Thapsigargin analogues for targeting programmed death of androgen-independent prostate cancer cells

Article abstract of DOI:10.1016/S0968-0896(99)00074-7

A number of analogues of thapsigargin, a selective inhibitor of the sarco-endoplasmic reticulum Ca²⁺-ATPases have been synthesized. In all of the prepared analogues the butanoyl residue at O-8 has been replaced with a residue containing an arom

Full text of DOI:10.1016/S0968-0896(99)00074-7

Bioorganic & Medicinal Chemistry 7 (1999) 1273±1280
Thapsigargin Analogues for Targeting Programmed Death of
Androgen-Independent Prostate Cancer Cells
S. Brùgger Christensen, a,* Annette Andersen a, Hasse Kromann, a Marek Treiman, b
Bertrand Tombal, c Sam Denmeade c and John T. Isaacs c
aDepartment of Medicinal Chemistry, Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen é, Denmark
bDepartment of Medical Physiology-Biotechnology Center for Cellular Communication, University of Copenhagen, The Panum Institute,
Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark
cJohns Hopkins Oncology Center, Johns Hopkins School of Medicine, 422 N. Bond Street, Baltimore, MD 21231, USA
Received 8 September 1998; accepted 18 November 1998
AbstractÐA number of analogues of thapsigargin, a selective inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPases have
been synthesized. In all of the prepared analogues the butanoyl residue at O-8 has been replaced with a residue containing an aro-
matic amine. The amine can be used as an anchoring point for attaching a peptide group sensitive to the proteolytic enzyme,
prostate speci®c antigen, secreted by prostate cancer cells. Like thapsigargin, the analogues are capable of elevating the cytoplasmic
Ca2+ concentration approximately sevenfold when tested at e€ective cytotoxic doses. The analogues in which the 8-O-butanoyl
group has been replaced with 3-(4-aminophenyl)propanoyl or 4-aminocinnamoyl were found potently to induce programmed cell
death of the prostate cancer cells. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
in many normal host cells. Consequently a method of
targeting the proliferation independent cytotoxicity of
Tg selectively to prostate cancer cells is needed. In order
to target Tg, advantage can be taken of the secretion of
prostate-speci®c antigen (PSA) by prostate cancer cells.7
PSA is a serine protease with an unusual speci®city.
Thus, PSA eciently liberates 7-amino-4-methylcou-
marin from a substrate in which the amino group of this
coumarin is coupled to the six-amino acid peptide His-
Ser-Ser-Lys-Leu-Gln. In contrast, this conjugate is a
very poor substrate for other puri®ed proteases and
proteases present in sera.8 The speci®city has further
been illustrated by incubating cancer cells with a pro-
drug in which the amino group of doxorubicin is cou-
pled to this six-amino acid. Only PSA producing cells
are killed, indicating that doxorubicin is only liberated
in the vicinity of such cells.9 A fast inactivation of PSA
outside the prostate ductal system means that the
hydrolytic activity only will be of signi®cance in the
close vicinity of prostate cells and prostate cancer cells.
The aim of the present project is to develop a prodrug
consisting of a primary amine containing Tg analogue,
which via the amine group is coupled to a PSA clea-
vable peptide.3 This paper describes design, synthesis
and SERCA inhibitory potencies of analogues of Tg
that can be coupled to promoiety groups.
Currently there is no treatment that signi®cantly pro-
longs survival in men with metastatic prostate cancer.1
Androgen ablation therapy eventually fails because the
metastatic prostate cancer within an individual patient
is heterogenously composed of clones of both androgen
dependent and independent cancer cells.2 Thapsigargin
(1, Tg, Fig. 1) is a sesquiterpene lactone3 that selectively
inhibits the sarcoplasmic and the endoplasmic reticulum
Ca2+-ATPases (SERCA).4,5 Inhibition of the SERCA
causes depletion of intracellular Ca2+ stores resulting in
an initial rise in cytoplasmic Ca2+ concentration, which
by capacitance in¯ux of extracellular Ca2+ a€ords a
secondary sustained elevation of cytoplasmic Ca2+
.
This sustained elevation in the cytosolic Ca2+ con-
centration activates the proliferation independent pro-
grammed death of susceptible cells including androgen
independent prostate cancer cells.6 Since the SERCA is
present in almost all cells Tg will also induce apoptosis
Key words: Prostate cancer; prodrug; thapsigargin; prostate-speci®c
antigen; apoptosis.
* Corresponding author. Tel.: +45-3537-0850; fax: +45-3537-2209;
e-mail: sbc@mail.dfh.dk
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00074-7

1274
S. B. Christensen et al. / Bioorg. Med. Chem. 7 (1999) 1273±1280
Figure 1. Structure of thapsigargin (1, Tg).
Synthesis
All the 13 analogues have been prepared from 8-O-
debutanoylthapsigargin (2). For the preparation of 4a
pimelic acid was reacted with 2 in the presence of N,N'-
dicyclohexylcarbodiimide (DCCI) to give 8-O-(6-carb-
oxyhexanoyl)-8-O-debutanoylthapsigargin (3a), which
in the presence of DCCI was coupled with 2,4-diamino-
toluene. The carboxylic acids (3b) and (3c) were prepared
using glutaric and succinic anhydride, respectively, as
starting materials. The analogues (4b) and (4c) were
obtained through dicyclohexylcarbodiimide (DCCI)
promoted coupling of 3b and 3c with 2,4-diaminotoluene
(Scheme 1).
The analogues 6a through 6e all have been prepared by
DCCI promoted esteri®cation of the BOC protected
aminocarboxylic acids 7a±7e with 2 (Scheme 2).
Results and Discussion
Scheme 1. Synthesis of the analogues containing a 2,4-diaminotoluene
residue (4a±4c).
