Synthesis and preliminary evaluation of mono-[123I]iodohypericin monocarboxylic acid as a necrosis avid imaging agent

Article abstract of DOI:10.1016/j.bmcl.2007.04.083

Hypericin monocarboxylic acid was synthesized in an overall yield of 25% in four steps and radiolabelled with iodine-123 in good yield (>75%). The resulting mono-[¹²³I]iodohypericin monocarboxylic acid was evaluated in normal mice and in rats with ethanol induced liver necrosis. In this model, tracer concentration in necrotic liver tissue was 14 times higher than in the viable liver tissue as quantified by autoradiography at 24 h post injection. The results indicate the feasibility of visualization of necrotic tissue with the novel tracer.

Full text of DOI:10.1016/j.bmcl.2007.04.083

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4001–4005
Synthesis and preliminary evaluation of mono-[123I]iodohypericin
monocarboxylic acid as a necrosis avid imaging agent
Humphrey Fonge,a Lixin Jin,a Huaijun Wang,b Yicheng Ni,b
Guy Bormansa and Alfons Verbruggena,*
aLaboratory of Radiopharmacy, Faculty of Pharmaceutical Sciences, K.U. Leuven, BE-3000 Leuven, Belgium
bDepartment of Radiology, Faculty of Medicine, K.U. Leuven, BE-3000 Leuven, Belgium
Received 19 March 2007; revised 20 April 2007; accepted 25 April 2007
Available online 30 April 2007
Abstract—Hypericin monocarboxylic acid was synthesized in an overall yield of 25% in four steps and radiolabelled with iodine-123
in good yield (>75%). The resulting mono-[123I]iodohypericin monocarboxylic acid was evaluated in normal mice and in rats with
ethanol induced liver necrosis. In this model, tracer concentration in necrotic liver tissue was 14 times higher than in the viable liver
tissue as quantified by autoradiography at 24 h post injection. The results indicate the feasibility of visualization of necrotic tissue
with the novel tracer.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Cell death occurs by two distinct processes, that is, pro-
grammed cell death (apoptosis) and necrosis.1,2 Apopto-
sis can be initiated by several physiological stimuli that
trigger a preprogrammed cellular set of events involving
the activation and release of caspases which in turn com-
mand a structured cell destruction and removal. Necro-
sis, on the other hand, is caused by mechanical, thermal,
electrical or noxious chemical injury and by profound
hypoxia, ischaemia or even respiratory poisons such as
cyanide. Both forms of cell death have been implicated
in some diseases, for example in acute myocardial
infarction (AMI). 99mTc-pyrophosphate, 111In-antimyo-
sin Fab antibody and 99mTc-glucarate have all been used
in nuclear medicine to locate and quantify infarct size
but none has optimal imaging characteristics.3,4
Our group has been focusing on infarct avid agents for
contrast enhancement in magnetic resonance imaging
(MRI) and single photon emission computer tomogra-
phy (SPECT).7–9 We have shown that mono-
[
123I]iodohypericin ([123I]MIH), an iodine-123 labelled
derivative of hypericin (Fig. 1A), is very avid for necro-
tic9 but not apoptotic (results unpublished) tissue in dif-
ferent animal models of necrosis and apoptosis. Because
of the slow plasma clearance of [123I]MIH, early in vivo
visualization (2 h post tracer injection) of necrotic tissue
in experimentally reperfused acute myocardial infarc-
tion (AMI) was not possible using [123I]MIH in combi-
nation with single photon emission computerized
tomography (SPECT).10 We hypothesized that a less
lipophilic derivative of hypericin would have a faster
clearance from circulation and therefore permit earlier
Percutaneous ethanol injection (PEI) is widely used to
kill small tumuors (especially hepatocellular carcino-
mas) but a radiopharmaceutical to monitor response
to this therapy5,6 has not yet been developed. Upon
PEI, tumuor cells die by necrosis and a radiolabelled
necrosis avid agent may enable quantification of the ne-
crotic tumuor volume after PEI treatment as well as
guide subsequent treatments.
Keywords: Mono-[123I]iodohypericin monocarboxylic acid; Necrosis
avidity; Autoradiography.
