Synthesis, in vitro pharmacology and biodistribution studies of new PD 156707-derived ETA receptor radioligands

Article abstract of DOI:10.1016/j.bmc.2005.10.039

It is assumed that the regulation of cardiac endothelin (ET) receptor density is abnormal in heart diseases. From that perspective, an ET receptor radioligand is needed to assess ET receptor density in vivo. The nonpeptidyl ET_A receptor antagon

Full text of DOI:10.1016/j.bmc.2005.10.039

Bioorganic & Medicinal Chemistry 14 (2006) 1910–1917
Synthesis, in vitro pharmacology and biodistribution studies
of new PD 156707-derived ETA receptor radioligands
Carsten Ho¨ltke,a,* Marilyn P. Law,a Stefan Wagner,a Hans-Jo¨rg Breyholz,a Klaus Kopka,a,c
Christoph Bremer,b,c Bodo Levkau,d Otmar Schobera and Michael Scha¨fersa,c
aDepartment of Nuclear Medicine, Albert-Schweitzer-Str. 33, University Hospital Mu¨nster, 48149 Mu¨nster, Germany
bDepartment of Clinical Radiology, Albert-Schweitzer-Str. 33, University Hospital Mu¨nster, 48149 Mu¨nster, Germany
cInterdisciplinary Center for Clinical Research (IZKF Mu¨nster), University of Mu¨nster, 48149 Mu¨nster, Germany
dInstitute of Pathophysiology, Hufelandstr. 55, University Hospital Essen, 45122 Essen, Germany
Received 12 January 2005; accepted 16 August 2005
Available online 10 November 2005
Abstract—It is assumed that the regulation of cardiac endothelin (ET) receptor density is abnormal in heart diseases. From that
perspective, an ET receptor radioligand is needed to assess ET receptor density in vivo. The nonpeptidyl ETA receptor antagonist
PD 169390 was labelled with radioiodine to give a putative radioligand for SPECT. Labelling with [125I]iodide and [123I]iodide was
accomplished with good to excellent radiochemical yields. The affinities of the nonradioactive reference and those of selected
precursor compounds for ETA receptors were determined, using [125I]iodine labelled endothelin-1 with mouse ventricular mem-
branes. All employed substances exhibited potent in vitro pharmacological characteristics with Ki values comparable to that of
the lead compound PD 156707. Biodistribution studies and scintigraphic imaging experiments in mice, however, showed no signif-
icant uptake of the [123I] derivative in the heart.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
receptor, however, is equal for all three isoforms.14–17
All native endothelins, therefore, represent nonselective
ET receptor substrates.
Since endothelin (ET) was first described by Hickey et al.1
and subsequently isolated by Yanagisawa et al.2 as a 21-
amino acid peptide with vasoactive potential, the role of
the endothelins in several diseases, including atheroscle-
rosis, congestive heart failure and pulmonary hyperten-
sion, has been increasingly investigated.3–7 At least
three isoforms of endothelin exist (ET-1, ET-2 and ET-
3), exerting their effects via two different G-protein cou-
pled receptors (ETA, ETB). ETA receptors are primarily
located on vascular smooth muscle cells and are respon-
sible for vasoconstriction and cell proliferation, whereas
ETB receptors are located on smooth muscle cells and
vascular endothelial cells, cause vasodilation by the re-
lease of nitric oxide and prostacyclin and are responsible
for the clearance of ET-1 from plasma.8–13 The affinity of
ET-1 and ET-2 to the ETA receptor is about a 100-fold
higher than the affinity of ET-3. The affinity to the ETB
Plasma levels of ET-1 are elevated in many cardiovascu-
lar diseases. In this connection, it has been shown that
ET receptor distribution is abnormally regulated in dis-
eased human tissue.18a,b In mouse models of human ath-
erosclerosis (i.e., ApoEꢀ/ꢀ) ETA receptor density has
been found to be upregulated in atherosclerotic
plaques.19a,b To overcome the unwanted effects of an
abnormally regulated ET system, ET receptor antago-
nists are used in the treatment of these diseases, and a
number of different peptidyl and nonpeptidyl ligands,
both selective and nonselective, have been developed in
attempts to improve efficacy.20–27 In addition to its role
in cardiovascular diseases, ET-1 is also an important
factor in the pathophysiology of certain human cancers,
especially prostate cancer,28 promoting tumour growth
by inducing cell proliferation and angiogenesis and
inhibiting apoptosis.29,30
Keywords: Radioiodination; Endothelin receptor antagonists; SPECT;
Biodistribution studies.
