Synthesis and evaluation of a [18F]BODIPY-labeled caspase-inhibitor

Article abstract of DOI:10.1016/j.bmc.2017.02.033

BODIPYs (boron dipyrromethenes) are fluorescent dyes which show high stability and quantum yields. They feature the possibility of selective ¹⁸F-fluorination at the boron-core. Attached to a bioactive molecule and labeled with [¹⁸F]fluorine, the resulting compounds are promising tracers for multimodal imaging in vivo and can be used for PET and fluorescence imaging. A BODIPY containing a phenyl and a hydroxy substituent on boron was synthesized and characterized. Fluorinated and hydroxy substituted dyes were coupled to an isatin-based caspase inhibitor via cycloaddition and the resulting compounds were evaluated in vitro in caspase inhibition assays. The metabolic stability and the formed metabolites were investigated by incubation with mouse liver microsomes and LC-MS analysis. Subsequently the fluorophores were labeled with [¹⁸F]fluorine and an in vivo biodistribution study using dynamic PET was performed.

Full text of DOI:10.1016/j.bmc.2017.02.033

Accepted Manuscript
Synthesis and evaluation of a [18F]BODIPY-labeled Caspase-inhibitor
Christian Paul Ortmeyer, Günter Haufe, Katrin Schwegmann, Sven Hermann,
Michael Schäfers, Frederik Börgel, Bernhard Wünsch, Stefan Wagner, Verena
Hugenberg
PII:
S0968-0896(16)31490-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.033
BMC 13563
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 December 2016
11 February 2017
14 February 2017
Please cite this article as: Ortmeyer, C.P., Haufe, G., Schwegmann, K., Hermann, S., Schäfers, M., Börgel, F.,
Wünsch, B., Wagner, S., Hugenberg, V., Synthesis and evaluation of a [18F]BODIPY-labeled Caspase-inhibitor,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.033
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Graphical Abstract.
Synthesis and evaluation of a [18F]BODIPY-labeled
Caspase-inhibitor
Leave this area blank for abstract info.
Christian Paul Ortmeyer, Günter Haufe,* Katrin Schwegmann, Sven Hermann, Michael Schäfers, Frederik Börgel, Bernhard Wünsch,
Stefan Wagner, Verena Hugenberg*
Departmental address here
ARTICLE INFO
ABSTRACT
BODIPYs (boron dipyrromethenes) are fluorescent dyes which show high stability and
quantum yields. They feature the possibility of selective 18F-fluorination at the boron-core.
Attached to a bioactive molecule and labeled with [18F]fluorine, the resulting compounds are
promising tracers for multimodal imaging in vivo and can be used for PET and fluorescence
imaging. A BODIPY containing a phenyl and a hydroxy substituent on boron was synthesized
and characterized. Fluorinated and hydroxy substituted dyes were coupled to an isatin-based
caspase inhibitor via cycloaddition and the resulting compounds were evaluated in vitro in
caspase inhibition assays. The metabolic stability and the formed metabolites were
investigated by incubation with mouse liver microsomes and LC-MS analysis. Subsequently
the fluorophores were labeled with [18F]fluorine and an in vivo biodistribution study using
dynamic PET was performed.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
BODIPY
Caspase inhibitor
Molecular imaging
18F-labeling
2016 Elsevier Ltd. All rights reserved.
Dual-function probe

Bioorganic & Medicinal Chemistry
Synthesis and evaluation of a [18F]BODIPY-labeled Caspase-inhibitor
Christian Paul Ortmeyera,b, Günter Haufeb,c*, Katrin Schwegmannd, Sven Hermannd, Michael Schäfersa,c,d
,
Frederik Börgele, Bernhard Wünsche, Stefan Wagnera, Verena Hugenbergf*
aDepartment of Nuclear Medicine, University Hospital Münster, Albert-Schweitzer-Campus 1, Building A1, D-48149 Münster, Germany
bOrganic Chemistry Institute, University of Münster, Corrensstr. 40, D-48149 Münster, Germany
cCells-in-Motion Cluster of Excellence, University of Münster, Waldeyerstr. 15, D-48149 Münster, Germany
dEuropean Institute for Molecular Imaging, University of Münster, Waldeyerstr. 15, D-48149 Münster, Germany
eInstitute for Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstr. 48, D-48149 Münster, Germany
fInstitute for Radiology, Nuclear Medicine and Molecular Imaging, HDZ NRW, Georgstr. 11, D-32545 Bad Oeynhausen, Germany
ARTICLE INFO
ABSTRACT
BODIPYs (boron dipyrromethenes) are fluorescent dyes which show high stability and quantum
yields. They feature the possibility of selective 18F-fluorination at the boron-core. Attached to a
bioactive molecule and labeled with [18F]fluorine, the resulting compounds are promising tracers
for multimodal imaging in vivo and can be used for PET and fluorescence imaging. A BODIPY
containing a phenyl and a hydroxy substituent on boron was synthesized and characterized.
