytic
3 (hK3)] Tg has been targeted towards cells of different tis-
sues.12–15 The strategy is based on the assumption that conjugation
of an amino-containing derivative of Tg to a peptide yields a pro-
drug, which does not penetrate the cell membrane, but after cleav-
age of the peptide moiety by a tissue characteristic proteolytic
enzyme a drug is formed, which can reach and block the intracel-
lular located SERCA and eventually induce apoptosis.16 The concept
of selectively targeting prostate cancer cells has been proven valid
in a mouse model, where systemic administration of a Tg derived
prodrug selectively controlled the growth of prostate cancer
cells.17 Conversion of Tg into a derivative, which can be used for
conjugation with a peptide, necessitates introduction of a long lin-
ker moiety.14 Introduction of the linker into a critical position
might afford an inactive derivative. Consequently, extensive stud-
ies of the topography of the Tg binding site in SERCA have been
performed14,18–21 and reviewed.11,22 Empirical structure–activity
relationships reveal that a large flexible linker is allowed at O-8
and at O-2 (for numbering see Scheme 1) the lactone carbonyl at
C-12 can be replaced with a methylene group, and the hydroxyl
group at C-11 can be alkylated without loss of SERCA inhibitory
activity. A GRID analysis of the Tg binding site in SERCA as revealed
by X-ray structures of SERCA1a stabilized with Tg23 or with Tg, in
which the O-8 group has been replaced with a N-Boc-12-aminod-
odecanoate21 to some extent rationalized these findings.24 This
pharmacophore model suggests lipophilic interactions between
* Corresponding author. Tel.: +45 3533 6253; fax +45 3533 6401.
E-mail address: sbc@farma.ku.dk (S.B. Christensen).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.032

D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
5635
a minor importance.25 The present study was undertaken in order
to elaborate on the topographies of the binding cavity around the
angeloyl group at O-3, the acetyl group at O-10, and of the hydro-
xyl group at O-11.
2. Results
2.1. Chemistry
To save our stock of thapsigargin (1a) thapsigargicin (1b,
Scheme 1) obtained from Thapsia garganica as previously de-
scribed26 was used as starting material for the model compounds,
in which hexanoate during the reaction sequence later was re-
placed with octanoic acid. Thus, the use of thapsigargicin only
had importance for the synthetic procedure. The described meth-
ods27–30 for removing the acyl groups at O-2, O-8, and O-10 and
for selective protection of O-8 and O-11 were used to get the iso-
propylidene diol 5 (Scheme 1). Selective acylation at O-2 was ob-
tained by reaction of 5 with octanoic or benzoic anhydride in the
presence of 4-N,N-dimethylaminopyridine (DMAP) to give 6a and
6b, respectively (Scheme 2). Acetylation at O-10 to give 7b pro-
ceeded smoothly when isopropenyl acetate and p-toluenesulfonic
acid (p-TsOH) were used as reagents. If, however, larger esters of
isopropenol like butyric or octanoic esters were used to acylate
6a significant amounts of side products like 13a and 14 were ob-
served beside 7a or 7c (Scheme 3). Since formation of the isopro-
penyl esters by reesterification of isopropenyl acetate with the
appropriate acid is known to give acid anhydrides as side prod-
ucts,31 it was assumed that the formed acid anhydrides caused
the side products. Surprisingly, however, treatment of 7a with
anhydrides like butyric, 4-biphenylacetic or octanoic acid anhy-
dride in the presence of p-TsOH smoothly and selectively afforded
the O-10 esters 7a, 7c, and 7d. Consequently, this method was used
for preparation of these compounds. Acidic hydrolysis of the iso-
propylidene acetal followed by selective esterification of O-8 was
performed analogous to published procedures.21 Acylation of
O-10 in 6a by reaction with benzoic acid anhydride and p-TsOH
was not possible, apparently according to steric hindrance from
the isopropylidene acetal. Alternatively, the isopropylidene acetal
of 6a was hydrolyzed under acidic conditions to give 10 (Scheme
2). Selective acylation of O-8 with butyric acid anhydride and
DMAP gave 11a which subsequently was acylated at O-10 by reac-
tion with benzoic acid anhydride and p-TsOH under prolonged
reaction time to give 12. Advantage was taken of the availability
of the side products 13a and 14 to test the importance of substit-
uents at O-7 and O-11. Furthermore, the reaction was used to pre-
pare the O-10 octanoic or butanoic acid esters 7a, 9a, 9c, and 14
using 5 as starting material. For replacement of the angeloyl resi-
due advantage of the trisubstituted double bond was taken. Selec-
tive oxidative cleavage of this double bond was obtained using
osmium tetraoxide and periodate as reagents to give the pyruvate,
which selectively could be hydrolyzed to give the 3-hydroxy deriv-
ative 15 (Scheme 4). The secondary hydroxy group was selectively
esterified using either acid anhydrides and DMAP as catalyst or
DCC and DMAP promoted esterification of free acids to give ana-
logues 16a–16f.
Scheme 1.
2.2. SERCA inhibitory activity
SERCA and the acetyl group at O-10, the angeloyl group at O-3, and
the butanoyl group at O-8. In addition a minor interaction between
the methyl-15 and the pump is suggested. The octanoyl group at
O-2 might stabilize the complex by interacting with the lipophilic
membrane. Surprisingly no hydrophilic interactions were re-
vealed24 even though a X-ray crystal structure of the SERCA-Tg
The activity of SERCA was measured spectrophotometrically
with a NADH coupled and ATP regenerating assay medium.32
For this an assay protocol was established in which SERCA1a
(purified from sarcoplasmic reticulum vesicles of rabbit skeletal
muscle by treatment with a low concentration of deoxycholate)
at a protein concentration of 1 mg/mL was pre-equilibrated with
complex indicates that
a potential hydrogen bond between
Ile829 and the carbonyl group of the butanoyl moiety might have

5636
D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
Scheme 2.
different inhibitor concentrations for 2.5–6 min in a medium con-
taining 1 mM EGTA, 1 mM Mg2+
10 mM Tes (pH 7.5), and
100 mM KCl. Activities were measured from the rate of absorp-
tion decrease at 340 nm. Following addition of 10 L of the
pre-equilibrated mixture to a cuvette, containing 3 mL of 0.1
mM Ca2+, 1 mM Mg2+, 5 mM MgATP, 1 mM phosphoenolpyruvate,
0.15 mM NADH, phosphoenolpyruvate kinase, and lactate dehy-
drogenase. The inhibitor (thapsigargin or thapsigargin derivative),
solubilized in DMSO, was added cumulatively to the ATPase pre-
equilibration mixture in increments of usually 0.2–0.4 mol/mg
protein. We found that the gradual increase in inhibitor concen-
tration in the cuvette sample, effectuated by the incremental
addition, facilitated the attainment of binding equilibrium, which
often was a slow process, requiring up to 6 min preincubation.
The assay was often carried out with glassware instead of plastic
to safeguard as much as possible against unspecific adsorption of
the inhibitor by the side walls of containers, pipettes, and
cuvettes. The inhibition of activity is given as IC50 values in Table
1. Thapsigargin was used as a positive control in all exper-
iments.
,
l

D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
5637
Scheme 3.
(9d). The stiff and equally voluminous 4-phenylbenzoate (12b)
failed to exert SERCA inhibitory activity in the concentration range
studied. This suggests that as expected the O-10 acetyl pocket is
not large enough to contain the 4-phenylbenzoyl group (12b). Con-
sequently the high affinity of the bisphenylacetyl (9d) must be as-
cribed to the flexibility retained, due to rotation of the two single
bonds attaching the methylene group to the carbonyl- and the
bisphenyl group, respectively. This flexibility enables part of the
side chain either to avoid sterical interaction with the backbone
of the transmembrane segments or to engage in interaction with
other parts of the ATPase or with the lipid phase.
Very similar characteristics as with the O-10 acetyl group was
observed by replacement of the angeloyl residue with volumi-
nous acyl groups. The benzoyl analogue 16a is bound with al-
most the same affinity as Tg. The meta-methylbenzoyl analogue
16b, even the very voluminous, but flexible para-bisphenylace-
tate 16c and ortho-phenylbenzoyl analogue 16d (an ortho-phen-
3. Discussion
The pharmacophore model for Tg24 suggests extensive hydro-
phobic interactions between SERCA and the angeloyl, the butanoyl,
and the acetyl moieties of Tg. These interactions are localized to
the 3rd, 5th, and 7th (M3, M5, and M7) transmembrane helices
of SERCA which form the bottom and sides of the binding cavity,
while the front of the bound Tg molecule is exposed towards the
cytoplasmic leaflet of the lipid phase (Fig. 1). The pharmacophore
model implies that removal of the three mentioned side chains
should cause a significant reduction in the affinity for SERCA. This
prediction was confirmed by measuring the inhibitory effect on en-
zyme activity of Tg analogues with removed side chains. Thus, the
debutanoylated thapsigargin derivative possesses an IC50 value of
10 nM (Table 1, compound 9e), compared to an IC50 value of
0.2 nM estimated for the unmodified Tg. Analoguously, the des-
acetylated analogues 11a and 11b give rise to IC50 values of
20 nM. All of these analogues, and in particular the desangeloylat-
ed 15, confirm the importance of the hydrophobic interactions be-
tween the acyl group and the pump. In continuation of these
findings we have substituted the angeloyl and acetyl groups of
thapsigargin with other acyl groups in order to explore the topog-
raphy of the binding cavity.
Replacement of the acetyl group at O-10 with flexible acyl
groups like butanoyl (9a), octanoyl (9c), the non-flexible benzoyl
(12a), and even the large bisphenylacetyl group (9d) reduced the
IC50 values, but not nearly as much as expected for a ‘lock and
key’ mechanism. This suggests flexibility at the O-10 binding site
for accommodation of large ligands. From a structural perspective
this calls attention to Phe 834 which closely covers and interacts
with the acetyl group of Tg.21 Comparisons of E2 conformations
of ATPase, with and without bound Tg, indicate a slight retraction
of Phe 834 upon Tg bind in accordance with the idea that the 834
phenyl group is flexible and by rotation adjusts to the volume size
of the O-10 side chain. Furthermore, Tyr 837 upon Tg binding
undergoes a dramatic 110 degree rotation out of the binding cavity
to make room for accommodation of Tg. There is thus clear evi-
dence that this part of the binding site which is localized between
the M5 and M7 helices has induced-fit characteristics (Fig. 1B). We
also found that in contrast to the more flexible bisphenylacetyl
ylbenzoyl) only to
a limited extent displayed reduced IC50
values. In contrast introduction of a para-methylbenzoate (16e)
and even more pronounced the stiff 4-phenylbenzoate (16f) dra-
matically reduced the affinities. These different binding proper-
ties can also be accounted for on the basis of the interactions
between Tg and the SERCA pump revealed by the X-ray diffrac-
tion data. In the Tg bound pump the angeloyl side chain is lo-
cated in an internal cavity between M3, M4, and M5, the
bottom of which is formed by the proline residue of the
A(306)IPEGL(311) motif in M4, forming part of one of the Ca2+
binding sites. This cavity would be sufficiently large for accom-
modation of a benzoyl group; it can also adjust to the 16b
meta-methyl, but not so well to the 16e para-methyl derivative,
because the length of the substituent exceeds the length of the
cavity. However, the cavity is too small to accept the bulkier sub-
stituents. Furthermore, the stiff phenylbenzoyl group (16f) is
bound with a low, but perceptible affinity (ꢀ0.1
lM). We there-
fore conclude that the side chains with bulky flexible and stiff
substituents at O-2 probably are bound differently than in the
internal cavity of the angeloyl moiety, but without disrupting
other interactions of the ligand with the ATPase.
Remaining data in Table 1 deal with Tg analogues close to the
lactone ring.

