Carbon-11 labelling of an N-sulfonylamino acid derivative: A potential tracer for MMP-2 and MMP-9 imaging

Article abstract of DOI:10.1002/jlcr.695

Matrix metalloproteinases (MMPs) are a family of proteinases that play an important role in cancer. Non invasive imaging of MMPs would allow the evaluation of MMP activity in cancer and the assessment of the response of MMP inhibitor based therapies. In t

Full text of DOI:10.1002/jlcr.695

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 539–553.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.695
Research Article
Carbon-11 labelling of an N-sulfonylamino acid
derivative: a potential tracer for MMP-2
and MMP-9 imaging
.
Bertrand Kuhnast*, Claudia Bodenstein, Hans Jurgen Wester, and
Wolfang Weber
Department of Nuclear Medicine, Klinikum rechts der Isar,
.
.
Technische Universitat Munchen, 81675 Munich, Germany
Summary
Matrix metalloproteinases (MMPs) are
a family of proteinases that
play an important role in cancer. Non invasive imaging of MMPs
would allow the evaluation of MMP activity in cancer and the assessment
of the response of MMP inhibitor based therapies. In this paper, we
investigated the synthesis and labelling by methylation with [11C]CH3I
of an N-sulfonylamino acid derivative, the (2R)-3-methyl-2-[[4-[(4-methox-
ybenzoyl)amino]benzenesulfonyl]amino] butanoic acid, a selective and high
potent MMP-2 and MMP-9 inhibitor, for cancer imaging with positron
emission tomography. Labelling of (2R)-3-methyl-2-[[4-[(4-hydroxybenzoyl)
amino]benzenesulfonyl] amino] butanoic acid was carried out in a radio-
chemical yield of 50%–60% within 40 min with a specific activity of 11–26
GBq/mmol (EOS). In vitro inhibitory activity studies, biodistribution and in
vivo serum stability in normal mice are also described. Copyright # 2003 John
Wiley & Sons, Ltd.
Key Words: matrix metalloproteinases inhibitors; carbon-11; positron emis-
sion tomography
! !
*Correspondence to: B. Kuhnast, Service Hospitalier Frederic Joliot /CEA/DSV, 4 place du
! !
General Leclerc, 91401 ORSAY Cedex, France. E-mail: kuhnast@shfj.cea.fr
Copyright # 2003 John Wiley & Sons, Ltd.
Received 30 October 2002
Revised 13 December 2002
Accepted 6 January 2003

540
B. KUHNAST ET AL.
Introduction
Matrix metalloproteinases are a family of zinc-dependent endopepti-
dases. According to the structure of the enzymes and their substrate
specificity, the 20 members described so far are classified into 5 groups.
They are all collectively capable of degrading the components of the
extracellular matrix to ensure its remodelling and repairing.1–3 Their
activity is tightly controlled by the endogenous co-expression of tissue
inhibitors.4 The implication of MMPs in cancer, and particularly of
MMP-2 and MMP-9, also called gelatinases, has been demonstrated by
a large number of preclinical and clinical studies.5–8 The abnormal
degradation of the basement membrane due to the over-expression of
gelatinases: (i) is necessary for angiogenesis; (ii) promotes tumour
growth by increasing the bioavailability of the growth factors released
during the degradation and (iii) facilitates the invasion of tumour cells
through the connective tissue and blood vessel walls resulting in the
establishment of metastases.9–14 Therefore gelatinases are potential
targets for therapeutic intervention in cancer as well as for imaging of
malignant tumours.