Previously, it has been shown that Tg (1) can induce
programmed cell death in androgen independent pros-
tate cancer cell lines with a LC50 value of approximately
100 nM.6 In order to irreversibly commit cells to
undergo apoptosis chronic exposure for greater than
24 h is needed; exposure to 500 nM for up to 12 h did
not result in loss of clonogenic ability or viability.6 In
the present study TSU-Pr1 human prostate cancer cells
were exposed for 72 h to Tg or a series of Tg analogues,
in which the butanoyl group of the 8-O-butanoyl group
has been replaced with an acyl group containing an
amino group. The amino group might be used as an
anchoring point for attachment of a promoiety peptide.
It was decided to replace the butanoyl group because a
model of the binding site indicates that when Tg is
bound to the SERCA this group is situated in a cave.10
Consequently, replacement of the butanoyl group with
a more voluminous group should preserve the anity.
After exposure, cells were assayed for the loss of clono-
genic survival (i.e. cytotoxic response) and LC50 were
determined for the various analogues, Table 1. Table 1
also gives the IC50 values of the analogues for inhibition
of skeletal muscle Ca2+-ATPase (SERCA1). The a-
nity of SERCA1 towards Tg equals that of SERCA2b,
a housekeeping SERCA isoform expressed in all cell
types.4 The SERCA1 activity was assayed as the ATP-
dependent 45Ca2+ uptake in a preparation of sarco-
plasmic reticulum (SR) membranes isolated from rabbit
skeletal muscle (see the Experimental). At least 50%
of the protein in this preparation consisted of Ca2+
-ATPase, as determined by densitometric scanning of
SDS-polyacrylamide gels. The data in Table 1 were
obtained with either 2 or 5 mg/mL of the SR mem-
brane protein in the assay. Two di€erent preparations
have been used to give SERCA1 enzymes, in which the
concentration of SERCA di€ers. The low IC50 value of
Tg5 causes Tg to bind to the binding site in reality in an
irreversible way. Consequently di€erent concentrations
of SERCA in the assay will appear to give di€erent IC50
values. To overcome this problem activities relative to
that of Tg are given. Notice, that in the case of 4a the
IC50 value for inhibition of the SERCA is only
approximately 4.5 times larger than that of Tg, whereas
the ability to kill TSU cells is approximately 100 times
smaller.
For analogues 4a and 4b whereas the IC50 value for
inhibition of the SERCA is only approximately 4±5
times larger than that of Tg the concentration needed to
kill TSU cells is approximately 100±300 times higher.
More encouraging results were obtained with the ami-
nobenzenecarboxylic acid esters 6a±6e. The analogues
6c±6e possess submicromolar LC50 values. The corre-
sponding IC50 values are of the same order of magnitude

S. B. Christensen et al. / Bioorg. Med. Chem. 7 (1999) 1273±1280
1275
as that of Tg. Surprisingly the LC50 value of 6a was 100
times less active than Tg and 6b only showed minor
activity in concentrations up to 10 mM.
The cytotoxicity of Tg on prostate cancer cells is due to
its ability to sustain an increase of the cytosolic Ca2+
concentration ([Ca2+]i) more than 10 h leading to the
subsequent activation of the programmed death path-
way in these cells.6 Several of the analogues were tested
for their ability to elevate [Ca2+]i in TSU cells using
appropriate drug levels as based upon their LC50 values.
The 4 analogues 4a and 6c±6e produced the same initial
rise in [Ca2+]i as Tg and also like Tg sustained an
approximately sevenfold elevation in [Ca2+]i for more
than 4 h of treatment in TSU cells, Table 2. If EGTA
was added to lower the extracellular Ca2+ concentra-
tion to below 100 nM in the media, then the [Ca2+
]
i
levels rapidly (i.e. minutes) return to below 50 nM. After
4 h of exposure to Tg or the analogue, further addition
of 1 mM Tg to the media did not result in any further
increase in [Ca2+]i. This indicates that theses analogues
6
like Tg itself deplete the ER calcium stores leading to
the capacitive entrance of extracellular Ca2+ into the
cells to elevate [Ca2+]i.11 The analogue 6b that did not
induce programmed cell death was also unable to
increase the [Ca2+]i up to a concentration of 10 mM,
Table 2.
In conclusion, coupling of the analogues 6c±6e to pep-
tides, which are selectively cleaved by PSA will allow for
targeted delivery of these cytotoxic agents to sites of
PSA producing metastatic prostate cancer. Currently we
are coupling the analogues to the peptide carrier for
further in vitro and in vivo testing for activity against
PSA producing prostate cancers.
Experimental
Chemistry
The spectra have been recorded on a AF200X Bruker
spectrometer in deuterated chloroform solutions using
tetramethylsilane as an internal standard. In all the
spectra the signals originating in the acetyl, angeloyl,
Scheme 2. Synthesis of the analogues containing a 4-aminobenzene
carboxylic acid (5a±5e and 6a±6e).
Table 1. IC50 values for inhibition of the skeletal muscle Ca2+-ATPase and LC50 values for loss of clonogenic survival for Tg (1) and some ana-
logues against TSU-Pr1 human prostate cancer cells
Compound
To inhibit Ca2+-ATPase 50%a
(IC50 in nM)
To inhibit Ca2+-ATPase 50%b
(IC50 in nM)
Activity relative
to Tg (1)c
To kill 50% of clonogenic cells
(LC50 in mM)
Tg (1)
3.9‹0.2 (6)
21‹1 (3)
17.2‹2.4 (3)
316‹22 (3)
1278‹91 (3)
11.0‹1.1 (4)
1
0.030‹0.004 (5)
10.0‹0.5 (5)
3.1‹0.2 (5)
16.6‹0.4 (5)
>25
dBTg (2)
4a sch1
4b sch1
4c sch1
6a sch2
6b sch2
5c sch2
6c sch2
5d sch2
6d sch2
5e sch2
6e sch2
0.19
0.22
0.012
0.0030
0.59
18.4‹0.3 (3)
n.d.
