11C-Radiosynthesis and preliminary human evaluation of the disposition of the ACE inhibitor [11C]zofenoprilat

Article abstract of DOI:10.1016/j.bmc.2003.10.054

(4S)-1-[(S)-3-Mercapto-2-methylpropanoyl]-4-phenylthio-L-proline (Zofenoprilat, 2), the active metabolite of the potent ACE inhibitor Zofenopril Calcium (1), was labelled with carbon-11 (t_1/2=20.4 min) to evaluate its pharmacokinetics behaviour in human body using Positron Emission Tomography (PET). [¹¹C]2 labelling procedures were based on the use of immobilized Grignard reagent and the acylation of (S)-4-phenylthio-L-proline methyl ester (5) with ¹¹C-labelled methacryloyl chloride, followed by a Michael addition with thiobenzoic acid. The radiochemical yield was 5-10% (EOB, decay corrected) and specific radioactivity ranged from 0.5 to 1.5 Ci/μmol (18.5-55.5 GBq/μmol). Preliminary in vivo human evaluation of [¹¹C]2 showed that the drug accumulates in organs which express high levels of ACE, like lungs and kidneys, and in organs involved in drug metabolism such as the liver and gall bladder. Results of the distribution of [¹¹C]2 showed a measurable concentration of the drug in the target tissues such as the kidney and to a minor extent, the heart, where it can afford organ protection.

Full text of DOI:10.1016/j.bmc.2003.10.054

Bioorganic & Medicinal Chemistry 12 (2004) 603–611
11C-Radiosynthesis and preliminary human evaluation of the
disposition of the ACE inhibitor [11C]zofenoprilat
Mario Matarrese,a Aldo Salimbeni,b Elia Anna Turolla,a Damiano Turozzi,b
Rosa Maria Moresco,a Davide Poma,b Fulvio Magni,c Sergio Todde,a Claudio Rossetti,a
Maria Teresa Sciarrone,a Giuseppe Bianchi,a Marzia Galli Kienlec and Ferruccio Fazioa,*
aInstitute of Molecular Bioimaging and Physiology-CNR; University of Milano/Bicocca, Institute San Raffaele;
Via Olgettina 60, 20132 Milan, Italy
bR&D Department, Lusochimica S.p.A., via Giotto 9, 23871 Lomagna (Lecco), Italy
cUniversity of Milano/Bicocca, DIMESAB, via Cadore 48, 20052 Monza (MI), Italy
Received 10 July 2003; accepted 22 October 2003
Abstract—(4S)-1-[(S)-3-Mercapto-2-methylpropanoyl]-4-phenylthio-l-proline (Zofenoprilat, 2), the active metabolite of the potent
ACE inhibitor Zofenopril Calcium (1), was labelled withcarbon-11 ( t1/2=20.4 min) to evaluate its pharmacokinetics behaviour in
human body using Positron Emission Tomography (PET). [11C]2 labelling procedures were based on the use of immobilized
Grignard reagent and the acylation of (S)-4-phenylthio-l-proline methyl ester (5) with 11C-labelled methacryloyl chloride, followed
by a Michael addition with thiobenzoic acid. The radiochemical yield was 5–10% (EOB, decay corrected) and specific radioactivity
ranged from 0.5 to 1.5 Ci/mmol (18.5–55.5 GBq/mmol). Preliminary in vivo human evaluation of [11C]2 showed that the drug
accumulates in organs which express high levels of ACE, like lungs and kidneys, and in organs involved in drug metabolism such as
the liver and gall bladder. Results of the distribution of [11C]2 showed a measurable concentration of the drug in the target tissues
such as the kidney and to a minor extent, the heart, where it can afford organ protection.
# 2003 Elsevier Ltd. All rights reserved.
1.Introduction
developed and introduced into medical practice. These
agents (Captopril, Analapril, Fosinopril, etc.) differ in
Positron Emission Tomography (PET) enables the in
vivo study of different pharmacological variables in
human subjects using methods originally developed for
pre-clinical studies in rodents.1 The radiolabelling of
drugs withpositron emitters allows teh in vivo
measurement of drug concentration in different areas of
the human body and the in vivo characterisation of a
drug’s pharmacokinetic behaviour in the target organ.
Limits of this strategy are the short half-lives of
positron emitting isotopes that ranges from 20.4 min for
carbon-11 to 109.8 min for fluorine-18.2 Thus, the
synthetic procedures must be selected in a way that
introduction of the label occurs at the final steps of the
procedure. After the discovery of Captopril, the
prototype of orally active angiotensin converting
enzyme (ACE) inhibitors, several new ACE ligands were
their chemical structure, functional groups (presence of
sulfhydryl, carboxyl or phosphinyl), active moiety,
pharmacological potency and pharmacokinetics. These
characteristics differentiate ACE inhibitors from each
other and influence their ability to inhibit the enzyme in
various organs.3 Among the various ACE agents, the
18Fꢀ radiolabelled fluoro analogue of captopril, 4-
[18F]fluoro-1-(3-mercapto-2-methyl-1-oxopropyl)-l-pro-
line (4-cis-[18F]fluorocaptopril) has been evaluated as a
potential radioligand for PET and was indicated as a
new radiopharmaceutical for the in vivo ACE measure-
ment.4
(4S)-1-[(2S)-3-Benzoylthio-2-methyl-1-oxopropyl]-4-
(phenylthio)-l-proline (Zofenopril, 1) (EC50=6–80 nM)
is an ACE inhibitor that is characterised by an high
cardio-protective activity supposed to be due to a potent
and selective inhibition of myocardial ACE as well as to
the free radical scavenging properties of its sulfhydryl
group.5ꢀ10 The cardio-protective activity of Zofenopril
Keywords: Zofenoprilat; ACE inhibitor; 11C-radioligand; PET.
* Corresponding author. Tel.: +39-02-2643-2716; fax: +39-02-2641-
5202; e-mail: fazio.ferruccio@hsr.it
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.054

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M. Matarrese et al. / Bioorg. Med. Chem. 12 (2004) 603–611
is of relevance for its use in the prevention and therapy
of cardiovascular diseases.11
diastereomer with higher Rf was Zofenopril methyl ester
[(S,S,S)7]. Zofenoprilat (2) and its (R,R,S) diastereomer
were finally obtained by hydrolysis with NaOH in
MeOH/H2O. The whole process was then repeated
‘one-pot’ (see Experimental) without isolation and
purification of the intermediates or separation of the
two diastereomers 7 in order to reduce the time of the
synthesis. The results (<40 min required for the overall
process) were considered satisfactory for transferring to
the radiosynthesis.