Phage display,15 substrate based or structure based design16,17 and
combinatorial chemistry18–20 have been extensively used to design and
develop MMP inhibitors (MMPIs). Today, about half a dozen MMPIs
are currently under clinical trials in patients.21–24 The opportunity to
label MMPIs with positron emitters (carbon-11 or fluorine-18) would
offer a tool to image MMP activity in cancer and to assess the response
to an MMP inhibitor based therapy,25,26 with positron emission
tomography.27,28 Among the different chemical classes of MMPIs, a
family of N-sulfonylamino acid derivatives29–34 has been described as
selective and orally active gelatinases inhibitors (general structure,
Figure 1). Some members of this MMPI family display high selectivity
and nanomolar IC50 for MMP-2 and MMP-9. Furthermore, the
versatility of the para-substituent of the benzoylaminobenzene side
chain (Figure 1, substituent R2) provides a straightforward strategy for
isotopic labelling via carbon-11 methylation without the need for
protected precursors.
Fei et al. 27,28 recently described the synthesis of 6 analogues of CGS
27023A,29ꢀ34 an MMPI from Ciba Geygy/Novartis under clinical trials.
CGS 27023A is a broad spectrum MMPI1–3 belonging to the family of
the N-sulfonylamino acid derivatives.29 These compounds were
successfully labelled with carbon-11. Furthermore, they demonstrated
Copyright # 2003 John Wiley & Sons, Ltd.
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CARBON-11 LABELLING OF AN
541
R
1
O
S
HOOC
N
H
X
R
2
O
R = hydrophobic amino acid side chain
1
R = SMe, OMe, NO , H....
2
2
X = tetrazolyl or amide
Figure 1. General structure of the N-sulfonylamino acid derivatives
that the analogues exhibited the same inhibitory activity as the parent
compound, CGS 27023A, at a concentration of 200 nM. However, no
detailed data on the affinity of these compounds as well as in vivo
stability and biodistribution are available.
In this study, we prepared the (2R)-3-methyl-2-[[4-[(4-methoxyben-
zoyl)amino] benzenesulfonyl]amino] butanoic acid, a member of the N-
sulfonyl amino acid derivative family and tested its MMP-2 and MMP-
9 inhibitory activity in vitro. Starting from the corresponding
4-hydroxybenzoyl analogue, the N-sulfonylamino acid derivative was
labelled with carbon-11 by methylation with [11C]CH3I. Biodistribution
and serum stability studies of the tracer were evaluated in vivo in normal
mice.
Results and discussion
Chemistry
There are at least 3 requirements for a molecule to be an effective MMP
inhibitor: (i) the presence of a functional group capable of participating
in the chelation of the zinc atom in the active site of the enzyme
(hydroxamic, carboxylic, sulfhydryl groups) is required; (ii) at least one
functional group providing hydrogen bonds to the enzyme backbone
and (iii) one or more side chains fitting with the subsites of the active
site.16,17
The N-sulfonylamino acid derivatives meet most of these criteria. The
carboxylic group is able to participate in the chelation of the zinc atom,
the presence of the sulfonyl group provides the hydrogen bonds35,36 and
the benzoyl aminobenzene side chain makes hydrophobic contacts with
the S’1 subsite of the enzyme.37–40 Furthermore, analogues of
compound 7 have demonstrated selective gelatinases inhibition in vitro
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542
B. KUHNAST ET AL.
and anti-tumour activity in mice after oral administration.29 The
synthesis of 7 was conducted according to the preparation of other bi-
phenyl-N-sulfonylamino acid derivatives described by Tamura et al.29
Starting from the protected commercially available d-valine, it was
possible to synthesize 7 in 4 steps with an overall yield of 52%
(Scheme 1). d-valine 1 was first reacted with 4-nitrobenzene sulfo-
nylchloride 2. The nitro function was then reduced to the corresponding
amine 4 by catalytic hydrogenation. After purification, compound 4 was
acylated either with 4-methoxybenzoyl chloride 5 to provide the cold
reference 7 after hydrolysis of 6 with sodium hydroxide (Scheme 1) or
with acetoxy benzoylchloride 9 to provide the labelling precursor
(Scheme 2). To demonstrate MMP inhibitory activity, in vitro gelatin
degradation assay were carried out with 7 as MMP-2 and MMP-9
inhibitor.
