Design, synthesis, and initial evaluation of a high affinity positron emission tomography probe for imaging matrix metalloproteinases 2 and 9

Article abstract of DOI:10.1021/jm400156p

The activity of matrix metalloproteinases (MMPs) is elevated locally under many pathological conditions. Gelatinases MMP2 and MMP9 are of particular interest because of their implication in angiogenesis, cancer cell proliferation and metastasis, and atherosclerotic plaque rupture. The aim of this study was to identify and develop a selective gelatinase inhibitor for imaging active MMP2/MMP9 in vivo. We synthesized a series of N-sulfonylamino acid derivatives with low to high nanomolar inhibitory potencies. (R)-2-(4-(4-Fluorobenzamido) phenylsulfonamido)-3-(1H-indol-3-yl)propanoic acid (7) exhibited the best in vitro binding properties: MMP2 IC₅₀ = 1.8 nM, MMP9 IC₅₀ = 7.2 nM. Radiolabeling of 7 with no carrier added ¹⁸F-radioisotope was accomplished starting from iodonium salts as precursors. The radiochemical yield strongly depended on the iodonium counteranion (ClO₄ ^- > Br^- > TFA^- > tosylate). ¹⁸F-7 was obtained in up to 20% radiochemical yield (decay corrected), high radiochemical purity, and >90 GBq/μmol specific radioactivity. The radiolabeled compound showed excellent stability in vitro and in mice in vivo.

Full text of DOI:10.1021/jm400156p

Article
pubs.acs.org/jmc
Design, Synthesis, and Initial Evaluation of a High Affinity Positron
Emission Tomography Probe for Imaging Matrix Metalloproteinases
2 and 9
Svetlana V. Selivanova,*,†,∥ Timo Stellfeld,‡,∥ Tobias K. Heinrich,‡ Adrienne Muller,† Stefanie D. Kramer,†
̈
̈
P. August Schubiger,† Roger Schibli,† Simon M. Ametamey,*,† Bernhard Vos,‡ Jorg Meding,‡
̈
Marcus Bauser,‡ Joachim Hutter,‡ and Ludger M. Dinkelborg‡,§
̈
†Center for Radiopharmaceutical Sciences, ETH Zurich, CH-8093 Zurich, Switzerland
‡Global Drug Discovery, Bayer Healthcare, 13353 Berlin, Germany
S
* Supporting Information
ABSTRACT: The activity of matrix metalloproteinases (MMPs) is elevated locally under many pathological conditions.
Gelatinases MMP2 and MMP9 are of particular interest because of their implication in angiogenesis, cancer cell proliferation and
metastasis, and atherosclerotic plaque rupture. The aim of this study was to identify and develop a selective gelatinase inhibitor
for imaging active MMP2/MMP9 in vivo. We synthesized a series of N-sulfonylamino acid derivatives with low to high
nanomolar inhibitory potencies. (R)-2-(4-(4-Fluorobenzamido)phenylsulfonamido)-3-(1H-indol-3-yl)propanoic acid (7)
exhibited the best in vitro binding properties: MMP2 IC50 = 1.8 nM, MMP9 IC50 = 7.2 nM. Radiolabeling of 7 with no
carrier added 18F-radioisotope was accomplished starting from iodonium salts as precursors. The radiochemical yield strongly
depended on the iodonium counteranion (ClO4− > Br− > TFA− > tosylate). 18F-7 was obtained in up to 20% radiochemical yield
(decay corrected), high radiochemical purity, and >90 GBq/μmol specific radioactivity. The radiolabeled compound showed
excellent stability in vitro and in mice in vivo.
quantification of active MMPs by high-sensitivity imaging.
Positron emission tomography (PET) offers noninvasive
quantitative analysis at nanomolar concentrations both, in
preclinical and clinical settings.
INTRODUCTION
■
Matrix metalloproteinases (MMPs) are extracellular enzymes
which are synthesized in the rough endoplasmic reticulum and
excreted into the extracellular space as inactive pro-forms.1
Activation of the excreted MMPs by proteolytic cleavage results
in the exposure of the catalytic domain containing a Zn2+ ion.
Tissue inhibitors of metalloproteinases (TIMPs) serve as
endogenous MMP inhibitors and control the amount of active
enzyme by blocking its catalytic site. The activity of several
MMPs is elevated locally during many pathologies, for example,
cancer, atherosclerosis, and chronic inflammatory diseases, as a
result of a disrupted balance between synthesis, activation,
inhibition, and degradation. Noninvasive imaging of increased
MMP activity could aid early diagnosis of a pathological state.
Targeting activated MMPs can be achieved with appropriate
MMP substrates or inhibitors. The substrates, which are
cleaved by MMP to products with altered fluorescent
properties, have been evaluated for optical imaging with some
success.2,3 However, optical imaging cannot be translated to
clinical applications due to the limited depth of tissue
penetration by light. MMP inhibitors interact with the enzyme
at the catalytic site in a stoichiometric fashion and allow
Several research groups attempted to develop PET probes
for active MMPs based on inhibitors.4−10 A decade ago, a range
of 18F- or 11C-radiolabeled sulfonamides 1−3 (Figure 1) were
reported.5−7 Despite promising in vitro results, none of the
proposed tracers was advanced further as an imaging agent.
