A novel cardenolide photoaffinity label for the Na/K-ATPase

Article abstract of DOI:10.1016/S0040-4020(00)00916-9

A benzoylbenzoyloxy derivative of digitoxigenin has been prepared which is the first cardenolide with an affinity label in the lactone ring. It inhibits the Na⁺/K⁺-ATPase almost as effectively as digitoxigenin itself. The activity was lost when in addition a biotin tag was attached to ring A. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00916-9

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 9625±9632
A Novel Cardenolide Photoaf®nity Label for the Na/K-ATPase
a
Jorg Zotzmann, Lothar Hennig, Peter Welzel, Dietrich Muller, Claudia Schafer,
a
a,
b
c
*
È
È
È
Stefan Zillikens,c Hermann Pusch,c Helfried G. Glitschc and Ralf Regenthald
a
È
È
Institut fur Organische Chemie der Universitat Leipzig, Johannisallee 29, D-04103 Leipzig, Germany
È
b
È
Lehrstuhl fur Analytische Chemie der Ruhr-Universitat, D-44780 Bochum, Germany
Arbeitsgruppe Muskelphysiologie der Ruhr-Universitat, D-44780 Bochum, Germany
c
È
d
È È È
Institut fur Klinische Pharmakologie der Universitat Leipzig, Hartelstr. 16-18, D-04107 Leipzig, Germany
This paper is dedicated with great admiration and graditude to Professor Koji Nakanishi on the occasion of his 75th birthday
Received 7 September 2000; accepted 29 September 2000
AbstractÐA benzoylbenzoyloxy derivative of digitoxigenin has been prepared which is the ®rst cardenolide with an af®nity label in the
lactone ring. It inhibits the Na1/K1-ATPase almost as effectively as digitoxigenin itself. The activity was lost when in addition a biotin tag
was attached to ring A. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
structure of the protein and of the conformational inter-
mediates in the catalytic cycle which is not available. All
what is known today has been extracted from indirect
informations such as twodimensional crystals, site-directed
mutagenesis, ¯uorescence methods, af®nity labeling,
competition experiments with antibodies, structure-activity
relationships (as far as the cardiotonic steroids are involved)
and molecular modeling.6±9
The Na1/K1-ATPase (Na1 pump) is a dimeric (ab) trans-
membrane complex that is expressed in virtually all animal
cells. It couples ATP hydrolysis with the export of Na1 and
the import of K1. The ion gradients thus established over the
membrane drive numerous co- and countertransporters that
supply the cells inter alia with glucose and amino acids,
regulate cell volume, Ca21 concentration, and underlie
nearly all electrical activity in the peripheral and central
nervous system and in cardiac and skeletal muscle.1±3 In
addition, the Na1/K1-ATPase is the receptor for the cardio-
active steroids. These compounds speci®cally inhibit the
Na1 pump, thereby raising the intracellular Na1 concentra-
tion. The resulting decrease in the Na1 gradient across the
cell membrane of cardiac myocytes reduces the energy
available for transport of Ca21 out of the cell by Na1/
Ca21 exchange. This ultimately leads to the (medicinally
used) positive inotropic effect of these substances.4
The ATPase consists of a large catalytic a subunit and a
smaller b subunit the function of which is still not com-
pletely understood. The complete amino-acid sequences of
a subunits of various isoforms have been determined.5 The
protein chain spans the plasma membrane ten times
(H1±H10 spans).
According to the present knowledge the major cytoplasmic
loop of the a subunit contains the ATP binding and the
phosphorylation sites. The cation binding sites are provided
by amino acid residues of the intramembrane segments of
the a unit. The cardioactive steroids (see digitoxigenin (1)
as an example) interact with the extracellular portion. Both
from site-directed mutagenesis and from af®nity labeling
experiments it has been inferred that the H1±H2 extra-
cellular region is involved in binding. There are other
labeling experiments that indicate that the H3±H4 region
is involved in binding the lactone moiety.10 These results
Mechanistic understanding both of the normal function of
the enzyme and of the inhibition by the cardioactive steroids
would demand knowledge of the three-dimensional
Keywords: cardenolides; Na,K-ATPase; af®nity label; benzophenone.
* Corresponding author. 149-341-97-36551; fax: 149-341-97-36599;
e-mail: welzel@organik.chemie.uni-leipzig.de
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00916-9

9626
J. Zotzmann et al. / Tetrahedron 56 (2000) 9625±9632
Scheme 1.
È
have been summarized by Holtje and Anzali in a binding
model9 which, however, has been shown to be unable to
explain all the experimental results.11,12
derivatives having an acetate and a benzoate group, respec-
tively, attached to C-22 (see, for example formula 2b)
inhibit the Na1/K1-ATPase quite effectively.11,12 It seemed
promising, to study derivatives of 2b with reactive groups
attached to the aromatic ring (Scheme 1).
New results point to the major involvement of the H5±H6
domain in the inhibition of the enzyme by cardiotonic
steroids (ouabain).13,14
With this in mind we prepared compound 3b. The aryl
tri¯uoromethyl azirine is known to decompose to a carbene
on irradiation at 350 nm.16 Thus, the known derivative 2a
of 22-hydroxy-digitoxigenin11 was esteri®ed with 4-(3-
With respect to af®nity labeling, most labeling experiments
have identi®ed binding sites in the H1±H2 region.15 In all
cases the af®nity label was in some way attached to ring A
of the steroid. The only labeling experiment we are aware
of, in which the af®nity label was part of the side chain at
C-17 was reported by McParland and coworkers who used a
tritiated lactone analogue with a 24-azido-21-nor-20(22)-
en-23-one side chain. Degradation of the labeled protein
and sequence analysis showed the label to be in the extra-
cellular domain between the H3 and H4 hydrophobic
domains.10
tri¯uoromethyl-3-H-diazirinyl)benzoic
acid17
using
dicyclohexylcarbodiimide in the presence of Steglich's
base18 to give 3a in 84% yield. Removal of the silyl protect-
ing groups was achieved by acid treatment in methanolic
solution.11 Unfortunately, 3b did not at all inhibit the Na1/
K1-ATPase (see Table 1).