5638
D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
as well as from a medicinal chemical aspect appears to be compli-
cated and will be a subject of a future communication.21,25,33
4. Conclusions
Selective acylation of OH-10 in the presence of free hydroxyl
groups at O-7 in a thapsigargin analogue can be obtained using
an acid anhydride as reagent and p-TsOH as a catalyst. The as-
sumed crucial effect of a lipophilic acyl group at O-3, O-8 and
O-10 has been confirmed. Introduction of flexible voluminous
groups at O-3 and O-10 only to a minor extent reduces the affinity
for the analogues to the pump. In contrast introduction of the volu-
minous and stiff 4-phenylbenzoic acid at O-3 dramatically reduces
the ability to inhibit the pump. Our data suggest that stiff benzoyl
and 4-methylbenzoyl derivatives can be used as rulers to estimate
available space in the binding pockets of the side chains, for exam-
ple, the latter substituent at O-3 (with a distance from carbonyl
carbon atom to the carbon atom of the methyl group 0.58 nm) dra-
matically reduces the affinity compared to introduction of benzoic
acid (distance from carbonyl carbon atom to para carbon atom
0.43 nm). The present findings substantiate the previously sug-
gested pharmacophore model.24
5. Experimental
5.1. General methods for chemistry
1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were re-
corded on a Varian Mercury spectrometer or a Varian Gemini
2000 spectrometer using CDCl3 as a solvent. 2D NMR spectra were
recorded using standard pulse sequences. Splitting patterns are de-
scribed as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), broad singlet (br s), double doublet (dd), double double double
doublet (dddd), double quartet (dq) or quartet of quartets (qq).
High-resolution MS was recorded on a Q-Tof1 (Micromass). Col-
umn chromatography was performed on Silica Gel 60 (Merck
107734). Reversed phase chromatography was performed over
LiChroprep RP-18 (Merck 1.13900).
Octanoic anhydride: Dicyclohexylcarbodiimide (DCC, 6.5 g,
31.6 mmol) was added to a solution of octanoic acid (10 mL,
63.1 mmol) in CH2Cl2. The mixture was stirred at room tempera-
ture for 1½ h, filtered and concentrated in vacuo.
1H NMR (CDCl3)
OCOCH2CH2–); 1.66 (4H, dddd, J = 7.5 Hz, 2 ꢁ –OCOCH2CH2CH2–);
1.28–1.37 (16H, m, 4 ꢁ –CH2–); 0.88 (6H, t, J = 6.9 Hz, 2 ꢁ –
CH2CH3).28
d
(ppm): 2.44 (4H, t, J = 7.5 Hz, 2 ꢁ –
Scheme 4.
4-Biphenylacetic anhydride: 4-Biphenylacetic acid (2.00 g,
9.4 mmol) was dissolved in (60 mL) and DCC (0.97 g, 4.7 mmol)
was added. The reaction mixture was stirred at room temperature
over night, filtered and concentrated in vacuo. The product was
recrystallized from heptane and EtOAc. White solid (1.32 g, 69%).
Melting point 145–147 °C. 1H NMR (CDCl3) d (ppm): 7.51–7.56
(m, 8H, H-8, H-9, H-11, and H-12); 7.39–7.42 (m, 4H, H-3 and
H-5); 7.34 (dddd, J = 2.4 and 7.5 Hz, 2H, H-10); 7.27 (d, J = 8.1 Hz,
4H, H-2 and H-6); 3.78 (s, 4H, COCH2-Ph).
The IC50 value of the isopropylidene derivative 7a was 19 nM
and comparable to the affinity of the debutanoylated analogue
(9e), which may be accounted for by assuming that the loss of
affinity compared to Tg is caused by the absence of the butanoyl
group and that the isopropylidene group does not interact with
the SERCA pump. Furthermore, docking of 7a with the guaianolide
skeleton of 12ADT (structure 2BY4)21 reveals that the two methyl
groups of the isopropylidene group are placed distant from the
helices of the pump. The pharmacophore model does not predict
the importance of the two hydroxyl groups of Tg and previous data
have revealed that acetylation of O-7 or O-11 only moderately re-
duces the affinity for the SERCA pump approximately 2.5 times,
whereas acetylation of both hydroxy groups reduces the affinity
15 times.19 Introduction of a large acyl groups at O-11 as in 14 con-
firms the previous findings by showing that this only causes some
reduction in affinity. Substituents at O-7 as in compound 13 reveal
that substituents in this position have importance for the affinity.
Substitution in this position, however, from a chemical synthetic
5.1.1. Debutanoyl thapsigargicin (2)
Thapsigarcin (1b) (1.5 g, 2.4 mmol) was dissolved in dry MeOH
(150 mL). Et3N (6.8 mL, 48 mmol) was added and the mixture was
stirred at room temperature for 4½ h. The reaction was quenched
with saturated aqueous NH4Cl-solution (200 mL) and extracted
five times with EtOAc (100 mL). The combined organic layer was
dried over Na2SO4, filtered and concentrated in vacuo. Compound
2 (1.11 g, 83%) was purified by column chromatography using hep-
tanes–EtOAc (3:2) as an eluent.

D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
5639
1H NMR (CDCl3) d (ppm): 5.81 (br s, 1H, H-6); 5.69 (br s, 1H, H-
5.1.2. Isopropylidene (3)
3); 5.45 (dd, J = 3.6 Hz, 1H, H-2); 5.00 (s, 1H, OH); 4.36 (br m, 1H, H-
8); 4.22 (br s, 1H, H-1); 4.13 (s, 1H, OH); 3.53 (s, 1H, OH); 2.84 (dd,
J = 2.1 Hz and 14.1 Hz, 1H, H-9a); 2.47 (dd, J = 3.3 Hz and 14.1 Hz,
1H, H-9b), 1.90 (br s, 3H, H-15), 1.49 (3H, s, H-13); 1.44 (3H, s, H-
14). Acetate 1.90 (s, 3H, H-2). Angeloate 6.12 (1H, qq, J = 1.5 and
7.2 Hz, 1H, H-3), 1.98 (3H, dq, J = 1.5 Hz; 7.2 Hz, 3H, H-4), 1.84 (s,
3H, Me-2). Hexanoate 2.20–2.38 (2H, m, H-2), 1.60 (m, 2H, H-3);
1.30 (m, 4H, H-4 and H-5); 0.88 (t, J = 6.9 Hz, 3H, H-6).
Compound 2 (1.09 g, 1.97 mmol) was dissolved in acetone
(16 mL) and 2,2-dimethoxypropane (30 mL). p-Toluenesulfonic
acid monohydrate (p-TsOH, 0.055 g, 0.29 mmol) was added and
the mixture was stirred at room temperature over night. The reac-
tion was quenched with a saturated aqueous NaHCO3-solution
(100 mL) and extracted three times with EtOAc (100 mL). The com-
bined organic layer was washed with brine (100 mL), dried over
Na2SO4, filtered and concentrated in vacuo. Compound 3 (1.01 g,
Table 1
Inhibition constants (IC50 values) for thapsigargin analogues towards SERCA1a
O
R1
H C
H3
O
H3C
O
O
R2
H3C
O
O
Compd
IC50 (nM)
OH
OH
CH3
CH
3 H3C
O
O
1a
R
1 = –COCH3
R2 = –CO(CH2)2CH3
R2 = –CO(CH2)2CH3
R2 = –CO(CH2)6CH3
R2 = –CO(CH2)2CH3
R2 = –CO(CH2)2CH3
R2 = –CO(CH2)2CH3
R2 = –CO(CH2)2CH3
R2 = –CO(CH2)2CH3
R2 = –H
0.2
20
11a
11b
9a
R1 = –H
R1 = –H
20 8.5
2.5 0.3
16 2.7
15.6 0.1
>70
R1 = –CO(CH2)2CH3
R1 = –CO(CH2)6CH3
R1 = –COC6H5
9c
12a
12b
9d
R1 = –COC6H4–C6H5
R1 = –COCH2C6H4C6H5
R1 = –COCH3
27 1.6
10
9e
O
CH3
O
H C
H3
O
O
O
O
9b
1.6
H3C
O
O
CH3
OH
OH
CH3
CH
3 H3C
O
O
O
O
H C
H3
CH3
O
O
O
H3C
O
O
H3C
H3C
H3C
O
O
13
7a
14
>70
CH3
CH3
O
O
CH3
H3C
CH3
O
CH3
O
O
O
O
O
O
H C
H3
CH3
H3C
O
O
O
O
OH
O
19 1.1
CH3
CH3
CH
CH
3 H3C
O
CH3
O
O
O
H C
H3
CH3
H3C
O
O
O
O
O
OH
O
CH3
CH3
11.1 1.9
3 H3C
O
CH3
(continued on next page)

5640
D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
Table 1 (continued)
O
CH3
14
O
H C
H3
H3C
O
O
O
10
9
2
1
Compd
IC50 (nM)
8
O
O
CH3
5
3
7
6
4
R
OH
H3C
15
11OH
O
CH3
12
O
15
R = H
>70
O
16a
0.8 0.7
R =
O
16b
16c
9
4.5
H3C
R =
R =
O
18 2.5
16d
3
2.0
O
R =
R =
R =
O
H3C
16e
16f
44 8.6
>70
O
Figure 1. Thapsigargin (carbons as green sticks and oxygen as red sticks) in the binding cavity of SERCA1a in the E2 conformation. A: The binding cavity with adjacent amino
acid residues accentuated. B: Superimpositions of structures of SERCA1a in the E2 conformation in the absence (PDB code 3B9R, blue) and in the presence of bound
thapsigargin (PDB code 1XP5, yellow).
87%) was purified by column chromatography using heptanes–
ethyl acetate (3:1) as an eluent.
H-8); 4.06 (s, 1H, OH); 3.99 (br s, 1H, H-1); 2.75 (dd, J = 4.8 and
14.7 Hz, 1H, H-9a); 2.64 (dd, J = 2.7 and 14.7 Hz, 1H, H-9b), 1.88
(s, 3H, H-15), 1.56 (s, 3H, H-13); 1.47 (3H, s, H-14). Acetate 1.88
(s, 3H, H-2). Angeloate 6.10 (qq, J = 1.5 Hz; 7.2 Hz, 1H, H-3), 1.99
1H NMR (CDCl3) d: 5.78 m, 1H, H-6); 5.73 (m, 1H, H-3); 5.49 (dd,
J = 4.2 Hz and 5.4 Hz, 1H, H-2); 4.30 (dd, J = 3.0 Hz and 4.2 Hz, 1H,

D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
5641
(dq, J = 1.5 and 7.2 Hz, 3H, H-4), 1.91 (m, 3H, Me-2). Hexanoate
2.30 (m, 2H, H-2), 1.60 (m, 2H, H-3), 1.30 (m, 4H, H-4 and H-5);
0.89 (t, J = 6.9 Hz, 3H, H-6). Isopropylidene, 1.54 (s, 3H, H-1), 1.42
(s, 3H, H-3), (H, H-3).
2.10 (m, 2H, H-2), 1.55 (m, 2H, H-3), 0.91 (t, J = 7.2 Hz, 3H, H-4).
Isopropylidene 1.53 (s, 3H, H-3) 1.42 (s, 3H, H-1). Octanoate 2.32
(dt, J = 3.0 and 7.5 Hz, 2H, H-2), 1.60 (m, 2H, H-3), 1.28 (m, 8H,
H-4–H-7), 0.87 (t, J = 6.9 Hz, 3H, H-8).
For the data of 8a, see below.
5.1.3. Isopropylidene 5
Compound 3 (1.00 g, 1.7 mmol) was stirred in 10% KOH in
MeOH (15 mL) for 5 min. The mixture was neutralized with Dowex
50WX8-100 ion exchange resin, filtered and concentrated in vacuo.
Compound 5 (0.61 g, 79%) was purified by column chromatography
using heptanes–ethyl acetate (1:3?1:6) as eluents.
5.1.6. Triol (8a) (general procedure B)
Butanoate (7a) (18.2 mg, 0.028 mmol) was dissolved in MeOH
(2 mL). Three molar of HCl (39 lL) was added dropwise under stir-
ring and the mixture was heated to 45 °C for 45 min. The reaction
mixture was cooled to room temperature and a saturated aqueous
solution of NaHCO3 (10 mL) and EtOAc (10 ml) was added. The lay-
ers were separated and the aqueous layer was extracted three
times with EtOAc (10 mL). The combined organic layer was dried
over Na2SO4, filtered and concentrated in vacuo. Compound 8a
(15.5 mg, 87%) was purified by column chromatography using hep-
tanes–ethyl acetate (5:2) as an eluent.