Selected analogue 4 (Figure 1) was evaluated in breast cancer
mouse models but did not significantly accumulate in the MCF-
7 transfected with IL-1α or MDA-MB-435 xenografts.8 Very
recently, novel MMP inhibitors were introduced having the
pyrimidine-2,4,6-trione (5, Figure 1) structural core.9,10
Preliminary PET experiments using 5 in healthy mice showed
low background radioactivity in nontargeted tissues, but
clearance was slower than desired for an 18F-labeled tracer.9
It should be noted that all of the above-mentioned compounds
are broad spectrum MMP inhibitors, binding to MMP2,
Received: January 30, 2013
© XXXX American Chemical Society
A
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and the same approach was adapted for the syntheses of
analogues 7−16 (Scheme 2). Final acylation of the amine
function was performed according to two different methods,
using corresponding acid or acyl chloride. Compounds 17−19
were synthesized by coupling D-tryptophan methyl ester and
appropriate sulfonyl chloride under basic conditions (Scheme
2). Hydrolysis of the methyl ester bond with sodium or lithium
hydroxide resulted in the free acid function. The t-butyl ester
group in compounds 13, 15, and 16 was cleaved under acidic
conditions.
Synthesized compounds 6−19 were assayed for their
inhibitory potencies. MMP enzyme activity was determined
by recording the initial slopes of fluorescence increase, which
resulted from enzymatic cleavage of an internally quenched
fluorogenic substrate. MMP2 and MMP9 inhibitors were
identified by a decrease in initial slopes and compared to the
control reaction without the test compound. The obtained IC50
values are presented in Table 1. Compound 6, which was
reported previously,11 shows equally high binding affinity to
both MMP2 and MMP9. In an attempt to shift selectivity
toward MMP9, we modified the aromatic ring, adjacent to the
sulfonyl moiety, by introducing different halogens and alkoxy
substituents. None of the modifications we attempted resulted
in MMP9 selectivity. Often, MMP2 inhibitory potency
remained in a low nanomolar range, while MMP9 inhibition
decreased, indicating that MMP2 is tolerant to variations in this
part of the molecule. In contrast, the inhibitory potency of
MMP9 was retained only when modifications on the aromatic
ring were carried out with bromo, fluoro, and iodo substituents.
Increasing the bulkiness and lipophilicity by replacing the
methoxy substituent with iso-propoxy and iso-butoxy moieties
(9−12) clearly diminished the inhibitory activity, although it
remained below 1 μM. Replacement of benzene by pyridine
ring (13−16) gave derivatives with slightly increased IC50
values for both MMPs but still in the low nanomolar range
(e.g., 14). However, an exception in this series was compound
15; positioning of the fluoro-substituent in the pyridine ring
ortho to the carbonyl group resulted in a drastic loss of affinity,
which, we speculate, could be due to steric and/or electronic
hindrance.12,13 Removal of the amide linker between the two
benzene rings (17−19) was tolerated better by MMP2 than by
MMP9. Selected compounds, namely, 6, 7, and 14, were tested
also against MMP1. The IC50 values for all the three
compounds for MMP1 were >1000 nM. Because of a favorable
inhibitory potency and the possibility of labeling the structure
with 18F radioisotope, we selected MMP2/MMP9 inhibitor 7 as
a potential radiotracer.
Figure 1. Previously reported radiolabeled broad spectrum MMP
inhibitors. X = 18F/19F, NO2, O11CH3/O12CH3; Y = OH, O11CH3/
O12CH3; R = CH2CH2SCH3, CH2(3-indolyl), CH2(CH3)2.
MMP8, MMP9, and MMP13. There is currently no successful
MMP selective radiotracers available for the PET imaging.
Deregulation of the gelatinases MMP2 (gelatinase A, 72 kDa
type IV collagenase) and MMP9 (gelatinase B, 92 kDa type IV
collagenase) are of particular interest because they are
implicated in angiogenesis, cancer cell proliferation and
metastasis, and atherosclerotic plaque rupture. In this work,
we were looking to identify and develop a selective MMP2/
MMP9 inhibitor for imaging and quantification of gelatinase
activity in vivo by PET. We synthesized a series of novel
compounds containing (R)-3-(1H-indol-3-yl)-2-sulfonamido-
propanoic acid pharmacophore and tested their inhibitory
potencies (IC50) in vitro using a fluorescence-based assay
(Table 1). MMP2/MMP9 inhibitor 7 was chosen as a potential
PET imaging agent due to its promising binding characteristics.