A noninvasive method for the visualisation of ET recep-
tor density in affected tissue would be invaluable for
clinical diagnosis and the evaluation of therapy. The
*
Corresponding author. Tel.: +49 251 8347362; fax: +49 251
8347363; e-mail: carsten.hoeltke@uni-muenster.de
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.039

C. Ho¨ltke et al. / Bioorg. Med. Chem. 14 (2006) 1910–1917
1911
methods of choice for imaging receptor distribution
in vivo are positron emission tomography (PET) and
single photon emission computed tomography
(SPECT).31–33 Radiolabelled ET receptor antagonists
could be used to assess ET receptor density with these
techniques. The radiolabelling of ET receptor agonists
and antagonists in connection with in vivo studies has
been reported.34–42 Very recently, Johnstroem et al.40b
have shown the importance of ETB receptors for the
clearance of ET-1 from circulation using [18F]ET-1 for
PET imaging in rats. Aleksic et al.42a have used
[11C]L-753037, a 1,3-bisarylindane-2-carboxylic acid
mixed ETA/ETB antagonist, for the imaging of ET
receptors in a dog heart in vivo. Johnstroem et al.42b
have [18F]fluorinated another representative of this class
of compounds (SB-209670) and imaged ET receptor dis-
tribution in rats. Furthermore, the here described lead
compound PD 156707 has been synthesised as a
[11C]methoxy analogue, but no imaging experiments
were performed.39 Interestingly, none of these
approaches have been utilised for the in vivo imaging
of ET receptors in humans so far, so there is still a
strong motivation for research towards new ET receptor
radioligands for use in the human body.
tissue.35 Subsequently the 123I-derivative 8 was prepared
for biodistribution studies and initial scintigraphic imag-
ing experiments using wild type mice. The affinities of
the nonradioactive 4-hydroxy-3-iodo-derivative 6, the
benzyloxy-precursor 4 and PD 169390 5 to ET receptors
were evaluated. Compound 6 is the corresponding non-
radioactive counterpart of the radioiodinated and struc-
turally identical target radioligands 7 and 8 and is used
for the evaluation of its binding potency to ET receptors
in vitro. PD 156707 3 was also synthesised and used for
comparison of the affinity values.
2. Results and discussion
2.1. Chemistry
PD 156707 3 (Scheme 1) and related butenolide deriva-
tives are known to be potent ETA receptor antago-
nists.43–48 To use butenolide-derived structures for the
development of a radioiodinated ETA ligand for SPECT,
we used the 4-hydroxy-derivative PD 169390 5 as a pre-
cursor for radioiodination. The synthesis of PD 169390
5 was accomplished via a route using a benzyloxy-protect-
ing group, starting from keto-ester 1 and 3,4,5-trimeth-
oxybenzaldehyde 2 (Scheme 2). Deprotection of the
benzyloxy butenolide 4 was achieved by hydrogenolysis
in alcoholic solution, resulting in precursor PD 169390 5
in moderate yield (39%). Hydrogenation of the double-
bond of the butenolide five-membered ring occurred as
a side reaction. PD 169390 5 was iodinated with iodine
and ammonia in alcoholic solution, yielding the monoio-
dinated compound 6 as a nonradioactive reference for the
target radioligands 7 and 8 (Scheme 3). The method de-
scribed below for the ortho-radioiodination resulting in
compounds 7 and 8 could not be transferred to this reac-
tion. Because of longer reaction times needed for the com-
plete conversion of the reactants to give compound 6,
unwanted byproducts resulted. To be able to compare
the receptor binding potencies of the newly developed
compounds, PD 156707 3 was synthesised as a reference,
using literature procedures.45
PD 156707 3 (3-benzo[1,3]dioxol-5-yl-5-hydroxy-5-(4-
methoxyphenyl)-4-(3,4,5-trimethoxybenzyl)-5H-furan-2-