The preparation of the labelling precursor (Scheme 2) by activation of
the 4-acetoxybenzoic acid 8 with thionyl chloride takes advantage of a
simple purification step of 9 by evaporation of non-reacted thionyl
chloride, by-products and solvents before reaction with the amine 4.
The double ester 10 was hydrolysed in one step with sodium hydroxide
to provide the precursor 11. Compared to this two step method, trials to
produce 10 in one step starting from 4 and 8 using the standard
carbodiimide coupling reagents for peptide synthesis DCC (N,N0-
dicyclohexylcarbodiimide) and WSC (1-ethyl-3-(30-dimethylaminopro-
pyl) carbodiimide) resulted in low transformation yields (less than
O
S
O
S
H , cat
2
CH Cl
2
2
+
Cl
NO
2
MeOOC
N
H
NO
2
MeOOC
NH
2
NMM
RT, 2 hrs
80%
MeOH/EtOAc
RT, 4 hrs
90%
O
O
3
1
2
O
OMe
Cl
O
S
O
S
5
MeOOC
N
H
NH
MeOOC
N
H
NH CO
OMe
2
CH Cl , NMM
2
2
O
H
O
RT, 16 hrs
75%
6
4
O
S
NaOH
HOOC
N
H
NH CO
OMe
MeOH/THF
60 ˚C, 4 hrs
95%
O
7
Scheme 1. Synthesis of the cold reference 7, (2R)-3-methyl-2-[[4-[(4-methoxy-
benzoyl)amino]benzenesulfonyl]amino] butanoic acid
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CARBON-11 LABELLING OF AN
543
O
O
SOCl /CH Cl
2
2
2
O
RT, 16hrs
O
HO
CH
3
8
OAc
O
S
Cl
9
O
S
MeOOC
N
H
NH
2
MeOOC
N
H
NH CO
OAc
CH Cl , NMM
RT, 16 hrs
60%
2
2
O
O
10
4
O
NaOH
HOOC
N
H
S
NH CO
OH
MeOH/THF
60 ˚C, 4 hrs
100%
O
11
Scheme 2. Synthesis of compound 11, (2R)-3-methyl-2-[[4-[(4-hydroxy-
benzoyl)amino]benzenesulfonyl]amino] butanoic acid
20%). Alternative methods to prepare the precursor 11, such as the
transformation of the 4-methoxybenzene group of 6 (Scheme 1) into the
corresponding 4-hydroxynezene with EtSNa41 or l-selectride,42 fol-
lowed by the hydrolysis of the carboxylate, were not investigated but
seem to be promising routes .
Radiochemistry
The synthesis of [11C]7 was performed by carbon-11 methylation of the
precursor 11 with [11C]CH3I in DMF in the presence of NaH for 5 min
at 808C (Scheme 3). The product [11C]7 was purified by RP-HPLC. The
total synthesis time (from end of bombardment) was about 40 min
including HPLC purification and formulation. The decay corrected
yield for the methylation, determined by HPLC, was 50–60%. Radio-
chemical purity was >98% and the specific radioactivity of [11C]7 was
11–26 GBq/mmol EOS (0.3–0.7 Ci/mmol).