The next compound to be studied was the benzoylbenzoate
4a. Again, the DCC method was employed, and the yield
was 90%. Deprotection under the conditions described
above provided 4a, but to a major extent the dimethyl acetal
at the benzophenone unit (formula not shown) was
formed.19 The unwanted side product was omitted when
after the cleavage in methanolic solution the crude reaction
product was treated with aqueous acid.20 The overall yield
was 94%. 4b turned out to inhibit the Na1/K1-ATPase very
effectively (see Table 1). This is an exciting result since for
the ®rst time an active inhibitor of the Na1/K1-ATPase was
found with the photo-reactive group in the lactone ring. In
addition, it can be concluded that in the binding pocket
around the lactone ring there must be even more space
From many structure-activity studies it is quite clear, that
the lactone side chain of the cardiotonic steroids is a main
structural prerequisite for their interaction with the Na1/K1-
ATPase.7 It appeared to us, therefore, worthwhile to develop
af®nity labels with the reactive groups attached to the
lactone ring. These studies will be detailed below.
Results and Discussion
Our work started from the observation, that digitoxigenin

J. Zotzmann et al. / Tetrahedron 56 (2000) 9625±9632
9627
Scheme 2.
than previously assumed.11 It should be remembered in this
context that obviously the ester group is involved in binding
to the enzyme since many other 22-substituted digitoxigenin
derivatives have been shown to be inactive.11
C-3 of 4b. In a ®rst series of experiments (Scheme 2) it was
tried to prepare compound 5b. Unfortunately, attempted
esteri®cation of 4b with biotin using the DCC method was
unsuccessful. Then it was tried to use the N-hydroxy suc-
cinimide derivative of biotin as well as the acid chloride22 as
acylating reagents, again without success. The Staab
method failed, too.23 The same results were obtained
when digitoxigenin was employed as a model compound.
Finally after much experimentation it was shown that
the Yamaguchi method24 provided 5a in 54% yield. But
even this method did not reliably provide 5b. In one
experiment a yield of 35% was obtained but many other
experiments failed. Instead the trichlorobenzoate 4c was
isolated.
Usually, after photolabeling the enzyme is digested
enzymatically and the labeled fragments are isolated and
submitted to structural analysis. Biotin-tagging is a power-
ful method for identifying labeled protein fragments and
isolating them by af®nity chromatography based on the
strong interaction of biotin with either avidin or
streptavidin.21
We attempted, therefore, to add a biotin tag to the oxygen at

9628
J. Zotzmann et al. / Tetrahedron 56 (2000) 9625±9632
Table 2. Recognition of compound 1, 4b, 5a by anti-digitoxin antibodies
Table 1. K0.5 values for Na1/K1 pump inhibition by digitoxigenin and
derivatives 2b, 3b, 4b, 5a, 5b, 8a, 8b in cardiac cells (The K0.5 value
represents the drug concentration required for half-maximal Na1/K1
pump inhibition. The pump exchanges three intracellular Na1 for two
extracellular K1 per ATP molecule split and generates thereby the pump
current Ip. This current was measured as an indicator of pump activity in
isolated guinea pig cardiac ventricular cells or sheep cardiac Purkinje cells
(from the conducting system) by means of whole-cell recording.25 The
measurements were performed in a bathing solution including 144 mM
NaCl and 5.4 mM KCl. The patch pipette solution contained inter alia
50 mM NaCl and 110 mM CsCl. pH was 7.4 in both media. The compounds
were added to the external solution from 1022 M ethanolic or DMSO stock
solutions and their inhibitory effect on Ip was measured. The ®nal concen-
trations of the solvents had no effect on Ip. The experiments were carried out
at 328C and 0 mV membrane (holding) potential. Note the higher sensitivity
of the Purkinje cells to cardiac steroids. `n' indicates the number of
measurements.)
Compound
Sample concentration (M)
Result in digitoxin
concentrations
1
4b
5a
1028
1027
1027
9.80 ng/ml (1.28£1028 M)
No effect
61.76 ng/ml (8.07£1028 M)
directed against the steroid moiety. We used the ABBOTT
AxSYM Digitoxin system which is based on a ¯uorescence
polarisation immuno assay in which digitoxigenin and a
¯uorescein-labeled digitoxin derivative compete for the
binding sites at the polyclonal antibodies (canin serum). It
turned out that the biotinylated digitoxin derivative 5a was
identi®ed by the antibodies at a concentration of 1027 mol/l.
In the control experiment digitoxigenin was recognized at a
concentration of less than 1027 mol/l. Unfortunately, 4b was
not recognized by the antibodies (see Table 2). This result
probably means that the lactone part of the cardenolides
interacts with the antibody as has been found for mono-
clonal anti-digoxin antibodies.26
Compound
Guinea-
pig
ventricular
cells
K0.5 [M]
n
Sheep
cardiac
Purkinje
cells
n
K0.5 [M]
Digitoxigenin (1)
3.0£1026
±
50
1.9£1027
8
17
2b
3b
4b
5a
5b
8a
8b
4.1£1026
5.0£1025
1.2£1025
1.0£1026
1.4£1025
8.0£1026
No effect
2
28
4
4
11
5
6.0£1027
8
The result of our investigations is that neither the biotin±
avidin/streptavidin technology nor af®nity chromatography
using anti-digitoxin antibodies can be used for the isolation
of labeled fragments of the Na1/K1-ATPase. In any case,
the compounds described here should be valuable tools for
further work in this area. For the af®nity labeling studies
presumably recourse has to be made to analytical methods
based on radioactive isotopes. Since the synthesis of
p-benzoylbenzoic acid is very simply achieved, a radio-
actively labeled derivative should be easily accessible.27
5b only weakly inhibited the Na1/K1-ATPase. On the other
hand, the biotinylated digitoxigenin 5a was quite active. In
order to ®nd out whether a longer spacer arm would lead to
better results, both digitoxigenin (1) and 4b were converted
into the biotinylated derivatives 6a and 6b, respectively.
Again, the Yamaguchi method was only partly successful.
The yields were only in the range of 10±14% and we could
not prepare suf®cient amounts for the biological tests. In a
last set of experiments digitoxigenin (1) was converted into
the isothiocyanatobenzoate 7a on treatment with 4-isothio-
cyanatobenzoyl chloride. This reaction worked quite well
and provided 7a in 68% yield. 7a was in turn converted into
8a on treatment with the appropriate amine derivative of
biotin. The yield of thiourea 8a was 74%. In the same
way 4b was converted to 7b (69%) in a very slow reaction
and thence to the rather unstable compound 8b (44%). 8b is
Experimental
General
NMR: GEMINI 200 (Varian), GEMINI 2000 (Varian),
GEMINI 300 (Varian), DRX 400 (Bruker); chemical shifts
are given in d values, CH3, CH2, CH groups and quaternary
carbons when identi®ed by APT are indicated by (2) (CH3,
CH) and (1) (CH2, Cq), respectively. Mass spectrometry:
FAB MS: VG Autospec (Fisons, 3-nitrobenzylalcohol
matrix), ESI MS: FT-ICR-MS Apex II (Bruker Daltonics,
acetonitrile±formic acid, positive ion mode). IR spectra:
FT-IR ATI Mattson (Genesis Series, KBr). UV spectra:
DU-650 (Beckman, acetonitrile).