one, Scheme 1) and related butenolide derivatives are
potent ET receptor antagonists with high affinity and
ETA selectivity.43–48 In this study, we describe the radio-
iodination of PD 169390 5 (Scheme 1), the 4-hydroxy-
phenyl-analogue of 3. The structure of 5 has been
described but its synthesis has not been reported.39
[
125I]NaI was used to develop the radiosynthesis, yield-
ing derivative 7. A related compound, [125I]PD 164333,
with an ortho-radioiodinated hydroxyphenyl group at-
tached via a small amide-linker to a butenolide based
structure, was synthesised by Davenport et al. and used
for in vitro determination of ET receptors in human
H3CO
OCH3
H3CO
2.2. Radiochemistry
O
HO
R1
The radioiodination of PD 169390 5 was performed
using chloroamine T as oxidant and [125I]NaI or
O
X
O
[
123I]NaI (Scheme 3). The radiolabelled products were
purified by reversed-phase radio-HPLC to yield the
compounds 7 or 8 with radiochemical yields between
70% and 90%. The radiochemical purities of the com-
pounds were >99%, with specific radioactivities of
121.7 MBq/lg (75.0 GBq/lmol) in case of the 125I-de-
rivative 7 and 14.04 GBq/lg (8.63 TBq/lmol) for the
123I-derivative 8 at the end of synthesis (EOS). The
iodinated nonradioactive analogue 6 was used to verify
the chemical identities of the radiolabelled compounds 7
and 8 (tR,(6/7/8) = 21.5 min).
O
O
R1= CH3, X = H
3 (PD 156707)
R
1 = CH2C6H5, X = H
4
R1 = H, X = H
5 (PD 169390)
= H, X = I
6
7
8
R1
R
1 = H, X = 125I
2.3. Biology
R1 = H, X = 123
I
2.3.1. In vitro assays. The affinities of the prepared but-
enolide derivatives 3–6 towards endothelin receptors
Scheme 1. Lead structure PD 156707 3 and its derivatives.

1912
C. Ho¨ltke et al. / Bioorg. Med. Chem. 14 (2006) 1910–1917
O
CHO
pounds were found to have a significantly higher affinity
to ETA than to ETB receptors (Table 1). The high- and
low-affinity IC50 values were converted into the high-
and low-affinity inhibition constants (Ki/ETA and
Ki/ETB) by the method of Cheng–Prusoff47,48 using the
experimentally determined KD value of [125I]ET-1
(208 2 pM). The ratios of the low- to high-affinity
inhibition constants (Ki/ETB/Ki/ETA) yield the ETA selec-
tivities of the derivatives 3–6 (Table 1). Additionally, the
calculated logP values (LogD Suite, ACD-software) for
compounds 3–6 are also listed in Table 1 to indicate the
changes in lipophilicities caused by the chemical modifi-
cations of the lead compound PD 156707.
+
O
O
H3 CO
OCH3
BnO
MeOOC
OCH3
1
2
a, b
H3CO
OCH3
Recent structure–activity relationship (SAR)stud-
ies45,49–51 have shown that at least two aromatic moieties
surrounding a carboxylic group are necessary for ET
receptor binding, although most ligands, like the lead
compound 3, contain a third aromatic group. The inves-
tigation of substitution patterns of some butenolide
derivatives45 indicates that additional increase of the ste-
ric bulk on these aromatic moieties leads to a decrease in
receptor affinity. As predicted, the benzyloxy derivative
4 shows the poorest affinity to the ETA receptor
(Ki/ETA = 32.3 22.1 nM) and also the poorest ETA
selectivity (Ki/ETA/Ki/ETB = 141 32). The affinity values
of the compounds 3, 5 and 6 to the ETA receptor all lie
in the subnanomolar range, whereas the affinity to the
ETB receptors is only micromolar. Compound 5, as an
exception, also shows nanomolar affinity to the ETB
receptor, probably due to the polar phenolic hydroxy
group, although selectivity is still >500. The nonradioac-
tive reference compound 6 shows Ki values of ꢁ1 nM for
the ETA receptor and ꢁ1.25 lM for the ETB receptor,
respectively. Therefore, with the corresponding highly
affine and selective ETA receptor radioligand 8 in hand,
first in vivo evaluation studies were performed.