O
S
O
S
11
[
C]CH I
3
11
HOOC
N
H
NH CO
O
CH
3
HOOC
N
H
NH CO
OH
DMF, NaH
80˚C, 5 min
O
O
11
[
C]7
11
Scheme 3. Synthesis of (2R)-3-methyl-2-[[4-[(4-[11C]methoxybenzoyl)amino]ben-
zenesulfonyl]amino] butanoic acid
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544
B. KUHNAST ET AL.
In vitro gelatin degradation assay
The phage display selected cyclic decapeptide (CTTHWGFTLC),
containing the amino acids sequence HWGF responsible for its selective
MMP-2 and MMP-9 inhibitory activity (12),15 the non selective
metalloproteinases inhibitor 1,10 phenanthroline (13) and 7 were
evaluated for their inhibitory activity towards MMP-2 and MMP-9 in
a gelatin degradation assay. The compounds 7, 12 and 13 were first
dissolved in DMF before dilution in the assay buffer. We tested that
the presence of DMF had no influence on the degradation of the
gelatin by the purified activated MMP-2 and MMP-9. Among
these three compounds, 7 displayed the highest inhibitory activity
(Figure 2a, b). We determined an IC50 for 7 of 110 nM for MMP-2
and 200 nM for MMP-9. Peptide 12 displayed an inhibitory activity
of 10 mM for MMP-2 but a more than 10 fold lower inhibitory
activity for MMP-9 (IC50>100 mM). Compound 13 showed a typical
inhibitory profile for a non specific and low potent metalloproteinases
inhibitor.
Inhibitory activity towards MMP-2
120%
100%
80%
Cpd 13
60%
40%
20%
0%
Cpd 7
Cpd 12
0.001
0.01
0.1
1
10
100
1000
(a)
Inhibitor concentration [µM]
Inhibitory activity towards MMP-9
120%
100%
80%
60%
40%
20%
0%
Cpd 7
Cpd 13
Cpd 12
0.001
0.01
0.1
1
10
100
1000
(b)
Inhibitor concentration [µM]
Figure 2. (a) and (b) Inhibitory activity of compounds 7, 12 and 13 towards
MMP-2 and MMP-9. The results are the mean of 2 to 3 independent experiments
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CARBON-11 LABELLING OF AN
545
5%
4%
3%
2%
1%
0%
Organs
Figure 3. Biodistribution in normal mice (n=3) of [11C]7. Radioactivity
concentration was expressed as a percentage of injected dose per gram of tissue
(% ID/g) and reported as the mean 6 standard deviation (SD)
Biodistribution and Serum stability.
In the biodistribution study, 30 min post injection (Figure 3), there was
only a moderate uptake of [11C]7 in the liver and kidneys
(2.15 ꢁ 0.45%ID/g and 3.29 ꢁ 1.1%ID/g, respectively). The corre-
sponding concentration of radioactivity in the plasma was
0.93 ꢁ 0.17%ID/g. In the heart, lungs, spleen, muscles and brain, the
uptake of radioactivity was lower than 1%ID/g.
For metabolite analyses, blood was collected and centrifuged. To
evaluate the partition of the [11C]7 between blood cells and serum, a
blood pellet and aliquots of serum were counted showing that 75%
( ꢁ 5%) of the radioactivity was bound to the blood cells. The serum
was deproteinized by acetonitrile precipitation. Less than 10% of the
radioactivity precipitates with the proteins. No metabolites were
detectable by HPLC analyses of the extractable fraction of the
radioactivity. All the radioactivity was recovered in one peak co-eluting
with the cold reference 7. The compound was found to be rapidly
excreted and stable towards degradation up to 30 minutes post injection
and thus seems to be suitable for PET imaging of gelatinases activity.
The evaluation of the [11C]7 derivate in Lewis lung tumour bearing mice
is in progress.
Conclusion
(2R)-3-methyl-2-[[4-[(4-methoxybenzoyl)amino]benzenesulfonyl]amino]
butanoic acid (7) and the corresponding labelled hydroxy precursor
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B. KUHNAST ET AL.
(2R)-3-methyl-2-[[4-[(4-hydroxybenzoyl)amino] benzenesulfonyl]amino]
butanoic (11) have been synthesized. [11C]7 was prepared by methyla-
tion with [11C]CH3I in high yields. The time required for labelling and
purification of the tracer is compatible with the half life of carbon-11. In
the gelatine degradation assay (2R)-3-methyl-2-[[4-[(4-methoxybenz-
oyl)amino]benzenesulfonyl]amino] butanoic acid (7) exhibited a strong
inhibitory effectiveness for gelatinases. Furthermore early in vivo studies
demonstrated favourable pharmacokinetics for MMP-2 and MMP-9
activity monitoring and stability towards enzymatic degradation within
the first 30 min after injection. Further in vivo evaluations in tumour
bearing mice models are currently in progress.