4.0‹0.3 (5)
>10
21.2‹0.8 (3)
16.2‹0.9 (3)
20.3‹0.2 (3)
18.7‹0.2 (3)
14.4‹0.8 (3)
18.4‹1.4
0.52
0.68
0.54
0.59
0.76
0.60
0.87‹0.02 (5)
0.28‹0.06 (5)
1.9‹0.1 (5)
0.11‹0.03 (5)
n.d.
0.23‹0.02 (5)
Results expressed as mean‹standard error (number of experiments).
a
The concentration of SR protein in the test solution was 2 mg/mL.
The concentration of SR protein in the test solution was 5 mg/mL.
The ratio of the IC50 value of Tg/IC50 value of the analogue.
b
c

1276
S. B. Christensen et al. / Bioorg. Med. Chem. 7 (1999) 1273±1280
Table 2. Increase in cytosolic Ca2+ concentration ([Ca2+]i) in TSU-
Pr1 cells induced by Tg and the Tg analogues at e€ective cytotoxic
concentrationsa
8-O-(4-Carboxybutanoyl)-8-O-debutanoylthapsigargin
(3b). A solution of 8-O-debutanoylthapsigargin (2,13
100 mg, 0.17 mmol), glutaric anhydride (400 mg, 3.5
mmol) and 4-dimethylaminopyridine (100 mg, 0.82
mmol) in methylene chloride (10 mL) was left for 21 h at
room temperature and ®ltered. The ®ltrate was added 4
M hydrochloric acid (10 mL) and extracted twice with
ethyl acetate (10 mL). The organic phase was con-
centrated in vacuo to give 320 mg of a residue from
which 68 mg (58%) of 3b was isolated by column chro-
matography using an eluent consisting of toluene/ethyl
acetate/acetic acid (5/1/0.01), to which increasing
amounts of ethyl acetate were added. 1H NMR (CDCl3)
d guaianolide 4.22 (br s, H-1), 5.46 (br t, J=2 Hz, H-2),
5.7±5.5 (br, H-3, H-8, and H-6), 2.95 (dd, J=15 and
3 Hz, H-9), 1.78 (br s H-15), 1.43 (s, H-13), 1.38 (s, H-
14); glutaryl 2.2±2.4 (m, H-2 and H-4), 1.5±1.7 (m, H-3);
13C NMR (CDCl3) d. The guaianolide nucleus 57.6 (C-
1), 83.9 (C-3), 141.2 (C-4), 130.1 (C-5), 78.3 (C-7/C-11),
66.2 (C-8), 37.9 (C-9), 84.2 (C-10), 78.5 (C-11/C-7),
176.3 (C-12), 15.6 (C-13), 21.8 (C-14), 12.5 (C-15); glu-
taroyl 171.2 (C-1), 33.4 (C-2), 20.5 (C-3), 31.6 (C-4). MS
FAB m/z 693 [M H+].
Compound
Concentration in mM
[Ca2+]i in nM
Control
Tg (1)
4a
6b
6c
Ð
0.10
10
51‹20
283‹19
275‹32
54‹6
253‹26
295‹56
233‹11
10
0.80
0.30
0.70
6d
6e
a
The cells were treated wi th Tg or the analogue for 1 h prior to the
determinations of [Ca2+]i. All experiments were performed in tripli-
cate. Results expressed as mean‹standard error.
butanoyl, and octanoyl residues have been found as
1
previously reported12 and are not listed. The H NMR
spectra were recorded at 200 MHz. The signals of H-90
have in many cases been overlapped by the signals from
the a protons in the octanoyl residue. The 13C NMR
spectra were recorded at 50 MHz. In the 13C NMR
spectra the assignments of signals with similar chemical
shift values might be interchanged. The signals origi-
nating in C-2 and C-6 are hidden by the signals of
chloroform, but have been visualised in a few cases by
recording the DEPT spectra. The small amounts of
compounds available have in some cases precluded
observation of signals of poor intensities. Unless other-
wise stated column chromatography has been per-
formed over silica gel 60, 0.040±0.063 mm, Merck. The
purity of all target compounds has been veri®ed by
HPLC to be more than 95%.
8 - O - {4 - (N - [3 - Amino - 4 - methylphenyl] - carboxamido)
butanoyl}-8-O-debutanoylthapsigargin (4b). A solution
of 3b (14 mg, 20 mmol), 2,4-diaminotoluene (10 mg,
80 mmol), and DCCI (7.9 mg, 40 mmol) in methylene
chloride (2 mL) was left for 2 h at room temperature.
The mixture was ®ltered, and the ®ltrate concentrated in
vacuo to give a residue from which 8 mg (50%) of 4b
was isolated as a colourless amorphous powder by col-
umn chromatography using an eluent consisting of tol-
uene/ethyl acetate (9/1), to which increasing amounts of
1
8-O-{6-(N-[3-Amino-4-methylphenyl]-carboxamido)hexa-
noyl}-8-O-debutanoylthapsigargin (4a). A solution of 213
(103 mg, 0.177 mmol), pimelic acid (185 mg, 1.16 mmol),
DCCI (233 mg, 1.15 mmol) and dimethylaminopyridine
(100 mg, 0.71 mmol) in dichloromethane is left for 3 h.