H3CO
BnO
HO
O
O
O
4
O
c
H3CO
OCH3
H3CO
HO
HO
O
O
2.3.2. Biodistribution studies. The 123I-radioiodinated
compound 8 was chosen for biodistribution studies
and scintigraphic imaging experiments in BL6 mice.
With the exception of the liver, uptake indices for the
tissues dissected were lower than those for plasma at
all time points (5, 10, 20, 30, 40 and 130 min). Values
for the liver were comparable to those for plasma. Up-
take indices decreased with time after injection, for
example indices for myocardium and liver were 0.19–
0.46 and 0.9–1.5, respectively, at 20–30 min and fell to
0.07–0.11 and 0.3–0.4 at 130 min. Predosing with PD
156707 had no effect (Fig. 2). Radioactivity accumulated
in the urine which showed uptake indices between 5 and
8 at 20–30 min and 12 and 20 at 130 min. Scintigraphic
imaging confirmed uptake of radioactivity in the liver
and bladder but the heart was not visualised (Fig. 3).
Radioactivity in the three regions of interest, chest, liver
and bladder, are shown in Figure 3. Predosing with PD
156707 had no effect on tissue radioactivity assessed
either ex vivo (Fig. 2) or by scintigraphic imaging
(Fig. 3). Radioactivity accumulated in the urine and
HPLC analysis of urine taken from one mouse at
5 min after injection of 8 (data not shown) predominant-
ly showed a radioactive polar fraction and, to a lesser
extent, the parent radioligand (ratio ꢂ 9:1).
O
O
PD169390,
5
Scheme 2. Synthesis of PD 169390 via the benzyloxy derivative.
Reagents: (a) NaOMe, methanol; (b) HOAc, methanol; (c) H2/Pd/C,
ethanol/isopropanol 2:1.
were determined by competition binding studies using
[
125I]iodine labelled endothelin-1 ([125I]ET-1) (Perkin-El-
mer Live Sciences Inc., Billerica, MA, USA) and mouse
ventricular membrane preparations (Fig. 1). The bind-
ing of the nonselective ET receptor antagonist
[
125I]ET-1 to ventricular membranes was specific, satu-
rable and of high affinity. Scatchard46,47 transformation
of the saturation data yielded values for the dissociation
constant (KD = 208 2 pM) and the maximum number
of binding sites (Bmax = 300 3 fmol/mg protein). Dif-
ferent concentrations of the prepared compounds
(1.0 pM–100 lM) were incubated with 40 pM [125I]ET-
1 and a fixed amount of mouse ventricular membrane
preparation (10 lg) at 37 ꢁC for 4 h. The membrane
bound radioactivity was evaluated using a c-scintillation
counter. Nonlinear regression analysis showed that the
data of the compounds fitted a two-site model. All com-

C. Ho¨ltke et al. / Bioorg. Med. Chem. 14 (2006) 1910–1917
1913
H3CO
OCH3
H3CO
HO
HO
O
O
O
5
O
a
b
H3CO
H3CO
OCH3
OCH3
H3CO
H3CO
HO
HO
HO
O
HO
O
125/123I
I
O
O
6
7/8
O
O
O
O
Scheme 3. Iodination of PD 169390 5. Reagents: (a) I2, NH3, ethanol; (b) [123/125I]NaI n.c.a., chloroamine T, K2HPO4-buffer, water, methanol.
Table 1. Receptor affinities and calculated logP values (LogD Suite,
ACD-software) of prepared compounds
3
logPa
4
Compound Ki/ETA (nM) Ki/ETB (lM) Selectivity
5
6
3
4
5
6
0.04 0.02
32.3 22.1
0.11 0.09
0.97 0.13
4.5 3.1
4.6 3.1
>50,000
140 32
3.30
4.95
0.06 0.05
1.3 0.2
780 170 2.65
1300 43 3.81
ET receptor affinities were determined by competition binding studies
using [125I]ET-1 and mouse ventricular membrane preparations. Ki
values were calculated by nonlinear regression analysis using the
XMGRACE program (Linux software), given as means SEM.