To evaluate whether the cardio-protective effect could be
linked to a preferential accumulation of the drug in
myocardial tissue, we have developed a strategy for the in
vivo PET measurement of myocardial uptake of Zofenopril
in humans. Zofenopril is a pro-drug that is deesterified
at the thioester group to give the active product Zofe-
noprilat, (4S)-1-[(S)-3-mercapto-2-methylpropanoyl]-4-
phenylthio-l-proline (2) (EC50=1–8 nM) (Fig. 1).
2.2. Radiochemistry
To avoid confusing effects resulting from the radi-
olabelled circulating pro-drug, we decided to label the
active compound Zofenoprilat. Finally, to test the
hypothesis of myocardial targeting, the kinetics of
myocardial distribution of [11C]zofenoprilat was inves-
tigated in a pilot study carried out on a patient with
moderate hypertension.
The one-pot procedure developed for the preparation of
cold Zofenoprilat was adapted for the synthesis of
[11C]zofenoprilat ([11C]2) by converting [11C]4 to radio-
synthon 8 to provide the [11C]6 intermediate. The 11C-
carboxylation (Scheme 3) procedure described by
McCarron12 for the radiosynthesis of the [carbo-
nyl-11C]cyclohexanecarbonyl chloride, using a modified
11C-methylation radiosynthesis module, was adopted.13
The described strategy consists of the deposition of a
thin film of the Grignard reagent on the inner surface of
narrow bore tubing. [11C]Carbon dioxide is then passed
through the tube in a stream of inert gas. The reaction
to give [11C]4 occurs at the gas–liquid interface.
Formation and recovery of the [11C]acyl chloride 8 is
achieved by passing thionyl chloride through the tube,
which is directly connected to the reaction vial contain-
ing the appropriate precursor 5 for the subsequent
[11C]carboxylation reaction.12 THF was found to be the
solvent of choice for [11C]6 radiosynthesis, provoding a
55% EOS (end of synthesis) radiochemical yield.
To this aim a new synthetic approach of Zofenoprilat was
designed. It was based on the acylation of (S)-4-phe-
nylthio-l-proline methyl ester (5) with 11C-labelled
methacryloyl chloride followed by a Michael addition with
thiobenzoic acid. A key feature of the radiochemical
synthesis was the use of an immobilized Grignard
reagent to obtain 11C-labelled acyl chloride (as
published by McCarron et al.12) employing a partially
modified Nuclear Interface PET Tracer Synthesizer.
2.Results and discussion
2.1. Chemistry
The Michael adduct [11C]7 was synthesized by addition
of thiobenzoic acid (150 mL) to the methacrylamide
The short-lived positron emitting 11C radionuclide
requires that the labelling be introduced in one of the
last steps of the synthesis. Hence, the current process (to
be published elsewhere) to obtain the precursor
Zofenopril 1 was modified. As shown in Scheme 1, th e
retrosynthetic pathway implies the introduction of the
11C label in a carbonyl group.
The synthesis (Scheme 2) involves the reaction of com-
mercially available isopropenylmagnesium bromide (3)
withCO to give magnesium bromide methacrylate (4).
2
This compound was joined to the proline derivative 5 to
give the methacrylamide 6 which can alternatively be
prepared starting from methacrylic acid (see Experi-
mental). After purification by flashchromatography ( n-
hexane/EtOAc 6:4), methacrylamide 6 was reacted with
thiobenzoic acid in the presence of N,N-diisopropyl-
ethylamine (DIPEA) to give a mixture of the two
diastereomers 7 as products of the Michael addition.
These isomers were separated by flash chromatography
1
(n-hexane/AcOEt 65:35). On the basis of H NMR the
Figure 1. Zofenoprilat and its precursor Zofenopril.
Scheme 1. Retrosynthetic analysis of Zofenoprilat.

M. Matarrese et al. / Bioorg. Med. Chem. 12 (2004) 603–611
605
ꢁ
.
Scheme 2. Synthesis of Zofenoprilat via Michael addition. Reagents and conditions: (a) CO2, THF, ꢀ10 C to rt; (b) EDAC HCl, DIPEA, HOBt,
THF, rt; (c) HATU, DIPEA, DMF 55 ꢁC; (d) PhCOSH, DIPEA, 60–95 ꢁC; (e) separation of diastereomers by flash chromatography (n-hexane/
AcOEt 65:35); (f) 1 N NaOH, H2O, MeOH or 1,4-dioxane.
Scheme 3. Radiosynthesis of [11C]Zofenoprilat.
[11C]6 with a radiochemical yield of 65%. Also in this
part of the radiosynthesis a mixture of two labelled
diastereomers was obtained (as described in the Experi-
mental). An HPLC pre-purification step of the [11C]7
adduct was performed, thus allowing elimination of the
large excess of thiobenzoic acid. This technical solution
and subsequent solid phase extraction (SPE) for recovery
of the pure Michael adduct [11C]7 required manual per-
forming of the hydrolysis and final radiopharmaceutical
formulation for intravenous administration.
authentic standards on reverse-phase HPLC and by the
mass spectrometric analyses of the products of their
carrier-added radiosyntheses (see Experimental).
2.3. PET study
A rapid and transient increase of radioactivity was
observed immediately post-injection in the myocardial
wall and in the ventricular cavity (Fig. 2). In these
regions maximal radioactivity uptake was observed
approximately 1 min after injection. At this time,
radioactivity concentration in the myocardial tissue
(MT) and ventricular cavity (VC) was equal to 0.008%
I.D./mL (0.37 pmol/mL) and 0.014% I.D./mL (0.56
pmol/mL), respectively. Time courses of blood radio-
activity concentrations whether measured by PET or
direct counting of blood samples gave similar results
(Fig. 3) indicating the reliability of PET measurements.
Myocardial radioactivity concentration remained below
that measured in the LV for the entire period of obser-
vation. At the early stages, it displayed a kinetic
behaviour similar to that in blood suggesting that,
immediately after injection, radioactivity uptake in
The overall radiochemical yield (EOB, decay corrected)
of the [11C]2 was 5–10%. The specific radioactivity ranged
from 0.5 to 1.5 Ci/mmol (18.5–55.5 GBq/mmol), (EOS)
withan average time of radiosyntehsis of 60 min,
including radiopharmaceutical formulation. In a typical
experiment starting from 400 to 600 mCi (14.8–22.2
GBq) of [11C]CO2, 5–10 mCi (185–270 MBq) of the
formulated [11C]Zofenoprilat was obtained with100%
radiochemical purity.