Figure 1. Structures of (A) R = H = hypericin and R = 123I = mono-
[
123I]iodohypericin and (B) R = H = hypericin monocarboxylic acid
*
Corresponding author. Tel.: +32 16330441; fax: +32 16330449;
e-mail: alfons.verbruggen@pharm.kuleuven.be
and R = 123I = mono-[123I]iodohypericin monocarboxylic acid.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.083

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H. Fonge et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4001–4005
visualization of necrotic tissue. This study reports the
synthesis of mono-[123I]iodohypericin monocarboxylic
acid ([123I]MIHA, Fig. 1B), and its preliminary evalua-
tion in normal mice and in rats with ethanol induced
liver necrosis. Avidity of [123I]MIHA for necrotic liver
was assessed ex vivo by gamma counting, autoradiogra-
phy and triphenyltetrazolium (TTC), haematoxylin and
eosin (H&E) staining techniques.
time point). The organs were weighed and radioactivity
was counted in a NaI(Tl) gamma counter and expressed
as percentage of injected dose (ID)/organ and % ID/g of
organ. Table 1 shows the biodistribution of [123I]MIHA.
Blood clearance of [123I]MIHA (0.6% ID/g at 4 h pi) was
much faster than for [123I]MIH (28.2% ID/g at 4 h pi).
Low blood clearance of [123I]MIH has been attributed
to its high affinity for plasma lipoproteins (data unpub-
lished). The present data suggest that less the lipophilic
Protohypericin monocarboxylic acid (4) was obtained
starting from emodin (1) by a modification of a
reported four-step procedure11 in an overall yield of
25% (Scheme 1a). Emodin was first acetylated to yield
mainly tri-acetyl-emodin (2)12 which upon oxidation
yielded triacetyl-emodic acid (3).13 Dimerization of 1
and 3 in the presence of hydroquinone as a free rad-
ical scavenger yielded protohypericin monocarboxylic
acid (4)14 which was readily converted to hypericin
monocarboxylic acid (5) by photocyclization during
irradiation of 4 with a 400-W halogen lamp for
30 min. 1H NMR of 4 was recorded on a Gemini
200 MHz spectrometer and corresponded with previ-
ously reported d-values.11 Hypericin monocarboxylic
acid (5) was used as precursor for the synthesis of
the unlabelled mono-iodo-derivative (6) by a standard
electrophilic substitution reaction employing iodide in
the presence of peracetic acid as an in situ oxidizing
agent.15,16 The resulting compound was purified by
HPLC and its identity was confirmed by mass spec-
[
123I]MIHA has much less affinity for plasma proteins
resulting in enhanced plasma clearance. The tracer is
mainly cleared via the hepatobiliary pathway resulting
in high percentages excreted to the intestines and faeces.
A significant amount of [123I]MIHA was also cleared via
the kidneys resulting in 21.1% ID of [123I]MIHA in urine
at 24 h pi compared to <6% ID of [123I]MIH.
Preliminary evaluation of affinity of [123I]MIHA for
necrotic tissue has been performed in male adult Wistar
rats (350–450 g) with hepatic necrosis. Hepatic necrosis
was induced under laparotomy in seven rats by gradual
infusion of 0.2 mL of ethanol into the left liver lobe.
After closing the abdominal cavity with a two-layered
suture, animals were allowed to recover for at least
12 h prior to tracer injection, during which the left liver
lobe became necrotic. The rats were injected under
anaesthesia via a tail vein with 22 MBq [123I]MIHA
and then sacrificed under anaesthesia by decapitation
at 4 h (n = 4) or 24 h (n = 3) pi. The necrotic (left) and
viable (right) liver lobes were harvested, rinsed with cold
(4 ꢁC) saline, weighed and counted in a gamma counter.