1H NMR (CDCl3) d (ppm): 6.21 (1H, qq, J = 1.5 Hz; 7.2 Hz,
CH3CH = C(CH3)COO–); 5.77–5.80 (1H, m, H-6); 5.33–5.35 (1H, m,
H-3); 4.53 (1H, s, –OH); 4.22–4.26 (2H, m, H-2 + H-8); 3.65 (s,
1H, OH); 3.25–3.28 (1H, m, H-1); 3.09 (1H, s, –OH); 2.29 (1H, dd,
J = 4.2 Hz; 15.0 Hz, H-9); 2.09 (1H, dd, J = 2.7 Hz; 15.0 Hz, H-9);
2.03 (3H, dq, J = 1.5 Hz; 7.5 Hz, CH3CH = C(CH3)COO–); 1.93 (6H,
m, CH3CH = C(CH3)COO– + H-15); 1.54 (3H, s, H-13); 1.53 (3H, s,
–OC(CH3)(CH3)O–); 1.41 (3H, s, –OC(CH3)(CH3)O–); 1.23 (3H, s,
H-14).20
1H NMR (CDCl3) d (ppm) Guaianolide: 5.81 (br s, 1H, H-6); 5.69
(br s, 1H, H-3); 5.42 (dd, J = 3.3 and 3.6 Hz, 1H, H-2); 4.86 (s, 1H,
OH); 4.36 (br s, 1H, H-8); 4.21 (br s, 1H, H-1); 4.05 (s, 1H, OH);
3.34 (s, 1H, OH); 2.80 (dd, J = 3.0 and 14.1 Hz, 1H, H-9a); 2.50
(dd, J = 3.3 and 14.4 Hz, 1H, H-9b), 1.84 (3H, m, H-15), 1.50 (3H,
s, H-13); 1.45 (3H, s, H-14). Angeloate 6.11 (qq, J = 1.2 and
7.2 Hz, 1H, H-3), 1.98 (dq, J = 1.5 and 7.2 Hz, 3H, H-4), 1.91 (dq,
J = 1.5 Hz, 3H, Me-2), butyrate 2.10 (m, 2H, H-2), 1.55 (m, 2H,
H-3), 0.90 (t, J = 7.5 Hz, 3H, H-4). Octanoate 2.32 (m, 2H, H-2),
1.60 (m, 2H, H-3), 1.28 (m, 8H, H-4–H-7), 0.87 (t, J = 6.9 Hz, 3H,
H-8).27
5.1.4. Isopropylidene 6a
Compound 5 (385 mg, 0.85 mmol) was dissolved in dichloro-
methane (55 mL). 4-Dimethylaminopyridine (DMAP, 10 mg,
0.085 mmol) and
a solution of octanoic anhydride (368 mg,
1.36 mmol) in CH2Cl2 (15 mL) was added. The mixture was stirred
at room temperature for 1 h, and further octanoic anhydride
(368 mg, 1.36 mmol) in CH2Cl2 (2 mL) and DMAP (10 mg,
0.085 mmol) was added. The reaction mixture was stirred over
night at room temperature. Saturated NH4Cl-solution (150 mL)
and EtOAc (150 mL) was added, and the layers were separated.
The aqueous layer was extracted three times with ethyl acetate
(75 mL). The combined organic layer was dried over Na2SO4, fil-
tered and concentrated in vacuo. Compound 6a (463 mg, 94%)
was purified by column chromatography using heptane–ethyl ace-
tate (4:1) as an eluent.
5.1.7. Dibutyrate (9a) (general procedure C)
Compound 8a (44.6 mg, 0.073 mmol) was dissolved in dicho-
romethane (0.5 mL) and DMAP (1.8 mg, 0.015 mmol) and 800
lL
of a solution of butanoic anhydride (150 L) in dichloromethane
l
(5.0 mL) was added. The mixture was stirred at room temperature
for 1 h. The reaction was quenched by addition of a saturated aque-
ous ammonium chloride solution (30 mL) and ethyl acetate
(30 mL). The layers were separated and the aqueous layer was ex-
tracted three times with ethyl acetate (15 mL). The combined or-
ganic layer was dried over Na2SO4, filtered and concentrated in
vacuo. Compound 9a (44.7 mg, 90%) was purified by column chro-
matography using heptane–ethyl acetate (9:2) as an eluent.
1H NMR (CDCl3) d: Guaianloide 5.68 (m, 1H, H-3); 5.66 (m, 1H,
H-6); 5.63 (dd, J = 3.6 Hz, 1H, H-8); 5.45 (t, J = 3.3 Hz, 1H, H-2); 4.29
(br s, 1H, H-1); 3.38 (s, 1H, OH); 3.02 (dd, J = 3.0 and 14.7 Hz, 1H,
H-9a); 2.94 (s, 1H, OH); 2.20 (overlapped, H-9b), 1.87 (s, 3H,
H-15); 1.48 (s, 3H, H-13); 1.40 (s, 3H, H-14). Angeloate 6.11 (qq,
J = 1.5 and 6.9 Hz, 1H, H-3), 1.98 (dq, J = 1.5 and 7.5 Hz, 3H, H-4),
1.92 (dq, J = 1.5 Hz, 3H, Me-2), butyrate 2.10 (m, 4H, H-2), 1.55
(m, 4H, H-3), 0.94 and 0.89 (each t, J = 7.5 Hz, 3H, H-4). Octanoate
2.32 (m, 2H, H-2), 1.60 (m, 2H, H-3), 1.28 (m, 8H, H-4–H-7), 0.87
(t, J = 6.9 Hz, 3H, H-8). 13C NMR (CDCl3) d Guaianolide: 175.5
(C-12), 141.3 (C-4), 130.3 (C-5), 84.5 (C-3); 84.1 (C-10); 78.4 (C-7
or C-11); 78.3 (C-7 or C-11); 77.8 (C-2); 76.8 (C-6); 66.0 (C-8);
57.2 (C-1); 38.2 (C-9), 23.0 (C-14), 15.8 (C-13), 12.9 (C-15). Angelo-
ate 166.8 (C-1) 138.5 (C-3), 127.2 (C-2), 20.6 (Me-2), 15.9 (C-4).
Butyrate 172.4 and 172.4 (C-1), 36.5 and 34.1 (C-2), 18.1 and
17.9 (C-3), 13.7 and 13.5 (C-4). Octanoate 173.4 (C-1), 37.3 (C-2)
31.6 (C-6), 29.0 (C-4), 29.0 (C-5), 24.7 (C-3), 22.6 (C-7), 14.1
1H NMR (CDCl3) d (ppm): 5.81 (s, 2H, H-3 and H-6); 5.38 (dd,
J = 4.2 and 5.4 Hz, 1H, H-2); 4.28 (dd, J = 3.0 and 4.5 Hz, 1H, H-8);
3.44 (br s, 1H, H-1); 2.27 (1H, dd, J = 4.2 and 15.3 Hz, H-9a), 2.05
(dd, J = 3.0 and 15.3 Hz, 1H, H-9b); 1.87 (3H, d, J = 1.8 Hz, H-15),
1.54 (3H, s, H-13), 1.25 (3H, s, H-14). Angeloate 6.11 (qq, J = 1.5
and 7.5 Hz, 1H, H-3), 1.99 (dq, J = 1.5 and 6.9 Hz, 3H, H-4), 1.91
(dq, J = 1.5 Hz, 3H, Me-2), Isopropylidene:; 1.55 (s, 3H, H-3) 1.42
(s, 3H, H-1). Octanoate 2.32 (dt, J = 3.0 and 7.5 Hz, 2H, H-2), 1.60
(m, 2H, H-3), 1.28 (m, 8H, H-4–H-7), 0.87 (t, J = 6.9 Hz, 3H,
H-8).27
5.1.5. Butanoate (7a) and triol (8a) (general procedure A)
Compound 6a (50 mg, 0.085 mmol) and butanoic anhydride
(0.35 mL, 2.1 mmol) was dissolved in CH2Cl2 (6 mL). p-TsOH acid
monohydrate (49 mg, 0.26 mmol) was added and the mixture
was stirred at room temperature for 45 min. A saturated aqueous
NaHCO3-solution (20 mL) and EtOAc (20 mL) was added and the
layers were separated. The aqueous layer was extracted three
times with EtOAc (15 mL) and the combined organic layer was
washed with brine (20 mL), dried over Na2SO4, filtered and concen-
trated in vacuo. The product was purified by column chromatogra-
phy using heptanes–ethyl acetate (9:1) as an eluent to give
compound 7a (18.2 mg, 33%) and compound 8a (33 mg, 63%).
Compound (7a): colorless syrup (18.2 mg, 33%).
(C-8). ½a 2D5:2
= ꢃ31.2 (c 0.36, acetone). HRMS calcd for C36H54NaO12
ꢂ
701.3507, found 701.3496.
1H NMR (CDCl3) d: 5.78 (s, 1H, H-6); 5.74 (s, 1H, H-3); 5.46 (dd,
J = 3.9 Hz, 1H, H-2); 4.27 (br s, 1H, H-8); 3.96 (br s, 1H, H-1); 3.29
(s, 1H, OH); 2.73 (dd, J = 3.9 and 14.7 Hz, 1H, H-9a); 2.66 (dd, J = 3.0
and 14.7 Hz, 1H, H-9b), 1.87 (s, 3H, H-15) 1.56 (s, 3H, H-13), 1.47 (s,
3H, H-14). Angeloate 6.10 (qq, J = 1.5 and 7.5 Hz, 1H, H-3), 1.98 (dq,
J = 1.5 and 7.2 Hz, 3H, H-4), 1.91 (dq, J = 1.5 Hz, 3H, Me-2), butyrate
5.1.8. Dioctanoate (7c)
Synthesized according to the general procedure A using isopro-
pylidene (6a) (50 mg, 0.085 mmol) and octanoic anhydride (27 mg,
0.100 mmol) as starting materials. Compound 7c was purified by

5642
D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
column chromatography using heptane–ethyl acetate (12:1) as an
eluent. The product was slightly contaminated with octanoic
anhydride.
1H NMR (CDCl3) d Guaianolide: 5.75 (br s, 1H, H-6); 5.67 (br s,
1H, H-3); 5.49 (t, J = 3.6 Hz, 1H, H-2); 4.71 (s, 1H, OH); 4.30 (br s,
1H, H-1); 4.21 (t, J = 3.0 Hz, 1H, H-8); 3.90 (s, 1H, OH); 3.00 (s,
1H, OH); 2.82 (dd, J = 2.4 and 13.5 Hz, 1H, H-9a), 2.20 (overlapped,
H-9), 1.82 (s, 3H, H-15), 1.42 (s, 3H, H-13); 1.35 (s, 3H, H-14) l).
Angeloate 6.11 (qq, J = 1.2 and 7.5 Hz, 1H, H-3), 1.99 (dq, J = 1.5
and 7.5 Hz, 3H, H-4), 1.91 (q, J = 1.5 Hz, 3H, Me-2). Biphenylace-
tate: 7.48–7.59 (m, 4H, H-8, H-9, H-11 and H-12); 7.38–7.43 (m,
2H, H-3 and H-5); 7.34 (m, 1H, H-10); 7.27 (m, 2H, H-2 and H-6),
1H NMR (CDCl3) d Guaianolide: 5.79 (1H, br s, H-6), 5.75 (br s,
1H, H-3), 5.46 (dd, J = and 5.4 Hz, 1H, H-2), 4.27 (dd, J = 3.6 Hz,
1H, H-8), 3.94 (br s, 1H, H-1), 2.70 (d, J = 3.6 Hz, 2H, H-9a), 1.87
(s, 3H, H-15, 1.56 (s, 3H, H-13) 1.47 (3H, s, H-14). Angeloate 6.10
(qq, J = 1.5 and 7.2 Hz, 1H, H-3), 1.99 (dq, J = 1.5 and 7.2 Hz, 3H,
H-4), 1.92 (m, 3H, Me-2. Isopropylidene 1.53 (s, 3H, H-1), 1.42 (s,
3H, H-3). Octanoate 2.32 and 2.12 (m, each 2H, H-2), 1.60 (m,
4H, H-3), 1.28 (m, 16H, H-4–H-7), 0.88 (t, J = 6.9 Hz, 6H, H-8).
3.55 (d, J = 15.6 Hz, 1H, H-aa); 3.47 (d, J = 15.6 Hz, 1H, H-ab). Octa-
noate 2.32 (m, 2H, H-2), 1.60 (m, 2H, H-3), 1.28 (m, 8H, H-4–H-7),
0.83 (t, J = 6.9 Hz, 6H, H-8).
5.1.9. Triol (8c)
Synthesized according to the general procedure B using dioct-
anoate (7c) as starting material. Compound 8c (40.4 mg, 70%)
was purified by column chromatography using heptanes–ethyl
acetate (3:1) as an eluent.
5.1.13. Biphenylacetate (9d)
Synthesized according to the general procedure C using triol 8d
(26.9 mg, 0.037 mmol) as
a starting material. Compound 9d
(18.5 mg, 63%) was purified by column chromatography using hep-
tane–ethyl acetate (7:1) as an eluent.