1
characterized by a nice ESI mass spectrum. The H NMR
spectra suffered from broad peaks obviously caused by
restricted rotation in some parts of the molecule. Heating
to 808C improved the quality of the NMR spectra.
The inhibition of the Na1/K1 pump by both 8a and 8b were
studied as summarized in Table 1. 8a had a reduced activity
whereas 8b was completely inactive.
22-[4-(3-Tri¯uoromethyl-3H-diazirinyl)benzoyloxy]-3b-
(tert-butyldimethylsilyloxy)-14-trimethylsilyloxy-5b,14b-
card-20(22)-enolide (3a). A solution of 2a (100 mg;
173 mmol)
and
4-dimethylaminopyridine
(2.8 mg;
Obviously, there is a binding pocket for the cardioactive
steroids which is accessible to both 4b and 5a with one
extra substituent but not to 5b that seems to be too big. In
the same way the results for 8a and 8b can be explained.
23 mmol) in dichloromethane (ca. 5 ml) was added to a
mixture of N,N0-dicyclohexylcarbodiimide (71 mg;
346 mmol) and 4-(3-tri¯uoromethyl-3H-diazirinyl)benzoic
acid (80 mg; 346 mmol). The reaction mixture was stirred
at 208C for 2 h (white precipitate) and then ®ltered. The
®ltrate was diluted with dichloromethane and the solution
washed with water then with 5 percent sodium hydrogen
carbonate. The aqueous phases were reextracted with
dichloromethane. Drying of the combined organic phases
with sodium sulfate, solvent evaporation and LC (petroleum
From these results summarized in Table 1 it has to be
concluded that the isolation of labeled peptides in af®nity
labeling experiments will not be possible making use of the
biotin±avidin method. An alternative would be to identify
labeled fragments of the Na1/K1-ATPase with antibodies

J. Zotzmann et al. / Tetrahedron 56 (2000) 9625±9632
9629
ether±ethyl acetate 12:1) provided 3a (115 mg, 84%). IR
(KBr): 1779, 1754, 1633, 1260, 1196, 1159, 1118, 1056,
®ltered off and the ®ltrate was worked up as usual. LC
(petroleum ether±ethyl acetate 8:1!6:1) furnished
244 mg (90%) of 4a. Mp 171±173 8C (from petroleum
ether). IR (KBr): 1754, 1633, 1254, 1124, 1056,
1
837 cm21. H NMR (200 MHz, CDCl3): dˆ0.01 (s, 6H,
tBuSi(CH3)2), 0.16 (s, 9H, (CH3)3Si), 0.87 (s, 9H,
(CH3)3C), 0.90 and 0.92 (2 s, respectively 3H, CH3-18,
CH3-19), 1.00±2.05 (om (overlapping multiplets)), 2.85±
2.95 (m, 1H, 17a-H), 4.02 (broad s, 1H, 3a-H),
1
837 cm21. H NMR (200 MHz, CDCl3): dˆ0.01 (s, 6H,
tBuSi(CH3)2), 0.18 (s, 9H, (CH3)3Si), 0.88 (s, 9H,
(CH3)3C), 0.91 (s, 3H, CH3-19), 0.96 (s, 3H, CH3-18),
1.05±2.10 (om), 2.90±3.01 (m, 1H, 17a-H), 4.03 (broad s,
2
4.9015.02 (AB, 2H, CH2-21, JABˆ17.2 Hz), 7.3018.15
3
2
(AA0BB0, 4H, arom. H, JABˆ8.5 Hz). 13C NMR
1H, 3a-H), 4.9315.04 (AB, 2H, CH2-21, JABˆ17.0 Hz),
(50.3 MHz, APT, CDCl3): dˆ24.39 (2) (tBuSi(CH3)2),
3.49 (2) (Si(CH3)3), 18.0 (2) (C-18), 18.6 (1) (C(CH3)3),
21.3 (1), 24.1 (1), 24.3 (2) (C-19), 25.8 (1), 26.3 (2)
7.46±7.57 (m, 2H, arom. Hm, ring G), 7.59±7.69 (m, 1H,
arom. Hp, ring G), 7.78±7.86 (m, 2H, arom. Ho, ring G),
3
7.8918.25 (AA0BB0, 4H, arom. H, ring F, JABˆ8.4 Hz).
2
(C(CH3)3), 27.2 (1), 28.9 (1) (q, C(CF3), JC,Fˆ41.1 Hz),
13C NMR (50.3 MHz, APT, CDCl3): dˆ24.51 (2) and
24.49 (2) (tBuSi(CH3)2), 3.41 (2) ((CH3)3Si), 17.9 (2)
(C-18), 18.5 (1) (C(CH3)3), 21.2 (1), 24.0 (1), 24.2 (2)
(C-19), 25.8 (1), 26.2 (2) (C(CH3)3), 27.1 (1), 28.9 (1),
30.2 (1), 34.4 (1), 34.8 (1), 36.1 (1), 36.2 (2) and 37.3
(2) (C-5, C-9), 41.2 (2) (C-8), 42.3 (1) (C-12), 48.2 (2)
(C-17), 52.3 (1) (C-13), 67.6 (2) (C-3), 70.0 (1) (C-21),
91.8 (1) (C-14), 129.1 (2) (C-3G), 130.4 (2) and 130.8 (2)
(C-2F, C-3F), 130.6 (2) (C-2G), 131.5 (1) and 137.2 (1)
(C-1F, C-1G), 133.7 (2) (C-4G), 135.3 (1) (C-22), 142.9 (1)
(C-4F), 155.0 (1) (C-20), 162.9 (1) (COArF), 168.0 (1)
(C-23), 196.3 (1) (COArG). UV (CH3CN): lmax.