Experimental
General
Chemicals and enzymes were purchased from Aldrich, Sigma, Fluka
(Neu-Ulm, Germany) or Chemicon (Hofheim, Germany) and were used
without further purification.
Analytical methods
Thin Layer Chromatography (TLC) was run on precoated plates of
silica gel 60F54 (Merck, Darmstadt, Germany). The compounds were
localized at 254 nm using a UV lamp. Flash chromatography was
conducted on silica 63–200 mm (Merck, Darmstadt, Germany) under
compressed air. Semi-preparative HPLC was performed using a S1020
pump (Sykam, Gilching, Germany) equipped with a detector to monitor
radioactivity. Analytical HPLC was performed using a S1121 Solvent
Delivery System (Sykam, Gilching, Germany), a UV detector and an
EG&G detector to monitor radioactivity. Metabolite analyses were
performed on an HPLC system composed of a S1121 Solvent Delivery
System (Sykam, Gilching, Germany), a UV detector and a Flow
Scintillation Analyzer 500TR Series (Packard, Germany) equipped with
a PET cell for radioactivity monitoring. For HPLC, the following
columns were used : for semi-preparative HPLC : Multospher 100 RP18
5 mm 250 ꢂ 10 mm (CS Chromatographie Service GmBH, Germany),
for analytical HPLC : Phenomemex C18 150 ꢂ 4.60 mm (CS Chroma-
tographie Service GmBH, Germany), for metabolite analyses: Nucleosil
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CARBON-11 LABELLING OF AN
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100C18 5 mm 150 ꢂ 10 mm (CS Chromatographie Service GmBH,
Germany). Mass spectra were recorded on the LC-MS system LCQ
from Finnigan (Bremen, Germany) using the Hewlett Packard series
1100 HPLC system. NMR spectra were recorded on an Bruker AMX
250 MHz instrument using the hydrogenated residue of the deuterated
1
solvents as internal standards for H NMR. The chemical shifts were
reported in ppm.
Chemistry
Synthesis of (2R)-3-methyl-2-[(4-nitrobenzenesulfonyl)amino] butanoic
acid methyl ester (3). To a solution of D-valine methyl ester (1) (2 g,
12 mmol) in CH2Cl2 (120 ml) were added 2.6 ml (24 mmol) of N-
methylmorpholine (NMM) and 3.18 g (14.3 mmol) of 4-nitrobenzensul-
fonyl chloride (2) at 08C. The mixture was allowed to warm to room
temperature and stirred for 2 h. The mixture was twice washed with
100 ml of 1 N HCl solution, brine, dried over MgSO4 and evaporated.
The residue was purified by chromatography on silica gel using 80:20
hexane-ethyl acetate to give 3.2 g (80%) of 3 as a yellow solid. TLC Rf:
1
0.36 (7/3/1, hexane/EtOAc/Et3N, v/v/v). H NMR (250 MHz, CDCl3):
d: 8.32 (d, J=8.7 Hz, 2 H), 8.01 (d, J=8.7 Hz, 2 H), 5.33 (d, J=10.0 Hz,
1 H), 3.81 (dd, J=4.9 Hz, 1 H), 3.48 (s, 3 H), 2.08 (m, 1 H), 0.94 (d,
J=6.8 Hz, 3 H), 0.84 (d, J=6.8 Hz, 3 H).
Synthesis of (2R)-3-methyl-2-[(4-aminobenzenesulfonyl)amino] buta-
noic acid methyl ester (4). A solution of 3 (2.7 g, 8.5 mmol) in 30 ml of
MeOH and 30 ml of EtOAc was hydrogenated using 200 mg of 10% Pd/
C catalyst for 4 h. The solution was filtered on Celite and the filtrate was
concentrated under reduced pressure. The residue was crystallized from
1/1 (v/v) CH2Cl2-hexane to give 2.23 g (91%) of 4 as colourless crystals.