The reaction mixture was ®ltered and the ®ltrate washed
twice with hydrochloric acid (0.5 M, 10 mL). The organic
phase was concentrated and 8-O-(6-carboxyhexanoyl)-8-
O-debutanoylthapsigargin (3a, 63 mg, 49%) isolated by
repeated chromatography using an eluent consisting of
toluene/ethyl acetate (6/1) added 1% of acetic acid, to
which increasing amounts of ethyl acetate were added. A
solution of 8-O-{6-carboxyhexanoyl}-8-O-debutanoyl-
thapsigargin (52 mg, 72mmol), 2,4-diaminotoluene
(75 mg, 635 mmol), and DCCI (27.3 mg, 132 mmol) in
methylene chloride (6 mL) was left for 1 h at room tem-
perature. The mixture was ®ltered, and the ®ltrate con-
centrated in vacuo to give a residue from which 26mg
(43%) of 4a was isolated as as a colourless amorphous
powder by column chromatography using an eluent con-
sisting of toluene/ethyl acetate (9/1). 1H NMR
((CD3)2CO) d guaianolide 4.38 (br s, H-1), 5.52 (dd, J=4.0
and 3.2 Hz, H-2), 5.80 (br q, J=3Hz, H-3), 5.67 (m, H-6),
5.70 (t, J=3.6 Hz, H-8), 3.02 (dd, J=15 and 3 Hz), 1.86 (br
s H-15), 1.43 (s, H-13), 1.42 (s, H-14); diaminotoluene 2.00
(br s, CH3), (7.12, d J=2.0Hz, H-2), 7.08 (d, J=8.2Hz, H-
5), 7.18 (dd, J=8.2 and 2.0 Hz, H-6), pimeloyl 2.25±2.35
(m, H-2 and H-6), 1.55±1.65 (m, H-3 and H-5), 1.35±1.40
(m, H-4). MS FAB m/z 825 [M H+].
ethyl acetate were added. H NMR (CDCl3) d guaiano-
lide 4.17 (br s, H-1), 5.46 (br t, J=2 Hz, H-2), 5.6±5.4
(br, H-3, H-8, and H-6), 2.80 (dd, J=15 and 3 Hz, H-9),
1.78 (br s H-15), 1.43 (s, H-13), 1.38 (s, H-14); diami-
notoluyl 2.00 (br s, CH3), (7.03, d J=2.0 Hz, H-2), 6.90
(d, J=8.2 Hz, H-5), 6.72 (dd, J=8.2 and 2.0 Hz, H-6),
glutaroyl 2.2±2.4 (m, H-2 and H-4), 1.5±1.7 (m, H-3); 13C
NMR (CDCl3) d the guaianolide nucleus 57.6 (C-1),
83.4 (C-3), 141.4 (C-4), 130.3 (C-5), 78.2 (C-7), 66.2 (C-
8), 37.9 (C-9), 84.2 (C-10), 78.2 (C-11), 175.9 (C-12),
15.5 (C-13), 22.0 (C-14), 12.8 (C-15); glutaroyl 170.6 (C-
1), 33.2 (C-2), 22.2 (C-3), 31.3 (C-4), 161.9 (C-5); dia-
minotoluyl 138.4 (C-1), 106.5 (C-2), 136.9 (C-3), 118.4
(C-4), 129.6 (C-5), 110.0 (C-6). MS FAB m/z 797
[M H+].
8 - O - {3 - (N - [3 - Amino - 4 - methylphenyl] - carboxamido)
propanoyl}-8-O-debutanoylthapsigargin (4c). The inter-
mediate
8-O-{6-carboxypropanoyl}-8-O-debutanoyl-
thapsigargin (3c) was prepared as described above for 3b
using 2 and succinic anhydride as starting materials. A
solution of 3c (25 mg, 33 mmol), 2,4-diaminotoluene
(14 mg, 120 mmol), and DCCI (6 mg, 30 mmol) in meth-
ylene chloride (3 mL) was left for 4.5 h at room tem-
perature. The mixture was ®ltered and the ®ltrate
concentrated in vacuo to give a residue from which 8 mg
(33%) of 4c was isolated as a colourless amorphous
powder by column chromatography using an eluent
consisting of toluene/ethyl acetate (9/1), to which

S. B. Christensen et al. / Bioorg. Med. Chem. 7 (1999) 1273±1280
1277
increasing amounts of ethyl acetate were added. 1H NMR
(CDCl3) d guaianolide 4.20 (br s, H-1), 5.46 (br t, J=2Hz,
H-2), 5.59 (br t, H-8), 5.62 (br, H-3, and H-6), 2.92 (dd,
J=15 and 3 Hz, H-9), 2.42 (dd, J=15 and 3 Hz, H-9'),
1.85 (br s H-15), 1.40 (s, H-13), 1.38 (s, H-14); diamino-
toluyl 1.95 (br s, CH3), (7.00, d J=2.0 Hz, H-2), 6.90 (d,
J=8Hz, H-5), 6.72 (dd, J=8 and 2.0 Hz, H-6), succinyl
2.68 (m, H-2 and H-3); 13C NMR (CDCl3) d the guaia-
nolide nucleus 57.6 (C-1), 83.4 (C-3), 141.4 (C-4), 130.1
(C-5), 78.6 (C-7), 67.1 (C-8), 38.1 (C-9), 84.6 (C-10), 176.0
(C-12), 15.9 (C-13), 22.6 (C-14), 12.9 (C-15); succinyl
172.6 (C-1), 29.1 (C-2), 29.0 (C-3), 167.1 (C-5); diamino-
toluyl 138.8 (C-1), 136.4 (C-3), 119.7 (C-4), 127.4 (C-5).
(C-2 and C-6 not seen). MS FAB m/z 783 [M H+].