(emax.)ˆ255 nm (18500). C46H64O7Si2 (785.18), FAB MS:
m/z (%)ˆ807 (4; [M1Na]1), 727 (3; [M1H2C4H10]1),
563 (8; [M1H2tBuMe2SiOH±Me3SiOH]1), 209 (100,
[C6H5COC6H4CO]1). HRMS: calcd for C46H64O7Si2Na
([M1Na]1) 807.4088, found 807.4090.
29.0 (1), 30.3 (1), 34.4 (1), 34.9 (1), 36.2 (1), 36.3 (2)
and 37.4 (2) (C-5, C-9), 41.3 (2) (C-8), 42.4 (1) (C-12),
48.3 (2) (C-17), 52.3 (1) (C-13), 67.6 (2) (C-3), 70.0 (1)
(C-21), 91.8 (1) (C-14), 122.3 (1), (q, CF3, 1JC,Fˆ275 Hz),
4
127.1 (2) (q, C-3F, JC,Fˆ1.5 Hz), 129.6 (1) (C-1F), 131.2
(2) (C-2F), 135.2 (1) and 135.5 (1) (C-22, C-4F), 154.8
(1) (C-20), 162.5 (1) (COAr), 167.8 (1) (C-23). 19F NMR
(188.2 MHz, CDCl3): dˆ12.9 (s, CF3). UV (CH3CN): lmax.
(emax.)ˆ233 nm (19500). C41H59F3N2O6Si2 (789.10), FAB
MS: m/z (%)ˆ811 (5; [M1Na]1), 789 (2; [M1H]1), 567
(31; [M1H2tBuMe2SiOH±Me3SiOH]1), 213 (48;
[COC6H4CN2CF3]1), 185 (100; [COC6H4CFvCF2]1).
22-[4-(3-Tri¯uoromethyl-3H-diazirinyl)benzoyl]oxy-3b,14-
dihydroxy-5b,14b-card-20(22)-enolide (3b). To 3a
(99 mg; 125 mmol) a solution of p-toluenesulfonic acid
monohydrate (10 ml of a 10 mM solution in methanol)
was added. The mixture was stirred at 208C for 14 h.
Usual work-up and LC (petroleum ether±ethyl acetate
2:3) furnished 3b (72 mg, 95%). IR (KBr): 1756, 1632,
22-(4-Benzoylbenzoyloxy)-3b,14-dihydroxy-5b,14b-card-
20(22)-enolide (4b). To 4a (244 mg; 311 mmol) p-toluene-
sulfonic acid monohydrate (20 ml of a 10 mM solution in
methanol) was added and the mixture was stirred at 208C for
24 h. After usual work-up the residue was redissolved in 4:1
dioxane±water (15 ml). HCl (1 ml of a 0.02 M aqueous
solution) was added and the mixture was stirred at 408C
for 6 h. After solvent evaporation, usual work-up and LC
(petroleum ether±ethyl acetateˆ1:1!1:2) 175 mg (94%) of
4b were obtained. IR (KBr): 1761, 1658, 1256, 1118, 1022,
1344, 1264, 1230, 1197, 1160, 1121 cm21 1H NMR
.
(200 MHz, CDCl3): dˆ0.94 (s, 6H, CH3-18, CH3-19),
1.15±2.20 (om), 2.88±2.97 (m, 1H, 17a-H), 4.01 (broad s,
2
1H, 3a-H), 4.9415.20 (AB, 2H, CH2-21, JABˆ17.8 Hz),
3
7.3118.11 (AA0BB0, 4H, arom. H, JABˆ8.5 Hz). 13C
NMR (50.3 MHz, APT, CDCl3): dˆ15.7 (2) (C-18), 21.5
(1), 21.7 (1), 24.1 (2) (C-19), 25.6 (1), 26.8 (1), 28.3
2
1
(1), 28.8 (1) (q, C(CF3), JC,Fˆ40.8 Hz), 30.0 (1), 33.6
710 cm21. H NMR (200 MHz, CDCl3): dˆ0.83±1.01 (m)
(1), 33.7 (1), 35.8 (1), 35.9 (2) and 36.3 (2) (C-5, C-9),
40.3 (1) (C-12), 42.0 (2) (C-8), 47.6 (2) (C-17), 50.4 (1)
(C-13), 67.2 (2) (C-3), 70.3 (1) (C-21), 85.8 (1) (C-14),
therein 0.94 and 0.96 (2 s, CH3-18, CH3-19), 1.15±2.22
(om), 2.93±3.02 (m, 1H, 17a-H), 4.11 (broad s, 1H, 3a-
2
H), 4.9615.22 (AB, 2H, CH2-21, JABˆ17.8 Hz), 7.46±
1
122.3 (1) (q, CF3, JC,Fˆ275 Hz), 127.2 (2) (q, C-3F,
7.57 (m, 2H, arom. Hm, ring G), 7.59±7.69 (m, 1H, arom.
Hp, ring G), 7.78±7.84 (m, 2H, arom. Ho, ring G),
4JC,Fˆ1.5 Hz), 129.6 (1) (C-1F), 131.3 (2) (C-2F), 135.4
(1) and 135.6 (1) (C-22, C-4F), 155.5 (1) (C-20), 162.8
(1) (COAr), 168.0 (1) (C-23). 19F NMR (188.2 MHz,
CDCl3): dˆ12.9 (s, CF3). UV (CH3CN): lmax.
(emax.)ˆ231 nm (20300). C32H37F3N2O6 (602.65), FAB
MS: m/z (%)ˆ625 (5; [M1Na]1), 603 (2; [M1H]1), 585
(9; [M1H2H2O]1), 567 (7; [M1H22H2O]1). HRMS:
calcd for C32H38F3N2O6 ([M1H]1) 603.2682, found
603.2692.
3
7.8818.25 (AA0BB0, 4H, arom. H, ring F, JABˆ8.6 Hz).
13C NMR (50.3 MHz, APT, CDCl3): dˆ15.9 (2) (C-18),
21.6 (1), 21.9 (1), 24.2 (2) (C-19), 25.7 (1), 26.9 (1),
28.4 (1), 30.1 (1), 33.7 (1), 33.8 (1), 35.9 (1), 36.0 (2)
and 36.5 (2) (C-5, C-9), 40.4 (1) (C-12), 42.2 (2) (C-8),
47.7 (2) (C-17), 50.5 (1) (C-13), 67.3 (2) (C-3), 70.3 (1)
(C-21), 85.8 (1) (C-14), 129.1 (2) (C-3G), 130.4 (2) and
130.9 (2) (C-2F, C-3F), 130.7 (2) (C-2G), 131.5 (1) and
135.4 (1) and 137.2 (1) (C-22, C-1F, C-1G), 133.7 (2) (C-
4G), 142.9 (1) (C-4F), 155.4 (1) (C-20), 163.0 (1)
(COArF), 168.0 (1) (C-23), 196.3 (1) (COArG). UV
(CH3CN): lmax. (emax.)ˆ255.0 nm (25800). C37H42O7
(598.74).