Fluorinated and hydroxy substituted dyes were coupled to an isatin-based caspase inhibitor via
cycloaddition and the resulting compounds were evaluated in vitro in caspase inhibition assays.
The metabolic stability and the formed metabolites were investigated by incubation with mouse
liver microsomes and LC-MS analysis. Subsequently the fluorophores were labeled with
[18F]fluorine and an in vivo biodistribution study using dynamic PET was performed.
2016 Elsevier Ltd. All rights reserved.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
BODIPY
Caspase inhibitor
Molecular Imaging
18F-labeling
Dual-labeled probe
1. Introduction
Fluorescent probes based on boron dipyrromethene
(BODIPYs) are a subject of numerous areas of research.1,2 Their
unique properties make them excellent fluorophores for a lot of
different applications. Depending on the substituents on the
ligand, BODIPYs usually possess high quantum yields,3 variable
Stokes shifts[1,4] and offer different synthetic pathways to
influence the absorption and emission wavelength.5-7 These
comparatively apolar dyes often show high (photo)stability in a
broad range of pH and polarity values as well as low toxicity.
These features make them suitable for medicinal research and in
vivo applications.8 The main drawback is the often low solubility
in water and physiological liquids. The implementations so far
include chemo- and ionic sensors,9-17 fluorescent switches,18-20
laser dyes21-24 as well as protein and DNA labeling.2,25-33 Also the
possibility to radiolabel these dyes with fluorine-18 to get dual-
function probes has gained considerable interest.34-39 Most recent
publications using 19F/18F isotopic exchange reactions or
abstraction of one of the two fluorine atoms to yield radiolabeled
BODIPYs suffer from low specific activity or unsuitable emission
wavelengths for in vivo fluorescence imaging applications.
Figure 1. BODIPY-labeled caspase inhibitor 1.
The enzyme family of caspases (cysteinyl-aspartate-specific
proteases) is crucial for the regulation of apoptotic processes
within organisms.40 Dysregulation of apoptosis, or programmed
cell death, is related to many human diseases.41 Therefore this
group of enzymes represents an important target for in vivo
imaging applications. Apart from other approaches, small
molecules based on isatin have been extensively studied for in
vitro and in vivo applications.42-51 [18F]radiolabeling of isatins was
already successful using different approaches.52-63
2. Results and discussion
2.1. Chemistry and radiochemistry
Synthesis of BODIPY 9 started from p-hydroxybenzaldehyde
(2) by two substitution reactions via bromoethoxy compound 3
and 4-(2-azidoethoxy)-benzaldehyde (4) using literature-known
procedures.64,65 The second component, 2,4-dimethylpyrrole (7),
was synthesized from ethyl acetoacetate (5) using Paal-Knorr
pyrrole synthesis via the diester 6 followed by saponification and
decarboxylation to 7.66 Aldehyde 4 and pyrrole 7 were then
Herein we describe the synthesis of the first hydroxy- or
monofluoro-substituted BODIPY with an azido group, its
conjugation to an isatin-based caspase inhibitor to form
compound 1 (Figure 1), and first proof-of-principle investigations
with [18F]1 as a dual-function probe for imaging.

condensed, complexed in situ1 with dichlorophenylborane and
hydrolyzed to obtain compound 8 in 21% yield. BODIPY 8 was
fluorinated using potassium hydrogen fluoride to get fluorophore
9 in 92% yield. To obtain a high yield, activation using Lewis-
acids (preferably trimethylsilyl trifluoromethanesulfonate) was
necessary (Scheme 1). As an internal standard for the
determination of the metabolic stability of 1, a difluorinated
BODIPY-derivative 10 was prepared utilizing a literature known
procedure.65
derivative 11 were selectively labeled with [18F]fluoride in two
steps applying a modified method of Hudnall et al.67 under
previously optimized conditions to form [18F]9 and [18F]1,
respectively (Schemes 3 and 4).