8-O-([2-t-Butoxycarbonylaminophenyl]acetyl)-8-O-de-
butanoylthapsigargin (5b). A solution of 213 (60.0 mg,
100 mol), 4-t-butoxycarbonylaminophenyla-cetic acid
(7b, 100.0 mg, 0.40 mmol), DCCI (39.1 mg, 0.19 mmol)
and DMAP (23.0 mg, 0.19 mmol) in dichloromethane
(5 mL) was left for 90 min at room temperature. The
solution was ®ltered and the ®ltrate was evaporated in
vacuo. The residue was dissolved in a few milliliters of
cold ethyl acetate and the solution was ®ltered. The ®l-
trate was concentrated in vacuo to give a residue from
which 5b (54.0 mg, 66%) was isolated as a colourless
amorphous powder by column chromatography using
toluene/ethyl acetate/acetic acid (4/1/0.01) to which
increasing amounts of ethyl acetate were added as an
1
eluent. H NMR (CDCl3) guaianolide 4.27 (br.s, H-1),
8-O-(4-t-Butoxycarbonylaminobenzoyl)-8-O-debutanoyl-
thapsigargin (5a). A solution of 213 (52.9 mg, 91 mmol)
4-t-butoxycarbonylaminobenzoic acid (7a, 40.0 mg,
169 mmol), DCCI (24.5 mg, 122 mmol) and DMAP
(7.3 mg, 60 mmol) in dichloromethane (5 mL) was left for
7 h at room temperature. The solution was ®ltered and
the ®ltrate was concentrated in vacuo. The residue was
dissolved in a few milliliters of cold ethyl acetate and the
solution ®ltered and concentrated in vacuo. Compound
5a (40.1 mg, 55%) was isolated from the residue as a
colourless amorphous powder by chromatography using
toluene/ethyl acetate (5/1) to which increasing amounts of
ethyl acetate were added as an eluent followed by chro-
matography over silanized silica gel 60 RP 2 (0.070±
5.42 (t, J=2 Hz, H-2), 5.48 (br.s, H-3), 5.63 (br.s, H-6),
5.55 (br.s, H-8), 2.94 (dd, J=15 and 3 Hz, H-9), 2.20
(dd, J=15 and 3 Hz, H-90), 1.80 (br.s H-15), 1.38 (s, H-
13), 1.18 (s, H-14); 4-aminophenylacetyl 7.30 (d,
J=7 Hz, H-2 and H-6), 7.11 (d, J=7 Hz, H-3 and H-5),
3.55 (s, CH2); boc 1.50 (s, CH3); 13C NMR (CDCl3)
guaianolide 57.4 (C-1), 76.7 (C-2), 84.1 (C-3), 141.4 (C-
4), 130.3 (C-5), 77.6 (C-6), 78.4 (C-7), 66.7 (C-8), 37.9
(C-9), 84.7 (C-10), 78.4 (C-11), 176.1 (C-12), 15.9 (C-
13), 22.5 (C-14), 12.8 (C-15); 2-aminophenylacetyl 171.1
(CˆO), 130.7 (C-1), 130.2 (C-2 and C-6), 118.6 (C-3 and
C-5), 137.5 (C-4), 40.9 (CH2); boc 28.4 (CH3), 80.7
(C O), 153.1 (C O). MS FAB Found 812.3879. Calc.
for C43H58NO14 812.3857.
1
0.230 mm) using methanol/water (9/1) as an eluent. H
NMR (CDCl3) d guaianolide 4.20 (br s, H-1), 5.50 (br t,
J=2Hz, H-2), 5.8±5.85 (H-3 and H-6), 5.72 (H-8), 3.14
(dd, J=15 and 3 Hz, H-9), 2.45 (dd, J=15 and 3 Hz, H-
9'), 1.87 (br s H-15), 1.48 (s, H-13), 1.42 (s, H-14); 4-ami-
nobenzoyl 7.85 (H-2 and H-6), 7.42 (H-3 and H-5); boc
1.52 (CH3); 13C NMR (CDCl3) d the guaianolide nucleus
57.2 (C-1), 84.0 (C-3), 141.2 (C-4), 130.2 (C-5), 78.6 (C-7),
67.1 (C-8), 38.1 (C-9), 84.5 (C-10), 78.5 (C-11), 175.6 (C-
12), 15.9 (C-13), 22.5 (C-14), 12.9 (C-15); 4-aminobenzoyl
165.4 (CˆO), 123.6 (C-1), 130.9 (C-2 and C-6), 117.6 (C-3
and C-5), 138.6 (C-4); boc 28.1 (CH3), 81.3 (C-O), 152.2
(CˆO). MS FAB m/z 798 [M H+].
8-O-(2-[4-Aminophenyl]acetyl)-8-O-debutanoylthapsi-
gargin (6b). A solution of 5b (63.4 mg, 78 mol) and tri-
¯uoroacetic acid (500 L) in dichloromethane (5 mL) was
left for 3 h at room temperature and concentrated in
vacuo. Compound 6b (46.3, 83%) was isolated as an
amorphous powder by chromatography using toluene/
ethyl acetate/acetic acid (4/1/0.01) to which increasing
1
amounts of ethyl acetate were added as an eluent. H
NMR (CDCl3) d guaianolide 4.26 (br.s, H-1), 5.43 (br.t,
J=2 Hz, H-2), 5.52 (s, H-3 and H-8) 5.64 (s, H-6), 2.97
(dd, J=15 and 3 Hz, H-9), 2.20 (dd, J=15 and 3 Hz, H-
90), 1.82 (br.s H-15), 1.34 (s, H-13), 1.21 (s, H-14); ami-
nophenylacetyl 6.98 (d, J=8 Hz, H-2 and H-6), 6.62 (d,
J=8 Hz, H-3 and H-5), 3.47 (s, CH2); 13C NMR
(CDCl3) guaianolide 57.3 (C-1), 76.7 (C-2), 84.1 (C-3),
141.4 (C-4), 130.5 (C-5), 77.7 (C-6), 78.4 (C-7), 66.6 (C-
8), 37.1 (C-9), 84.8 (C-10), 78.4 (C-11), 176.2 (C-12),
15.7 (C-3), 22.5 (C-14), 12.6 (C-15); 2-aminophenylace-
tyl 171.3 (CˆO), 40.7 (CH2), 131.0 (C-1), 130.5 (C-2
and C-6), 115.6 (C-3 and C-5), 141.4 (C-4). FABMS:
Found 712.3347. Calc. for C38H51NO12 712.3333.
8-O-(4-Aminobenzoyl)-8-O-debutanoylthapsigargin (6a).
A solution of 5a (18.1 mg, 23 mmol) and tri¯uoroacetic
acid (100 mL) in dichloromethane (2 mL) was left for
3.5 h at room temperature and concentrated in vacuo.