The ratios of the low- over the high-affinity inhibition constants
(Ki/ETA/Ki/ETB) yield the ETA selectivities of the derivatives, given as
means SEM. Note the differences in concentration dimensions in
Ki/ETA and Ki/ETB
.
a Calculated by LogD Suite program (ACD-software).
Antagonist [M]
Figure 1. Competition of
125I]ET-1 binding. Mouse ventricular
[
membranes were incubated with 40 pM [125I]ET-1. Membrane bound
radioactivity is expressed as percentage of [125I]ET-1 binding. Lines for
best fit (nonlinear regression analysis) are shown.
>99% with specific radioactivities of 121.7 MBq/lg
(75.0 GBq/lmol) for the 125I-derivative and 14.04 GBq/
lg (8.63 TBq/lmol) for the 123I-compound (EOS). The
affinities of the nonradioactive reference as well as those
of selected precursor compounds towards ET receptors
were determined. Inhibition constants were all in the
nanomolar range and are comparable to that of the
known ETA receptor antagonist PD 156707. The affinity
and selectivity of the nonradioactive ligand 6 to ETA
receptors evidence the potency of this compound. The
derived radioligands 7 and 8, therefore, are promising
3. Conclusion
The endothelin receptor antagonist 3-benzo[1,3]dioxol-
5-yl-5-hydroxy-5-(4-hydroxy-phenyl)-4-(3,4,5-trimetho-
xybenzyl)-5H-furan-2-one (PD 169390) was radiola-
belled with [125I]iodide and [123I]iodide, respectively.
The radioligands were prepared in good to excellent
radiochemical yields. The radiochemical purities were

1914
C. Ho¨ltke et al. / Bioorg. Med. Chem. 14 (2006) 1910–1917
more stable derivatives of PD 156707 and use these
compounds for further in vivo studies with the aim to
develop a suitable radioligand for human in vivo imaging.
4. Experimental
4.1. General
All chemicals, reagents and solvents for the synthesis of
the compounds were of analytical grade and purchased
from commercial sources. PD 156707 3 and 2-(benzo-
[1,3]-dioxol-5-yl)-1-(4-benzyloxyphenyl)-4-oxobutyric acid
methyl ester 1 were synthesised as reported.45 Melting
points (uncorrected) were determined with a Stuart Scien-
Figure 2. Radioactivity in tissues 20–30 min after iv injection of 8
,
given alone ( , 4 mice) or 5 min after iv injection of 1 lmol kgꢀ1 3 (
1 mouse). Bars indicate standard errors.
1
tific SMP3 capillary melting point apparatus. H NMR
and 13C NMR spectra were recorded on a Bruker ARX
300, and AMX 400 spectrometer, respectively. Mass spec-
trometry was performed using a Varian MAT 212
(EI = 70 eV) spectrometer and a Bruker MALDI-TOF-
MS Reflex IV (matrix: DHB). Elemental analysis was
realised by a Vario EL III analyser. Separation of the
radiosynthesised compounds, analyses of radiochemical
yields and radiochemical purities were performed by gra-
dient radio-RP-HPLC with a RP-HPLC column Nucleo-
sil 100 C-18 5 l 250 · 4.6 mm2, a corresponding
20 · 4.6 mm2 precolumn, a Knauer K-500 and a Latek
P 402 pump, a Knauer K-2000 UV-detector (wavelength
254 nm) and a Crismatec Na(Tl) Scintibloc 51 SP51 c-
detector. Sample injection was carried out using a Rheo-
dyne injector block (type 7125 incl. 200 ll loop). The
tools for in vitro and in vivo studies. First biodistribu-
tion studies with the 123I-derivative 8 in mice, however,
showed no significant uptake of the 123I-labelled com-
pound in the heart after different periods of time. With
the exception of the liver, uptake indices for the tissues
dissected were lower than those for plasma. Altogether,
scintigraphic imaging confirmed uptake of radioactivity
in the liver and bladder but the heart was not visualised.