1H NMR (CDCl3) d Guaianolide: 5.81 (br s, 1H, H-6); 5.68 (br s,
1H, H-3); 5.42 (dd, J = 3.3 Hz, 1H, H-2); 4.81 (s, 1H, OH); 4.35 (br
s, 1H, H-8); 4.20 (br s, 1H, H-1); 4.02 (s, 1H, OH); 3.18 (s, 1H,
OH); 2.81 (dd, J = 3.0 and 14.4 Hz, 1H, H-9a), 2.48 (dd, J = 3.3 and
14.4 Hz, 1H, H-9b), 1.84 (s, 3H, H-15), 1.50 (s, 3H, H-13); 1.44 (s,
3H, H-14). Angeloate 6.10 (qq, J = 1.5 and 7.5 Hz, 1H, H-3), 1.98
(dq, J = 1.5 and 7.2 Hz, 3H, H-4), 1.91 (t, J = 1.5 Hz, 3H, Me-2). Octa-
noate 2.32 and 2.12 (m, each 2H, H-2), 1.60 (m, 4H, H-3), 1.28 (m,
16H, H-4–H-7), 0.87 (t, J = 6.9 Hz, 6H, H-8).
1H NMR (CDCl3) d: 5.68 (s, 1H, H-3), 5.63 (s, 1H, H-6), 5.57 (t,
J = 3.6 Hz, 1H, H-8), 5.53 (dd, J = 3.0 Hz, 1H, H-2), 4.32 (br s, 1H,
H-1), 3.07 (s, 1H, OH); 2.97 (dd, J = 3.0 and 15.0 Hz, 1H, H-9a),
2.73 (s, 1H, OH), 1.86 (s, 3H, H-15), 1.42 (s, 3H, H-13); 1.41 (s,
3H, H-14). Angeloate 6.11 (qq, J = 1.2 and 7.5 Hz, 1H, H-3), 2.01
(dq, J = 1.5 and 7.5 Hz, 3H, H-4), 1.92 (q, J = 1.5 Hz, 3H, Me-2). Biph-
enylacetate: 7.51–7.58 (m, 4H, H-8, H-9, H-11 and H-12); 7.39–
7.45 (m, 2H, H-3 and H-5); 7.33 (m, 1H, H-10), 3.54 (d,
J = 15.9 Hz, 1H, H-aa); 3.46 (d, J = 15.9 Hz, 1H, H-ab). Butyrate
5.1.10. Octanoate (9c)
2.20 (m, 2H, H-2), 1.55 (m, 2H, H-3), 0.91 (t, J = 7.5 Hz, 3H, H-4).
Octanoate 2.32 (m, 2H, H-2), 1.60 (m, 2H, H-3), 1.28 (m, 8H,
H-4–H-7), 0.84 (t, J = 6.9 Hz, 6H, H-8). 13C NMR (CDCl3) d: 175.0
(C-12), 142.0 (C-4), 129.6 (C-5), 85.0 (C-10), 84.2 (C-3), 78.5 (C-7
and C-11), 77.7 (C-2), 77.4 (C-6), 65.9 (C-8), 58.0 (C-1), 38.2
(C-9), 23.0 (C-13), 16.1 (C-14), 13.1 (C-15). Angeloate 166.9 (C-1),
139.8 (C-3), 127.3 (C-2), 20.7 (Me-2), 15.9 (C-4). Biphenylacetate
171.2 (C@O), 140.6 (C-4), 138.8 (C-7), 132.7 (C-1), 130.2 (C-2 and
Synthesized according to the general procedure C using triol 8c
(40.4 mg, 0.061 mmol) as starting material. Compound 9c
(44.0 mg, 98%) was purified by column chromatography using hep-
tane–ethyl acetate (5:1) as an eluent.
1H NMR (CDCl3) d: Guaianloide 5.68 (br s, 1H, H-3); 5.66 (br s,
1H, H-6); 5.63 (t, J = 3.9 Hz, 1H, H-8); 5.45 (t, J = 3.3 Hz, 1H, H-2);
4.28 (br s, 1H, H-1); 3.28 (s, 1H, OH); 3.00 (t, J = 3.3 and 14.7 Hz,
1H, H-9a), 2.91 (s, 1H, OH); 2.20 (overlapped, H-9), 1.87 (s, 3H,
H-15); 1.57–1.71 (4H, m, –OCOCH2CH2-2-octanoyl and butanoyl);
1.49 (3H, s, H-13); 1.45–1.49 (2H, m, –OCOCH2CH2-10-octanoyl);
1.40 (3H, s, H-14). Angeloate 6.11 (qq, J = 1.2 and 7.2 Hz, 1H, H-
3), 1.99 (dq, J = 1.5 and 7.5 Hz, 3H, H-4), 1.92 (dq, J = 1.5 Hz, 3H,
Me-2), Butyrate 2.10 (m, 4H, H-2), 1.55 (m, 4H, H-3), 0.95 (t,
J = 7.5 Hz, 3H, H-4). Octanoate 2.32 and 2.10 (each m, 2H, H-2),
1.60 (m, 4H, H-3), 1.28 (m, 8H, H-4–H-7), 0.87 (t, J = 6.9 Hz, 6H,
H-8).
C-6), 128.6 (C-3 and C-5), 127.2 and 126.9 (C-8–C12), 41.9 (C-a).
Butyrate 172.5 (C-1), 36.5 (C-2), 18.0 (C-3), 13.8 (C-4). Octanoate
172.3 (C-1), 34.3 (C-2) 31.7 (C-6), 29.1 (C-4), 29.1 (C-5), 24.9
+
(C-3), 22.7 (C-7), 14.2 (C-8). HRMS calcd for [C46H58NaO12
825.3820, found 825.3806.
]
5.1.14. Tetraol (10)
Synthesized according to the general procedure B using isopro-
pylidene 6a (50 mg, 0.086 mmol) as starting material. Compound
10 (41.4 mg, 89%) was purified by column chromatography using
heptane–ethyl acetate (1:2) as an eluent.
13C NMR (CDCl3) d: Guaianloide 175.4 (C-12), 141.3 (C-5), 130.3
(C-4), 84.3 (C-10), 84.1 (C-3), 78.5 (C-7 or C-11), 78.4 (C-7 or C-11),
77.8 (C-2), 76.7 (C-6), 66.0 (C-8), 57.4 (C-1), 38.2 (C-9), 22.9 (C-14),
16.0 (C-13), 12.9 (C-15). Angeloate 166.9 (C-1) 138.5 (C-3), 127.2
(C-2), 20.6 (Me-2), 15.8 (C-4). Butyrate 172.4 (C-1), 36.5 (C-2),
18.0 (C-3), (C-4). Octanoate 173.4 and 172.4 (C-1), 35.4 and 34.1
(C-2) 31.7 (C-6), 29.1 and 29.0 (C-4), 29.0 and 28.9 (C-5), 24.7
1H NMR (CDCl3) d: 5.80 (br s, 1H, H-6); 5.73 (br s, 1H, H-3); 5.37
(s, 1H, OH); 5.23 (t, J = 3.0 Hz, 1H, H-2), 4.28 (s, 2H, H-8 and OH),
3.79 (s, 1H, OH); 3.51 (br s, 1H, H-1); 3.43 (s, 1H, OH); 2.19 (dd,
J = 1.2 and 13.5 Hz, 1H, H-9a); 2.04 (dd, J = 1.2 and 13.5 Hz, 1H, H-
9b), 1.86 (s, 3H, H-15); 1.47 (s, 3H, H-13), 1.20 (s, 3H, H-14). Ange-
loate 6.14 (qq, J = 1.2 and 7.2 Hz, 1H, H-3), 1.98 (dq, J = 1.5 and
7.5 Hz, 3H, H-4), 1.89 (q, J = 1.5 Hz, 3H, Me-2). Octanoate 2.32 (m,
2H, H-2), 1.60 (m, 2H, H-3), 1.28 (m, 8H, H-4–H-7), 0.86 (t,
J = 6.9 Hz, 6H, H-8). 13C NMR (CDCl3) d Guaianolide: 61.9 (C-1),
78.8 (C-2), 83.5 (C-3), 141.1 (C-4), 131.3 (C-5), 76.7 (C-6), 78.6 (C-
7), 66.3 (C-8), 44.9 (C-9), 72.8 (C-10), 78.6 (C-11), 174.9 (C-12),
16.4 (C-13), 24.8 (C-14), 13.1 (C-15). Angeloate: 166.9 (C-1), 127.1
(C-2), 139.4 (C-3), 16.0 (C-4), 20.0 (Me-2). Butyrate: 172.4 (C-1),
36.6 (C-2), 18.1 (C-3), 13.9 (C-4). Octanoate: 173.8 (C-1), 34.5 (C-
2), 25.0 (C-3), 29.0 (C-4), 29.0 (C-5), 31.7 (C-6), 22.7 (C-7), 14.2
(C-8). HRMS calcd for [C32H48NaO11]+: 631.3089, found 631.3073.
(C-3), 22.6 and 22.6 (C-7), 14.1 (C-8). ½a D26:2
= ꢃ32.2 (c 0.35, ace-
ꢂ
tone). HRMS calcd for C40H62NaO12 757.4133, found 757.4126.
5.1.11. Biphenylacetate (7d)
Synthesized according to the general procedure A using isopro-
pylidene 6a (65.2 mg, 0.113 mmol) and 4-biphenylacetic anhy-
dride as starting materials. The reaction mixture was stirred at
room temperature over night. The crude reaction product was used
in the next step.
5.1.12. Triol (8d)
Synthesized according to the general procedure B using 4-biph-
enylacetate (7d) as starting material. Compound 8d (26.9 mg, 33%)
was purified by column chromatography using heptane–ethyl ace-
tate (7:1?5:1) as an eluent.
5.1.15. Triol (11a)
Synthesized according to the general procedure C using tetraol
10 (41.4 mg, 0.077 mmol) as starting material. Compound 11a

D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
5643
(38.2 mg, 82%) was purified by column chromatography using
heptane–ethyl acetate (3:1) as an eluent.
and C-6), 128.2 (C-3 and C-5). Butyrate 172.3 (C-1), 36.6 (C-2),
18.0 (C-3), 13.7 (C-4). Octanoate 172.5 (C-1), 33.6 (C-2) 31.5 (C-
1H NMR (CDCl3) d Guaianolide: 5.73 (s, 1H, H-6); 5.68 (s, 1H,
H-3); 5.55 (dd, J = 3.3 Hz, 1H, H-8); 5.24 (dd, J = 2.4 Hz, 1H, H-2),
3.76 (s, 1H, OH), 3.57 (br s, 1H, H-1), 3.40 (s, 1H, OH), 3.05 (s, 1H,
OH), 2.35 (overlapped, H-9a), 1.97 (dd, J = 3.9 and 11.4 Hz, 1H, H-
9b), 1.88 (s, 3H, H-15), 1.47 (s, 3H, H-13), 1.16 (s, 3H, H-14). Ange-
loate 6.14 (qq, J = 1.5 and 7.2 Hz, 1H, H-3), 2.00 (dq, J = 1.8 and
6.9 Hz, 3H, H-4), 1.90 (q, J = 1.5 Hz, 3H, Me-2). Butyrate 2.26 (t,
J = 7.5 Hz, 2H, H-2), 1.60 (m, 2H, H-3), 0.93 (t, J = 7.5 Hz, 3H, H-4).
Octanoate 2.32 (m, 2H, H-2), 1.60 (m, 2H, H-3), 1.28 (m, 8H, H-
4–H-7), 0.87 (t, J = 6.9 Hz, 6H, H-8). 13C NMR (CDCl3) d: Guaiano-
lide: 61.9 (C-1), 78.8 (C-2), 83.5 (C-3), 141.1 (C-4), 131.3 (C-5),
76.7 (C-6), 78.6 (C-7), 66.3 (C-8), 44.9 (C-9), 72.8 (C-10), 78.6 (C-
11), 174.9 (C-12), 16.4 (C-13), 24.8 (C-14), 13.1 (C-15). Angeloate:
166.9 (C-1), 127.1 (C-2), 139.4 (C-3), 16.0 (C-4), 20.0 (Me-2). Buty-
rate: 172.4 (C-1), 36.6 (C-2), 18.1 (C-3), 13.9 (C-4). Octanoate:
6), 28.8 (C-4), 28.2 (C-5), 24.2 (C-3), 22.6 (C-7), 14.1 (C-8). HRMS
+
calcd for [C39H52NaO12
]
735.3351, found 735.3330.