1
TLC: Rf: 0.0 (7/3/1, hexane/EtOAc/Et3N, v/v/v). H NMR (250 MHz,
MeOD): d: 7.48 (d, J=8.8 Hz, 2 H), 6.66 (d, J=8.8 Hz, 2 H), 3.52 (d,
J=6.5 Hz, 1 H), 3.42 (s, 3 H), 1.90 (m, 1 H), 0.90 (d, J=0.9 Hz, 3 H),
0.88 (d, J=0.9 Hz, 3 H). MS (C12H18N2O4S) calculated 286, measured:
m/z: 286.8 [M+H]+.
Synthesis of (2R)-3-methyl-2-[[4-[(4-methoxybenzoyl)amino]benzene-
sulfonyl]amino] butanoic acid methyl ester (6). To a solution of 4
(400 mg, 1.4 mmol) in CH2Cl2 (20 ml), 380 ml (2.8 mmol) of N-
methylmorpholine and 586 mg of 4-methoxybenzoylchloride (5)
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B. KUHNAST ET AL.
(2.1 mmol) were added at room temperature and stirred overnight. The
mixture was poured into water and extracted with EtOAc. The organic
solution was twice washed with 100 ml of 1 N HCl solution, brine, dried
over MgSO4 and evaporated. The residue was purified by chromato-
graphy on silica gel using 70:30 hexane-ethyl acetate to give 441 mg
(75%) of 6 as a white solid. TLC: Rf: 0.45 (6/4, hexane/EtOAc, v/v). 1H
NMR (250 MHz, DMSO-d6): d: 10.37 (s, 1 H), 7.96 (d, J=8.7 Hz, 2 H),
7.92 (d, J=8.7 Hz, 2 H), 7.82 (d, J=9.7 Hz, 1 H), 7.70 (d, J=8.1 Hz,
2 H), 7.09 (d, J=8.2 Hz, 2 H), 3.83 (s, 3 H), 3.52 (dd, J=7.8, 8.9 Hz,
1 H), 3.37 (s, 3 H), 1.87 (m, 1 H), 0.80 (d, J=6.8 Hz, 3 H), 0.77 (d,
J=6.7 Hz, 3 H). MS (C20H24N2O6S) calculated 420, measured: m/z:
421.1 [M+H]+, 863.0 [2M+Na]+.
Synthesis of (2R)-3-methyl-2-[[4-[(4-methoxybenzoyl)amino]benzene-
sulfonyl]amino] butanoic acid (7). To a solution of 6 (150 mg, 0.3 mmol)
in 4 ml of MeOH and 4 ml of THF, 2.5 ml of 1 M NaOH solution were
added at room temperature. The mixture was stirred for 4 h at 608C.
After being cooled to room temperature, the solution was acidified with
1 N HCl solution and extracted with EtOAc. The organic solution was
washed with brine, dried over MgSO4 and evaporated. The residue was
crystallized from EtOAc-hexane to give 120 mg (>95%) of 7 as
colourless crystals. 1H NMR (250 MHz, DMSO-d6): d: 12.56 (br s, 1 H),
10.37 (s, 1 H), 7.96 (d, J=8.8 Hz, 2 H), 7.92 (d, J=8.7 Hz, 2 H), 7.82 (d,
J=9.7 Hz, 1 H), 7.72 (d, J=8.7 Hz, 2 H), 7.06 (d, J=8.8 Hz, 2 H), 3.83
(s, 3 H), 3.47 (dd, J=7.2 Hz, 6.1 Hz, 1 H), 1.94 (m, 1 H), 0.81 (d,
J=6.9 Hz, 3 H), 0.77 (d, J=6.9 Hz, 3 H). MS (C19H22N2O6S) calcu-
lated: 406, measured : m/z: 406.9 [M+H]+, 812.8 [2M+H]+.
Synthesis of 4-acetoxybenzoylchloride (9). To a solution of 1 g
(5.5 mmol) of 4-acetoxybenzoic acid (8) in CH2Cl2 (20 ml), 10 ml of
thionyl chloride were added. After the solution was stirred overnight,
the solvents were removed under vacuum to give a colourless oil (9) that
was used for the next step without further purification.