Predosing with PD 156707 had no significant effect. The
radioligand supposably metabolises in vivo very rapidly
without approaching the target receptors. The use of the
125I-radioligand 7 as a substitute for [3H]BQ-123 as a
ETA selective radioligand for in vitro studies is currently
under investigation. Also, work is in progress to design
a
b
Alone
PD156707
0- 10 min
0- 10 min
A
B
A
B
C
C
50- 60 min
50- 60 min
A
B
C
A
B
C
Figure 3. (a) Representative scintigraphic images of two mice after retro-orbital injection of 8 given alone or 30 min after retro-orbital injection of 3
at 1 lmol kgꢀ1. Two time frames (1–10 min and 51–60 min) are shown. Regions of interest were drawn over the chest (A), liver (B) and bladder (C) in
the early images for each of 6 mice (3 mice with 8 alone, 3 mice with 8 after administration of unlabelled 3) and radioactivity in each region for each of 5
time frames was calculated using in-house software. (b) The uptake of radioactivity (arbitrary units) is shown in the right-hand panel; solid symbols, 7
alone (d mouse 1, m mouse 2 and . mouse 3): open symbols, 8 after administration of unlabelled 3 (s mouse 4, n mouse 5 and , mouse 6). Lines are
drawn between mean values; solid line, 8 alone; dashed line, 8 after administration of unlabelled 3. Note the differences in the scales for radioactivity.

C. Ho¨ltke et al. / Bioorg. Med. Chem. 14 (2006) 1910–1917
1915
following conditions were used: eluent A: CH3CN/H2O/
TFA (950:50:1); eluent B: CH3CN/H2O/TFA (50:950:1);
isocratic time-programme with 55% eluent B for 40 min;
flow rate: 2.0 ml/min; k = 254 nm. The recorded data were
processed by the NINA radio-HPLC software (GE Func-
tional Imaging GmbH). Radioactivity of tissue was deter-
mined using an automated c-counter (Wallac Wizard 300,
Perkin-Elmer Life Sciences, Boston, USA). All animal
experiments were conducted in accordance with Local
Institutional Guidelines for the Care and Use of Labora-
tory Animals.
[MꢀH2O]+. Anal. calcd for C27H24O9: C, 65.85; H,
4.91. Found: C, 65.70; H, 4.55.
4.2.3. 3-Benzo[1,3]dioxol-5-yl-5-hydroxy-5-(4-hydroxy-3-
iodophenyl)-4-(3,4,5-trimethoxybenzyl)-5H-furan-2-one 6.
Two hundred forty-six milligram (0.5 mmol) 3-benzo
[1,3]dioxol-5-yl-5-hydroxy-5-(4-hydroxyphenyl)-4-(3,4,5-
trimethoxybenzyl)-5H-furan-2-one 5 was dissolved in
15 ml of a 2.0 M solution of ammonia in ethanol. 2.5 mil-
lilitres of a 0.4 N solution of iodine in ethanol (1.0 mmol)
was added dropwise. After completion of the addition, the
mixture was stirred for 60 min at ambient temperature.
The solvent was evaporated and the residue was adsorbed
on silica gel and purified by silica gel chromatography
(light petroleum/ethyl acetate 3/2!1/1) to yield 170 mg
(0.275 mmol, 55%)of an off-white powder, mp 216 ꢁC (de-
comp). 1H NMR (DMSO-d6) d = 10.43 (s, br, 1H), 8.13 (s,
br, 1H), 7.67 (s, 1H), 7.19 (d, 1H, J = 8.5 Hz), 6.94 (m,
3H), 6.76 (d, 1H, J = 8.5 Hz), 6.00 (s, 2H), 5.96 (s, 2H),
3.53 (s, 6H), 3.51 (s, 3H) ppm. 13C NMR d = 170.8,
161.3, 157.4, 152.5, 147.7, 147.4, 136.9, 136.0, 131.8,
129.8, 127.9, 127.2, 123.6, 123.3, 114.7, 109.5, 108.5,
106.3, 105.2, 101.5, 84.2, 60.2, 55.9 ppm. MS: m/z = 641
[M+Na]+, 618 [M+], 601 [MꢀH2O]+. Anal. calcd for
C27H23IO9: C, 52.44; H, 3.75. Found: C, 52.60; H, 3.90.