Two asymmetric iodonium salt precursors were synthesized
for further labeling with 18F radioisotope and four different
iodonium counteranions were evaluated. The radiofluorinated
product, 18F-7, was prepared in good and reproducible yields.
Metabolites studies were performed to determine the in vivo
stability of the radiotracer. Its clearance profile was evaluated in
mice using PET.
Synthesis of Precursors for Radiolabeling. The
introduction of nucleophilic 18F-fluoride into an aromatic ring
during the last steps of radiosynthesis, as required for short-
lived PET tracers, is not always easy. Usually, a very good
leaving group such as nitro or trimethylammonium group is
needed as well as an activating electron-withdrawing group
ortho or para to the leaving group. In this work, we selected an
approach which utilizes aryliodonium salts as precursors
without the need to introduce an activating group. It is
documented that aryliodonium salts are highly reactive due to
the excellent leaving ability of the hypervalent aryliodonium
group.14
RESULTS AND DISCUSSION
■
Chemistry. A series of 14 compounds 6−19, containing
(R)-3-(1H-indol-3-yl)-2-sulfonamidopropanoic acid pharmaco-
phore, were synthesized. The preparation of the key
intermediates, aniline I and aniline II, is shown in Scheme 1.
The anilines were synthesized according to the published
procedure.7,11 Compound 6 was prepared as described earlier,11
The synthesis of asymmetrical iodonium salt precursors 22
and 23, having two distinct leaving groups, thiophene or
anisole, proceeded according to Scheme 3. The procedure
involving the oxidative formation of iodonium compounds (20,
B
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a
Table 1. Chemical Structures of Derivatives 6−19 and Their Inhibitory Potencies
a
IC50 values are represented as average of four independent determinations with standard deviation below 5%.
21) was adapted from the literature.15 Coupling of the resulting
diaryliodonium synthon to the pharmacophore was performed
under basic conditions. Purification of the iodonium salts was
not trivial. Conventional workup and column chromatography
resulted in partial counterion exchange due to the presence of
other anions in the mobile phase. To ensure the integrity of the
structure, the counterion was introduced using reversed-phase
HPLC ion-exchange protocol. All iodonium salts were prepared
in low to moderate yields.
rate, trifluoroacetate, tosylate) were evaluated (Scheme 4).
Thiophene and anisole leaving groups, both, were displaced by
18F in the presence of cesium carbonate in DMF containing
trace amounts of water at 130 °C for 3 min. Interestingly, the
radiochemical yield (RCY) was higher with a shorter reaction
time: 70% fluoride incorporation was observed at 3 min,
decreasing to 55% at 5 min, and further to 40% at 15 min
reaction time. The reaction did not occur or gave low and
unreliable yields when dry (anhydrous) solvents (acetonitrile,
DMF, DMSO) were used (data not shown). Similar
observations were reported in the literature.16,17 The exact
mechanism of this reaction is still under discussion.13 We
speculate that traces of water are beneficial because they
Radiochemistry. Radiolabeling with nucleophilic 18F-
fluoride was achieved in one step followed by deprotection of
the carboxylic acid function. The reaction conditions were
optimized and different leaving groups (anisole, thiophene) as
well as different iodonium counteranions (bromide, perchlo-
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Scheme 1. Synthesis of Key Intermediates Aniline I and Aniline II
Scheme 2. Synthesis of Compounds 6−19
catalyze the formation of the intimate ion−molecule pair
(where the molecule is p-iodoanisole or 2-iodothiophene),
making the nonactivated aromatic ring positively charged and
ready for a nucleophilic attack (Scheme 5).14 Ionizing solvent,
such as water, reduces the energy required for heterolytic bond
dissociation, thereby favoring this pathway over a homolytic
one. The use of radical scavengers like TEMPO (2,2,6,6-
tetramethylpiperidine 1-oxyl) did not improve RCY (data not
shown), as was reported by others.18,19 This suggests that there
is no important formation of radicals or they do not interfere
with the radiolabeling reaction. In general, the precursor having
anisole leaving group gave higher radiochemical yields than the
precursors containing thiophene, thus further optimization of
the reaction was performed using the anisole containing
precursor. It was shown previously for small model compounds
that inorganic counterions give higher RCYs.20 The influence of
the counteranion on RCY was evaluated under optimized
radiolabeling conditions and found to be substantial. For our
heavily functionalized precursors, the RCYs were also
considerably higher when inorganic counteranions were used
and decreased in the following order: perchlorate > bromide >
tosylate > triflate (Chart 1). We speculate that bulkier organic
anions that have in addition more pronounced covalent
bonding properties hinder solvolysis of an iodonium salt,
limiting the formation of ion−molecule pair. Other possible
reasons for the advantage of inorganic counterions are
extensively discussed in the literature.20
Cleavage of the methyl ester functionality was performed
under basic conditions. To confirm that there was no
racemization of 18F-7, we performed the same reaction on a
larger scale using nonradioactive methyl ester of compound 7.