5.1.18. 4-Phenylbenzoate (12b)
To a solution of 11a (20 mg, 33 lmol) and DMAP (10 mg,
82
lmol) in dichloromethane (1 mL) was added 4-phenylbenzoic
anhydride (110 mg, 290
l
mol). The solution was stirred at room
temperature overnight, filtered and concentrated. Compound 12b
(6 mg, 25%) was isolated from the residue by column chromatogra-
phy over silica gel using toluene–ethyl acetate (3:1) added acetic
acid (0.5%) as an eluent. 1H NMR data (CDCl3): d Guaianolide:
4.70 (br s, 1H, H-1), 5.56 (dd, J = 4.3 and 3.8 Hz, 1H, H-2), 5.76
(br s, 1H, H-3), 5.76 (s, 1H, H-6), 5.79 (overlapped, 1H, H-8), 3.65
(dd, J = 13.9 and 2.06 Hz, 1H, H-9a), 2.22 (dd, J = 13.9 and 4.1 Hz,
1H, H-9b), 1.55 (s, 3H, H-13), 1.59 (s, 3H, H-14), 1.93 (br s, 1H,
H-15). Angeloate: 6.10 (dq, J = 7.3 and 1.5 Hz, 1H, H-3), 1.99 (dd,
J = 7.4 and 2.3 Hz, 3H, H-4), 1.93 (br s, 1H, 3H, Me-2). Butanoate:
2.32 (t, 7.3 Hz,, 2H, H-2), 1.66 (overlapped, 2H, H-3), 0.97 (t,
J = 7.2 Hz, 3H, H-4). Octanoate: 2.32 (overlapped, 2H, H-2), 1.61
(overlapped, 2H, H-3), 1.28 (br s, 8H, H-4–H-7), 0.78 (t, J = 7.0 Hz,
3H, H-8). 4-Phenylbenzoate 8.10 (m, 2H, H2 and H6), 7.61 (m,
2H, H-3 and H-5), 7.61 (m, 2H, H-8 and H-12), 7.46 (m, 3H, H-9,
H-10 and H-11). 13C NMR (CDCl3) d Guaianolide: 54.7 (C-1), 78.4
(C-2), 83.9 (C-3), 141.6 (C-4), 138.4 (C-5), 77.2 (C-6), 78.6 (C-7),
66.3 (C-8), 38.5 (C-9), 85.4 (C-10), 78.6 (C-11), 175.4 (C-12), 15.8
(C-13), 23.7 (C-14), 13.0 (C-15). Angeloate: 165.7 (C-1), 129.3 (C-
2), 139.7 (C-3), 16.2 (C-4), 20.6 (Me-2). Butyrate: 172.3 (C-1),
33.8 (C-2), 18.1 (C-3), 13.8 (C-4). Octanoate: 172.8 (C-1), 36.6 (C-
2), 24.3 (C-3), 29.0 (C-4), 29.7 (C-5), 31.6 (C-6), 22.5 (C-7), 13.7
(C-8). 4-Phenylbenzoate 167.0 (C@O), 129.1 (C-1), 128.9 (C-2 and
C-6), 127.1 (C-3 and C-5), 145.8 (C-4), 129.3 (C-7), 127.2 (C-8
and H-12), 127.5 (H-9 and H-11), 130.0 (H-10). HRMS calcd for
173.8 (C-1), 34.5 (C-2), 25.0 (C-3), 29.0 (C-4), 29.0 (C-5), 31.7 (C-
+
6), 22.7 (C-7), 14.2 (C-8). HRMS calcd for
631.3089, found 631.3073.
C
32H48NaO11
:
5.1.16. Dioctanoate (11b)
Synthesized according to the general procedure C using tetraol
10 (41.4 mg, 0.077 mmol) as starting material. Compound 11b
(40 mg, 80%) was purified by column chromatography using hep-
tane–ethyl acetate (3:1) as an eluent. 1H NMR data (CDCl3): d Guai-
anolide: 3.56 (br s, 1H, H-1), 5.24 (br s, 1H, H-2), 5.74 (br s, 1H, H-
3), 5.68 (s, 1H, H-6), 5.55 (bf s, 1H, H-8), 2.30 (overlapped, 1H, H-
9a), 1.96 (overlapped, 1H, H-9b), 1.48 (s, 3H, H-13), 1.17 (s, 3H,
H-14), 1.90 (br s, 1H, H-15), 3.77, 3.24, 2.89 (each s, 1H, OH). Ange-
loate: 6.16 (dq, J = 7.0 and 1.5 Hz, 1H, H-3), 2.02 (overlapped, 3H,
H-4), 1.99 (br s, 1H, 3H, Me-2). 2 times octanoate: 2.35 and 2.28
(overlapped, 4H, H-2), 1.61 and 1.59 (overlapped, 4H, H-3), 1.28
(br s, 16H, H-4–H-7), 0.88 (t, J = 7.5 Hz, 6H, H-8). 13C NMR (CDCl3)
d Guaianolide: 61.6 (C-1), 79.1 (C-2), 83.5 (C-3), 140.5 (C-4), 131.5
(C-5), 76.9 (C-6), 78.6 (C-7), 66.3 (C-8), 44.6 (C-9), 72.9 (C-10), 78.6
(C-11), 175.3 (C-12), 16.3 (C-13), 24.9 (C-14), 13.0 (C-15). Angelo-
ate: 167.0 (C-1), 127.1 (C-2), 139.5 (C-3), 16.0 (C-4), 20.7 (Me-2).
Two times octanoate: 175.0 and172.8 (C-1), 35.0 and 34.9 (C-2),
24.8 and 24.6 (C-3), 29.1 and 29.0 (C-4), 29.2 (C-5), 31.7 (C-6),
22.7 (C-7), 14.2 (C-8), HRMS calcd for C36H56NaO11+: 687.3715,
found 687.3693.
C
45H56NaO12+: 811.3664, found 811.3638.
5.1.19. Compound (6b)
To a solution of 5 (53 mg, 0.12 mmol) and DMAP (23 mg,
0.19 mmol) in DCM (9 mL) was added a solution of benzoic anhy-
dride (81.5 mg, 0.36 mmol) in dichloromethane (1 mL). After stir-
ring for 20 h a saturated aqueous solution of ammonium chloride
(15 mL) and ethyl acetate (15 mL) was added. The aqueous phase
was extracted two times with ethyl acetate (15 mL) and the com-
bined organic phases were dried (sodium sulfate) and concen-
trated. Compound 6b (31 mg, 48%) was isolated by column
chromatography over LiChroprep RP-18 using methanol–water
(4:1) as an eluent. 1H NMR data (CDCl3): d Guaianolide: 3.56 (br
s, 1H, H-1), 5.58 (br t, J = 4.5 Hz, 1H, H-2), 5.98 (br s, 1H, H-3),
5.83 (s, 1H, H-6), 4.19 (br t, J = 3 Hz, 1H, H-8), 2.20 (dd, J = 14 and
2 Hz, H-9a), 2.03 (dd, J = 14 and 2 Hz, 1H, H-9b), 1.28 (s, 3H,
H-13), 1.52 (s, 3H, H-14), 1.93 (br s, 1H, H-15), 3.22 (1H, OH). Ange-
loate: 6.11 (q, J = 7.1 Hz, 1H, H-3), 2.00 (d, J = 7 Hz, 3H, H-4), 1.96
(br s, 1H, 3H, Me-2). Benzoate: 8.02 (m, 2H, H-2 and H-6), 7.54
(m, 1H, H-4), 7.40 (m, 2H, H-3 and H-5), Isopropanylen: 1.51 (s,
5.1.17. Benzoate (12a)
Synthesized according to the general procedure A using triol
(11a) (38.2 mg, 0.063 mmol) as starting material and stirring for
10 days. Compound 12 (17.3 mg, 39%) was purified by column
chromatography over silica gel using heptane–ethyl acetate (4:1)
as an eluent followed by column chromatography over LiChrosorp
RP-18 using methanol–water (6:1) as an eluent.
1H NMR (CDCl3) d: Guaianloide 5.74–5.77 (overlapped, 3H, H-3,
H-6 and H-8), 5.52 (dd, J = 3.9 and 4.8 Hz, 1H, H-2), 4.68 (br s, 1H,
H-1), 3.63 (dd, J = 3.0 and 15.0 Hz, 1H, H-9a); 3.38 (s, 1H, OH), 3.15
(s, 1H, OH), 2.18 (dd, J = 3.6 and 14.7 Hz, 1H, H-9b), 1.87 (s, 3H, H-
15), 1.56 (s, 3H, H-14); 1.52 (s, 3H, H-13). Angeloate 6.07 (qq,
J = 1.2 and 7.2 Hz, 1H, H-3), 1.97 (dq, J = 1.5 and 7.2 Hz, 3H, H-4),
1.92 (q, J = 1.5 Hz, 3H, Me-2). Benzoate 7.87 (m, 2H, H-2 and H-
6), 7.52 (m, 1H, H-4); 7.39 (m, 2H, H-3 and H-5). Butyrate 2.32
(t, J = 7.2 Hz, 2H, H-2), 1.60 (m, 2H, H-3), 0.95 (t, J = 7.2 Hz, 3H,
H-4). Octanoate 2.00 (m, 2H, H-2), 1.60 (m, 2H, H-3), 1.20 (m,
8H, H-4–H-7), 0.85 (t, J = 7.2 Hz, 6H, H-8).
3H, Me-b), 1.42 (s, 3H, Me-a).
5.1.20. Compound (7b)
To a solution of 6b (29 mg, 52 mmol) in isopropenyl acetate
(2 mL) was added p-toluenesulfornic acid (30 mg). After 2 h a solu-
tion of sodium hydrogen carbonate (M, 15 mL) and ethyl acetate
(15 mL) was added. The aqueous phase was extracted two times
with ethyl acetate (15 mL) and the combined organic phases dried
and concentrated. Compound 7b (25 mg, 79%) was isolated from
the residue by column chromatography over Sigel using toluene–
ethyl acetate (3:1) as an eluent. 1H NMR data (CDCl3): d Guaiano-
lide: 4.17 (br s, 1H, H-1), 5.74 (br t, J = 6 Hz, 1H, H-2), 5.91 (br s,
13C NMR (CDCl3) d Guaianolide: 175.2 (C-12), 141.4 (C-4), 129.0
(C-5), 85.3 (C-10); 83.3 (C-3); 78.5 (C-7 and C-11); 78.3 (C-2); 77.1
(C-6); 66.1 (C-8); 54.6 (C-1); 38.4 (C-9), 23.7 (C-13), 16.1 (C-14),
13.0 (C-15). Angeloate 166.7 (C-1), 138.2 (C-3), 127.3 (C-2), 20.6
(Me-2), 15.7 (C-4). Benzoate 165.6 (C@O), 137.9 (C-4), 129.3 (C-2

5644
D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
1H, H-3), 5.83 (s, 1H, H-6), 4.25 (br t, J = 3 Hz, 1H, H-8), 2.81 (dd,
J = 14 and 2 Hz, H-9a), 2.57 (dd, J = 14 and 2 Hz, 1H, H-9b), 1.53
(s, 3H, H-13), 1.43 (s, 3H, H-14), 1.91 (br s, 1H, H-15), 3.51 (1H,
OH). Acetate: 1.61 (s, 3H, H-2). Angeloate: 6.1 (qq, J = 6.1 and
1.5 Hz, 1H, H-3), 2.00 (d, J = 7 Hz, 3H, H-4), 1.96 (br s, 1H, 3H,
Me-2). Benzoate: 8.03 (m, 2H, H-2 and H-6), 7.56 (m, 1H, H-4),
7.42 (m, 2H, H-3 and H-5), Isopropanylen: 1.51 (s, 3H, Me-b),
eluent to give compound 8a (14 mg, 16%), compound 14 (16 mg,
17%), and compound 13a (14 mg, 16%).