Radioactivity concentration was expressed in counts per
min (CPM) per gram tissue. The tissues were immedi-
ately stained in TTC (1.5% solution in saline) for
15 min at 37 ꢁC and digitally photographed. They were
then frozen to ꢀ80 ꢁC and serial microtome sections
(5–50 lm) were made from these frozen samples that
were mounted on slides. Autoradiograms were made
by exposing the slides to a high performance phosphor
screen for 48 h. The screens were read with a phosphor
imager and analysed using Optiquantꢂ software. After-
wards the same slices were stained with H&E following
the conventional procedure. Figure 3 shows representa-
tive ex vivo images after TTC staining (Fig. 3a) and
autoradiography (Fig. 3b) of a 30-lm slice and the same
slice after H&E staining (Fig. 3c) of the necrotic lobe
(top row) and viable liver lobe (lower row) of a rat
sacrificed at 24 h pi. Necrosis was confirmed by TTC
staining (TTC negative areas) even though the images
did not match with those of autoradiography and
H&E staining due to disruptions that occurred during
freezing and microtome sectioning. As can be seen from
the images, there was a good match between the autora-
diograms (high tracer uptake red spots) and H&E
stained sections, thus confirming that uptake of
trometry.
[
Synthesis
of
radiolabelled
mono-
123I]iodohypericin monocarboxylic acid ([123I]MIHA)
was carried out by radioiodination of protohyperin
monocarboxylic acid (4) followed by cyclization of
the proto-derivative by irradiation of the reaction
mixture for 30 min with a 400-W halogen lamp
(Scheme 1b).17 The cyclization reaction proceeded
quantitatively as monitored by HPLC analysis. The
overall radiolabelling yield was >75% relative to start-
ing iodine-123 activity. The radiolabelled compound
([123I]MIHA) was purified by reversed phase HPLC
after which the solvents were evaporated by a gentle
flush of nitrogen and the tracer agent was formulated
in water/polyethylene glycol 400 (PEG 400) 80/20 V/V
for animal studies. The unlabelled compound was
used for identity confirmation of the radioiodinated
compound [123I]-6 after co-injection with the radiola-
1
belled derivative on HPLC (Fig. 2). H NMR of unla-
belled MIH showed that the iodination occurs ortho
to the ‘bay’ phenol15 (most acidic phenol, pKa = 1.7).
We therefore suppose that the position of iodine in
[
123I]MIHA is as presented in Figure 1B.
The log octanol/buffer (0.025 M phosphate buffer, pH
7.4) partition coefficient (logP) of [123I]MIHA was
found to be 1.47 0.06 (n = 6). The logP of [123I]MIH
is 3.08.15 As expected the substitution of a carboxylic
function for a methyl group has a profound effect on
the lipophilicity of the compound.
[
123I]MIHA was concentrated in the necrotic sections
of the liver. On H&E stained slices, pink areas
(eosinophilic) correspond to severely necrotic tissue
while purple areas (haematoxyphilic) correspond to
viable tissue. The ratio of necrotic:viable was 1.8 and
14 at 4 h and 24 h pi, respectively, as quantified by
autoradiography. The ratio of necrotic:viable was 1.2
and 1.9 at 4 h and 24 h, respectively, by gamma count-
Biodistribution of [123I]MIHA was studied in normal
NMRI mice after tail vein injection of 185 KBq. The
mice were sacrificed by decapitation under anaesthesia
at 30 min, 4 h or 24 h post injection (pi, n = 4 mice per

H. Fonge et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4001–4005
4003
Scheme 1. Reagents and conditions: (a) (i) (CH3COO)2, 60 ꢁC, 30 min; H2SO4; (ii) CrO3/CH3COOH + (CH3COO)2, 70 ꢁC 3 h; (iii) 3 + 1 + C6H6O,
0.8 M KOH, 155 ꢁC, 5 days; (iv) irradiation with 400-W halogen lamp for 30 min. (b) (i) [123I+], irradiation.
ing. The ratio was much less on gamma counting but
this is due to the fact that viable cells are intermixed with
necrotic cells, making sampling (separation of necrotic
from viable tissue) very difficult.
In a liver model of reperfused hepatic infarction, the
concentration of [123I]MIH was 3.1 times higher in ne-
crotic liver tissue compared to intact liver tissue as as-
sessed by autoradiography at 24 h pi. [123I]MIH has
been shown to be very useful in delineation and sizing
of infarcts in animal models of acute myocardial infarc-
tion (AMI).10 In reperfused AMI, infarct: remote tissue
ratios were as high as 81:1 and infarcts were well delin-
eated at 9 h pi. Avidity of [123I]MIHA for necrosis was
higher than that of [123I]MIH in a rat model with reper-
Figure 2. HPLC chromatogram after co-injection of unlabelled mono-
iodohypericin monocarboxylic acid with mono-[123I]iodohypericin
monocarboxylic acid.