22-(4-Benzoylbenzoyloxy)-3b-(tert-butyldimethylsilyl-
oxy)-14-trimethylsilyloxy-5b,14b-card-20(22)-enolide
(4a).
A solution of 2a (200.0 mg; 346 mmol) and
4-dimethylaminopyridine (5.5 mg; 45 mmol) in dichloro-
methane (5 ml) was added to a mixture of N,N0-
dicyclohexylcarbodiimide (128.7 mg; 624 mmol) and
4-benzoylbenzoic acid (141.2 mg; 624 mmol). The reaction
mixture was stirred at 208C for 4 h. A white precipitate was
14-Hydroxy-3b-[5-((3aS,4S,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl)pentanoyloxy]-5b,14b-card-
20(22)-enolide (5a). To a solution of biotin (73.3 mg;

9630
J. Zotzmann et al. / Tetrahedron 56 (2000) 9625±9632
300 mmol) and triethylamine (45.5 mg; 450 mmol) in THF
(ca. 6 ml, freshly distilled under Ar) 2,4,6-trichlorobenzoyl
chloride (73.2 mg; 300 mmol) was added dropwise. The
mixture was stirred at 208C for 4 h. After solvent evapora-
tion the residue was redissolved in toluene (5 ml) and a
solution of digitoxigenin (74.9 mg; 200 mmol) and 4-(1-
pyrrolidinyl)-pyridine (PPP, 59.3 mg; 400 mmol) in toluene
(1 ml) was added. The reaction mixture was stirred at 408C
for 4 h and at 208C for 16 h. Usual work-up and LC (ethyl
acetate±methanol 1!5:1) furnished 65.1 mg (54%) of 5a.
11.0 mg (15%) of digitoxigenin were recovered. IR (KBr):
(C-2G), 131.5 (1) and 137.2 (1) and 142.9 (1) (C-1F, C-4F,
C-1G), 133.7 (2) (C-4G), 135.4 (1) (C-22), 155.5 (1)
(C-20), 163.0 (1) (COArF), 164.0 (1) (C-2A), 168.0 (1)
(C-23), 173.6 (1) (C-1B), 196.3 (1) (COArG). UV
(CH3CN): lmax. (emax.)ˆ255.5 nm (12200). C47H56N2O9S
(825.04), FAB MS: m/z (%)ˆ847 (2; [M1Na]1), 825 (1;
[M1H]1).
22-(4-Benzoylbenzoyloxy)-14-hydroxy-3b-(2,4,6-tri-
chlorobenzoyloxy)-5b,14b-card-20(22)-enolide (4c). 1H
NMR (200 MHz, CDCl3): dˆ0.93 and 0.97 (2 s, 3H each,
18-CH3 and 19-CH3), 1.15±2.22 (om), 2.94±3.04 (m, 1H,
17a-H), 4.9715.23 (AB, 2H, CH2-21, 2JABˆ17.7 Hz), 7.35
(s, 2H, arom. trichlorobenzoyl-H), 7.46±7.57 (m, 2H, arom.
Hm, ring G), 7.59±7.69 (m, 1H, arom. Hp, ring G), 7.78±
7.85 (m, 2H, arom. Ho, ring G), 7.8918.25 (AA0BB0, 4H,
1739, 1706, 1453, 1263, 1151, 1024 cm21 1H NMR
.
(200 MHz, CDCl3): dˆ0.85 (s, 3H, CH3-18), 0.94 (s, 3H,
CH3-19), 1.10±2.22 (om), 2.31 (t, 2H, CH2-2B,
3JB2,3ˆ7.3 Hz), 2.7412.89 (AB (ABX), 2H, CH2-6A,
3
2JABˆ13.2 Hz, JBXˆ4.8 Hz) therein 2.70±2.82 (m, 1H,
17a-H), 3.07±3.19 (m, 1H, CH-4A), 4.24±4.33 (m, 1H,
arom. H, ring F, JABˆ8.4 Hz). C44H43O8Cl3 (806.18).
3
CH-3aA), 4.44±4.53 (m, 1H, CH-6aA), 4.8014.99 (AB
2
4
(ABX), 2H, CH2-21, JABˆ18.2 Hz, JAXˆ1.5 Hz), 5.05
(bs, 1H, 3a-H), 5.68 (broad s, 1H, NH), 5.85 (s, 1H,
22-H), 5.94 (broad s, 1H, NH). 13C NMR (50.3 MHz,
APT, HETCOR, CDCl3): dˆ16.3 (2) (C-18), 21.6 (1),
21.7 (1), 23.2 (1), 24.3 (2) (C-19), 25.4 (1) (C-3B),
25.5 (1), 26.8 (1), 27.4 (1), 28.8 (1), 28.9 (1), 31.0
(1), 33.6 (1), 34.9 (1) (C-2B), 35.6 (1), 36.1 (2) and
37.4 (2) (C-5, C-9), 40.4 (1) (C-12), 41.0 (1) (C-6A),
42.2 (2) (C-8), 50.1 (1), 51.4 (2) (C-17), 56.0 (2)
(C-4A), 60.6 (2) (C-6aA), 62.5 (2) (C-3aA), 70.8 (2) (C-
3), 74.0 (1) (C-21), 85.8 (1), 118.0 (2) (C-22), 164.3 (1)
(C-2A), 173.7 (1) (C-1B), 175.1 (1) and 175.4 (1) (C-23,
C-20). UV (CH3CN): lmax. (emax.)ˆ214.5 nm (11200).
C33H48N2O6S (600.82), FAB MS: m/z (%): 623 (9;
[M1Na]1), 601 (8; [M1H]1), 583 (11; [M1H2H2O]1),
245 (58; [biotin1H]1). HRMS: calcd for C33H49N2O6S
([M1H]1) 601.3311, found 601.3294.