Scheme 3: Radiosynthesis of BODIPY [18F]9.
Scheme 4: Radiosynthesis of dual-labeled probe [18F]1.
Compounds [18F]9 and [18F]1 were obtained in radiochemical
yields of 21% ± 2% (n = 4) and 11% ± 6% (n = 4) with both high
specific activity and radiochemical purity of 98% ± 2% (HPLC)
and 97% ± 2% (HPLC), respectively. The experimental logD
value (1.57) for [18F]1 is considered as medium lipophilic. The
compound is stable in human blood serum for at least 2 h.
Unfortunately, the emission wavelength of 1 (max,em = 509 nm) is
too short for fluorescence imaging in vivo. Currently, compounds
with increased emission wavelengths are rationally designed and
are under investigation.
Scheme 1: Synthesis of BODIPYs 8 and 9.
From N-propargylisatin ISA43 and the BODIPYs 8, 9 and 10,
the fluorescent-labeled isatin derivative 11, the target compound
1 and the reference compound 12 were prepared in copper-
catalyzed [3+2]-cycloaddition reactions (“click reactions”) with
yields of 34%, 38% and 52%, respectively (Scheme 2).
2.2. In vitro inhibition assay
In order to evaluate the effects of labeling, compound 1 was
compared with the lead compound ISA43 using an in vitro caspase
affinity test (see 4.4. In vitro enzyme inhibition assay). The
results are summarized in Table 1.
Table 1: Caspase inhibition potencies (IC50-values) of the lead
structure ISA and the probe 1 towards caspases-1, -3, -6, and -7.
IC50 (nM)
compound
caspase-1
>50000
11800 ± 9600
caspase-3
2.5 ± 1.5
34 ± 0.3
caspase-6
>50000
>50000
caspase-7
10 ± 0.1
422 ± 8
ISA
1
The isatin ISA shows high selectivity towards caspase-3 and -
7 with IC50-values in the nanomolar range. As expected, the
labeling with a rather sterically demanding and lipophilic
fluorophore BODIPY 9 resulting in inhibitor 1 led to an overall
diminished activity of the tracer (see Table 1). Future experiments
Scheme 2: Synthesis of labeled inhibitors 1, 11 and 12.
Bearing a phenyl and a hydroxy substituent on the boron
atom, the fluorophore 8 and the fluorescent labeled isatin

Figure 2: Distribution of radioactivity in an adult C57/Bl6 mouse after intravenous injection of [18F]1 visualized by small-animal PET.
Maximum intensity projections (MIP, upper row) of selected time frames display hepatobiliary excretion of [18F]1. Whole-body horizontal
slices from fused PET/CT imaging (one selected slice, lower row) allow for better anatomical correlation and localization of radioactivity.
Labeled structures are as follows: heart (ht), lung (lu), liver (lv), small bowel (sb), and gallbladder (gb).
60
60
liver
blood
50
50
kidney
40
40
intestine
bladder
30
30
lung
20
10
0
muscle
20
10
0
0
2
4
6
8
-10
10
10
30
50
70
90
time [min.]
Figure 3: Time-activity concentration (TAC) curves illustrating the clearance of [18F]1 from the blood due to hepatobiliary tracer elimination.
Control ROI in reference tissues (lung, muscle) show low levels of radioactivity according to their blood fraction but no tracer accumulation.

will show, whether the effect of labeling is less pronounced,
when spacer units are introduced between a bioactive moiety and
the fluorophore.
2.3. In vivo biodistribution studies
After intravenous injection of ~10 MBq of [18F]1 an in vivo
biodistribution study in adult C57/Bl6 mice was performed as a
90 min dynamic PET scan in combination with CT. Figure 2
shows representative maximum intensity projections (MIP) of
whole body images at selected time points after tracer injection
demonstrating notable and early accumulation of radioactivity in
the liver followed by transport of radioactivity into the intestines.