Chiral HPLC confirmed the formation of the R-enantiomer
only, which was obtained in quantitative yield, and no S-
enantiomer could be detected, indicating that no racemization
D
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Scheme 3. Synthesis of the Labeling Precursors
Scheme 4. Radiosynthesis of 18F-7 under Optimized Reaction Conditions
Scheme 5. Formation of Intimate Ion−Molecule Pair
reproducibly synthesized and formulated for in vivo application
in 10−20% RCY (decay corrected to EOB, n > 10) with
specific radioactivities >90 GBq/μmol (>2.5 Ci/μmol) (EOS)
under optimized radiolabeling and purification conditions.
Radiochemical and chemical purity was >95%, as confirmed by
HPLC.
Chart 1. Influence of the Counter-Anion on the
Radiolabeling Yield
Metabolic Stability. The tracer 18F-7 was stable in rodent
and human plasma in vitro. No decomposition occurred up to 2
h of incubation at 37 °C. Metabolic stability in vivo was
evaluated in C57BL/6 mice. Only parent compound was
identified in blood, and 85% of the radioactivity in urine
corresponded to intact radiotracer 60 min after injection.
PET/CT Imaging. In a preliminary study, PET/CT scan of a
C57BL/6 mouse (Figure 2) showed mainly hepatobiliary
clearance of the tracer. Radioactivity was also relatively high in
urinary bladder. The absence of bone uptake confirmed that
there is no defluorination of the tracer in vivo.
of 18F-7 occurred under the reaction conditions. The final
product was purified using reversed-phase semipreparative
HPLC. Initially, 0.1% aqueous trifluoroacetic acid (TFA) was
used with acetonitrile as the mobile phase for the semi-
preparative HPLC. Later we found the radiolabeled product
was unstable under strongly acidic conditions. For this reason,
the use of TFA as an additive was discontinued and replaced by
10 mM ammonium bicarbonate buffer. 18F-7 could be
E
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FCCl3 was used as an internal standard (δ 0.0). Low-resolution mass
spectra (MS) were recorded on a Waters ZQ 4000 mass spectrometer.
High-resolution mass spectra (HRMS) were recorded on a Waters Q-
TOF Premier mass spectrometer. Optical rotations were recorded on a
Jasco P-2000 polarimeter. Radiolabeling reactions were monitored and
radiometabolites were detected using UPLC. The system was Acquity
UPLC from Waters, equipped with a FlowStar LB 513 radiodetector
(Berthold Thecnologies) and a UPLC dedicated reversed-phase
Acquity BEH C18, particle size 1.7 μm, 100 mm × 2.1 mm column.
The mobile phase consisted of a gradient of acetonitrile in water with
0.1% TFA or a gradient of acetonitrile in aqueous 10 mM NH4HCO3
buffer (pH 8) from 5% to 95% over 2 min, flow rate 0.7 mL/min.
Semipreparative HPLC for radiolabeled product purification was
carried out on an HPLC system equipped with a Merck−Hitachi L-
6200A intelligent pump, a 5 mL injection loop, a Knauer Variable
Wavelength Monitor UV detector, and an Eberline RM-14 radio-
detector on a reversed-phase Phenomenex Gemini C18 column,
particle size 5 μm, 250 mm × 10 mm. The mobile phase consisted of
isocratic 40% acetonitrile/60% 10 mM NH4HCO3 buffer (pH 8) v/v,
eluted at 4 mL/min flow rate. UV absorbance was detected at 254 nm.
Analytical HPLC was performed on an Agilent 1100 series system
equipped with a Raytest Gabi Star radiodetector. Analytical reversed-
phase column was Phenomenex Luna C18, particle size 5 μm, 250 mm
× 4.6 mm. A gradient of acetonitrile in aqueous 10 mM NH4HCO3
buffer (pH 8) from 5% to 95% over 20 min at a flow rate of 1 mL/min
Figure 2. PET/CT image of a C57BL/6 mouse. Transversal (A),
sagittal (B), and coronal (C) sections (indicated by crosshairs),
averaged 8−38 min after injection of 5.3 MBq 18F-7, 2 bed positions,
15 min each starting with anterior part. Color scale, PET; white, CT.
PET maximum intensity projection (D). SUV, standardized uptake
value; L, liver; I, intestine; U, urinary bladder.
1
was used. The synthesized compounds were characterized by H, 13C,
19F NMR, MS, and HPLC. Purity was exceeding 95%, unless specified
otherwise.
Chemistry. Acylation of Anilines I, II: General Procedure.
Method A. The aniline was dissolved in dichloromethane (10 mL)
and N-methylmorpholine (2 equiv) was added at 0 °C followed by
acyl chloride (1.5 equiv). The reaction was slowly warmed to room
temperature. After 12 h, the reaction mixture was poured into water
(20 mL) and extracted with ethyl acetate (3 × 20 mL). The organic
phases were combined, washed with brine (20 mL), dried (MgSO4),
and concentrated. The crude product was purified by flash column
chromatography on silica gel with hexane/ethyl acetate as eluent.