[11C]2 and intermediate Michael adduct [11C]7 identities
were confirmed by co-injection of their non-radioactive

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M. Matarrese et al. / Bioorg. Med. Chem. 12 (2004) 603–611
Figure 4. Time–activity curve of heart tissue (MT) to left ventricle
cavity (LV) radioactivity concentration ratio measured in a patient
with moderate hypertension after the iv injection of [11C]2.
Figure 2. Time–activity curve of [11C]2 uptake, expressed in absolute
radioactivity concentration units, in the heart tissue (MT) and left
ventricle cavity (LV) of a patient withmoderate hypertension.
Figure 5. Time–activity curve of [11C]2 radioactivity uptake expressed
in absolute radioactivity concentration units, in lungs and kidneys of a
patient withmoderate hypertension.
Figure 3. Time–activity curve of [11C]2 radioactivity uptake, expressed
in absolute radioactivity concentration units, in the blood of a patient
withmoderate hypertension. Blood concentration was measured using
bothPET (LV) or direct sampling of serial blood aliquots (Blood).
myocardium depends on tissue perfusion or on the
spill-over effects of circulating radioactivity in the LV.
However, at a later times we observed a progressive
increase in myocardial tissue to blood radioactivity
concentration ratio (0.9 at 50 min after injection; see
Fig. 4) suggesting a slow cardiac accumulation.
A rapid rise in radioactivity concentration was observed
in lungs and kidneys. In these organs, radioactivity
concentration peaked at 2–3 min after injection and
remained fairly stable thereafter (Fig. 5). At the time of
maximum uptake, radioactivity concentration in lungs
and kidneys was 0.0072 and 0.0073% I.D./mL, respec-
tively. In the liver and gall bladder a progressive rise in
radioactivity concentration was also observed, more
markedly in the gall bladder.
Figure 6. Uptake of [11C]2 expressed as %I.D./mL, measured at
approximately 60 min in different organs of a patient withmoderate
hypertension.
study versus [11C]zofenoprilat in the human one) and
they differed also for the route of administration used
(oral versus intravenous). In spite of that, it should be
noted that zofenoprilat is the main and the only active
metabolite of Zofenopril.
Radioactivity distribution measured at 50 min after
injection is shown in Figure 6. The rank order of radio-
activity uptake was: gall bladder> >liver>kidney=
lung>blood=myocardium.
Pre-clinical data showed that Zofenopril was able to
accumulate in the heart and vascular tissue14 and exert a
long-lasting local ACE inhibitory activity.15 Local
activation of the renin–angiotensin system and the for-
mation of angiotensin II are reported as risk factors in
the development of cardiac hypertrophy and heart
failure, myocardial infarction, atherosclerosis and so
on16,17 and therefore the prolonged local inhibition of
cardiac ACE is thought beneficial for the prevention of
these cardiovascular events. The amount of radio-
activity observed in MT during this study and the low
concentration of Zofenoprilat (3 nM) necessary to inhibit
cardiac ACE16 suggest that, at pharmacological doses,
local enzyme inhibition may be effective.11
Quantitative organ distribution substantially overlaps
that previously observed in rats with oral administra-
tion of [14C]Zofenopril.14 In this study, 30 min after
drug administration, radioactivity concentration in all
tissue except the kidney, small intestine and urinary
bladder was lower than that found in plasma while after
24 hthe tissue to blood ratios were markedly increased
in excretory and cardiovascular tissues. Similarly, in our
study at 50 min after injection, the radioactivity con-
centration in most of the organs sampled was higher or
equal to blood concentration. In the two studies different
chemical forms were administered ([14C]zofenopril in rat

M. Matarrese et al. / Bioorg. Med. Chem. 12 (2004) 603–611
607
Likewise, the high radioactivity concentration found in
the kidney (peak=0.0073% I.D./mL) and the ability of
Zofenopril to inhibit local ACE in the 1–3 nM range15
may be the basis for the organ-protection afforded by
this drug.
Bruker CXP-400 spectrometer in indicated solvent;
chemical shifts are reported as d (ppm) relative to TMS
as external standard. The mass spectra were recorded in
positive ion mode witha VG 7070 EQ-HF mass spec-
trometer. The infrared spectra were recorded on a Perkin-
Elmer FT-IR 1600 spectrometer. Elemental analyses
were performed on Fison Instrument EA-1108. TLC
was performed on silica-coated glass plates (Merck,
Silicagel 60 F254) in the indicated solvent mixture, using
UV light as detector unless otherwise noted. Flash
chromatography was performed with silica gel 0.040–
0.063 mm using the indicated solvent mixture. Analytical
HPLC was performed with a Merck-Hitachi LaChrom
system equipped witha C-18 column (Waters mBonda-
pack, 10 mm, 300ꢂ3.9 mm); flow rate 1 mL/min, l 238
nm; mobile phase MeOH/0.05 M KH2PO4, pH 3 70:30
(v:v); temperature 31 ꢁC. Semi-preparative HPLC was
performed witha Waters 600 E system equipped witha
Waters 486 UV detector, a Shimatzu C-R6A Chromatopac
integrator and a C-18 column (Waters mBondapack, 10
mm, 300ꢂ19 mm); flow rate 17 mL/min; l 238 nm;
mobile phase MeOH/0.05 M KH2PO4, pH 3 50:50 (v:v);
temperature 25 ꢁC.
Extrapolating these data in a person (where the
common therapeutic antihypertensive dose of 30 mg
o.d. produced a Cmax of 341 ng/mL18 corresponding to
a concentration of 1 mM) we can put forward the
hypothesis that in the target tissues such as heart and
kidney the concentration reached by the drug is able to
block the local production of the ACE.
In conclusion, the results of this study, which for the first
time demonstrate the in vivo measurements of [11C]Zofe-
noprilat in the human body, confirm those previously
reported in rodents indicating that the drug accumulates
in target organs where ACE is more expressed.16,17 In
fact, in spite of the obvious limitation of a pilot study
conducted on one single patient, PET data indicate that
also in the human body the drug accumulates in organs
that express high levels of ACE, like lungs, kidneys and
to a minor extent in myocardium.
[11C]Carbon dioxide was produced by the 14N(p,a)11C
reaction on a CTI-Siemens RDS-112 cyclotron, using a
11.5MeV proton beam at currents of 10–30 mA, and
trapped in a hollow stainless steel loop cooled with liquid
nitrogen. Radiochemical syntheses were performed on
modified fully automated synthesis module (PET Tracer
Synthesizer, Nuclear Interface Datentechnik GmbH,
Munster, Germany). HPLC of labelled compounds was
performed witha Waters Millennium system equipped
withUV detector set at 238 nm, a flow radioactivity
detector and a reversed-phase analytical HPLC column
(Shandon Hypersil BDS C-18, 5 mm, 250ꢂ4.6 mm). pH of
solutions was measured witha Shott-Gera te pH-meter.