In contrast, no activity was observed in the kidneys and the
urinary bladder reflecting that renal elimination of [18F]1 is
negligible compared to hepatobiliary elimination of the tracer
and/or potential metabolites. In the first minutes after injection,
accumulation of radioactivity was observed in large vessels and
well perfused tissues such as liver, lung, and heart followed by a
continuously decreasing signal until the end of the study. Some
uptake of radioactivity in bone was observed, which likely
originates from free [18F]fluoride. Paulus et al. also observed for
18F-labelled BODIPYs an accumulation of radioactivity in bone,
which might indicate insufficient metabolic stability of the
boron-fluorine bond of the tracer in vivo.68 In all in vitro tests the
bond seemed to be stable. Selected ROIs were analysed
quantitatively using PET image data (Figure 3).
Figure 5: Representative FRI (fluorescence reflectance imaging)
images of dissected organs (vibratome sections) of a C57/Bl6 mouse.
Animals were sacrificed 2 h after intravenous injection of 2 nmol of
1 (A, white light image; B, FRI image overlay). C, Data of
corresponding FRI measurements after injection of 2 nmol of 1 (dark
grey bars) and untreated mice (light grey bars, autofluorescence).
Compared to the autofluorescence signal fluorochrome accumulation
was detected mainly in liver tissue. Lower to no intensity is found in
muscle, lung, and spleen. (1 brain, 2 heart, 3 muscle, 4 lung, 5 liver,
6 spleen, 7 kidney, 8 femur).
Time curves of regional radioactivity concentrations
expressed as percentage of injected dose per volume (% ID/mL)
reveal a rapid washout of [18F]1 from blood. In fact, blood levels
reach a peak 38 sec p.i. (36% ID/mL) with a subsequent
decrease. [18F]1 is taken up by the liver rapidly with a peak at 8
min p.i. (50% ID/mL). Thereafter, a slow and steady decrease of
radioactivity in the liver to 27% ID/mL at 90 min p.i. was
observed. The kidneys present an uptake of radioactivity to a
level of 3% ID/mL at 3 min p.i., which is maintained until the
end of the imaging study. Concurrently, almost no radioactivity
accumulated in the urinary bladder until 90 min p.i., excluding
the renal route as an elimination pathway of [18F]1. Further
regional analysis of brain, heart, spleen (data not shown), lung,
and muscle tissue reveal a similar distribution kinetics. After the
initial perfusion phase in the first 5 min the radioactivity
concentration remains constant over 90 min.
Finally, we analyzed the biodistribution of the non-
radioactive compound injected in healthy C57/Bl6 mice by
measuring the fluorescence intensity in various organs. Figure 5
(A + B, white light and FRI image overlay) shows the tracer
accumulation 2 hours after injection of 2 nmol per mouse. The
highest uptake of the optical tracer compared to the
autofluorescence signal of the organs was determined in liver
tissue, indicating an elimination that was mainly driven by
hepatic excretion in comparison to the lower uptake in the
kidneys.
2.4. Determination of metabolic stability
In consideration of the predominant hepatobiliary elimination
of 1, a metabolism study using mouse liver microsomes was
performed.69,70 Compound 1 was incubated in a buffered
suspension of mouse liver microsomes, NADPH/H+ and UDPGA
(UDP-activated glucuronic acid). The loss of tracer over time and
the formation of possible metabolites were monitored by LC/MS.
In order to investigate the stability in the presence of mouse liver
microsomes, 1 was incubated for different periods of time using
NADPH/H+ as cofactor. The concentration of 1 was determined
via external calibration using 12 as internal standard (ISTD).
Compound 1 and the ISTD 12 were measured as formate adducts
[M+HCOO]- in the negative scan mode.
After PET/CT imaging, mice were sacrificed, organs of interest
were harvested, and the specific uptake of the tracer was
determined by -counting, showing a similar distribution of the
radioactivity as observed by TAC analysis (Figure 4)
35
30
25
20
15
10
5
0
Figure 4: Quantitative ex vivo biodistribution data comparing
radioactivity in various major organs at the termination time point,
confirming the high uptake of [18F]1 in the liver found by PET in
vivo.

Further metabolites with similar structures as those detected by
Baumann et al. were not found for 1 in this study. In order to
confirm these results, ISA was treated in the same way as 1 with
mouse liver microsomes. The metabolites found for ISA are
shown in Scheme 6.