4.2. Synthesis of butenolide derivatives
4.2.1. 3-Benzo[1,3]dioxol-5-yl-5-hydroxy-5-(4-benzyloxy-
phenyl)-4-(3,4,5-trimethoxybenzyl)-5H-furan-2-one 4. To
a solution of 700 mg (13.0 mmol) sodium methoxide in
50 ml methanol, 5.00 g (11.9 mmol) 2-(benzo[1,3]dioxol-
5-yl)-1-(4-benzyloxyphenyl)-4-oxobutyric acid methyl es-
ter 1 and 2.45 g (12.5 mmol) 3,4,5-trimethoxybenzalde-
hyde 2 were added. The solution was refluxed for 12 h
and treated with 6.5 ml aceticacid. After another 4 h of re-
flux, the solvent was removed by evaporation. Water and
ethyl acetate were added and the mixture was neutralised
by the dropwise addition of 2.5 N hydrochloric acid. The
organic phase was separated and the aqueous phase was
extracted twice with ethyl acetate. The combined organic
extracts were washed with water and brine and dried over
sodium sulfate. After filtration and evaporation, an off-
white foam remained, which was purified by silica gel
chromatography (light petroleum/ethyl acetate 2:1),
yielding 5.13 g (8.81 mmol, 74%) of a white crystalline sol-
id, mp 123 ꢁC. 1H NMR (DMSO-d6) d = 8.18 (s, br s, 1H),
7.39 (m, 7H), 6,94 (m, 5H), 5.99 (s, 2H), 5.97 (s, 2H), 5.07
(s, 2H), 3.60 (s, br s, 1H), 3.40 (s, 6H), 3.19 (s, 3H), 3.06 (s,
br s, 1H) ppm. 13C NMR d = 171.4, 162.0, 159.5, 152.9,
148.0, 147.7, 137.6, 136.4, 132.4, 130.1, 129.2, 128.6,
128.5, 128.4, 128.3, 127.5, 124.1, 123.8, 115.2, 110.0,
108.8, 107.0, 101.9, 70.1, 60.5, 56.2, 32.2 ppm. MS:
m/z = 582 [M+], 91 [C7H7]+. Anal. calcd for
4.3. Radiosyntheses
4.3.1. General procedure for the synthesis of [125/123I]3-
benzo[1,3]dioxol-5-yl-5-hydroxy-5-(4-hydroxy-3-iodophenyl)-
4-(3,4,5-trimethoxybenzyl)-5H-furan-2-one 7, 8. To a
solution of 57 lg (0.115 lmol) 5 in 39 ll MeOH in a
conical glass-vial, 39 ll of 0.1 M K2HPO4-buffer (pH
7.34),
a solution of 32 lg (0.141 lmol) chloro-
amine-TÆH2O in 39 ll of 0.1 M K2HPO4-buffer and 3–
4 ll [123I]NaI n.c.a. solution or 6–22 ll [125I]NaI n.c.a.
solution (0.05 M NaOH) were added. The reaction mix-
ture was vortexed and allowed to stand for 5 min at RT.
Then 50 ll of 10% Na2S2O5-solution was added. After
5 min at RT, the mixture was injected into a radio-RP-
HPLC-chromatograph to isolate the radiolabelled com-
pounds 7 or 8, respectively. Yields: [125I] 7: 84% (average,
n = 3, decay-corrected, EOS), [123I] 8: 74% (average,
n = 4, decay-corrected, EOS). HPLC: tR = 21.5 min.
1
2
C34H30O9 ꢃ EtOAc: C, 69.00; H, 5.47. Found: C, 69.07;
H, 5.41.