Table 1. Biodistribution of mono-[123I]iodohypericin monocarboxylic acid ([123I]MIHA) in normal NMRI mice
Organ
% injected dose ( SD)
4 h
% injected dose/g ( SD)
4 h
30 min
24 h
30 min
24 h
Bladder
Kidneys
Liver
0.3 ( 0.1)
2.9 ( 0.3)
64.4 ( 3.0)
1.3 ( 0.3)
1.4 ( 0.4)
0.5 ( 0.1)
13.2 ( 2.4)
0.0 ( 0.0)
3.3 ( 1.5)
13.2 ( 3.8)
7.1 ( 6.2)
2.6 ( 0.2)
22.4 ( 5.8)
0.5 ( 0.2)
0.3 ( 0.1)
0.3 ( 0.1)
50.5 ( 12.5)
0.0 ( 0.0)
1.6 ( 1.3)
10.3 ( 6.6)
21.1 ( 9.8)
1.0 ( 0.2)
4.7 ( 1.1)
0.2 ( 0.0)
0.1 ( 0.0)
0.1 ( 0.0)
69.4 ( 9.4)
0.0 ( 0.0)
0.1 ( 0.0)
2.8 ( 0.5)
na
na
na
4.4 ( 0.4)
33.7 ( 2.5)
3.9 ( 0.4)
5.0 ( 1.7)
3.1 ( 0.8)
na
4.8 ( 0.3)
13.2 ( 3.5)
1.6 ( 0.6)
1.2 ( 0.3)
1.9 ( 0.6)
na
1.9 ( 0.4)
2.7 ( 0.9)
0.5 ( 0.1)
0.2 ( 0.1)
0.7 ( 0.5)
na
Spleen + pancreas
Lungs
Heart
Intestines + faeces
Brain
Blood
0.0 ( 0.0)
1.3 ( 0.6)
na
0.0 ( 0.0)
0.6 ( 0.5)
na
0.0 ( 0.0)
0.0 ( 0.0)
na
Carcass
SD, standard deviation; na, not applicable.

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H. Fonge et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4001–4005
ments will involve in vivo imaging to investigate the time
during which the best target (necrosis) to non-target
(viable tissue) ratios can be obtained. Single photon
emission computer tomography (SPECT) imaging with
[
123I]MIHA in this model of necrosis may find applica-
tions in the monitoring of response to PEI therapy.
PEI therapy has been monitored until now by computer
tomography (CT), which measures total tumuor volume
rather than the percentage of cells killed by therapy,
with a possibility of over- or underestimating the resid-
ual viable tumuor mass. Expanding infarct avid scintig-
raphy to PEI imaging would therefore be beneficial to
both patients and clinicians. Imaging of AMI presents
an even more challenging clinical task for which
[
123I]MIHA will also be evaluated for its potential
usefulness.
Figure 3. Ex vivo images of rat liver at 24 h post injection. (a) TTC
stained liver lobes of necrotic (1st row) and viable (2nd row) liver
tissue, (b) autoradiograms of 30-lm slice of necrotic (1st row) and
viable (2nd row) tissue and (c) H&E stained slices of necrotic (1st row)
and viable (2nd row) tissue. Arrows indicate regions of severe necrosis.
Note perfect match between autoradiograms and H&E stained
sections. Colour scale corresponds to autoradiograms.
Acknowledgments
We appreciate the invaluable contribution of Peter
Vermaelen in the animal experiments. This work was
supported by a grant awarded by Geconcerteerde
Onderzoeksactie (GOA) of the Flemish Government,
FWO-Vlaanderen Grant G. 0257.05 and in part by the
EC-FP6-project DiMI, LSHB-CT-2005-512146.
fused hepatic infarction. Whether this difference was due
to the different models used still has to be evaluated. In
the occlusion–reperfusion model of liver infarction both
apoptosis and necrosis can be induced with the extent of
tracer uptake being influenced by the balance between
both forms of cell death. Ethanol injection, on the other
hand, specifically induces necrosis. Early delineation
(2 h pi) of AMI using [123I]MIH was not possible given
the very high blood pool activity of the tracer agent at
this time point (in mice there was namely 30.0, 28.2
and 0.7% ID/g at 30 min, 4 h and 24 h pi, respectively,
assuming blood accounts to 7% of total body mass).