100
98
96
94
92
90
0
20
40
60
t [min]
80
100
120
Figure 6: Degradation of 1 by mouse liver microsomes over time
Figure 6 shows the remaining parent compound 1 during
incubation with mouse liver microsomes over a period of 120
min. This experiment indicates a high metabolic stability of 1,
since even after 120 min, more than 92% of 1 remained intact.
For comparison and to prove the activity of the microsome
preparation, imipramine was incubated with the same materials.
After 120 min only ~30% of the starting material was left
confirming the high activity of the microsomes.
Scheme 6: Detected metabolites of ISA.
In order to analyze metabolites of 1, LC was coupled with a
quadrupole-time-of-flight-MS (qTof) system, which allowed
identification and structure elucidation by analysis of the exact
masses of metabolites and their fragments.
Two monooxygenated metabolites ISA-Ia and ISA-Ib were
identified. ISA-Ia was formed by hydroxylation of the benzene
ring, since the ion [M-H]‾ was found in the negative scan mode.
The second monooxygenated metabolite ISA-Ib resulted from
oxidation of the pyrrolidine ring, as an ion [M-H]‾ was not found
in the negative scan mode. Additionally, the O-demethylated
metabolite ISA-Ic was observed. However, a metabolite
correlating with the hemiacetal 1a could not be detected.
3. Conclusions
The BODIPY derivatives 8 and 9 were prepared and used to
label the isatin-based caspase inhibitor ISA to get compounds 11
and 1. Both the fluorophore 8 and the labeled isatin 11 were 18F-
labeled in acceptable yields to get [18F]9 as well as the dual-
labeled probe [18F]1. An in vitro inhibition assay unveiled
slightly altered selectivity and diminished inhibitory activity
towards the target enzymes caspase-3 and caspase-7. First in vivo
experiments using PET/CT and FRI led to the conclusion that the
tracer is predominantly eliminated by a hepatobiliary pathway.
However, incubation with mouse liver microsomes and
NADPH/H+ indicated a rather high metabolic stability. Our
proof-of-principle investigation demonstrates the comparability
of results from different imaging modalities using a BODIPY-
based dual-labeled probe. In future work the fluorophores will be
altered to emit light in the near infrared region, attain an
increased water solubility and increase interaction with the target
enzymes.
Scheme 5: Postulated metabolite 1a.
After incubation of 1 with mouse liver microsomes,
NADPH/H+ and UDPGA only one metabolite 1a could be
detected by LC-MS (Scheme 5), which was also detected using
only NADPH/H+ as cofactor. Compared with the mass of 1, the
mass of the metabolite is higher by 18 (H2O), which should result
from addition of H2O or oxidation (+O) and reduction (+2H).
Scheme 5 shows the postulated structure of metabolite 1a. This
structure was postulated, as a similar metabolite was formed
during electrochemical oxidation of similar isatins.[71]
Furthermore, a metabolite with a mass higher by 16 (+O) or a
metabolite with the mass higher by 2 (+2H) could not be
detected. Due to low intensity of 1 and 1a online dilution method
was used to inject up to 100 µL of the sample, but the resulting
intensities were still too low for fragmentation experiments.