Synthesis of (2R)-3-methyl-2-[[4-[(4-acetoxybenzoyl)amino]benzene-
sulfonyl]amino] butanoic acid methyl ester (10). To a solution of 9
(300 mg, 1.5 mmol) in 30 ml of CH2Cl2, 400 mg of 4 (1.4 mmol) and
380 ml (2.8 mmol) of N-methylmorpholine were added at room
temperature and the solution stirred overnight. The mixture was poured
into water and extracted with EtOAc. The organic layer was twice
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CARBON-11 LABELLING OF AN
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washed with 100 ml of 1 N HCl solution, brine, dried over MgSO4 and
evaporated. The residue was purified by chromatography on silica gel
using 70:30 hexane-EtOAc to give 376 mg (60%) of 10 as a white solid.
1
TLC : Rf : 0.40 (6/4 hexane/EtOAc v/v). H NMR (250 MHz, DMSO-
d6 + MeOD) : d: 10.26 (s, 1 H), 7.94 (d, J=8.6 Hz, 2 H), 7.86 (d,
J=8.8 Hz, 2 H), 7.68 (d, J=8.8 Hz, 2 H), 7.21 (d, J=8.6 Hz, 2 H), 3.52
(d, J=6.7 Hz, 1 H), 3.32 (s, 3 H), 2.22 (s, 3 H), 1.86 (m, 1 H), 0.80 (d,
J=2.8 Hz, 3 H), 0.77 (d, J=2.8 Hz, 3 H). MS (C21H24N2O7S) calcu-
lated: 448, measured: m/z: 449.1 [M+H]+, 918.9 [2M+Na]+.
Synthesis of (2R)-3-methyl-2-[[4-[(4-hydroxybenzoyl)amino]benzene-
sulfonyl]amino] butanoic acid (11). To a solution of 10 (200 mg,
0.45 mmol) in 4 ml of MeOH and 4 ml of THF, 2.5 ml of 1M NaOH
solution were added at room temperature. The mixture was stirred for
4 h at 608C. After being cooled to room temperature, the solution was
acidified with 1 N HCl solution and extracted with EtOAc. The organic
solution was washed with brine, dried over MgSO4 and evaporated. The
residue was crystallized from EtOAc-hexane to give 172 mg (99%) of 7
1
as colourless crystals. H NMR (250 MHz,DMSO-d6) d: 12.53 (br s,
1 H), 10.22 (s, 1 H), 10.15 (br s, 1 H), 7.91 (d, J=8.8 Hz, 2 H), 7.84 (d,
J=8.7 Hz, 2 H), 7.70 (d, J=8.8 Hz, 2 H), 6.86 (d, J=8.7 Hz, 2 H), 3.48
(d, J=6.6 Hz, 1 H), 1.91 (m, 1 H), 0.81 (d, J=7.0 Hz, 3 H), 0.78 (d,
J=7.0 Hz, 3 H). MS (C18H20N2O6S) calculated: 392, measured: m/z:
392.9 [M+H]+, 823.0 [2M+K]+.
Radiochemistry
No carrier added [11C]CO2 was produced on an RDS 112 cyclotron
(Siemens, Germany) via the 14N(p,a)11C reaction using nitrogen with
1% oxygen. [11C]CO2 was converted into [11C]CH3I using the PET
Trace MeI Microlab remote system (GE Medical System, Germany).
Synthesis of (2R)-3-methyl-2-[[4-[(4-[11C]methoxybenzoyl]amino]-
benzenesulfonyl] amino] butanoic acid [11C]7. Precursor 11 (0.5 mg)
was dissolved in 300 ml of dry DMF with NaH (0.5 mg) in a 1.1 ml Screw
Neck Micro-Vial (Alltech, Germany). [11C]CH3I was allowed to bubble
in to a vial containing the precursor for 2–3 min. The mixture was
heated for 5 min at 808C. The purification was performed using the
semi-preparative system (see Analytical methods) eluted with a water/
MeCN/TFA mixture (60/40/0.1 v/v/v) at a flow rate of 6 ml/min
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(tR: 13–13.5 min). The product fraction was collected and concentrated
under vacuum. Radiochemical purity and specific radioactivity were
then determined using the analytical HPLC system eluted with a water/
MeCN/TFA mixture (60/40/0.1 v/v/v) at a flow rate of 1 ml/min (tR:
4.9 min).