In contrast, [123I]MIHA showed a rapid blood clearance
with 0.6% ID/g blood at 4 h pi. The rapid blood clear-
ance of [123I]MIHA could be an advantage over
References and notes
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Dymarkowski, S.; Verbruggen, A.; Marchall, G. Acad.
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[
123I]MIH for imaging AMI as a good contrast could
be obtained early enough to visualize AMI within the
clinically relevant time window for thrombolysis (usu-
ally within 6 h of the acute event). This faster clearance
from plasma and the major organs makes [123I]MIHA
an attractive and more favourable alternative for
[
123I]MIH for imaging of AMI.
Several infarct avid imaging agents have been proposed
and used in nuclear medicine, but none of them meets
the optimal imaging characteristics. The most interest-
ing has been 99mTc-glucarate which has been shown in
several studies to be very useful in delineating and
sizing myocardial infarcts in animals and in humans.
99mTc-glucarate imaging nonetheless presents with two
limitations18 which can be overcome by imaging using
8. Ni, Y.; Bormans, G.; Chen, F.; Verbruggen, A.; Marchal,
G. Invest. Radiol. 2005, 40, 526.
9. Ni, Y.; Huyghe, D.; Verbeke, K.; de Witte, P. A.; Nuyts,
J.; Mortelmans, L.; Chen, F.; Marchal, G.; Verbruggen,
A. M.; Bormans, G. M. Eur. J. Nucl. Med. Mol. Imaging
2006, 33, 595.
[
123I]MIH and [123I]MIHA: (1) scan positivity with
10. Fonge, H.; Vunckx, K.; Ni, Y.; Nuyts, J.; Mortelmans, L.;
Wang, H.; de Groot, T.; Vermaelen, P.; Bormans, G.;
Verbruggen, A. Abstract of Papers, 54th Annual Meeting
of the Society of Nuclear Medicine, Washington, DC, The
Society of Nuclear Medicine: Reston, VA, 2007; Abstract
610.
99mTc-glucarate is limited to the early hours of the acute
injury (typically < 9 h after AMI) and (2) it is rapidly
washed out from the infarcted myocardium. [123I]MIH
as well as [123I]MIHA have longer half lives in necrotic
tissue, making it potentially feasible to image old as well
as new infarcts.
11. Gruszecka-Kowalik, E.; Zalkow, L. H. Org. Prep. Proc.
Int. 2000, 32, 57.
12. To a mixture of 1 g (3.85 mmol) emodin (1) and 10 mL
acetic anhydride was added dropwise 200 lL of 96%
H2SO4under stirring. The reaction mixture was refluxed at
This preliminary evaluation presents a good indication
for the necrosis avidity of [123I]MIHA. Further experi-

H. Fonge et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4001–4005
4005
65 ꢁC for 30 min. The resulting yellow solution was added
to 50 mL of water and the yellow precipitate (2) so formed
was washed with water and dried under reduced pressure.
The precipitate 2 consisted of a mixture of di- and tri-
acetylated emodin. Yield 99%. Accurate mass di-acety-
lated emodin [C19H14O7]ꢀ theoretical 352.0739 Da, found
352.0694 Da.
for 30 min to give compound 5. 1H NMR 4 (DMSO) d
7.96 (s, CH-11), 7.67 (s, CH-9), 7.28 (s, CH-14), 7.18 (s,
CH-2, CH-5) 6.76 (s, CH-12), 2.50 (s, CH3). Accurate
mass of 5 [C30H14O10]ꢀ theoretical 533.0510 Da, found
533.0478 Da, accurate mass of 4 [C30H16O10]ꢀ theoretical
535.0703 Da, found 535.0671 Da.
15. Bormans, G.; Huyghe, D.; Christiaen, A.; Verbeke, K.; de
Groot, T.; Vanbilloen, H.; de Witte, P.; Verbruggen, A. J.