4. Experimental section
4.1. General
All chemicals were purchased in highest quality and used
without further purification. Dichloromethane for BODIPY-
synthesis was dried over calcium hydride and was distilled
freshly before use. Acetonitrile was dried over molecular sieves 3
Ǻ. Melting points were determined with a Stuart SMP 3
(STUART SCIENTIFIC) and are uncorrected. H, 13C, 11B and 19F
1
NMR spectra were measured on an AV400 (400 MHz) or an
AV300 (300 MHz) spectrometer (BRUKER) using TMS or solvent
signals as internal standard (11B NMR, 96.3 MHz, 19F NMR,
282.4 MHz). ESI-MS spectra were recorded on a MicroTof Mass

spectrometer (BRUKER DALTONICS). UV Spectra were recorded
on an U3010 Spectrometer (HITACHI). Fluorescence spectra
were recorded on a F4500 Spectrometer (HITACHI). Quantum
yields were determined in cooperation with the center for
nanotechnology on a HAMAMATSU PHOTONICS absolute PL
quantum yield measurement system (C9920-02), the software
U6039-05 PLQY (HAMAMATSU PHOTONICS) was used for
evaluation. Gas chromatography/EI spectra were recorded on a
GC/MS QP2010 Series (SHIMADZU). Analytical and semi-
preparative HPLC was conducted on two different systems:
4.2. Syntheses
4.2.1. 8-[4-(2-Azidoethoxy)phenyl]-4-hydroxy-4-phenyl-
1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (8)
2,4-Dimethylpyrrole (7) (0.861 mL, 8.4 mmol), 4-(2-
azidoethoxy)benzaldehyde (4) (0.800 g, 4.2 mmol) and molecular
sieve 3Ǻ (10 g) were added to carefully dried and degassed
dichloromethane (250 mL) and trifluoroacetic acid (1 drop) was
added. The mixture was stirred at RT for 12 h and then a solution
of 2,3,5,6-tetrachloro-1,4-benzochinone (p-chloranil, 1.030 g,
4.2 mmol) in dichloromethane (200 mL) was added quickly. The
deep-red suspension was stirred at RT for 15 min and
triethylamine (2.32 mL, 16.8 mmol) was added dropwise. The
mixture was stirred for additional 15 min before
dichlorophenylborane (1.63 mL, 12.6 mmol) was added
dropwise. After stirring for 24 h the mixture was filtered and
water (150 mL) was added. The layers were separated and the
organic layer was washed with water (3  300 mL), dried over
anhydrous sodium sulfate and solvents were removed under
reduced pressure. The residue was dissolved in a minimum
amount of trichloromethane and put on a small silica-pad. The
pad was eluted quickly with trichloromethane to pre-purify the
compound and to remove polymeric byproducts. The solvent was
removed under reduced pressure and the residue was purified by
column chromatography using neutral alumina (100 g,
6 cm  7 cm, 35 mL fractions, cyclohexane/ethyl acetate, 1:1,
v/v). All fluorescent fractions were combined, the solvents were
removed under reduced pressure and the residue was dissolved in
a minimum amount of dichloromethane. The solution was added
dropwise to an excess of cooled n-pentane (100 mL, -15°C) and
the precipitate was filtered off and dried in vacuum to obtain the
pure product as an orange solid. Yield: 0.408 g (21%) After this
purification procedure, the product can be recrystallized from
trichloromethane to obtain green-black, fluorescent crystals. Mp:
complete decomposition starting at 137 °C. 1H NMR (400 MHz,
A. Two Smartline 1000 pumps (KNAUER), a Smartline 2500 UV-
detector (KNAUER) and a GabiStar γ-detector (RAYTEST
ISOTOPENMESSGERÄTE). A Nucleosil 100-5 C18 analytical
column with the dimensions 4 mm х 250 mm was used.
B. A K-500 and a K-501 pump (KNAUER), a K-2000 UV-
detector (KNAUER) and a NaI(Tl) Scintibloc 51 SP51 γ-
detector (CHRISMATEC). A PHENOMENEX Gemini 5µm C18
column with the dimensions 10 mm х 250 mm was used.
The recorded data was processed by the GINA Star software.
(RAYTEST ISOTOPENMESSGERÄTE).
Column chromatography was performed using either silica
60 (40-63 μm, MERCK) or neutral Alumina (MP BIOMEDICALS
GERMANY GMBH).
No carrier added [18F]fluoride was generated on a RDS111e
cyclotron (CTI-SIEMENS) utilizing the 18O(p,n)18F reaction using
oxygen-18 enriched water and 10 MeV proton energy.
Radiosynthesis was conducted on a TRACERLab FxFDG
synthesizer (GE HEALTHCARE). Radiochemical yields are based
on the [18F]fluoride initial activity and are given decay corrected.