Enzyme inhibition assays
Gelatine labelling. Gelatine was dissolved in saline to a concentration of
1 mg/ml. 125 ml of this solution was transferred to an Eppendorf cap
coated with 150 mg iodogen. 10 ml of n.c.a. [125I]NaI (60 MBq) was
added to the solution. After 30 min reaction, the solution was removed
and eluted through a NAP-10 column (Amersham Pharmacia Biotech,
Freiburg, Germany) to separate non-reacted sodium iodide, according
to the manufacturer instructions. The radiochemical purity was
controlled by TLC on precaoted plates of silica gel 60F254 (Merck,
Darmstadt, Germany) eluted with a mixture of acetone, water, butanol
and ammonia (6.5:0.5:2:1) (v/v/v/v). The labelled gelatine was detected
with a radioTLC analyser Trace Master 20 (Berthold, Germany). Rf
gelatine: 0.0; Rf [125I]NaI: 1. The radiochemical purity was >98%.
Inhibition assay. Compound 7 (1 mg, 2.46 mmol in 5 ml DMF), HWGF-
peptide (12) (1 mg, 0.74 mmol in 10 ml DMF) and 1,10 phenanthroline
(13) (9.9 mg, 50 mmol in 25 ml DMF) were dissolved and diluted in the
assay buffer (50 mM Tris-HCL, pH 7.6, 150 mM NaCl, 5 mM CaCl2,
2 mM NaN3) to the appropriate concentrations (0.001–200 mM final
concentration). For each assay, 100 ng of MMP-2 or MMP-9 were
activated for 1 h at 378C with APMA (p-aminophenylmercuric acetate)
(5 mM). Compounds 7, 12 and 13, at the desired concentrations, were
added to the enzyme solution and incubated for 1 h at 378C. Finally,
1mg of labelled gelatine was added to each assay and incubated for 1 h at
378C. The degradation of labelled gelatine was determined by counting
the radioactivity in the supernatant after precipitation of the
undegraded gelatine with 20% trichloroacetic acid. The 1,10 phenan-
throline (13) and CTTHWGFTLC (12) peptide served as control MMP
inhibitors.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 539–553

CARBON-11 LABELLING OF AN
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Biodistribution and in vivo serum stability
All procedures involving animals were in conformity with the German
law for protection of animals that are in compliance with European laws
and policies.
Female Balb/c mice, aged 5–7 weeks, were injected in to the tail vein
with 22–26 GBq (600–700 mCi) of [11C]7. Animals were sacrificed
30 minutes after injection. Tissues of interest were immediately
dissected, weighed and counted for radioactivity with a g-counter.
Radioactivity concentration was expressed as a percentage of injected
dose per gram of tissue (% ID/g) and reported as the mean ꢁ standard
deviation (S.D.).
Serum stability of [11C]7 was studied by analytical radioHPLC. After
sacrifice, blood was immediately collected and centrifuged. Serum was
deproteinised by protein precipitation with acetonitrile. The samples
were then centrifuged for 15 min at 12 000 g. Aliquots of blood, serum
and supernatant after acetonitrile precipitation were counted for
radioactivity with a g-counter to evaluate the partition of the tracer.
The supernatants were concentrated and injected onto an HPLC
column (see Analytical methods) eluted with a water/MeCN/TFA
mixture (60/40/0,1 v/v/v).
Acknowledgements
.
This study was supported by the Konto fur Klinische Forschung
.
(KKF), Klinikum rechts der Iasr, Technische Universitat Munchen.
.
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