14-Hydroxy-3b-{6-[5-((3aS,4S,6aR)-2-oxohexahydro-
1H-thieno[3,4-d]imidazol-4-yl)pentanamido]hexanoyl-
oxy}-5b,14b-card-20(22)-enolide (6a). To a suspension of
N-biotinylaminocapronic acid (50.0 mg; 140 mmol) in THF
(5 ml) triethylamine (21.2 mg; 210 mmol) and 2,4,6-
trichlorobenzoyl chloride (34.2 mg; 140 mmol) were
added dropwise and the mixture was stirred at 208C for
4 h. After evaporation the residue was redissolved in toluene
(5 ml) and a solution of digitoxigenin (37.5 mg; 100 mmol)
and 4-(1-pyrrolidinyl)pyridine (29.6 mg; 200 mmol) in
toluene (2 ml) was added. The mixture was stirred at 208C
for 20 h. Usual work-up and LC (ethyl acetate±methanol
5:1) provided 6a (12.2 mg, 17%). IR (KBr): 1731, 1684,
1
1632, 1457, 1375 cm21. H NMR (200 MHz, HH COSY,
CDCl3): dˆ0.87 (s, 3H, CH3-18), 0.96 (s, 3H, CH3-19),
3
1.15±2.38 (om) therein 2.32 (t, CH2-2C, J2C,3ˆ7.3 Hz),
2.72±3.00 (m, 3H, CH2-6A, 17a-H), 3.10±3.30 (m, 3H,
CH-4A, CH2-2B), 4.30±4.38 (m, 1H, CH-3aA), 4.50±4.59
(m, 1H, CH-6aA), 4.8115.00 (AB (ABX), 2H, CH2-21,
22-(4-Benzoylbenzoyloxy)-14-hydroxy-3b-[5-((3aS,4S,
6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-
pentanoyloxy]-5b,14b-card-20(22)-enolide (5b). 4b
(63.6 mg; 106 mmol) was converted into 5b using the pro-
cedure described for 5a. LC (ethyl acetate±methanol
1!5:1) provided 5b (31.0 mg, 35%). In addition,
23.5 mg (27%) of trichlorobenzoate 4c were isolated and
15.1 mg (24%) of 4b were recovered. IR (KBr): 1773,
4
2JABˆ17.8 Hz, JAXˆ1.5 Hz), 5.09 (broad s, 3a-H), 5.88
(broad s, 1H, 22-H). 13C NMR (50.3 MHz, APT, CDCl3):
dˆ16.2 (2) (C-18), 21.5 (1), 21.7 (1), 24.2 (2) (C-19),
25.0 (1), 25.5 (1), 26.0 (1), 26.8 (1), 27.3 (1), 28.4 (1),
29.6 (1), 30.9 (1), 33.5 (1), 35.0 (1), 35.6 (1), 36.0 (2)
and 37.3 (2) (C-5, C-9), 36.2 (1), 39.8 (1), 40.4 (1)
(C-12), 40.9 (1) (C-6A), 42.2 (2) (C-8), 50.0 (2) (C-17),
51.3 (1) (C-13), 55.8 (2) (C-4A), 60.8 (2) (C-6aA), 62.3
(2) (C-3aA), 70.7 (2) (C-3), 73.9 (1) (C-21), 85.9 (1)
(C-14), 118.2 (2) (C-22), 164.0 (1) (C-2A), 173.8 (1),
175.1 (1) (C-20). C47H56N2O9S (825.04).
1700, 1252, 1109 cm21 1H NMR (200 MHz, CDCl3):
.
dˆ0.75±1.02 (m) therein 0.95 and 0.97 (2 s, CH3-18,
CH3-19), 1.10±2.35 (om) therein 2.33 (t, 2H, CH2-2B,
3JB2,3ˆ7.1 Hz), 2.69±3.02 (m, 3H, CH2-6A, 17a-H), 3.10±
3.21 (m, 1H, CH-4A), 4.27±4.35 (m, 1H, CH-3aA), 4.47±
4.57 (m, 1H, CH-6aA), 4.9715.24 (AB, 2H, CH2-21,
2JABˆ17.8 Hz), 5.07 (bs, 1H, 3a-H), 5.48 (bs, 1H, NH),
5.77 (bs, 1H, NH), 7.46±7.57 (m, 2H, arom. Hm, ring G),
7.60±7.69 (m, 1H, arom. Hp, ring G), 7.78±7.84 (m, 2H,
arom. Ho, ring G), 7.8918.25 (AA0BB0, 4H, arom. H, ring
22-(4-Benzoylbenzoyloxy)-14-hydroxy-3b-{6-[5-((3aS,
4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanamido]hexanoyloxy}-5b,14b-card-20(22)-
enolide (6b). 4b (60 mg; 100 mmol) was converted into 6b
as described for 6a. LC (petroleum ether±ethyl acetate±
methanol 1:1:0!0:5:1) furnished 6b (14 mg, 14%).