Labelled Compd. Radiopharm. 2004, 47, 191.
13. 1.34 g (3.38 mmol) of 2 was dissolved in a mixture of
30 mL CH3COOH and 30 mL acetic anhydride at 55–
60 ꢁC. A solution of 3 g CrO3 in a mixture of 2.5 mL H2O
and 35 mL CH3COOH was added dropwise over 30 min.
The mixture was stirred at 70 ꢁC for 3 h during which the
brown solution turned green. It was then added to 1 L of
water and the mixture was cooled overnight at 4 ꢁC to
precipitate the desired product (3) as a yellow powder. The
powder was washed thoroughly with H2O and dried over
P2O5. Yield 98%. Accurate mass of tri-acetylated emodic
acid [C21H14O10]ꢀ: theoretical 425.0514 Da, found
425.0547 Da.
14. 0.1 g of 1 (0.37 mmol), 0.32 g of 3 (0.75 mmol) and
0.162 g of hydroquinone (1.5 mmol) were dissolved in
5 mL of 0.8 M KOH protected from light in a capped
vial placed in a heavy steel reaction vial holder. The vial
was placed in an oven at 155 ꢁC and allowed to react for
5 days during which it was agitated every 24 h to allow
mixing. After cooling, the reaction mixture was acidified
to pH 4–4.5 with 1 M HCl and the dark coloured
precipitate was separated by centrifugation, after which
the filtrate was extracted three times with 2% NaHCO3
and EtOAc. The aqueous layer was evaporated in vacuo
and the residue was dissolved in 25 mL methanol and
applied on a silica gel column. Purification was afforded
by gradient elution starting from 100% CH2Cl2 to
CH2Cl2/MeOH (80:20 V/V). The first major fraction
was collected, evaporated in vacuo and repurified on
silica gel. Evaporation of solvents yielded the deep purple
solid 4. The residue was dissolved in dry methanol and
the solution was irradiated with a 400-W halogen lamp
16. Unlabelled-6: To 15 mL of a 0.5 mg/mL solution of
compound (5) in ethanol were successively added 2.5 mL
of 0.5 M H3PO4, 5 mL of 0.2 M peracetic acid and 5 mL
ethanol followed by dropwise addition of 1.1 mL of a
1 mg/mL solution of NaI in 0.01 M NaOH and stirring for
a further 30 min. The volume of the solution was reduced
in vacuo followed by purification of the residue using
reversed phase HPLC (LaChrom Elite, Hitachi, Darms-
tadt, Germany) on a semi-preparative column (XTerraꢂ
prep RP18 10 lm, 10 mm · 250 mm) using 0.05 M
NH4OAc/acetonitrile 60:40 V/V at a flow rate of 2.5
mL/min as mobile phase. Accurate mass [C30H13IO10]ꢀ:
theoretical 658.9453 Da, found 658.9481 Da.
17. [123I]-6 was synthesized by successively adding 150 lL of a
solution of 0.5 mg/mL of 4 in ethanol, 25 lL of 0.5 M
H3PO4, 50 lL of 0.2 M peracetic acid and 50 lL ethanol
to 37 MBq sodium[123I]iodide (Amersham Biosciences,
Diegem, Belgium) and incubating at room temperature for
30 min followed by photocyclization using a 400-W
halogen lamp for 30 min. The resulting [123I]MIHA was
purified on reversed phase HPLC coupled with a radio-
metric detector (3-inch NaI(Tl) crystal) using an XTerraꢂ
C18 column (5 lm, 4.6 mm · 250 mm) with 0.05 M
NH4OAc/acetonitrile 60:40 V/V at a flow rate of 1 mL/
min as mobile phase (K0 = 3.2). [123I]-6 was obtained in
high specific activity (950 GBq/lmol).
18. Mariani, G.; Villa, G.; Rossettin, P. F.; Spallarossa, P.;
Bezante, G. P.; Brunelli, C.; Pak, K. Y.; Khaw, B. A.;
Strauss, H. W. J. Nucl. Med. 1999, 40, 1832.