Compound 14: 1H NMR data (CDCl3): d Guaianolide: 4.25 (br s,
1H, H-1), 5.43 (dd, J = 3.5 and 2.1 Hz, 1H, H-2), 5.66 (br s, 1H, H-3),
5.66 (s, 1H, H-6), 5.48 (t, J = 3.6 Hz, 1H, H-8), 2.87 (dd, J = 14.9 and
3.5 Hz, 1H, H-9a), 2.49 (dd, J = 14.9 and 3.5 Hz, 1H, H-9b), 1.67 (s,
3H, H-13), 1.36 (s, 3H, H-14), 1.88 (br s, 1H, H-15), 3.50 (s, 1H,
OH). Angeloate: 6.11 (dq, J = 7.2 and 1.3 Hz, 1H, H-3), 2.00 (dd,
J = 7.2 and 1.3 Hz, 3H, H-4), 1.91 (br s, 1H, 3H, Me-2). Three times
butyrate: 2.16–2.35 (overlapped, 6H, H-2), 1.75–1.45 (overlapped,
6H, H-3), 1.03–0.90 (t, overlapped, 9H, H-4). Octanoate: 2.10 (over-
lapped, 2H, H-2), 1.61 (overlapped, 2H, H-3), 1.28 (br s, 8H, H-4–H-
7), 0.90 (t, J = 7.5 Hz, 3H, H-8). 13C NMR (CDCl3) d Guaianolide: 57.9
(C-1), 77.2 (C-2), 83.8 (C-3), 142.0 (C-4), 129.5 (C-5), 77.8 (C-6),
78.9 (C-7), 67.6 (C-8), 37.4 (C-9), 84.0 (C-10), 82.0 (C-11), 173.0
(C-12), 18.4 (C-13), 20.7 (C-14), 13.1 (C-15). Angeloate: 166.9 (C-
1), 129.5 (C-2), 138.7 (C-3), 16.0 (C-4), 22.7 (Me-2). Three times
butyrate: 172.3, 172.2, 170.9 (C-1), 36.1, 36.0, 34.3 (C-2), 18.4,
18.4, 18.1, (C-3), 13.8, 13.7, 13.5 (C-4). Octanoate: 172.3 (C-1),
37.4 (C-2), 24.8 (C-3), 29.2 (C-4), 29.2 (C-5), 31.7 (C-6), 22.7 (C-
7), 14.2 (C-8), HRMS calcd for C40H60NaO13+: 771.3926, found
771.3912.
1.43 (s, 3H, Me-a).
5.1.21. Compound (8b)
To a solution of 7b (25 mg, 0.41 mmol) in methanol (1.5 mL)
was added 75 lmol hydrogen chloride and the solution was left
for 2 h at 45 °C. The reaction mixture was added a saturated aque-
ous solution of sodium acetate (10 mL) and ethyl acetate (10 mL).
The aqueous phase was three times extracted with ethyl acetate
(10 mL) and the combined organic phases concentrated and dried
(sodium sulfate). Compound 8b (17 mg, 74%) was isolated by col-
umn chromatography over LiChroprep RP-18 using methanol–
water (3:1) as an eluent. 1H NMR data (CDCl3): d Guaianolide:
4.40 (br s, 1H, H-1), 5.72 (br t, J = 4.5 Hz, 1H, H-2), 5.90 (br s, 1H,
H-3), 5.84 (s, 1H, H-6), 4.34 (br t, J = 3 Hz, 1H, H-8), 2.90 (dd,
J = 15 and 3 Hz, H-9a), 2.47 (dd, J = 15 and 3 Hz, 1H, H-9b), 1.48
(s, 3H, H-13), 1.51 (s, 3H, H-14), 1.88 (br s, 1H, H-15), 4.07 and
3.33 (each 1H, OH). Acetate: 1.70 (s, 3H, H-2). Angeloate: 6.12
(qq, J = 6.1 and 1.5 Hz, 1H, H-3), 1.99 (d, J = 7 Hz, 3H, H-4), 1.95
(br s, 1H, 3H, Me-2). Benzoate: 8.01 (m, 2H, H-2 and H-6), 7.55
(m, 1H, H-4), 7.42 (m, 2H, H-3 and H-5).
Compound 13a: 1H NMR data (CDCl3): d Guaianolide: 3.70 (br s,
1H, H-1), 5.42 (br t, J = 4.3 Hz, 1H, H-2), 5.77 (br s, 1H, H-3), 5.94 (s,
1H, H-6), 5.04 (br t, J = 3.5 Hz, 1H, H-8), 2.66 (d, J = 3.5 Hz, 2H, H-9),
1.56 (s, 3H, H-13), 1.49 (s, 3H, H-14), 1.90 (br s, 1H, H-15). Angelo-
ate: 6.10 (dq, J = 7.2 and 1.4 Hz, 1H, H-3), 2.00 (dd, J = 7.4 and
1.2 Hz, 3H, H-4), 1.92 (br s, 1H, 3H, Me-2). Two times butyrate:
2.39 and 2.08 (overlapped, 4H, H-2), 1.8–1.5 (overlapped, 4H, H-
3), 0.95 (t, J = 7.2 Hz, 6H, H-4). Octanoate: 2.28 (overlapped, 2H,
H-2), 1.61 (overlapped, 2H, H-3), 1.28 (br s, 8H, H-4–H-7), 0.88
(t, J = 7.5 Hz, 3H, H-8). Isopropanylen: 1.58 (s, 3H, Me-b), 1.43 (s,
5.1.22. Compound (9b)
To a solution of 8b (17 mg, 0.30 mmol) and DMAP (9 mg,
7 mmol) in DCM (5 mL) was added butanoic anhydride (10 mg,
0.70 mmol) dissolved in dichloromethane (1 mL). After stirring
for 75 min a saturated solution of ammonium chloride (15 mL)
and ethyl acetate (15 mL) was added. The aqueous phase was ex-
tracted two times with ethyl acetate (15 mL) and the combined or-
ganic phases were dried (sodium sulfate) and concentrated.
Compound 9b (13 mg, 66%) was isolated by column chromatogra-
phy over LiChroprep RP-18 using methanol–water (5:1) as an elu-
ent: 1H NMR data (CDCl3): d Guaianolide: 4.48 (br s, 1H, H-1), 5.76
(br t, J = 4 Hz, 1H, H-2), 5.90 (br s, 1H, H-3), 5.71 (s, 1H, H-6), 5.66
(br t, J = 2 Hz, 1H, H-8), 3.14 (dd, J = 14 and 2 Hz, H-9a), 2.31 (dd,
J = 14 and 2 Hz, 1H, H-9b), 1.47 (s, 3H, H-13), 1.48 (s, 3H, H-14),
1.86 (br s, 1H, H-15), 2.80 and 3.51 (each 1H, OH). Angeloate:
6.12 (q, J = 7.1 Hz, 1H, H-3), 2.00 (d, J = 7 Hz, 3H, H-4), 1.96 (br s,
1H, 3H, Me-2). Acetate: 1.90 (s, 3H, H-2), Benzoate: 8.02 (m, 2H,
H-2 and H-6), 7.42 (m, 2H, H-3 and H-5), 7.51 (m, 1H, H-4). Buty-
rate: 2.28 (t, J = 7.5 Hz, 2H, H-2), 1.63 (sex, J = 7.5 Hz, 2H, H-3), 0.95
(t, J = 7.5 Hz, 3H, H-4). 13C NMR (CDCl3) d Guaianolide: 56.7 (C-1),
78.9 (C-2), 83.9 (C-3), 141.8 (C-4), 129.8 (C-5), 77.4 (C-6), 78.9
(C-7), 66.4 (C-8), 38.6 (C-9), 84.9 (C-10), 79.1 (C-11), 175.5
(C-12), 16.6 (C-13), 23.3 (C-14), 13.4 (C-15). Angeloate: 167.2 (C-
1), 133.3 (C-2), 138.7 (C-3), 16.2 (C-4), 20.9 (Me-2). Acetate:
171.1 (C.-1), 22.8 (C-2). Butyrate: 172.8 (C-1), 36.9 (C-2), 18.3 (C-
3), 14.1 (C-4). Benzoate: 165.7 (C-1), 127.7 (C-2), 129.9 (C-3 and
C-7), 128.6 (C-4 and C-6), 133.3 (C-5). HRMS calcd for
3H, Me-a
). 13C NMR (CDCl3) d Guaianolide: 56.8 (C-1), 78.5 (C-2),
83.7 (C-3), 138.5 (C-4), 127.4 (C-5), 76.4 (C-6), 83.6 (C-7), 63.6
(C-8), 38.9 (C-9), 84.0 (C-10), 78.0 (C-11), 172.6 (C-12), 17.2 (C-
13), 22.6 (C-14), 12.5 (C-15). Angeloate: 167.2 (C-1), 127.4 (C-2),
138.5 (C-3), 15.8 (C-4), 20.6 (Me-2). Two times butyrate: 171.1
and 171.1 (C-1), 37.3 and 36.4 (C-2), 18.3 and 18.1 (C-3), 13.6 (C-
4). Octanoate: 172.4 (C-1), 34.3 (C-2), 24.7 (C-3), 29.0 (C-4), 29.1
(C-5), 31.7 (C-6), 20.6 (C-7), 14.2 (C-8). Isopropanylen: 101.2 (C-
2) 30.3 (C-1), 22.0 (C-3), HRMS calcd for C39H58NaO12+: 741.3820,
found 741.3797.
5.1.24. Compound (13b)
To a solution of 5 (42 mg, 73 lmol) in isopropenyl octanoate
(2 mL) is added p-TsOH acid (39 mg, 0.22 mmol) and the solution
is left for 70 h. The reaction was quenched with a saturated aque-
ous solution of sodium hydrogen carbonate (15 mL). The mixture
was extracted three times with ethyl acetate (15 mL) and the or-
ganic phases were combined, dried (sodium sulfate) and concen-
trated. Compound 13b (27 mg, 445 lmol) was isolated from the
residue by column chromatography over LiChroprep RP-18 using
methanol–water (25:1) as an eluent.
1H NMR data (CDCl3): d Guaianolide: 3.71 (br s, 1H, H-1), 5.42
(br s, 1H, H-2), 5.78 (br s, 1H, H-3), 5.94 (s, 1H, H-6), 5.06 (br s,
1H, H-8), 2.66 (m, 1H, H-9a), 2.46 (dd, J = 14.9 and 3.6 Hz, 1H, H-
9b), 1.58 (s, 3H, H-13), 1.43 (s, 3H, H-14), 1.89 (br s, 1H, H-15).
Angeloate: 6.09 (dq, J = 7.0 and 1.5 Hz, 1H, H-3), 1.99 (qd, J = 7.0
and 1.5 Hz, 3H, H-4), 1.92 (br s, 1H, 3H, Me-2). Isopropylidene
1.56 (s, 1H, H-1), 1.48 (s, 3H, H-3). Three times octanoate: 2.4–
2.1 (overlapped, 6H, H-2), 1.6 (overlapped, 6H, H-3), 1.26 (br s,
24H, H-4–H-7), 0.88 (t, J = 7.5 Hz, 9H, H-8).
C
33H40NaO12+: 651.2412, found 651.2408.
5.1.23. Compounds (8a, 13a, and 14)
To a solution of 7a (78 mg, 135 nmol) in isopropenyl acetate
(5 mL) is added p-TsOH acid (83 mg, 83 nmol) and the mixture is
left for 105 min. The reaction is quenched by addition of a satu-
rated aqueous solution of sodium hydrogen carbonate (20 mL).
The mixture is extracted three times with ethyl acetate (10 mL)
and the combined organic phases dried (sodium sulfate) and con-
centrated. The residue was fractionated by column chromatogra-
phy over LiChroprep RP-18 using methanol–water (6:1) as an
5.1.25. Compound (15)
To a solution of thapsigargin (1a, Tg, 500 mg, 0.786 mmol) and
4-methylmorpholino N-oxide (106 mg, 0.905 mmol) in acetone–

D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
5645
water (3:1, 4 mL) was added osmium tetraoxide (0.153 mL,
15 mol) and the solution was stirred for 4 h at room temperature.
1H NMR data (CDCl3): d Guaianolide: 4.37 (br s, 1H, H-1), 5.58 (t,
J = 3.3, 1H, H-2), 5.84 (br s, 1H, H-3), 5.70 (br s, 1H, H-6), 5.67 (over-
lapped, 1H, H-8), 3.10 (br d, J = 14.7, 1H, H-9a), 2.3 (overlapped, 1H,
H-9b), 1.50 (s, 3H, H-13), 1.48 (s, 3H, H-14), 1.91 (br s, 1H, H-15).