Compound 6a (14.0 mg, 88%) was isolated from the
residue as a yellow amophous powder by column chro-
matography using an eluent consisting of toluene/ethyl
acetate/acetic acid (4/1/0.01) to which increasing
amounts of ethyl acetate were added. 1H NMR (CDCl3) d
guaianolide 4.32 (br s, H-1), 5.51 (br t, J=2Hz, H-2), 5.8±
5.85 (H-3, H-6, and H-8), 3.10 (dd, J=15 and 3 Hz, H-9),
2.48 (dd, J=15 and 3 Hz, H-90), 1.88 (br s H-15), 1.48 (s,
H-13), 1.45 (s, H-14); 4-aminobenzoyl 7.72 (H-2 and H-6),
6.60 (H-3 and H-5); 13C NMR (CDCl3) d the guaianolide
nucleus 57.2 (C-1), 83.9 (C-3), 141.4 (C-4), 130.1 (C-5),
79.1 (C-7), 66.8 (C-8), 38.3 (C-9), 84.5 (C-10), 79.0 (C-11),
175.2 (C-12), 16.3 (C-13), 23.3 (C-14), 12.9 (C-15); 4-ami-
nobenzoyl 163.6 (CˆO), 118.3 (C-1), 131.8 (C-2 and C-6),
113.8 (C-3 and C-5), 151.3 (C-4). MS FAB m/z 698
[M H+].
8-O-(3-[4-t-Butoxycarbonylaminophenylpropionyl)-8-O-
debutanoylthapsigargin (5c). A solution of 213 (29.3 mg,
51 mmol), 3-(4-t-butoxycarbonylaminophenyl) propionic
acid (7c, 30.2 mg, 114 mmol), DCCI (14.2 mg, 71 mmol)
and DMAP (7.3 mg, 60 mmol) in dichloromethane
(2 mL) was left for 3 h at room temperature. The solu-
tion was ®ltered and the ®ltrate was evaporated in
vacuo. The residue was dissolved in a few milliliters of
cold ethyl acetate and the solution was ®ltered. The ®l-
trate was concentrated in vacuo to give a residue from
which 5c (30 mg, 72%) was isolated as a colourless

1278
S. B. Christensen et al. / Bioorg. Med. Chem. 7 (1999) 1273±1280
amorphous powder by column chromatography using
toluene/ethyl acetate (5/1) to which increasing amounts
of ethyl acetate were added as an eluent. 1H NMR
(CDCl3) d guaianolide 4.20 (br s, H-1), 5.39 (br t,
J=2 Hz, H-2), 5.5±5.6 (H-3, H-6, and H-8), 2.92 (dd,
J=15 and 3 Hz, H-9), 1.75 (br s H-15), 1.33 (s, H-13),
1.28 (s, H-14); aminophenylpropionyl 2.81 (br.t,
J=7.5 Hz, H-a), 2.51 (br.t, J=7.5 Hz, H-b), 7.19 (H-2
and H-6), 7.02 (H-3 and H-5), boc 1.42 (CH3); 13C
NMR (CDCl3) d the guaianolide nucleus 57.4 (C-1),
76.8 (H-2), 84.0 (C-3), 141.2 (C-4), 130.3 (C-5), 77.7 (C-
6), 78.4 (C-7), 66.2 (C-8), 38.0 (C-9), 84.6 (C-10), 78.3
(C-11), 175.7 (C-12), 15.7 (C-13), 22.5 (C-14), 12.8 (C-
15); 4-aminophenylpropionyl 171.9 (CˆO), 36.1 (C-a),
29.6 (C-b), 134.8 (C-1), 128.6 (C-2 and C-6), 119.2 (C-3
and C-5), 136.3 (C-4), boc 28.2 (CH3), 80.5 (C-O), 153.0
(CˆO). MS FAB m/z 826 [M H+].
175.4 (C-12), 15.7 (C-13), 22.5 (C-14), 12.9 (C-15); 4-ami-
nocinnamoyl 166.4 (CˆO), 117.7 (C-a), 140.7 (C-b), 131.1
(C-1), 127.4 (C-2 and C-6), 118.2 (C-3 and C-5), 139.6 (C-
4), boc 28.2 (CH3), 81.9 (C-O), 152.3 (CˆO). MS FAB
m/z 824 [M H+].
8- O- (4- aminocinnamoyl)- 8- O- debutanoylthapsigargin
(6d). A solution of 5d (65.5 mg, 79mmol) and tri-
¯uoroacetic acid (0.7 mL) in dichloromethane (7mL) was
left for 15min at room temperature and concentrated in
vacuo. Compound 6d (45.1 mg, 78%) was isolated as a
amorphous powder by chromatography using toluene/
ethyl acetate/acetic acid (4/1/0.01) as an eluent to which
increasing amounts of ethyl acetate were added as an elu-
ent. 1H NMR (CDCl3) d guaianolide 4.32 (br s, H-1), 5.48
(br t, J=2Hz, H-2), 5.65±5.8 (H-3, H-6, and H-8), 3.04
(dd, J=15 and 3 Hz, H-9), 2.40 (dd, J=15 and 3 Hz, H-90),
1.89 (br s H-15), 1.50 (s, H-13), 1.48 (s, H-14); 4-amino-
cinnamoyl 6.18 (d, J=16 Hz, H-a), 7.58 (d, J=16 Hz, H-
b), 7.32 (H-2 and H-6), 6.63 (H-3 and H-5); 13C NMR
(CDCl3) d the guaianolide nucleus 57.3 (C-1), 84.1 (C-3),
141.1 (C-4), 130.2 (C-5), 78.6 (C-7), 66.8 (C-8), 38.2 (C-9),
84.5 (C-10), 78.5 (C-11), 175.6 (C-12), 15.9 (C-13), 22.5 (C-
14), 12.5 (C-15); 4-aminocinnamoyl 167.0 (CˆO), 112.4
(C-a), 149.0 (C-b), 124.2 (C-1), 130.0 (C-2 and C-6), 115.0
(C-3 and C-5), 146.0 (C-4). MS FAB m/z 724 [M H+].