Our PET biodistribution study was performed on a
diastereomeric mixture of radiolabelled Zofenoprilat and
not on the (S,S,S) isomer which is the main metabolite of
the form currently used clinically. In vitro studies on the
inhibitory activity of (R,S,S)- and (S,S,S)-Zofenoprilat
on ACE indicate that (S,S,S)-Zofenoprilat is 156 times
more potent than (R,S,S)-Zofenoprilat [IC50 values of
10.9 and 1700 nM, respectively (Lusochimica, internal
report)]. Thus, on the basis of the above results, we can
reasonably hypothesise that the administration of a
mixture of radiolabelled epimers instead of [11C](S,S,S)-
Zofenoprilat has reduced the levels of specific uptake
and signal to noise ratios in organs expressing ACE.
Identity of the final 11C-labelled compounds was asses-
sed by MS. Dried samples were reconstituted with50 mL
methanol and aliquots were analysed by APCI/MS.
Samples were introduced by a Rheodyne injector with a
loop of 20 mL using water/methanol 1:1 (v:v) as
delivering solvent (flow rate, 300 mL/min). The APCI
spectra were recorded witha Finnigan MAT95. LC-
APCI/MS was performed witha Beckman Ultrasphere
ODS column (150ꢂ46 mm, 5 mm) using methanol/water
1:1 (v:v) containing 0.1% acetic acid at flow rate of 1
mL/min generated witha Jasco 880-PU HPLC pump
(Jasco Spectroscopic Co. LTD, Tokyo, Japan). The
eluent was directly introduced into the APCI source of a
Finnigan MAT95 double focusing mass spectrometer.
The corona discharge was set at 2 mA, the vaporizer at
350 ꢁC and the heated capillary at 200 ꢁC. Mass spec-
trometry conditions were as follows: capillary voltage
3.1 KV and 38 V, tube lens 60 V, skimmer voltage 4.2 V
and octapole voltage 1 V. Spectra were recorded by
scanning from m/z 100 to m/z 800 at 3 s/dec. Skimmer-
CID spectra were recorded increasing tube lens and the
heated capillary voltage, respectively.
However, due to the short half life of the radioisotopes,
PET-based pharmacokinetic studies do not allow
monitoring of drug distribution for more than 1 h after
injection and it is not possible to evaluate in depththe
further distribution of the drug.
3.Experimental
3.1. Chemistry
Reagents and solvents were purchased from common
commercial suppliers and used without purification.
Isopropenylmagnesium bromide (0.5 M in THF) was
purchased from Aldrich (Milano, Italy). (S)-4-Phe-
nylthio-l-proline was prepared by Lusochimica. Unla-
belled reference substances were prepared by
Lusochimica and used for comparison in all the HPLC
runs in the analyses of the 11C-labelled compounds.
Authentic sodium Zofenoprilat was obtained from
Berlin Chemie (Berlin, Germany). Identities of all the
unlabelled synthesised materials were confirmed using
1H NMR, MS and elemental analysis. 1H NMR spectra
were recorded at 200 MHz on a Bruker AC-200 or a
3.1.1. Magnesium bromide methacrylate (4). A 250-mL
four-neck round-bottom flask equipped withmagnetic

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M. Matarrese et al. / Bioorg. Med. Chem. 12 (2004) 603–611
stirrer and thermometer was inertized with N2 stream
and charged with 40 mL (20 mmol) of iso-
propenylmagnesium bromide (0.5 M solution in THF)
(3). An excess of CO2(g) was bubbled in the solution
which cooled to ꢀ10 ꢁC over 5 min. An exothermic
effect was observed which increased the temperature to
rt. The resulting solution of raw magnesium bromide
methacrylate (20 mmol) (4) was used without further
purification. For analytical purpose an amount was
treated with0.1 N HCl, extracted withCH 2Cl2 and the
organic solution was evaporated. The 1H NMR
spectrum of the residue agreed with literature data19
HPLC: tR 3.2 min. 1H NMR (CDCl3): d 6.25 (dq,
J=1.6, 1.0 Hz, 1H, HCH¼); 5.68 (dq, J=1.6, 1.6 Hz,
1H, HCH¼); 1.95 (dq, J=1.6, 1.0 Hz, 3H, CH3).
HCHCHCO2CH3); 2.03 (m, 1H, HCHCHCO2CH3);
1.93 (s, 3H, CH3C¼). MS (EI) m/z 305 [M]+. Elemental
analysis: C16H19NO3S (305.39): calcd C 62.93, H 6.27,
N 4.59; found C 63.08, H 6.40, N 4.47.
3.1.4.
(4S)-1-[(S)-3-Benzoylthio-2-methylpropanoyl]-4-
phenylthio-L-proline methyl ester [(S,S,S)]-7); (4S)-1-[(R)
-3-benzoylthio-2-methylpropanoyl]-4-phenylthio-L-proline
methyl ester [(R,S,S)]-7). 5.0 g (16 mmol) of (4S)-1-(2-
methylpropanoyl)-4-phenylthio-l-proline methyl ester
(6) was dissolved in 10 mL (82 mmol) thiobenzoic acid.
5 mL (29 mmol) DIPEA was added. The mixture was
heated to 60 ꢁC. The reaction was monitored by TLC
(n-hexane/EtOAc 1:1). After 5 min, no starting material
was detected. Then the reaction mixture was diluted
with 50 mL of diethyl ether. The resulting solution was
washed with water (50 mL), 1 N NaHCO3 (50 mL),
water (25 mL) and brine (25 mL). The solvent was
removed under vacuum. The two diastereomers in the
raw product were separated by flashchromatography
(n-hexane/EtOAc 65:35). 3.0 g of each diastereomer
were recovered (overall yield 85%). [(S,S,S)]-7. TLC (n-
hexane/EtOAc 1:1): Rf 0.42. HPLC: tR 11.0 min. 1H
NMR (CDCl3): d 8.0–7.2 (m, 10H, aromatic H); 4.53
(dd, J=8.0, 8.0 Hz, 1H; CHCO2CH3), 4.10 (dd,
J=10.1, 6.9 Hz, 1H; HCHN); 3.74 (s, 3H, CH3O); 3.72
(m, 1H, CHSPh); 3.49 (dd, J=10.1, 8.5 Hz, 1H,
HCHN); 3.22 (dd, J=13.4, 8.0 Hz, 1H, HCHSCOPh);
3.15 (dd, J=13.4, 6.1 Hz, 1H, HCHSCOPh); 2.88 (m,
1H, CHCH3); 2.64 (m, 1H, HCHCHCO2CH3); 2.01
(ddd, J=13.0, 9.2, 7.7 Hz, 1H, HCHCHCO2CH3); 1.30
(d, J=6.9 Hz, 3H, CH3CH). MS (FAB) m/z 444
[MH]+. Elemental analysis: C23H25NO4S2 (443.57):
calcd C 62.28, H 5.68, N 3.16; found C 62.32, H 5.75, N
3.09. [(R,S,S)]-7. TLC (n-hexane/AcOEt 1:1): Rf 0.36.