Acetate 1.91 (s, 3H, H-2). Butanoate: 2.3 (overlapped, 2H, H-2),
1.6 (overlapped, H-3), 0.96 (t, J = 7.5 Hz, 3H, H-4). 3-Methylbenzo-
ate: 7.84 (m, 1H, H-2 and H-6), 7.40 (m, 3H, H-4, and H-5), 2.41 (s,
3H, Me). Octanoate: 2.3 (overlapped, 2H, H-2), 1.6 (overlapped, 2H,
H-3), 1.3 (br s, 8H, H-4–H-7), 0.86 (t, J = 6.6 Hz, 3H, H-8). 13C NMR
(CDCl3) d Guaianolide: 57.9 (C-1), 77.9 (C-2), 85.3 (C-3), 141.6 (C-
4), 130.5 (C-5), 77.1 (C-6), 78.8 (C-7), 66.4 (C-8), 38.6 (C-9), 84.9
(C-10), 78.9 (C-11), 175.8 (C-12), 16.5 (C-13), 23.5 (C-14), 13.4
(C-15). Acetate 171.1 (C-1), 23.0 (C-2). Butyrate: 172.8 (C-1), 36.9
(C-2), 18.4 (C-3), 14.1 (C-4). 3-Methylbenzoate 166.2 (C@O),
129.8 (C-1), 130.5 (C-2), 138.9 (C-3), 134.2 (C-4), 128.5 (C-5),
127.1 (C-6), 21.7 (Me). Octanoate: 172.7 (C-1), 34.6 (C-2), 25.2
(C-3), 29.3 (C-4), 29.4 (C-5), 32.0 (C-6), 23.0 (C-7), 14.7 (C-8). HRMS
calcd for C37H50NaO12+: 709.3194, found 709.3172.
l
To the bright orange solution was added sodium periodate
(496 mg, 2.3 mmol) and the solution was left under stirring for
the night. The reaction mixture was quenched by adding an aque-
ous solution of sodium thiosulfate (1 M, 2 mL) and water (1 mL).
The greenish mixture was filtered and the filtrate acidified with
M hydrochloric acid and extracted with ethyl acetate until a color-
less aqueous phase was obtained. The combined organic phases
were dried (sodium sulfate) and concentrated in vacuo. The resi-
due was dissolved in methanol (7.5 mL), pyridine (6 mL), and
water (3.7 mL), and the solution was refluxed over night. Com-
pound 15 (191 mg, 44%) was isolated from the residue of the reac-
tion mixture by column chromatography over silica gel using
toluene–ethyl acetate (4:1) added 1% acetic acid as an eluent.
1H NMR data (CDCl3): d Guaianolide: 4.35 (br s, 1H, H-1), 4.88
(dd, J = 3.3 and 4.4 Hz, 1H, H-2), 4.41 (br s, 1H, H-3), 5.66 (s, 1H,
H-6), 5.64 (t, J = 3.30 Hz, 1H, H-8), 3.24 (dd, J = 14.7 and 3.3 Hz,
1H, H-9a), 2.16 (overlapped, H-9b), 1.47 (s, 3H, H-13), 1.37 (s,
3H, H-14), 1.94 (br s, 1H, H-15). Acetate 1.98 (s, 3H, H-2). Butano-
ate: 2.4 (overlapped, H-2), 1.6 (overlapped, H-3), 0.94 (t, J = 7.2 Hz,
3H, H-4). Octanoate: 2.3 (overlapped, 2H, H-2), 1.61 (overlapped,
2H, H-3), 1.28 (br s, 8H, H-4–H-7), 0.87 (t, J = 7.0 Hz, 3H, H-8).
13C NMR (CDCl3) d Guaianolide: 55.6 (C-1), 85.0 (C-2), 84.0 (C-3),
144.5 (C-4), 126.6 (C-5), 77.4 (C-6), 78.9 (C-7), 66.4 (C-8), 38.5
(C-9), 85.0 (C-10), 78.9 (C-11), 175.4 (C-12), 16.6 (C-13), 23.7 (C-
14), 13.3 (C-15). Acetate 170.5 (C-1), 23.0 (C-2). Butyrate: 172.8
(C-1), 34.5 (C-2), 18.3 (C-3), 14.1 (C-4). Octanoate: 176.0 (C-1),
36.8 (C-2), 25.2 (C-3), 29.4 (C-4), 29.3 (C-5), 32.0 (C-6), 22.9 (C-
7), 14.1 (C-8). HRMS calcd for C29H44NaO12+: 591.2776, found
591.2774.
5.1.28. Compound (16c)
Compound 16c (45 mg, 85%) was prepared as described above
for compound 16a using 4-biphenylacetic anhydride (56.3 mg,
0.139 mmol), DMAP (11.6 mg, 94
as starting materials.
lmol) and 15 (40.5 mg, 70 lmol)
1H NMR data (CDCl3): d Guaianolide: 4.28 (br s, 1H, H-1), 5.44 (t,
J = 3.4, 1H, H-2), 5.61 (overlapped, 1H, H-3), 5.61 (overlapped, 1H,
H-6), 5.61 (overlapped, 1H, H-8), 3.04 (dd, J = 14.3 and 2.7 Hz, 1H,
H-9a), 2.32 (overlapped, 1H, H-9b), 1.43 (s, 3H, H-13), 1.32 (s, 3H,
H-14), 1.77 (br s, 1H, H-15). Acetate 1.89 (s, 3H, H-2). Biphenylac-
etate 7.6 (m, 4H, H-4, H-8, H-10 and H-14), 7.4 (m, 2H, H-5 and H-
7), 7.3 (m, 3H, H-11, H-12 and H-13), 3.74 (s, 2H, H-2). Butanoate:
2.3 (overlapped, 2H, H-2), 1.6 (overlapped, H-3), 0.92 (t, J = 7.3 Hz,
3H, H-4). Octanoate: 2.3 (overlapped, 2H, H-2), 1.61 (overlapped,
2H, H-3), 1.28 (br s, 8H, H-4–H-7), 0.86 (t, J = 7.0 Hz, 3H, H-8).
13C NMR (CDCl3) d Guaianolide: 57.5 (C-1), 77.1 (C-2), 85.0 (C-3),
141.2 (C-4), 130.5 (C-5), 77.8 (C-6), 78.9 (C-7), 66.4 (C-8), 38.6
(C-9), 85.2 (C-10), 78.8 (C-11), 175.8 (C-12), 16.5 (C-13), 23.7 (C-
14), 13.3 (C-15). Acetate 171.1 (C-1), 23.0 (C-2). Biphenylacetate
172.9 (C@O), 41.1 (C-2), 133.0 (C-3), 130.0 (C-4 and C-8), 129.0
(C-5 and C-7), 140.2 (C-6), 140.9 (C-9), 127.2 (C-10 and C-14),
127.4 (C-11 and C-13), 129.9 (C-12). Butyrate: 172.7 (C-1), 34.5
(C-2), 18.4 (C-3), 14.1 (C-4). Octanoate: 172.7 (C-1), 36.9 (C-2),
25.2 (C-3), 29.3 (C-4), 29.5 (C-5), 32.0 (C-6), 23.1 (C-7), 14.5 (C-
8). HRMS calcd for C43H54NaO12+: 785.3507, found 785.3435.
5.1.26. Compound (16a)
To a solution of compound 15 (25 mg, 43
lmol) and DMAP
(14 mg, 115 mol) in dichloromethane (3 mL) was added benzoic
l
anhydride (19.8 mg, 0.88 mmol). The solution was stirred for
2 days at room temperature, concentrated and the residue dis-
solved in ethyl acetate (3 mL). The solution was washed with M
hydrochloric acid (2 mL), the organic phase dried over sodium sul-
fate and concentrated. Compound 16a (21.4 mg, 74%) was isolated
from the residue by column chromatography over silica gel using
toluene–ethyl acetate (9:1) as an eluent. 1H NMR data (CDCl3): d
Guaianolide: 4.39 (br s, 1H, H-1), 5.58 (t, J = 2.9, 1H, H-2), 5.83
(br s, 1H, H-3), 5.69 (s, 1H, H-6), 5.67 (t, J = 3.30 Hz, 1H, H-8),
3.12 (dd, J = 14.3 and 2.2 Hz, 1H, H-9a), 2.39 (overlapped, 1H, H-
9b), 1.49 (s, 3H, H-13), 1.46 (s, 3H, H-14), 1.90 (br s, 1H, H-15). Ace-
tate 1.90 (s, 3H, H-2). Benzoate 8.05 (m, 2H, H-2 and H-6), 7.58 (m,
1H, H-4), 7.45 (m, 2H, H-3 and H-5). Butanoate: 2.3 (overlapped, H-
2), 1.6 (overlapped, H-3), 0.95 (t, J = 7.3 Hz, 3H, H-4). Octanoate: 2.3
(overlapped, 2H, H-2), 1.61 (overlapped, 2H, H-3), 1.28 (br s, 8H, H-
4–H-7), 0.86 (t, J = 7.0 Hz, 3H, H-8). 13C NMR (CDCl3) d Guaianolide:
57.8 (C-1), 77.1 (C-2), 85.0 (C-3), 141.6 (C-4), 129.5 (C-5), 77.8 (C-
6), 78.9 (C-7), 66.4 (C-8), 38.6 (C-9), 85.4 (C-10), 78.9 (C-11), 175.8
(C-12), 16.4 (C-13), 23.7 (C-14), 13.3 (C-15). Acetate 171.7 (C-1),
23.0 (C-2). Benzoate 166.0 (C@O), 131.0 (C-1), 130.0 (C-2 and C-
6), 128.6 (C-3 and C-5), 133.4 (C-4). Butyrate: 172.8 (C-1), 34.5
(C-2), 18.4 (C-3), 14.1 (C-4). Octanoate: 172.7 (C-1), 36.9 (C-2),
25.2 (C-3), 29.3 (C-4), 29.5 (C-5), 32.0 (C-6), 23.1 (C-7), 14.5 (C-
8). HRMS calcd for C36H48NaO12+: 695.3038, found 695.3021.
5.1.29. Compound (16d)
Compound 16d (50 mg, 76%) was prepared as described above
for 16a using 2-phenylbenzoic acid (19 mg, 96
lmol) and com-
pound 15 (50 mg, 66
l
mol) as starting materials. 1H NMR data
(CDCl3): d Guaianolide: 4.17 (br s, 1H, H-1), 5.28 (t, J = 3.2, 1H, H-
2), 5.70 (br s, 1H, H-3), 5.54 (br s, 1H, H-6), 5.58 (t, J = 3.5 Hz, 1H,
H-8), 2.94 (br d, J = 14.7, 1H, H-9a), 2.3 (overlapped, 1H, H-9b),
1.45 (s, 3H, H-13), 1.15 (s, 3H, H-14), 1.60 (br s, 1H, H-15). Acetate
1.91 (s, 3H, H-2). Butanoate: 2.3 (overlapped, 2H, H-2), 1.6 (over-
lapped, H-3), 0.96 (t, J = 7.3 Hz, 3H, H-4). Octanoate: 2.3 (over-
lapped, 2H, H-2), 1.6 (overlapped, 2H, H-3), 1.3 (br s, 8H, H-4–H-
7), 0.88 (t, J = 6.6 Hz, 3H, H-8); 2-Phenylbenzoate 7.87 (dd, J = 7.3
and 1.2 Hz, 1H, H-3), 7.54 (td, J = 6.1 and 1.5 Hz, H-5), 7.43 (td,
J = 6.1 and 1.5 Hz, H-4), 7.4 (overlapped, 6H, H-6, H-8–H-12). 13C
NMR (CDCl3) d Guaianolide: 57.6 (C-1), 77.0 (C-2), 84.8 (C-3),
141.2 (C-4), 130.8 (C-5), 72.9 (C-6), 78.8 (C-7), 66.3 (C-8), 38.5
(C-9), 85.2 (C-10), 78.9 (C-11), 175.5 (C-12), 16.5 (C-13), 22.3 (C-
14), 13.0 (C-15). Acetate 170.9 (C-1), 23.0 (C-2). Butyrate: 172.7
(C-1), 36.9 (C-2), 18.3 (C-3), 14.1 (C-4). Octanoate: 172.7 (C-1),
34.6 (C-2), 25.2 (C-3), 29.3 (C-4), 29.3 (C-5), 32.0 (C-6), 23.2 (C-
7), 14.5 (C-8). 2-Phenylbenzoate 168.4 (C@O), 130.4 (C-1), 142.2
5.1.27. Compound (16b)
Compound 16b (24 mg, 53%) was prepared as described above
for compound 16a using 3-methylbenzoic anhydride (30 mg,
0.12 mmol), DMAP (13 mg, 0.1 mmol) and 15 (38.3 mg, 66 lmol)
as starting materials.