8-O-(3-[4-Aminophenyl]propionyl)-8-O-debutanoylthap-
sigargin (6c). A solution of 5c (40.5 mg, 49mmol) and tri-
¯uoroacetic acid (400 mL) in dichloromethane (4 mL) was
left for 45min at room temperature and concentrated in
vacuo. Compound 6c (33.7 mg, 96%) was isolated as an
amorphous powder by chromatography using toluene/
ethyl acetate/acetic acid (4/1/0.01) to which increasing
1
amounts of ethyl acetate were added as an eluent. H
NMR (CDCl3) d guaianolide 4.25 (br.s, H-1), 5.48 (br.t,
J=2Hz, H-2), 5.55±5.70 (H-3, H-6, and H-8), 2.85 (dd,
J=15 and 3 Hz, H-9), 1.75 (br.s H-15), 1.28 (s, H-13), 1.23
(s, H-14); aminophenylpropionyl 2.71 (br.t, J=7 Hz, H-a),
2.45 (br.t, J=7 Hz, H-b), 6.95 (d, J=7 Hz, H-2 and H-6),
6.65 (d, J=7 Hz, H-3 and H-5); 13C NMR (CDCl3) d
guaianolide 57.6 (C-1), 76.2 (C-2), 84.0 (C-3), 141.4 (C-4),
130.1 (C-5), 77.7 (C-6), 78.4 (C-7), 66.2 (C-8), 38.3 (C-9),
84.5 (C-10), 78.4 (C-11), 175.6 (C-12), 15.7 (C-13), 22.5 (C-
14), 12.9 (C-15); 4-aminophenylpropionyl 172.0 (CˆO),
36.3 (C-a), 29.8 (C-b), 131.0 (C-1), 129.1 (C-2 and C-6), 116.1
(C-3 and C-5), 143.4 (C-4). MS FAB m/z 726 [M H+].
8-O-(4-[4-t-Butoxycarbonylaminophenyl]butan-oyl)-8-O-
debutanoylthapsigargin (5e). A solution of 213 (57.7 mg,
100 mmol), 4-(4-t-butoxycarbonylaminophenyl)butanoic
acid (7e, 63.3 mg, 230 mmol), DCCI (25.8 mg, 120 mmol)
and DMAP (20.1 mg, 170 mmol) in dichloromethane
(3 mL) was left for 100 min at room temperature. The
solution was ®ltered and the ®ltrate was evaporated in
vacuo. The residue was dissolved in a few milliliters of
cold ethyl acetate and the solution was ®ltered. The ®l-
trate was concentrated in vacuo to give a residue from
which 5e (81.5 mg, 97%) was isolated as a colourless
amorphous powder by column chromatography using
toluene/ethyl acetate (2/1) to which increasing amounts of
ethyl acetate were added as an eluent. 1H NMR (CDCl3) d
guaianolide 4.25 (br s, H-1), 5.42 (br t, J=2Hz, H-2), 5.5±
5.6 (H-3, H-6, and H-8), 2.93 (dd, J=14 and 3 Hz, H-9),
1.80 (br s H-15), 1.38 (s, H-13), 1.35 (s, H-14); aminophe-
nylbytanoyl 2.53 (br.t, J=7Hz, H-a), 1.80 (m, H-b), 2.25
(t, J=7Hz, H-g), 7.18 (H-2 and H-6), 7.05 (H-3 and H-5),
boc 1.50 (CH3); 13C NMR (CDCl3) d the guaianolide
nucleus 58.1 (C-1), 76.7 (H-2), 84.0 (C-3), 141.2 (C-4),
130.3 (C-5), 77.5 (C-6), 78.4 (C-7), 66.7 (C-8), 38.0 (C-9),
84.6 (C-10), 78.3 (C-11), 175.6 (C-12), 15.7 (C-13), 22.5
(C-14), 12.8 (C-15); 4-aminopheylbutanoyl 172.4 (CˆO),
33.3 (C-a and C-g), 26.0 (C-b), 135.8 (C-1), 128.8 (C-2 and
C-6), 119.0 (C-3 and C-5), 136.1 (C-4), boc 28.2 (CH3),
80.5 (C O), 152.8 (CˆO). MS FAB Found 840.4093.
Calc. for C45H63NO14 840.4170.
8-O-(4-t-Butoxycarbonylaminocinnamoyl)-8-O-debutan-
A
solution of 213 (62.1 mg,
oylthapsigargin (5d).