Method B. The carboxylic acid (2 equiv) was dissolved in DMF (2
mL/mmol carboxylic acid) and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (HATU, 2 equiv, 0.5 M
solution in DMF), 1-hydroxy-7-azabenzotriazole (HOAt, 2 equiv, 0.5
M solution in DMF) and N,N-diisopropylethylamine (DIPEA, 4 equiv,
1 M solution in DMF) were added consequently. After 5 min, the
aniline (1 equiv) was added and the mixture was stirred for 3 h. The
reaction was poured into water (20 mL) and extracted with ethyl
acetate (3 × 20 mL), and the combined organic phases were washed
with 1N aqueous HCl (20 mL), 1N NaHCO3 (2 × 20 mL), dried
(MgSO4), and concentrated. The crude product was purified by
reversed-phase column chromatography with acetonitrile/water as
eluent.
Deprotection of Carboxylic Acid Function: General Procedure.
The α-sulfonylamino methyl ester was dissolved in THF/water (1:1, 5
mL) and LiOH was added. The solution was stirred at room
temperature for 12 h. The mixture was poured into 1N aqueous HCl
(30 mL) and extracted with ethyl acetate (3 × 20 mL). The organic
phases were combined, washed with brine (20 mL), dried (MgSO4),
and concentrated. The crude product was purified by preparative
reversed-phase HPLC using water/acetonitrile as eluent.
(R)-2-[4-(4-Fluoro-benzoylamino)-benzenesulfonylamino]-3-(1H-
indol-3-yl)-propionic Acid (7). Aniline I (198 mg, 0.53 mmol) was
reacted with 4-fluorobenzoyl chloride (95 μL, 0.80 mmol) according
to method A to yield the corresponding amide (182 mg, 0.37 mmol,
69%). A part of the resulted methyl ester (50 mg, 0.1 mmol) was
dissolved in MeOH/THF (1:1, 3 mL), and 0.5 mL 1N aqueous
NaOH was added. The solution was stirred for 4 h at 60 °C and
cooled to room temperature. The mixture was poured into 1N
aqueous HCl (20 mL) and extracted with ethyl acetate (3 × 20 mL).
The organic phases were combined, washed with brine (20 mL), dried
(MgSO4), and concentrated. Purification by preparative reversed-
phase HPLC gave the title compound (19 mg, 0.04 mmol, 39%);
CONCLUSION
■
Novel potent MMP2/MMP9 inhibitor 7 was identified based
on SAR studies. The compound was radiolabeled with 18F in
satisfactory radiochemical yields and high specific radioactivity.
18F-7 showed excellent in vivo stability in wild-type mice.
Further evaluation of the tracer suitability for cancer and
atherosclerosis imaging is in progress.
EXPERIMENTAL SECTION
■
All commercially available reagents and solvents were used as received.
Organic reactions were monitored by TLC on normal phase silica gel
plates (DC Kieselgel 60 F254, Merck). Mobile phase was a mixture of
hexane and ethyl acetate at suitable ratios. Developed TLCs were
visualized under UV light at 254 nm. Crude products were purified by
normal phase flash column chromatography on Biotage Isolera Prime
system using SNAP KP-Sil cartridges and an appropriate gradient of
ethyl acetate in hexane as mobile phase. An optimal gradient was
calculated automatically by Biotage Isolera Prime TLC-to-Gradient
software. Reversed-phase preparative HPLC was performed on an
Agilent Technologies 1200 series HPLC system using an XBridge Prep
C18 OBD, particle size 5 μm, 30 mm × 100 mm column. The mobile
phase consisted of 0.1% TFA in water and acetonitrile. The acetonitrile
gradient from 5% to 95% over 20 min at 30 mL/min flow rate was
applied. Ion exchange was performed on Biotage Isolera using RP-18
PuriFlash 15PT C18T cartridges (Interchim) and the mobile phase as
described below (see: Introduction of Counterions). The chiral HPLC
system consisted of a Dionex Pump 680, ASI 100, and Waters UV-
detector 2487. The enantiomers were resolved on a Chiralpak AD-H 5
μm 150 mm × 4.6 mm column using hexane/ethanol 70/30 + 0.1%
diethylamine as solvent at a flow rate of 1.0 mL/min and UV detection
at 254 nm. NMR spectra were recorded on Bruker Avance 300
1
(frequencies: H, 300 MHz; 13C, 75 MHz) or Bruker Avance 400
1
(frequencies: H, 400 MHz; 13C, 100 MHz) spectrometers with the
corresponding residual solvent signals as an internal standard (CDCl3:
1
1H NMR at δ 7.26, 13C NMR at δ 77.23. D2O: H NMR at δ 4.8.