5646
D. M. Skytte et al. / Bioorg. Med. Chem. 18 (2010) 5634–5646
(C-2), 127.4 (C-3), 131.1 (C-4), 130.3 (C-6), 141.6 (C-7), 128.9 (C-8
and C-12), 128.5 (C-9 and C-11), 127.5 (C-10). HRMS calcd for
C
phosphoenolpyruvate (1 mM), MgATP (concd) NADH (0.15 mM),
lactate dehydrogenase (60
g/mL), and Ca2+ (concd). The reaction,
which took place at 20 °C, was initiated by addition of SERCA to the
medium at concentration of 4 nM and a concentration of 5–10
l
42H52NaO12+: 771.3351, found 771.3325.
l
g
5.1.30. Compound (16e)
Compound 16e (25 mg, 71%) was prepared as described below
for compound 16f using 4-methylbenzoic anhydride (30 mg,
protein/mL. The reaction was followed by measuring the optical
density at 340 nm. The experiments were repeated two or three
times.
0.12 mmol), DMAP (10 mg, 80
as starting materials.
lmol) and 15 (30.5 mg, 53 lmol)
Acknowledgments
1H NMR data (CDCl3): d Guaianolide: 4.30 (br s, 1H, H-1), 5.53 (t,
J = 3.1, 1H, H-2), 5.78 (br s, 1H, H-3), 5.65 (br s, 1H, H-6), 5.61 (t,
J = 3.7 Hz, 1H, H-8), 3.03 (dd, J = 14.7 and 3.2 Hz, 1H, H-9a), 2.3
(overlapped, 1H, H-9b), 1.46 (s, 3H, H-13), 1.43 (s, 3H, H-14),
1.86 (br s, 1H, H-15). Acetate 1.86 (s, 3H, H-2). Butanoate: 2.3
(overlapped, 2H, H-2), 1.6 (overlapped, H-3), 0.93 (t, J = 7.5 Hz,
3H, H-4). 4-Methylbenzoate: 7.89 (m, 2H, H-2 and H-6), 7.40 (m,
2H, H-3 and H-5). 4-Methylbenzoate 7.89 (m, 2H, H-2 and H-6),
7.20 (m, 2H, H-3 and H-5), 2.38 (s, 3H, Me). Octanoate: 2.3 (over-
lapped, 2H, H-2), 1.61 (overlapped, 2H, H-3), 1.3 (br s, 8H, H-4–
H-7), 0.82 (t, J = 6.7 Hz, 3H, H-8). 13C NMR (CDCl3) d Guaianolide:
57.9 (C-1), 77.8 (C-2), 85.2 (C-3), 142.0 (C-4), 130.8 (C-5), 77.1
(C-6), 78.8 (C-7), 66.4 (C-8), 38.6 (C-9), 84.8 (C-10), 78.9 (C-11),
175.6 (C-12), 16.6 (C-13), 23.5 (C-14), 13.4 (C-15). Acetate 171.1
(C-1), 23.0 (C-2). Butyrate: 172.6 (C-1), 36.9 (C-2), 18.4 (C-3),
14.1 (C-4). 4-Methylbenzoate 166.0 (C@O), 144.1 (C-1), 130.0 (C-
2 and C-6), 129.3 (C-3 and C-5), 127.2 (C-4), 22.1 (Me). Octanoate:
172.8 (C-1), 34.6 (C-2), 25.2 (C-3), 29.3 (C-4), 29.4 (C-5), 32.0 (C-6),
23.0 (C-7), 14.5 (C-8). HRMS calcd for C37H50NaO12+: 709.3194,
found 709.3170.
This work has been supported by the Danish Cancer Society and
the Carlsbergfondet.
Supplementary data
Supplementary data (1H NMR spectra of the compounds 9a–9d,
11b, 12a, 12b, 13, 14, and 16a–16f) associated with this article can
be found, in the online version, at doi:10.1016/j.bmc.2010.06.032.
References and notes
1. Bobe, R.; Bredoux, R.; Corvazier, E.; Martin, V.; Gelebart, P.; Lacabaratz-Porret,
C.; Launay, S.; Fanchaouy, M.; Dally, S.; Chaabane, C.; Kovacs, T.; Enouf, J. Curr.
Top. Biochem. Res. 2005, 7, 1.
2. Thastrup, O.; Cullen, P. J.; Drøbak, B. K.; Hanley, M. R.; Dawson, A. P. Proc. Natl.
Acad. Sci. U.S.A. 1990, 87, 2466.
3. Papp, B.; Enyedi, A.; Kovacs, T.; Sarkadi, B.; Wuytack, F.; Thastrup, O.;
Gardos, G.; Bredoux, R.; Levy-Toledano, S.; Enouf, J. J. Biol. Chem. 1991,
266, 14593.
4. Lytton, J.; Westlin, M.; Hanley, M. R. J. Biol. Chem. 1991, 266, 17067.
5. Xu, C.; Ma, H. L.; Inesi, G.; Al-Shawi, M. K.; Toyoshima, C. J. Biol. Chem. 2004,
279, 17973.
6. Sagara, Y.; Inesi, G. J. Biol. Chem. 1991, 266, 13503.
7. Wootton, L. L.; Michelangeli, F. J. Biol. Chem. 2006, 281, 6970.
8. Furuya, Y.; Isaacs, J. T. Prostate 1994, 25, 301.
9. Furuya, Y.; Lundmo, P.; Short, A. D.; Gill, D. L.; Isaacs, J. T. Cancer Res. 1994, 54,
6167.
10. Lin, X. S.; Denmeade, S. R.; Cisek, L.; Isaacs, J. T. Prostate 1997, 33, 201.
11. Christensen, S. B.; Skytte, D. M.; Denmeade, S. R.; Dionne, C.; Moller, J. V.;
Nissen, P.; Isaacs, J. T. Anticancer Agents Med. Chem. 2009, 9, 276.
12. Janssen, S.; Jakobsen, C. M.; Rosen, D. M.; Ricklis, R. M.; Reinecke, U.;
Christensen, S. B.; Lilja, H.; Denmeade, S. R. Mol. Cancer Ther. 2004, 3, 1439.
13. Denmeade, S. R.; Lou, W.; Lovgren, J.; Malm, J.; Lilja, H.; Isaacs, J. T. Cancer Res.
1997, 57, 4924.
5.1.31. Compound (16f)
To a solution of 15 (50 mg, 66
was added 4-phenylbenzoic acid (19 mg, 96
160 mol), and DCC (20 mg, 97 mol). The reaction mixture was
l
mol) in dichloromethane (2 mL)
lmol), DMAP (20 mg,
l
l
stirred at room temperature overnight, filtered and concentrated.
Compound 16f (35 mg, 53%) was isolated from the residue by col-
umn chromatography over silica gel using toluene–ethyl acetate
(5:1) added acetic acid (1%) as an eluent. 1H NMR data (CDCl3): d
Guaianolide: 4.39 (br s, 1H, H-1), 5.62 (t, J = 3.3, 1H, H-2), 5.88
(br s, 1H, H-3), 5.72 (br s, 1H, H-6), 5.68 (t, J = 3.5 Hz, 1H, H-8),
3.11 (br d, J = 14.7, 1H, H-9a), 2.3 (overlapped, 1H, H-9b), 1.51 (s,
3H, H-13), 1.52 (s, 3H, H-14), 1.95 (br s, 1H, H-15). Acetate 1.92
(s, 3H, H-2). Butanoate: 2.3 (overlapped, 2H, H-2), 1.6 (overlapped,
H-3), 0.96 (t, J = 7.5 Hz, 3H, H-4). Octanoate: 2.3 (overlapped, 2H,
H-2), 1.6 (overlapped, 2H, H-3), 1.3 (br s, 8H, H-4–H-7), 0.86 (t,
J = 6.6 Hz, 3H, H-8); 4-phenylbenzoate 8.13 (m, 2H, H-2 and H-6,
7.68 (m, 2H, H-3 and H-5), 7.63 (m, 2H, H8 and H-12), 7.50 (m,
2H, H-9 and H-11), 7.41 (m, 1H, H-10). 13C NMR (CDCl3) d Guaiano-
lide: 57.6 (C-1), 76.6 (C-2), 84.6 (C-3), 141.5 (C-4), 130.7 (C-5), 77.6
(C-6), 78.7 (C-7), 66.2 (C-8), 38.3 (C-9), 85.1 (C-10), 78.6 (C-11),
175.3 (C-12), 16.3 (C-13), 22.6 (C-14), 13.2 (C-15). Acetate 170.8
(C-1), 23.2 (C-2). Butyrate: 172.4 (C-1), 36.6 (C-2), 18.1 (C-3),
13.8 (C-4). Octanoate: 172.4 (C-1), 34.3 (C-2), 24.9 (C-3), 29.0 (C-
4), 29.1 (C-5), 31.7 (C-6), 22.7 (C-7), 14.2 (C-8). 4-Phenylbenzoate
165.5 (C@O), 128.3 (C-1), 127.2 (C-2 and C-6), 128.8 (C-3 and C-
5), 145.8 (C-4), 139.9 (C-7), 128.0 (C-8 and C-12), 130.2 (C-7 and
C-11), 128.1 (C-9). HRMS calcd for C42H52NaO12+: 771.3351, found
771.3322.
14. Jakobsen, C. M.; Denmeade, S. R.; Isaacs, J. T.; Gady, A.; Olsen, C. E.; Christensen,
S. B. J. Med. Chem. 2001, 44, 4696.
[15]. Denmeade, S. R.; Isaacs, J. T.; Lilja, H. Patent US 7,053,042 B1, 2006.
16. Denmeade, S. R.; Isaacs, J. T. Cancer Biol. Ther. 2005, 4, e69.
17. Denmeade, S. R.; Jakobsen, C. M.; Janssen, S.; Khan, S. R.; Garrett, E. S.; Lilja, H.;
Christensen, S. B.; Isaacs, J. T. J. Natl. Cancer Inst. 2003, 95, 990.
18. Andersen, A.; Treiman, M.; Poulsen, J. C. J.; Cornett, C.; Moldt, P.; Olsen, C. E.;
Christensen, S. B. Bioorg. Med. Chem. Lett. 1994, 4, 657.
19. Christensen, S. B.; Andersen, A.; Poulsen, J. C.; Treiman, M. FEBS Lett. 1993, 335,
345.
20. Nielsen, S. F.; Thastrup, O.; Pedersen, R.; Olsen, C. E.; Christensen, S. B. J. Med.
Chem. 1995, 38, 272.
21. Sohoel, H.; Jensen, A. M.; Moller, J. V.; Nissen, P.; Denmeade, S. R.; Isaacs, J. T.;
Olsen, C. E.; Christensen, S. B. Bioorg. Med. Chem. 2006, 14, 2810.
22. Christensen, S. B.; Andersen, A.; Smitt, U. W. Prog. Chem. Natl. Prod. 1997, 71,
131.
23. Toyoshima, C.; Nakasako, M.; Nomura, H.; Ogawa, H. Nature 2000, 405, 647.
24. Sohoel, H.; Liljefors, T.; Ley, S. V.; Oliver, S. F.; Antonello, A.; Smith, M. D.; Olsen,
C. E.; Isaacs, J. T.; Christensen, S. B. J. Med. Chem. 2005, 48, 7005.
25. Toyoshima, C.; Nomura, H. Nature 2002, 418, 605.
26. Rasmussen, U.; Christensen, S. B.; Sandberg, F. Acta Pharm. Suec. 1978, 15, 133.
27. Andrews, S. P.; Ball, M.; Wierschem, F.; Cleator, E.; Oliver, S.; Högenauer, K.;
Simic, O.; Antonello, A.; Hüniger, U.; Smith, M. D.; Ley, S. V. Chem.-A Eur. J.
2007, 13, 5688.
28. Ball, M.; Andrews, S. P.; Wierschem, F.; Cleator, E.; Smith, M. D.; Ley, S. V. Org.
Lett. 2007, 9, 663.
29. Andersen, A.; Cornett, C.; Lauridsen, A.; Olsen, C. E.; Christensen, S. B. Acta
Chem. Scand. 1994, 48, 340.
30. Christensen, S. B. Acta Chem. Scand. 1988, B 42, 623.
31. Rothman, E. S.; Swern, D.; Perlstein, T.; Serota, S. J. Org. Chem. 1962, 27, 3123.
32. Moller, J. V.; Lind, K. E.; Andersen, J. P. J. Biol. Chem. 1980, 255, 1912.
33. Sorensen, T. L.; Moller, J. V.; Nissen, P. Science 2004, 304, 1672.
5.2. Assay for SERCA inhibition
Sarcoplasmic reticulum from rabbit skeletal muscle was pre-
pared as previously described.32 The incubation medium contained