107 mmol), 4-t-butoxycarbonlyaminocinnamic acid (7d,
69.7 mg, 265 mmol), DCCI (21.3 mg, 106 mmol) and
DMAP (12.8 mg, 106 mmol) in dichloromethane (5 mL)
was left for 3 h at room temperature. The solution was
®ltered and the ®ltrate was evaporated in vacuo. The
residue was redissolved in a few milliliters of cold ethyl
acetate and the solution was ®ltered and evaporated in
vacuo to give yellowish foam. Compound 5d (31.5 mg,
36%) was isolated as a white amorphous powder by
chromatography using toluene/ethyl acetate (5/1) to
which increasing amounts of ethyl acetate were added
followed by chromatography over silanized silica gel 60
RP-2 (0.070±0.230 mm, Merck) using methanol/water
(9/1) as an eluent. 1H NMR (CDCl3) d guaianolide 4.31
(br s, H-1), 5.51 (br t, J=2Hz, H-2), 5.65±5.8 (H-3, H-6,
and H-8), 3.08 (dd, J=15 and 3 Hz, H-9), 2.40 (dd, J=15
and 3 Hz, H-90), 1.87 (br s H-15), 1.48 (s, H-13), 1.28 (s, H-
14); 4-aminocinnamoyl 6.28 (d, J=16 Hz, H-a), 7.59 (d,
J=16 Hz, H-b), 7.42 (H-2 and H-6), 7.28 (H-3 and H-5),
boc 1.50 (CH3); 13C NMR (CDCl3) d the guaianolide
nucleus 57.4 (C-1), 84.0 (C-3), 141.4 (C-4), 130.2 (C-5),
78.6 (C-7), 66.7 (C-8), 38.1 (C-9), 84.5 (C-10), 78.6 (C-11),
8-O-(4-[4-Aminophenyl]butanoyl)-8-O-debutanoylthap-
sigargin (6e). A solution of 5e (33.1 mg, 39 mmol) and
tri¯uoroacetic acid (400 mL) in dichloromethane (4 mL)
was left for 45 min at room temperature and con-
centrated in vacuo. Compound 6e (27.7 mg, 96%) was
isolated as an amorphous powder by chromatography
using toluene/ethyl acetate/acetic acid (2/1/0.01) to

S. B. Christensen et al. / Bioorg. Med. Chem. 7 (1999) 1273±1280
1279
which increasing amounts of ethyl acetate were added as
an eluent. 1H NMR (CDCl3) d guaianolide 4.25 (br.s, H-
1), 5.68 (br.s, H-6), 5.63 (br.s. H-8), 5.59 (t, J=3Hz, H-3),
5.47 (br.t, J=2 Hz, H-2), 2.97 (dd, J=20 and 3 Hz, H-9),
1.84 (br.s H-15), 1.40 (s, H-13), 1.37 (s, H-14); aminophe-
nylbutanoyl 2.52 (t, J=9Hz, H-a), 2.24 (t, J=9Hz, H-g),
1.80 (m, H-b), 6.94 (d, J=7Hz, H-2 and H-6), 6.64 (H-3
and H-5); 13C NMR (CDCl3) d guaianolide 57.5 (C-1),
76.7 (C-2), 84.1 (C-3), 141.6 (C-4), 130.5 (C-5), 77.8 (C-6),
78.5 (C-7), 66.1 (C-8), 38.1 (C-9), 84.6 (C-10), 78.5 (C-11),
178.5 (C-12), 15.7 (C-13), 22.5 (C-14), 12.8 (C-15); 4-ami-
nophenylbutanoyl 172.7 (CˆO), 34.0 (C-a), 33.6 (C-g),
26.9 (C-b), 129.3 (C-1), 129.3 (C-2 and C-6), 115.7 (C-3
and C-5), 143.9 (C-4). MS FAB Found 740.3672. Calc.
for C40H55NO12 740.3646.
Determination of cytosolic Ca2+ concentration ([Ca2+]i).
TSU cells (106) were treated with the indicated analo-
gues for 1 h at 37 ꢀC. Cells were then loaded with Fura
2-acetoxymethyl ester (5 mM) (0.1% DMSO/0.001%
Plutonic) (Molecular Probes, Eugene OR) for 30 min at
33 ꢀC in phenol red free RPMI media. Cells were then
pelleted and resuspended in bu€er of (4-(2-hydro-
xyethyl)-1-piperazineethansulfonic
acid
(HEPES)-
Krebb's medium [108 mM NaCl, 6 mM KCl, 1.2 mM
MgSO4, 1.5 mM CaCl2, 11.5 mM glucose, and 10 mM
HEPES-KOH (pH 7.4)]) for an additional 20 min. Cells
were then repelleted and washed just prior to assay then
suspended in a quartz cuvette and ¯uorescence emission
at 510 nm was measured after excitation at 340 nm and
380 nm using a photomultiplier from PTI (New Bruns-
wick, NJ) at 25 ꢀC. [Ca2+]i were calculated from ratios
from ¯uorescence intensities obtained every 1 second
using the calculations of Grynkiewicz et al. and a Kd of
150 nM.17 Dye was considered saturated on addition of
10 mM ionomycin while the minimum ¯uorescence ratio
was determined in the presence of 5 mM EGTA.
Pharmacology
SERCA assay. Sarcoplasmic reticulum (SR) vesicles
from rabbit skeletal muscle were prepared according to
De Meis and Hasselbach14 and stored in N-tris(hydrox-
ymethyl)methyl-2-aminoethanesulphonic acid (TES)
20 mM (pH 7.0), sucrose 300 mM, at 80 ꢀC.
Cytotoxicity assays. Percentage clonogenic survival of
TSU-Pr1 (2Â105 cells) following 2 h exposure to varying
concentrations of Tg analogues was performed as
described previously.18
ATPase activity was measured as 45Ca2+ uptake to SR
vesicles at 25 ꢀC, pH 7.0, using 2±5 mg/mL of protein.
Triplicate samples of SR membranes were preincubated
for 10 min in the uptake medium minus ATP, in the
presence of the desired inhibitor concentrations (added
as 100- or 200-fold concentrated stock solutions in
DMSO; corresponding amount of DMSO was present
in control incubations). The medium composition was
then completed by adding ATP (or TES bu€er for the
blank determinations) to start the uptake reaction. The
complete medium contained (concentrations in mM):
TES 20; sucrose, 60; KCl 110; MgCl2 5; K+-oxalate 6;
EGTA 1; 45CaCl2 0.2 (about 2 mCi/mL); ATP 2.5 mM.
(The concentration of free Ca2+ ions was 0.1 mM, cal-
culated according to Foehr et al. 199315). The uptake
was stopped after 4 min by ®ltration through Whatman
GF/F ®lters (presoaked in 0.1% polyethyleneimine to
reduce the background radioactivity) on a Brandel
Harvester M24 apparatus (SEMAT Technical Ltd.,
UK). The ®lters were washed immediately in an ice-cold
bu€er containing (concentrations in mM): tris/HCl 20
(pH 7); NaCl 140; MgCl2 10; and counted using Opti-
Fluor scintillation liquid (Packard Instruments, DK) at
100% eciency. The time course of the uptake was lin-
ear at least up to 4 min.
Acknowledgements
CaPCURE foundation and PharmaBiotec are
acknowledged for ®nancial support. MT was supported
by the Danish Medical Research Council and the Dan-
ish Government Biotechnology Programme. We thank
Dr. C. E. Olsen, The Royal Veterinary and Agricultural
University, Copenhagen for recording the mass spectra.
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