1
DMSO-d6: H NMR at δ 2.5, 13C NMR at δ 39.51). For 19F-NMR,
F
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[α]20D −22.6°
1.32° (c 0.10, DMSO). 1H NMR (400 MHz,
reaction and without any workup, the reaction mixture was
concentrated and the residue was adsorbed on LiChroprep RP18
packing material (Merck), charged on a RP-18 PuriFlash 15PT C18T
column (Interchim) and eluted consecutively with the following
solvents:
DMSO-d6) δ, ppm: 10.77−10.86 (m, 1H), 10.53 (s, 1H), 8.15 (d, J =
8.59 Hz, 1H), 8.05 (dd, J = 5.56, 8.84 Hz, 2H), 7.81−7.87 (m, 2H),
7.58−7.63 (m, 2H), 7.40 (t, J = 8.84 Hz, 2H), 7.30 (t, J = 8.34 Hz,
2H), 7.08 (d, J = 2.27 Hz, 1H), 7.02 (t, J = 8.08 Hz, 1H), 6.90−6.96
(m, 1H), 3.88−3.95 (m, 1H), 3.05 (dd, J = 6.82, 14.40 Hz, 1H), 2.86
(dd, J = 7.58, 14.40 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ,
ppm: 172.5, 165.5, 164.8, 163.0, 158.0, 142.4, 136.0, 135.2, 131.0,
130.6, 130.5, 127.3, 126.9, 123.9, 120.8, 119.4, 118.3, 117.9, 115.5,
115.3, 111.4, 108.9, 56.6, 28.3. 19F NMR (376 MHz, DMSO-d6) δ,
ppm: −111.9 (m). HRMS (TOF MS ES+): calcd for C24H21FN3O5S
([M + H]+), 482.1186; found 482.1183.
(1) an aqueous 0.1% solution H-X (X = tosylate, bromide, triflate,
or perchlorate, according to the desired salt form) (500 mL/1 g
crude product);
(2) water (500 mL/1 g crude product);
(3) a gradient of acetonitrile in water, from 20% to 90% over 20
min.
The product was collected by monitoring UV signal (215−360
nm, PDA detector) and lyophilized.
Synthesis of Labeling Precursors. The reaction of oxidative
formation of iodonium compound was adapted from the published
procedure.15
Tosylate (23b). The analytical data correspond to those described
above; integrals of the tosylate signals indicate the presence of 0.9−1.0
equiv tosylate with respect to the cation.
(4-Carboxyphenyl)(4-methoxyphenyl)iodonium 4-methylbenze-
nesulfonate (21). 4-Iodobenzoic acid methyl ester (10 g, 38.2
mmol), 3-chloroperoxybenzoic acid (17.8 g, 72 mmol), and anisole
(6.2 g, 57.2 mmol) were dissolved in dichloromethane/trifluoroetha-
nol (1:1, 200 mL). 4-Toluenesulfonic acid (10.89 g, 57.2 mmol) was
added, and the reaction mixture was stirred at room temperature for 3
days. To the obtained suspension ether (1500 mL) was added and the
precipitate was filtered and washed with ether. The solvent was
evaporated to yield 22.8 g of the crude intermediate methyl ester. MS
(ES+): m/z 368.61 ([M]+) (iodonium cation).
The methyl ester was dissolved in TFA/water (1:1, 600 mL) and
heated at 120 °C (oil bath) for 48 h. After cooling to room
temperature, the solvents were removed and the oily residue was
triturated with ether (500 mL), filtered, and dried to give an off-white
Triflate (23c). The triflate could be qualitatively confirmed by 19F
NMR δ −77.7 and 13C NMR δ 120.7 (q, J = 322 Hz). Because tosylate
could not be detected, a complete exchange to triflate was presumed.
Bromide (23d). The presence of bromide was not directly
confirmed. Because the tosylate, which was present in the crude
product, could not be detected, a complete exchange to bromide was
presumed.
Perchlorate (23e). The presence of perchlorate was not directly
confirmed. Because the tosylate, which was present in the crude
product, could not be detected, a complete exchange to perchlorate
was presumed.
Enantiomeric Stability under Basic Hydrolysis Conditions. Methyl
ester of compound 7 was hydrolyzed under conditions similar to the
hydrolysis reaction during radiosynthesis. For this, 20 mg of the
methyl ester, 26 mg of Cs2CO3, 5 mL pf DMF, and 100 μL of water
were combined in a reaction vial. Then, 1 mL of 4N NaOH was added
and the mixture was heated on an oil bath at 100 °C for 10 or 20 min.
The reactions were removed from the oil bath and neutralized with 4
mL of 1N HCl to pH = 7.5. Analysis on chiral HPLC confirmed that
the reactions were chemically clean and full conversion occurred at
both time points. The product eluted at 4.0 min, which corresponds to
R-enantiomer. The expected retention time for the S-enantiomer was
6.25 min.