Furthermore, 26 mg (32%) of the trichlorobenzoate 4c
were isolated. IR (KBr): 1769, 1698, 1452, 1253, 1113,
3
F, JABˆ8.4 Hz). 13C NMR (50.3 MHz, APT, HETCOR,
CDCl3): dˆ15.9 (2) (C-18), 21.6 (1), 21.8 (1), 24.3 (2)
(C-19), 25.4 (1), 25.5 (1), 25.7 (1), 27.4 (1), 28.8 (1),
28.9 (1), 31.0 (1), 34.9 (1) (C-2B), 35.6 (1), 36.1 (2) and
37.3 (2) (C-5, C-9), 40.4 (1) (C-12), 41.0 (1) (C-6A), 42.1
(1) (C-8), 47.6 (2) (C-17), 50.5 (1) (C-13), 55.9 (2)
(C-4A), 60.6 (2) (C-6aA), 62.5 (2) (C-3aA), 70.4 (1)
(C-21), 70.7 (2) (C-3), 85.7 (1) (C-14), 129.0 (2)
(C-3G), 130.4 (2) and 130.8 (2) (C-2F, C-3F), 130.9 (2)
1
1017, 709 cm21. H NMR (200 MHz, CDCl3): dˆ0.82±
1.00 (m) therein 0.96 and 0.97 (2 s, CH3-18, CH3-19),
1.20±2.22 (om), 2.32 (t, CH2-2C, 3J2C,3Cˆ7.3 Hz),
2.64±3.01 (m, 3H) therein 2.7312.91 (AB (ABX), CH2-
2
3
6A, JABˆ13.0 Hz, JBXˆ4.9 Hz) and (m, 17a-H), 3.15±
3.20 (m, 1H, CH-4A), 4.27±4.35 (m, 1H, CH-3aA),

J. Zotzmann et al. / Tetrahedron 56 (2000) 9625±9632
9631
4.47±4.56 (m, 1H, CH-6aA), 4.9615.23 (AB, 2H, CH2-21,
2JABˆ17.7 Hz), 5.07 (broad s, 3a-H), 5.13 (broad s, 1H,
NH, exchanges with D2O), 5.40 (broad s, 1H, NH,
exchanges with D2O), 7.44±7.57 (m, 2H, arom. Hm, ring
G), 7.60±7.69 (m, 1H, arom. Hp, ring G), 7.78±7.84 (m,
2H, arom. Ho, ring G), 7.8818.25 (AA0BB0, 4H, arom. H,
2£ N±CH2, 4£ O±CH2), 4.22±4.33 (m, 1H, 3aA-H), 4.43±
4.56 (m, 1H, 6aA-H), 4.8215.00 (AB, 2H, CH2-21,
2JABˆ18.3 Hz), 5.31 (broad s, 1H, 3a-H), 5.62 (broad s,
1H, NHA), 5.87 (s, 1H, 22-H), 6.54 (broad s, NHA), 6.78
(broad s, NHC), 7.38 (broad s, NHC), 7.6517.99 (AA0BB0,
3
4H, arom. HD, JABˆ8.6 Hz), 9.63 (s, NHD). 13C NMR
3
ring F, JABˆ8.4 Hz). 13C NMR (50.3 MHz, APT, CDCl3):
(50.3 MHz, APT, (CD3)2SO): Many of the signals were
broad. The following signals could clearly be identi®ed:
dˆ16.4 (2) (C-18), 21.5 (1), 21.6 (1), 24.3 (2) (C-19),
25.9 (1), 26.9 (1), 28.6 (1), 28.8 (1), 35.4 (2), 35.6 (1),
35.7 (1), 44.2 (1), 50.1 (1) (C-13), 50.9 (2) (C-17), 56.1
(2) (C-4A), 59.8 (2) (C-6aA), 61.7 (2) (C-3aA), 69.0 (1),
69.8 (1), 70.2 (1), 71.2 (2) (C-3), 73.8 (1) (C-21), 84.4
dˆ15.7 (2) (C-18), 21.5 (1), 21.7 (1), 24.1 (2) (C-19),
25.3 (1), 25.4 (1), 25.6 (1), 26.7 (1), 28.7 (1), 28.8 (1),
30.1 (1), 30.9 (1), 33.6 (1), 34.7 (1), 35.6 (1), 36.0 (2)
and 37.3 (2) (C-5, C-9), 40.2 (1) and 40.9 (1) (C-12,
C-6A), 42.0 (2) (C-8), 47.6 (2) (C-17), 50.4 (1) (C-13),
55.7 (2) (C-4A), 60.5 (2) (C-6aA), 62.4 (2) (C-3aA), 70.3
(1) (C-21), 70.7 (2) (C-3), 85.7 (1) (C-14), 129.1 (2)
(C-3G), 130.4 (2) and 130.6 (2) (C-2F, C-3F), 130.9 (2)
(C-2G), 131.5 (1) and 137.2 (1) and 143.0 (1) (C-1F, C-4F,
C-1G), 133.7 (2) (C-4G), 135.5 (1) (C-22), 155.4 (1)
(C-20), 163.1 (1) and 163.8 (1) (C-2A, COArF), 168.0
(1) (C-23), 173.7 (1) (C-1C), 196.4 (1) (COArG). UV
(CH3CN): lmax. (emax.)ˆ254 nm (11700). C53H67N3O10S
(938.20).
²
(1) (C-14), 116.9 (2) (C-22), 130.4 (2), 144.2 , 163.3 (1),
165.3 , 172.8 (1), 174.5 , 177.1 (1). UV (CH3CN): lmax.
²
²
³
³
(emax.)ˆ262.5 nm (qual.) , 293.5 nm (qual.) . C47H67N5O9S2
(910.21), ESI HRMS: calcd for C47H67N5O9S2Na
([M1Na]1) 932.4248, found 932.4276, calcd for
C47H68N5O9S2 ([M1H]1) 910.4459, found 910.4436.
22-(4-Benzoylbenzoyloxy)-14-hydroxy-3b-(4-isothio-
cyanatobenzoyloxy)-5b,14b-card-20(22)-enolide (7b).
4b (73 mg, 122 mmol) was converted into 7b as described
for 7a. Since after 27 h the reaction was incomplete (TLC)
another crop of the acid chloride (10 mg, 50 mmol) was
added and stirring was continued at 208C for further 20 h.
Usual work-up and LC (petroleum ether±ethyl acetate
2:1!1:2) provided 63.6 mg (68%) of the very sensitive
7b, that could only be characterized by 1H NMR
(200 MHz, (CD3)2SO), characteristic signals: dˆ0.88 and
0.93 (2 s, 6H, CH3-18, CH3-19), 1.05±2.27 (om), 2.85±3.00
(m, 1H, 17a-H), 5.1915.24 (AB, 2H, CH2-21,
2JABˆ18.3 Hz), 7.47±7.64 (m, arom. H), 7.67±7.84 (m,
arom. H), 7.89±8.05 (m, arom. H), 7.23±7.31 (BB0
(AA0BB0), arom. H). C45H45NO8S (759.92).
14-Hydroxy-3b-(4-isothiocyanatobenzoyloxy)-5b,14b-
card-20(22)-enolide (7a). A solution of digitoxigenin
(37.5 mg, 100 mmol) and 4-dimethylaminopyridine
(12.2 mg, 100 mmol) in pyridine (2 ml) at 08C to a solution
of 4-isothiocyanatobenzoyl chloride (21.7 mg, 110 mmol) in
pyridine (2 ml) was added dropwise. A yellow colour
developed. The mixture was stirred at 208C for 3 h. Usual
workup and LC (petroleum ether±ethyl acetate 2:1!1:2)
yielded 7a (36.3 mg, 68%). IR (KBr): 2100, 1716, 1601,
1
1278, 1119 cm21. H NMR (200 MHz, CDCl3): dˆ0.88
(s, 3H, CH3-18), 1.01 (s, 3H, CH3-19), 1.13±2.27 (om),
2.75±2.85 (m, 1H, 17a-H), 4.8215.00 (AB (ABX), 2H,
2
4
CH2-21, JABˆ18.2 Hz, JAXˆ1.4 Hz), 5.34 (broad s, 1H,
3a-H), 5.88 (s, 1H, 22-H), 7.27±8.02 (AA0BB0, 4H, arom.