3.1.2. (S)-4-Phenylthio-L-proline methyl ester (5). 15.0 g
(67 mmol) of (S)-4-phenylthio-l-proline was dissolved
in 150 mL of methanol. 10 mL (79 mmol) of
trimethylsilyl chloride was added and the mixture was
heated to reflux. The reaction was monitored by TLC
(CH2Cl2/MeOH/TEA 88:10:2). After 10 h, no starting
material was detected. The solvent was evaporated
under vacuum. The resulting residue was dissolved in 90
mL of CH2Cl2. The mixture was neutralised by adding
70 mL of 1 N NaHCO3. The aqueous phase was
separated and extracted withCH Cl2 (2ꢂ80 mL). The
2
combined organic solutions were washed with brine (70
mL), dried over Na2SO4 and filtered. The solvent was
removed under vacuum to give 15.4 g (97%) of 5 as
yellow oil. TLC (EtOAc/MeOH 8:2): Rf 0.47. HPLC: tR
1
3.4 min. H NMR (CDCl3): d 7.4–7.2 (m, 5H, aromatic
H); 3.9–3.7 (m, 2H, CHCO2CH3, CHSPh); 3.75 (s, 3H,
CH3); 3.22 (dd, J=11.6, 6.1 Hz, 1H, HCHN); 3.05 (dd,
J=11.6,5.1 Hz, 1H, HCHN); 2.56 (ddd, J=13.7, 8.8,
7.3 Hz, 1H, HCHCHCO2CH3); 2.24 (bs, 1H, NH); 1.93
(ddd, J=13.7, 5.9, 5.9 Hz, 1H, HCHCHCO2CH3). MS
(EI) m/z 237 [M]+. Preparation of the hydrobromide
salt of 5 was reported in literature20 but no analytical
data were reported.
1
HPLC: tR 10.2 min. H NMR (CDCl3): d 8.0–7.2 (m,
10H, aromatic H); 4.50 (dd, J=8.0, 8.0 Hz, 1H;
CHCO2CH3), 3.97 (dd, J=12.9, 7.2 Hz, 1H, HCHN);
3.75 (s, 3H, CH3O); 3.72 (m, 2H, CHSPh, HCHN); 3.37
(dd, J=13.4, 7.3 Hz, 1H, HCHSCOPh); 3.11 (dd,
J=13.4, 6.8 Hz, 1H, HCHSCOPh); 2.87 (m, 1H,
CHCH3); 2.65 (m, 1H, HCHCHCO2CH3); 2.01 (m, 1H,
HCHCHCO2CH3); 1.22 (d, J=6.9 Hz, 3H, CH3CH).MS
(FAB) m/z 444 [MH]+. Elemental analysis:
C23H25NO4S2 (443.57): calcd C 62.28, H 5.68, N 3.16;
found C 62.35, H 5.76, N 3.07.
3.1.3. (4S)-1-(2-Methylpropenoyl)-4-phenylthio-L-proline
methyl ester (6). 7.5 g (32 mmol) of (S)-4-phenylthio-l-
proline methyl ester (5) was dissolved in 75 mL of THF.
To the resulting solution under stirring, 5.15 g (33
mmol) of 1-hydroxybenzotriazole, 17 mL (95 mmol)
DIPEA, 2.90 mL (33 mmol) methacrylic acid and 8.1 g
(41 mmol) EDAC.HCl were added in this order. The
reaction was monitored by TLC (EtOAc/MeOH 8:2).
After 5 h, no amine starting material was detected. The
mixture was evaporated to dryness under vacuum. The
resulting residue was dissolved in 100 mL EtOAc. The
organic solution was washed with water (2ꢂ50 mL) and
brine (50 mL), dried over Na2SO4 and filtered. The
solvent was evaporated at reduced pressure to give the
raw product, which was purified by flash chromato-
graphy (n-hexane/EtOAc 6:4). 8.0 g of 6 as white waxy
solid were obtained (yield 83%). TLC (n-hexane/EtOAc
3.1.5. (4S)-1-[(S)-3-Mercapto-2-methylpropanoyl]-4-phe-
nylthio-L-proline (Zofenoprilat 2). g (2.3 mmol) (4S)-1-
[(S)-3-benzoylthio-2-methylpropanoyl]-4-phenylthio-l-
proline methyl ester [(S,S,S)-7] was dissolved in 10 mL
of methanol. 15 mL (15 mmol) 1 N NaOH was added.
The resulting mixture was heated to 50 ꢁC. The reaction
was monitored bothby TLC ( n-hexane/EtOAc 1:1, to
detect the starting material) and HPLC. After 10 min,
the reaction was complete. Methanol was removed
under vacuum, 2.5 N HCl was added to pH 2 and the
aqueous solution was extracted withCH Cl2 (3ꢂ5 mL).
2
1
1:1): Rf 0.35. HPLC: tR 5.1 min. H NMR (CDCl3): d
The combined organic extracts were washed with brine
(5 mL) dried over Na2SO4 and filtered. The solvent was
removed. 935 mg of product 2 (mixture withequimolar
amount of benzoic acid) were obtained as white powder
(yield 91%). HPLC: tR 4.2 min (benzoic acid tR 3.6
7.45–7.25 (m, 5H, aromatic H); 5.35 (s, 1H, HCH¼),
5.30 (s, 1H, HCH¼), 4.57 (dd, J=8.6, 8.6 Hz, 1H,
CHCO2CH3); 4.03 (m, 1H, HCHN); 3.74 (s, 3H,
CH3O); 3.55 (m, 2H, CHSPh, HCHN); 2.68 (m, 1H,

M. Matarrese et al. / Bioorg. Med. Chem. 12 (2004) 603–611
609
min). A sample (30 mg) was dissolved in MeOH/0.05 M
KH2PO4 pH 3, 50:50 v/v (1 mL) and purified by semi-
preparative HPLC (2, tR 11.4 min; benzoic acid, tR 5.1
min.). The fraction containing 2 was collected in water
(20 mL). The product was recovered by SPE on Sep-Pak
tC18 plus cartridge (Waters, 400 mg). This was washed
withwater (10 mL), then 2 was eluted withMeOH (2
mL). The resulting solution was dried under vacuum to
give 20 mg withno detectable benzoic acid. 1H NMR
(CDCl3): d 7.5–7.3 (m, 5H, aromatic H); 4.50 (dd,
J=7.8, 7.8 Hz, 1H, CHCO2H), 4.37 (dd, J=10.4, 7.0
Hz, 1H, HCHN); 3.89 (dddd, J=8.7, 8.7, 7.0, 5.2 Hz,
1H, CHSPh); 3.47 (dd, J=10.4, 8.7 Hz, 1H, HCHN);
2.9–2.6 (m, 3H, CH2SH, CHCH3); 2.47 (ddd, J=13.0,
7.8, 5.2 Hz, 1H, HCHCHCO2H); 1.98 (ddd, J=13.0,
8.7, 7.8 Hz, 1H, HCHCHCO2H); 1.14 (d, J=6.6 Hz,
3H, CH3CH). MS (FAB) m/z 326 [MH]+. Elemental
analysis: C15H19NO3S2 (325.44): calcd C 55.36, H 5.88,
N 4.30; found C 55.32, H 5.81, N 4.39. [a]2D0 (as sodium
salt)=ꢀ63ꢁ (c 1, 0.05 N HCl in MeOH), mp 84–89 ꢁC
(as sodium salt).