Inhibitory Potency Determination (Fluorescent Assay). Recombi-
nant human full length MMP2 (902-MP, R&D Systems) or MMP9
(911-MP, R&D Systems) was chemically activated using p-amino-
phenylmercuric acetate (APMA) according to the manufacturer’s
protocol.21 To the activated enzyme (final concentration 0.1 nM, 24
μL) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl2, 150
mM NaCl, 0.05% Brij-35), compound of interest (in DMSO, suitable
concentrations, e.g., 1 nM to 30 μM, 1 μL) was added in a 384-MTP
white plate. Reaction was started by addition of internally quenched
substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH2 (final con-
centration 10 μM; Mca = (7-methoxycoumarin-4-yl)acetyl; Dpa(Dnp)
= N-3(2,4-dinitrophenyl)-L-2,3-diaminopropionyl, R&D Systems) to
yield a total volume of 50 μL. Progress of the MMP reaction was
monitored by fluorescence intensity measurement (excitation, 320 nm;
emission, 410 nm) over 120 min at 32 °C. IC50 values were
determined by plotting the compound concentration versus the
percentage of MMP activity by interpolation.
1
solid (18.5 g, 35.1 mmol, 92%). H NMR (400 MHz, DMSO-d6) δ,
ppm: 13.50 (br. s., 1 H), 8.34−8.25 (m, 2 H), 8.24−8.15 (m, 2 H),
8.04−7.94 (m, 2 H), 7.47 (d, J = 8.1 Hz, 2 H), 7.15−7.02 (m, 4 H),
3.80 (s, 3 H), 2.28 (s, 3 H). 13C NMR (101 MHz, DMSO-d6) δ, ppm:
166.1, 162.1, 145.8, 137.5, 137.4, 134.9, 133.6, 131.9, 128.0, 125.5,
121.3, 117.5, 105.4, 55.7, 20.8. MS (ES+): m/z 355.11 ([M]+)
(iodonium cation). MS (ES−): m/z 171.07 ([M]−) (tosylate anion).
Methyl N-{[4-({4-[(4-Methoxyphenyl)iodonio]benzoyl}amino)-
phenyl]sulfonyl}-D-tryptophanate Salts (23b−e). Aniline I (200
mg, 0.54 mmol), (4-carboxyphenyl)(4-methoxyphenyl)iodonium 4-
methylbenzenesulfonate 21 (423 mg, 0.8 mmol), and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.03 g,
5.4 mmol) were suspended in acetonitrile/pyridine (2:1, 30 mL),
and the reaction was stirred at room temperature for 4 days. The
resulted clear yellow solution was acidified to pH 1 using 2N aqueous
HCl. The solution was extracted with dichloromethane (3 × 200 mL),
and the organic phases were combined, dried (MgSO4), and
concentrated. The residue was dissolved in water/acetonitrile (1:1,
10 mL). Toluenesulfonic acid monohydrate (101 mg, 0.53 mmol) was
added, and the mixture was lyophilized (1H NMR indicated an excess
of tosylate in this material). The product (12 mg) was stirred in
dichloromethane, filtered, and dried to obtain the title compound as
approximately 35−40% tosylate salt based on 1H NMR. The
remaining 60−65% were assumed to be the corresponding chloride
salt. The yield, when calculated for both counterions together, was in a
1
range of 70−80%. H NMR (400 MHz, DMSO-d6) δ, ppm: 10.94−
10.81 (m, 1 H), 10.70 (s, 1 H), 8.41 (d, J = 8.6 Hz, 1 H), 8.30 (d, J =
8.3 Hz, 2 H), 8.19−8.09 (m, 2 H), 8.00−7.91 (m, J = 8.6 Hz, 2 H),
7.87−7.79 (m, J = 8.8 Hz, 2 H), 7.65−7.57 (m, J = 8.8 Hz, 2 H), 7.48
(d, J = 8.1 Hz, 0.8 H, tosylate), 7.26 (d, J = 7.8 Hz, 1 H), 7.29 (d, J =
8.1 Hz, 1 H), 7.13−6.99 (m, 5 H), 6.96−6.89 (m, 1 H), 3.96 (q, J =
7.6 Hz, 1 H), 3.78 (s, 3 H), 3.33 (s, 3 H), 3.04 (dd, J = 7.5, 14.3 Hz, 1
H), 2.88 (dd, J = 7.1, 14.4 Hz, 1 H), 2.28 (s, 1.1 H, tosylate). 13C
NMR (101 MHz, DMSO-d6) δ, ppm: 171.4, 164.9, 161.6, 145.7,
142.2, 137.6, 137.0, 137.0, 136.0, 135.1, 134.6, 130.3, 128.0, 127.3,
126.7, 125.5, 123.9, 123.1, 120.9, 119.6, 118.4, 117.6, 117.2, 111.5,
108.6, 108.3, 56.7, 55.6, 51.7, 28.2, 20.8. MS (ES+): m/z 710.22
([M]+) (iodonium cation). MS (ES−): m/z 171.03 ([M]−) (tosylate
anion).