3
4
HD, JABˆ8.4 Hz, JAA ˆ2.1 Hz). 13C NMR (50.3 MHz,
CDCl3): dˆ16.2 (C-18), 21.7, 21.8, 24.4 (C-19), 25.7,
26.9, 27.4, 31.1, 31.3, 33.6, 35.8, 36.2 and 37.7 (C-5,
C-9), 40.4 (C-12), 42.3 (C-8), 50.1 and 51.4 (C-13, C-17),
72.0 (C-3), 73.9 (C-21), 85.9 (C-14), 118.2 (C-22), 126.1
(C-3D), 130.0, 131.4 (C-2D), 136.0 (SCN), 165.1 (COD),
174.9 and 175.0 (C-20, C-23). UV (CH3CN): lmax.
(emax.)ˆ229 nm (23400), 283 nm (16800), 294 nm
(17300). C31H37NO5S (535.70), FAB MS: m/z (%): 558
(12; [M1Na]1), 536 (16; [M1H]1). HRMS: calcd for
C31H38NO5S ([M1H]1) 536.2471, found 536.2482.
22-(4-Benzoylbenzoyloxy)-14-hydroxy-3b-[4-(3-{8-[5-
((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imida-
zol-4-yl)pentanamido]-3,6-dioxaoctyl}thioureido)ben-
zoyloxy]-5b,14b-card-20(22)-enolide (8b). Isothiocyanate
7b (35.2 mg, 46 mmol) was converted into 8b as described
for 8a. Solvent evaporation and LC (ethyl acetate±iso-
propanol 1:1!1:2) furnished 23.2 mg (44%) of 8b. IR
(KBr): 1762, 1694, 1661, 1539, 1450, 1270, 1114,
0
1
1016 cm21. H NMR (300 MHz, (CD3)2SO, 808C): many
signals were broad, characteristic signals: dˆ0.91 (s, 3H,
CH3-18), 0.97 (s, 3H, CH3-19), 1.05±1.95 (om), 4.01 (broad
s, 1H, NH), 4.12±4.18 (m, 1H, CH-3aA), 4.28±4.36 (m, 1H,
CH-6aA), 5.07±5.28 (m, 3H, CH2-21, 3a-H), 6.14 (broad s,
2H, 2 NH), 7.49±7.64 (m, arom. H), 7.69±7.98 (om, arom.
H), 7.9218.24 (AA0BB0, 4H, arom. H, ring F,
14-Hydroxy-3b-[4-(3-{8-[5-((3aS,4S,6aR)-2-oxohexahydro-
1H-thieno[3,4-d]imidazol-4-yl)pentanamido]-3,6-dioxa-
octyl}thioureido)benzoyloxy]-5b,14b-card-20(22)-
enolide (8a). A solution of 7a (21.5 mg, 40 mmol) in DMF
(3 ml) was added dropwise within 10 min to a suspension of
N-(1)-biotinyl-3,6-dioxaoctane-1,8-diamine (EZ-Linke
Biotin PEO-Amin, PIERCE, 16.5 mg, 44 mmol) in DMF
(2 ml). The colour of the reaction mixture turned to
intensely yellow. After another 10 min at 208C the mixture
became colourless and clear. Solvent evaporation and LC
(ethyl acetate±isopropanol±methanol 3:6:1) provided 8a
1
3JABˆ5.6 Hz), 9.90 (broad s, NH), H NMR (400 MHz,
CDCl3) characteristic signals: dˆ0.97 and 0.98 (2 s, CH3-
18, CH3-19), 0.80±2.35 (om), 2.65±3.15 (om), 3.28±3.93
(om), 4.28 (broad s, 1H, CH-3aA), 4.48 (broad s, 1H, CH-
2
6aA), 4.9715.25 (AB, 2H, CH2-21, JABˆ8.9 Hz), 5.29
(broad s, 3a-H), 7.38 (broad s, 2H, 2x NH), 7.48±8.05
(om, arom. H) therein 7.80 (BB0 (AA0BB0), arom. HD),
3
7.8818.24 (AA0BB0, 4H, arom. H, ring F, JABˆ4.1 Hz),
(26.8 mg, 74%). IR (KBr): 1683, 1645, 1276, 694 cm21
.
8.65 (broad s, NH), 9.72 (broad s, NH). UV (CH3CN): lmax.
1H NMR (200 MHz, CDCl3): dˆ0.88 (s, 3H, CH3-18),
1.00 (s, 3H, CH3-19), 1.17±2.40 (om), 2.60±2.55 (m, 3H,
17a-H, CH2-6A), 3.03±3.17 (m, 1H, 4A-H), 3.30±3.92 (om,
²
Observable only in 13C, not in APT.
Qualitative (due to low solubility).
³

9632
J. Zotzmann et al. / Tetrahedron 56 (2000) 9625±9632
(emax.)ˆ258 nm (qual.)§, 302 (qual.)§. C61H75N5O12S2
(1134.43), ESI HRMS: calcd for C61H76N5O12S2
([M1H]1) 1134.4932, found 1134.4894.
11. Staroske, T.; Hennig, L.; Welzel, P.; Hofmann, H.-J.; MuÈller,
È
D.; Hausler, T.; Sheldrick, W. S.; Zillikens, S.; Gretzer, B.; Pusch,
H.; Glitsch, H. G. Tetrahedron 1996, 52, 12723±12744.
12. Erlenkamp, S.; Gretzer, B.; Zillikens, S.; Glitsch, H. G.; Pusch,
H.; Staroske, T.; Welzel, P. Naunyn-Schmiedeberg's
Arch.Pharmacol. 1998, 357, 54±62.
Acknowledgements
13. Palasis, M.; Kuntzweiler, T. A.; ArguÈello, J. M.; Lingrel, J. B.
J. Biol. Chem. 1996, 271, 14176±14182.
We wish to thank Dr Sabine Giesa for the ESI mass spectra.
Financial support by the Deutsche Forschungsgemeinschaft
and the Fonds der Chemischen Industrie is gratefully
acknowledged.
14. Lingrel, J. B.; ArguÈello, J. M.; van Huysse, J.; Kuntzweiler,
T. A. in Ref. 3, p.194±206.
15. See Refs. 6±9,11 and Antolovic, R.; Schoner, W.; Geering, K.;
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§
Qualitative (due to low solubility).