HCHCHCO2Na); 1.11 (d, J=6.7 Hz, 3H, CH3). 13C
NMR (CD3OD) of the major rotamer: d 176.6 (C);
167.9 (C); 136.1 (C); 132.7 (2 CH), 130.2 (2 CH); 128.3
(CH); 63.5 (CH); 54.1 (CH2); 43.9 (CH); 43.4 (CH2);
39.5 (CH2); 38.7 (CH); 17.4 (CH3).
3.1.7. One-pot synthesis of (4S)-1-[(S)-3-mercapto-2-me-
thylpropanoyl]-4-phenylthio-L-proline, 2.
1
mL of
magnesium bromide methacrylate solution (¼0.5 M,
0.50 mmol) (4) was evaporated in 2 min withN 2 stream.
To the resulting residue a solution of 150 mg (0.62
mmol) of (S)-4-(phenylthio)-l-proline methyl ester (5)
and 130 mL (0.74 mmol) of DIPEA in 500 mL of DMF
was added. Then 210 mg (0.55 mmol) of HATU was
poured into the mixture. The suspension was heated to
55 ꢁC under stirring. After 5 min a clear solution of the
methacrylamide 6 was obtained. 500 mL (4.0 mmol) of
thiobenzoic acid was added. The resulting solution was
heated to 95 ꢁC for 10 min to afford an equimolar
mixture of the two diastereomers 7. An amount of this
solution (500 mL, containing about 5 mg of pure 7 as
assessed by HPLC analysis) was diluted with1 mL
acqueous 10% NaHCO3 and set aside for 30 s while a
white solid (dibenzoyl disulfide) precipitated. The
solution was separated by suction witha syringe and
injected into a Sep-Pak tC18 Plus cartridge (Waters, 400
mg) previously activated by washing with methanol (5
mL), water (5 mL) and 10% NaHCO3 (5 mL). Then the
cartridge was flushed with 10% NaHCO3 (5 mL) and
water (5 mL) to remove thiobenzoic acid. The products
7 were recovered by washing the cartridge with 1,4-
dioxane (1.8 mL).
3.1.6. (4S)-1-[(R)-3-Mercapto-2-methylpropanoyl]-4-phe-
nylthio-L-proline (R,S,S of 2). Obtained from (4S)-1-
[(R)-3-benzoylthio-2-methylpropanoyl]-4-phenylthio-l-
proline methyl ester [(R,S,S)-13] as described for
Zofenoprilat (2). HPLC: tR 4.2 min (benzoic acid tR 3.6
min). 1H NMR (CDCl3): d 7.6–7.3 (m, 5H, aromatic H);
4.45 (dd, J=7.8, 7.8 Hz, 1H, CHCO2H), 4.08 (dd,
J=10.4, 7.0 Hz, 1H, HCHN); 3.89 (m, 1H, CHSPh);
3.65 (dd, J=10.4, 8.7 Hz, 1H, HCHN); 3.0–2.6 (m, 3H,
CH2SH, CHCH3); 2.35 (m, 1H, HCHCHCO2H); 1.95
(m, 1H, HCHCHCO2H); 1.12 (d, J=6.6 Hz, 3H,
CH3CH). MS (EI) m/z 325 [M]+. Elemental analysis:
C15H19NO3S2 (325.44): calcd C 55.36, H 5.88, N 4.30;
found C 55.29, H 5.79, N 4.38.
The organic solution of 7 was poured into 1 N NaOH
(1.3 mL). The resulting mixture was heated to 50 ꢁC for
5 min. to give 2, then cooled to rt and acidified to pH 2
by adding 4 M H3PO4 (400 mL). The solution was
diluted with10 mL of 0.1 M KH 2PO4 (pH 7.5) and
transferred into a new Sep-Pak cartridge. This was
washed with abundant water (10 mL) and the product
was eluted withMeOH (0.5 mL). The methanolic solution
was evaporated under vacuum to give 2.3 mg of 2
[equimolar mixture withits ( R,S,S) diastereomer]
(overall yield 46%). HPLC tR 4.2 min. The overall process
took less than 40 min. MS (FAB) m/z 326 [MH]+.