18F-Fluoride Production. No-carrier-added (nca) 18F-fluoride was
produced via the 18O(p,n)18F nuclear reaction in a fixed-energy
Cyclone 18/9 cyclotron (IBA, Belgium). For this, >98% isotopically
enriched 18O-water (Nukem GmbH, Germany) was irradiated by 18
MeV proton beam. Produced 18F-fluoride/18O-water solution was
transferred using a helium stream from the target to a shielded hot cell
equipped with a manipulator where radiosynthesis was performed.
Typical production of 18F-fluoride at end of bombardment (EOB) of
the 2.5 mL target for 20 mAh (∼45 min) was 47−60 GBq.
Radiochemistry. 18F-fluoride (∼ 80−100 GBq) was trapped on an
anion exchange Sep-Pak Light Accell Plus QMA cartridge (Waters)
preconditioned with 5 mL of 0.5 M potassium carbonate solution, 10
mL of water, and flushed with 10 mL of air. It was eluted with 1 mL
Introduction of Counterions. The synthesis was conducted as
described above (scale: 2 g of aniline I). After completion of the
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Journal of Medicinal Chemistry
Article
solution of cesium carbonate (2.6 mg) in acetonitrile/water (1:1).
Effluent was collected to a tightly closed 5 mL Reacti-Vial (Thermo
Fisher Scientific), and solvent was evaporated under a stream of
nitrogen and reduced pressure (50−80 mbar) at 100 °C for 10 min.
The residue was azeotropically dried by addition of 3 × 0.8 mL
anhydrous acetonitrile. Precursor (22a−b, 23b−e) (∼2 mg, 2.5 0.2
μmol) predissolved in 500 μL of DMSO, containing 10 μL of water,
was added to the dry 18F-fluoride, and the reaction mixture was heated
at 100 °C for 3 min. After cooling for 5 min at room temperature, the
reaction mixture was diluted with 1 mL of water and loaded into
semipreparative HPLC. The radioactive fraction containing the
product was collected into 30 mL of water and passed through a
Sep-Pak Light C18 cartridge (preconditioned in advance with 5 mL of
ethanol followed by 10 mL of water). The cartridge was washed with
additional 5 mL of water for injection to remove traces of 18F-fluoride.
The product was eluted with 0.5 mL of ethanol to a penicillin vial. The
ethanol fraction was diluted with 9.5 mL of PBS and passed through a
13 mm Nalgene 0.2 μm sterile filter (Thermo Fisher Scientific) to a
sterile vial, giving an injectable sterile solution containing 5% ethanol
by volume, which was used for biological experiments. An aliquot of
known volume and radioactivity of the final formulated solution was
injected into analytical HPLC for quality control, which required
approximately 20 min. The retention time of the radiolabeled product,
18F-7, was in a range 10.67−10.72 min. The specific radioactivity was
determined from the quality control run matching the area of the UV
absorbance peak at 254 nm, which coeluted with the radiolabeled
product, to a standard calibration curve calculated using known
concentrations of the nonradioactive reference compound 7.
Evaluation of the Stability in Human and Rodent Plasma in
Vitro. An aliquot of 50 μL (∼60 MBq) of 18F-7 formulated solution
was added to human or rodent plasma (∼0.5 mL) and vortexed. The
mixture was distributed into six Eppendorf microcentrifuge tubes, 70
μL (∼7 MBq) to each. The first tube, which represented zero time
point, contained 70 μL of methanol. Five other tubes were incubated
at 37 °C in an Eppendorf Thermomixer Compact (Vaudaux-
Eppendorf) shaker. The samples were quenched with 70 μL of ice-
cold methanol, one by one, at 5, 30, 60, 90, and 120 min. Plasma
proteins were precipitated by centrifugation at 10000g for 10 min. The
supernatants were analyzed using UPLC.
AUTHOR INFORMATION
■
Corresponding Author
*For S.M.A.: phone, +41 44 633 7463; E-mail, simon.
ametamey@pharma.ethz.ch. For S.V.S.: phone, +41 44 633
7492; E-mail, svetlana.selivanova@pharma.ethz.ch, s.
selivanova@usherbrooke.ca.
Present Address
§L.M.D.: Piramal Imaging, Berlin, Germany.
Author Contributions
∥These authors contributed equally.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
MMP, matrix metalloproteinase; TIMP, tissue inhibitor of
metalloproteinase; PET, positron emission tomography; CT,
computed tomography; EOB, end of bombardment; EOS, end
of synthesis; RCY, radiochemical yield; SUV, standardized
uptake value; APMA, 4-aminophenylmercuric acetate; TFA,
trifluoroacetic acid, DMF, dimethylformamide; DMSO, dime-
thylsulfoxide; HPLC, high performance liquid chromatography;
UPLC, ultra performance liquid chromatography; NMR,
nuclear magnetic resonance; HRMS, high resolution mass
spectrometry
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ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and spectroscopic characterization of
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13C, and 19F NMR spectra of MMP inhibitor 7 and its labeling
precursor 23d. UPLC chromatograms of mouse blood and
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