4.2.2. 3-Benzo[1,3]dioxol-5-yl-5-hydroxy-5-(4-hydroxy-
phenyl)-4-(3,4,5-trimethoxybenzyl)-5H-furan-2-one, PD
169390, 5. 6.35 g (10.9 mmol) 3-benzo[1,3]dioxol-5-
yl-5-hydroxy-5-(4-benzyloxyphenyl)-4-(3,4,5-trimethoxy-
benzyl)-5H-furan-2-one 4 was suspended in 100 ml abso-
lute ethanol and 50 ml isopropanol. 440 mg of catalyst
(Pd/C) was added and the mixture was stirred under
hydrogen atmosphere. After completion of the reaction
(tlc, 12 h), the catalyst was filtered off and the filtrate
was evacuated to remove the solvent. The remaining off-
white foam was purified by silica gel chromatography
(light petroleum/ethyl acetate, 1:1) to yield 2.09 g
(4.2 mmol, 39%) of the desired product as a white powder,
mp 172 ꢁC (decomp). 1H NMR (DMSO-d6) d = 9.80 (s, br
s, 1H), 8.25 (s, br s, 1H), 7.47 (m, 2H), 7.12 (m, 3H), 6,92
(m, 2H), 6.20 (s, 2H), 6.17 (s, 2H), 3.90 (m, 2H), 3.71 (s,
6H), 3.70 (s, 3H) ppm. 13C NMR d = 170.9, 161.7,
158.1, 152.3, 147.4, 147.0, 135.9, 131.9, 127.7, 127.4,
126.6, 123.6, 123.2, 115.1, 109.4, 108.2, 106.4, 106.1,
101.3, 60.0, 55.7, 31.6 ppm. MS: m/z = 492 [M]+, 474
4.3.2. Radioanalytical data
4.3.2.1. Quality control. Fifty to two hundred micro-
litres of the product fraction was re-injected into the
HPLC column. The quality control did not show any
impurities within the c-range. Only the injection peak
was detectable within the UV-range.
4.3.2.2. Reference control. The radioiodinated prod-
ucts 7 and 8 were verified by concentrating 200 ll of
the isolated c-fraction with 50 lg of the nonradioactive
reference 6. The concentrated mixtures were again
injected into the HPLC column. The radiolabelled prod-
uct and the nonradioactive reference co-eluted.
4.4. Biology
4.4.1. Tissue preparation. Microsomes were prepared by
homogenizing ventricles from DBA mice at 4 ꢁC for 90 s

1916
C. Ho¨ltke et al. / Bioorg. Med. Chem. 14 (2006) 1910–1917
in 1 ml buffer A (10 mM EDTA, 10 mM Hepes and
0.1 mM benzamidine, pH 7.4), using a Polytron PT
3000 (Kinematica, Lucerne, Switzerland). Homogenates
were centrifuged at 45,000gmax for 15 min at 4 ꢁC. The
pellets were resuspended in 1 ml buffer B (1 mM EDTA,
10 mM Hepes and 0.1 mM benzamidine, pH 7.4) and
recentrifuged at 45,000gmax for 15 min at 4 ꢁC. The pel-
lets were resuspended in 1 ml buffer B and centrifuged at
10,000gmax for 10 min at 4 ꢁC. The supernatants were
recentrifuged at 45,000gmax for 15 min at 4 ꢁC. The pel-
lets, partially enriched membranes, were resuspended in
buffer C (50 mM Tris Æ HCl, 5 mM MgCl2, pH 7.4) and
stored frozen at ꢀ80 ꢁC.
12.9 Mbq in 100 ll of 0.9% NaCl) was injected retro-
orbitally and the scan was started within 5 min. Images
(115 frames) were added in 10 min time frames, namely
1–10, 21–30, 51–60, 81–90 and 104–115 min. Rectangu-
lar regions of interest were drawn to represent the chest,
liver and bladder and radioactivity in each region was
expressed as arbitrary units using in-house software.
Acknowledgments
Financial support of the Deutsche Forschungsgemein-
schaft (SCHA 758/5-1) is gratefully acknowledged.
C.H. wishes to thank Sandra Schro¨er for the in vitro
experiments.
4.4.2. In vitro assays. For competition binding studies,
the prepared membranes were resuspended in buffer D
(10 mM Tris Æ HCl, 154 mM NaCl and 0.1 mM ascorbic
acid, pH 7.4) at 0 ꢁC. Ten micrograms of membranes
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