Compound 2 was converted to its sodium salt by the
following procedure: it was dissolved in a mixture of
methanol–water and the pH was adjusted to 7 with
0.5 N NaOH. Methanol was evaporated under vacuum
and the resulting solution was lyophilised to afford a
white powder. [a]2D0=ꢀ25ꢁ (c 1, 0.05 N HCl in MeOH),
mp 94–100 ꢁC. 1H NMR (CD3OD): presence of two
rotamers: major rotamer: d 7.5–7.2 (m, 5H, aromatic
H); 4.75 (broad, 1H, SH); 4.52 (dd, J=8.3, 7.9 Hz, 1H
CHCO2Na); 4.22 (ddd, J=11.8, 7.0, 1.4 Hz, 1H,
HCHN); 3.62 (dddd, J=10.1, 9.5, 7.3, 7.0 Hz, 1H,
CHSPh); 3.32 (dd, J=11.8, 9.6 Hz, 1H, HCHN); 2.76
(dddd, J=12.9, 7.9, 7.3, 1.4 Hz, 1H, HCHCHCO2Na);
2.73 (ddq, J=9.4, 5.2, 6.5 Hz, 1H, CHCH3); 2.65 (dd,
J=13.1, 9.4 Hz, 1H, HCHSH); 2.47 (dd, J=13.1, 5.2
Hz, 1H, HCHSH); 2.07 (ddd, J=12.9, 10.1, 8.3 Hz, 1H,
HCHCHCO2Na); 1.14 (d, J=6.5 Hz, 3H, CH3); minor
rotamer: 7.5–7.2 (m, 5H, aromatic H); 4.75 (broad, 1H,
SH); 4.30 (dd, J=8.9, 8.0 Hz, 1H, CHCO2Na); 4.03
(ddd, J=10.2, 6.6, 1.4 Hz, 1H, HCHN); 3.72 (dddd,
J=10.7, 9.5, 7.2, 6.6 Hz, 1H, CHSPh); 3.62 (dd,
J=10.2, 9.5 Hz, 1H, HCHN); 2.87 (dd, J=13.2, 7.1 Hz,
1H, HCHSH); 2.78 (ddq, J=7.1, 6.2, 6.7 Hz, 1H,
CHCH3); 2.72 (dddd, J=12.7, 8.0, 7.2, 1.4 Hz, 1H,
HCHCHCO2Na); 2.36 (dd, J=13.2, 6.2 Hz, 1H,
HCHSH); 1.91 (ddd, J=12.7, 10.7, 8.9 Hz, 1H,
3.2. Radiosynthesis
3.2.1. [11C]-(4S)-1-(2-methylpropenoyl)-4-phenylthio-L-
proline methyl ester, [11C]6. The procedures described
by McCarron12 for the preparation of [carbonyl-11C]cy-
clohexanecarbonyl chloride were applied in the radio-
synthesis 2-methyl-[1-11C]acryloyl chloride (8) using
isopropenylmagnesium bromide as Grignard reagent.
Isopropenylmagnesium bromide (3) (1–2 mL, 0.5 M in
THF) was passed at room temperature through a coil of
fluorinated polypropylene tube (FEP) (80 cm length; i.d.
0.50 mm; o.d. 1/160) previously flushed with argon, to
leave a thin film on the inner tube surface. [11C]Carbon
dioxide was then dispensed at room temperature from
the stainless steel loop of the Nuclear Interface module
through the FEP tube in a stream of argon of 2 mL/min
for 1.30 min to make the on-line formation of [11C]4.

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M. Matarrese et al. / Bioorg. Med. Chem. 12 (2004) 603–611
Thionyl chloride (3 mL, 41 mmol) in THF (400 mL) was
then washed through the tube into a reactor vessel
containing (S)-4-phenylthio-l-proline methyl ester (5)
(3.5 mg, 15 mmol) and DIPEA (25 mL, 144 mmol) dissolved
in 50 mL of THF, at 50 ꢁC. After raising the temperature
to 75 ꢁC, the reaction mixture reacted for 5 min and
[11C]6 was obtained witha radiochemical yield of 55%.
used for the [11C]acyl chloride formation. After semi-
preparative HPLC purification, the fraction corre-
sponding to the desired 11C-product was collected in 40
mL sterile water and the product was recovered by SPE
on pre-activated Sep-Pak tC18 cartridge by elution with
1 mL of methanol. The non-radioactive material with the
11C-labelled product was ensured by the strong UV peak
at the retention time of the target compound. The
peak was collected and its identity was confirmed by
mass spectrometry.
Quality control of 8 and [11C]6 were performed by an
analytical HPLC (Shandon Hypersil BDS C18, 5 m,
250ꢂ4.6 mm; CH3CN/50 mM NaH2PO4 buffer pH 3,
50/50 v/v; 1 mL/min).
[11C]7: The APCI mass spectrum displayed an intense
ion at m/z 444 corresponding to the protonated mole-
cular ion [M+H]+. Identity of the compound was
ensured by its skimmer-CID spectrum (+25V), which
shows ions at m/z 412 (M-CH3O), ions at m/z
3.2.2. [11C]-(4S)-1-[(R,S)-3-benzoylthio-2-methylpropa-
noyl]-4-phenylthio-L-proline methyl ester, [11C]7. Thio-
benzoic acid (150 mL, 1.27 mmol, diluted with50 mL
THF) was added at room temperature to the cooled
reaction mixture containing [11C]6. The mixture was
heated (95 ꢁC, 10 min), diluted with1.5 mL of mobile
phase and injected onto a semi-preparative reversed
phase column [Shandon Hypersil BDS C-18 (250ꢂ10
mm, 5 mm)]. Purification of the intermediate [11C]7 was
accomplished using CH3CN/50 mM NaH2PO4 buffer
pH 7.2, 55/45 v/v plus 0.2% TFA as mobile phase at 8
mL/min flow rate. Quality control was performed using
a Shandon Hypersil BDS C-18 (250ꢂ4.6 mm, 5 mm)
column withCH 3CN/50 mM NaH2PO4 buffer pH 3, 65/
35 v/v at 1.3 mL/min flow rate.
384
(MꢀCOOCH3)
and
at
m/z
207
[MꢀPheCOSCH2CH(CH3)CO].
[11C]2: the LC-APCI chromatogram of the reaction
mixture showed three main peaks at tr 3.34, 6.65 and of
9.08 min. The Mass spectrum of the first prominent
peak displayed an intense ion at m/z 326 corresponding
to the protonated molecular ion [M+H]+ of Zofeno-
prilat while the spectra of the other two peaks showed
ions at m/z 340 and at 354. The former compound with
Mr 339 could be the methyl ester of [11C]2 originating
from its incomplete hydrolysis, while the latter with Mr
353 could be either the methyl thioether and carboxy-
methyl ester or the ethyl ester of [11C]2 originating from
a transesterification reaction withethanol.
3.2.3.
[11C]-(4S)-1-[(R,S)-3-mercapto-2-methylpropa-
noyl]-4-phenylthio-L-proline, [11C]2. The effluent from
the column corresponding to [11C]7 was collected in 40
mL of sterile water and recovered by SPE on a Sep-Pak
tC18 plus cartridge (Millipore) pre-activated with5 mL
of ethanol followed by 20 mL of water. The Sep-Pak
was washed with water (10 mL) before eluting [11C]7
with ethanol (1 mL) into a sealed vial flushed by argon.
Hydrolysis proceeded by the addition of 1.2 mL of a
solution of 1 N NaOH and heating at 60 ꢁC for 8 min
and was quenched by addition of a solution of 1 N HCl
(1.3 mL). After cleaning the necessary parts of the
module, the solution was transferred manually with a
syringe containing 10 mL of a solution of NaH2PO4
(0.1 M; pH 7.3) into a new Sep-Pak tC18 cartridge
(Waters). The Sep-Pak was washed with water (10 mL)
and [11C]2 was eluted withethanol (0.5 mL) under stream
of argon into a vial containing 9.5 mL of saline solution,
which was sterilised through a sterile 0.22 mm filter (Gel-
man Acrodisc). The pH of the final solution was neutral.
Addition of authentic standard to the mixture before
HPLC analysis caused the expected increase in the UV
peak area coeluting withthe radioactive material.
3.3. PET study
A 46 year old male subject witha diagnosis of moderate
hypertension (95/150 mmHg) was recruited in the
nephrology department of the San Raffaele Hospital.
Before his participation, the patient signed a written
informed consent according to the study protocol
previously approved by the Ethical Committee of the
Scientific Institute H San Raffaele. A complete physical
examination, blood and urine laboratory routine tests
and ECG were performed at the time of recruitment.
The patient suspended ACE inhibitor intake 24 h before
the PET scan (5 drug half-lives). The PET study was
performed withan eighteen-ring (GE Advance; General
Electric Medical System, Milwaukee, WI, USA)
tomograph.
The final solution of known volume of [11C]2 and its
(R,S,S) diasteromer was assayed for total radioactivity
and a 20-mL aliquot was applied to an analytical HPLC
Shandon Hypersil BDS C-18 (250ꢂ4.6 mm, 5 mm) col-
umn using CH3CN/50 mM NaH2PO4 buffer pH3, 50/50
v/v as mobile phase at 1 mL/min flow rate. The amount
of carrier was calculated from the UV absorbance peak
area by means of the external standard calibration plot.
The subject was positioned on the scanner bed with
arms immobilized and the thorax centred in the scanner
ring. Before positioning, two catheters were inserted
bi-laterally in brachial veins (one for for tracer injection
and one for venous blood sampling). One 15-min
transmission scan was carried out withan external 68Ge
ring source. At the end of the transmission scan,
the subject received an intravenous injection of
approximately 4 mCi (148 MBq) of [11C]Zofenoprilat in
5 mL of saline. After tracer injection, simultaneous
3.2.4. Carrier-added radiosyntheses for identification of
[11C]7, [11C]2. The radiosynthesis was carried out as
described above for the no-carried-added preparation
withthe addition of cold CO
carrier to the [11C]CO2
2

M. Matarrese et al. / Bioorg. Med. Chem. 12 (2004) 603–611
611
acquisition of data from 35 transaxial slices (slice thick-
ness 4.25 mm; axial field of view 15.5 cm) was acquired
according to the following schedule: six scans of 0.5 min
each, two scans of 1 min each, two scans of 2.5 min
each, two scans of 5 min each, two scans of 10 min and
finally a 20-min scan (total acquisition time: 1 h). For
the evaluation of [11C]Zofenoprilat kinetics in blood,
serial samples (1 mL each) were collected, from the
brachial vein contra-lateral to the tracer injection site at
0.1, 1, 1.5, 3, 5, 11, 30 and 60 min after injection. Radio-
activity concentration in the blood and plasma was
measured by a gamma counter (LKB 1282 Compu-
gamma CS) and expressed as KBq/mL or percentage of
injected dose per mL of blood or plasma (%I.D./mL).
Todde, S.; Carla Gilardi, M.; Grimaldi, A.; Fazio, F.
Pharmacol. Res. 2001, 44, 151.
3. Subissi, A.; Evangelista, S.; Giachetti, A. Cardiovasc.
Drug. Rev. 1999, 17, 115.
4. Hwang, D. R.; Eckelman, W. C.; Mathias, C. J.; Petrillo,
E. W. J.; Lloyd, J.; Welch, M. J. J. Nucl. Med. 1991, 32,
1730.
5. Brilla, C. G. Cardiovasc. Res. 2000, 46, 324.
6. Ferrari, R.; Cargnoni, A.; Curello, S.; Ceconi, C.; Boraso,
A.; Visioli, O. J. Cardiovasc. Pharmacol. 1992, 20, 694.
7. Liu, X.; Engelman, R. M.; Rousou, J. A.; Cordis, G. A.;
Das, D. K. Cardiovasc. Drugs Ther. 1992, 6, 437.
8. Przyklenk, K.; Kloner, R. A. Am. Heart. J. 1991, 121,
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9. Sargent, C. A.; Sleph, P. G.; Dzwonczyk, S.; Smith, M. A.;
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Isot. 2002, 57, 675.
3.4. Data analysis
Irregular regions of interest (ROIs) were defined on the
left myocardial wall, left ventricular cavity, lungs, liver,
gall bladder and kidney (cortical aspect). ROIs were
drawn on the temporal frame with the maximum uptake
and then copied on the whole dynamic series to calculate
the time–activity curve of radioactivity concentration in
the various tissues examined. The external contour of
myocardial wall was defined on the transmission images.
14. Evangelista, S.; Criscuoli, M.; Manzini, S. Recent Res.
Drug Metab. Dispos. 2002, 1, 17.
All data were corrected for the radioactive decay of
carbon-11 and expressed in KBq/mL or percentage of the
injected dose per mL of tissue (%I.D./mL). Radioactivity
concentrations may be transformed in pmol/mL using the
specific radioactivity of the tracer calculated at the time of
injection.
15. Cusham, D. W.; Wang, F. L.; Fung, W. C.; Harvey,
C. M.; De Forrest, J. M. Am. J. Hypert. 1989, 2, 294.
16. Danser, A. H. J.; Saris, J. J.; Schuijlt, M. P.; van Kats,
J. P. Cardiovasc. Res. 1999, 44, 252.
17. Walmor, C.; De Mello, A. H.; Danser, A. H. J. Hyper-
tension 2000, 35, 1183.
18. Marzo, A.; Dal Bo, L.; Mazzucchelli, P.; Ceppi Monti,
N.; Crivelli, F.; Ismaili, S.; Giusti, A.; Uhr, M. R.
Arzneim-Forsch/Drug Res. 2002, 52, 233.
19. Pouchert, C.J.; Campbell, J.R.The Aldrich Library of
NMR Spectra; AldrichCo.; London, 1974.
References and notes
1. Lucignani, G.; Moresco, R. M.; Fazio, F. Cerebrovasc.
Brain Metab. Rev. 1989, 1, 271.
2. Moresco, R. M.; Messa, C.; Lucignani, G.; Rizzo, G. G.;
20. Smith, E. M.; Swiss, G. F.; Neustadt, B. R.; Gold, E. H.;
Sommer, J. A.; Brown, A. D.; Chiu, P. J. S.; Moran, R.;
Sybertz, E. J.; Baum, T. J. Med. Chem. 1988, 31, 875.