Radiochemical purity is the ratio of the fraction of product
radioactivity and total radioactivity and was determined by
analytical HPLC. Identification of radioactive products was
performed by co-injection of the non-radioactive reference
compound. Only solvents of pharmaceutical purity from ABX
and Milli-Q®-water or water for injection from B. BRAUN were
used for radiosyntheses. Following a method described by Prante
et al.,72 the distribution coefficient (logDexp) of the radioactive
compound was determined in a two-phase system consisting of
1-octanol and PBS-buffer (pH = 7.4) to determine the
lipophilicity. For this purpose, the corresponding ligand (~
20 kBq) was dissolved in buffer (500 μL). 1-Octanol (500 μL)
was added and the mixture was shaken at room temperature for
1 min. To achieve phase separation, the system was centrifuged
for 2 min at 3000 rpm. Subsequently a part of the octanol phase
(400 μL) was removed and buffer (400 μL) was added. The
combined phases were shaken and layers were separated
according to the description above. Aliquots of the buffer and
octanol layers (3 × 100 μL from every layer) were taken to
measure radioactivity in a -counter. The measurement provided
3
CDCl3): δ = 1.45 (s, 6H, CH3), 2.19 (s, 6H, CH3), 3.67 (t, 2H, J
3
= 5.0 Hz, CH2N3), 4.22 (t, 2H, J = 5.0 Hz, ArOCH2), 5.88 (s,
2H, ArpyrroleCH), 7.03 – 7.10 (m, 2H, Ar-CH), 7.10-7.17 (m, 2H,
Ar-CH), 7.18-7.24 (m, 2H, Ar-CH), 7.28-7.33 (m, 1H, Ar-CH),
7.37-7.44 (m, 2H, Ar-CH), B-OH not observed. 13C NMR (100
MHz, CDCl3): δ = 14.9, 15.4, 50.3, 67.1, 115.3, 121.2, 127.0,
128.5, 129.6, 129.8, 131.5, 132.1, 141.9, 155.6, 158.9. 11B NMR
(96 MHz, CDCl3): δ = 1.20 (s). MS (ESI): Calculated for
[C27H28BN5O2]Na+: 488.2228. Found: 488.2226. Calculated for
2[C27H28BN5O2]Na+: 953.4564. Found: 953.4573. UV: λmax
499 nm. FT: λmax,emission: 509 nm. QY: 22% ± 0.5% (acetonitrile).
:
4.2.2. 8-[4-(2-Azidoethoxy)phenyl]-4-fluoro-4-phenyl-1,3,5,7-
tetramethyl-4-bora-3a,4a-diaza-s-indacene (9)
A solution of trimethylsilyl trifluoromethanesulfonate in
acetonitrile (0.25 M, 13.75 mL, 3.4 mmol) was added to a
solution of compound 8 (80 mg, 0.2 mmol) in a mixture of
acetonitrile and dichloromethane (2:1, v/v, 15 mL). After 15 min
a solution of tert-butanol in acetonitrile (0.25 M, 13.75 mL,
3.4 mmol) was added at RT followed by solid potassium
hydrogendifluoride (134 mg, 1.7 mmol). After stirring at RT for
12 h water (20 mL) was added and organic compounds were
extracted with dichloromethane (3  40 mL). The combined
organic layers were dried over anhydrous sodium sulfate and
solvents were removed under reduced pressure. The residue was
purified by column chromatography with silica 60 (125 g, 13 cm
 5.5 cm, 50 mL fractions, cyclohexane/ethyl acetate, 2:1, v/v) to
obtain the pure product as a red solid. Yield: 74 mg (92%). Mp:
complete decomposition starting at 187 °C. 1H NMR (400 MHz,
the activity in counts per minute (cpm), the values were decay
corrected. By calculating the quotient of
the
logDexp can be determined as
.
The serum stability of radioactive compounds was evaluated
by incubation in human serum at 37 °C for up to 120 min. An
aliquot of radioactive product (20 μL, ~5 MBq) in PBS-buffer
was added to a sample of human serum (200 μL), and the mixture
was incubated at 37 °C. Samples of 20 μL each were taken after
periods of 10, 30, 60, 90 and 120 min and quenched in
MeOH/CH2Cl2 (1:1 (v/v), 100 μL) followed by centrifugation for
2 min. The clear solution was analyzed by analytical radio-HPLC
system A.
3
CDCl3): δ = 1.45 (s, 6H, CH3), 2.22 (s, 6H, CH3), 3.67 (t, 2H, J
3
= 5.0 Hz, CH2N3), 4.22 (t, 2H, J = 5.0 Hz, ArOCH2), 5.88 (s,

2H, ArpyrroleCH), 7.03 – 7.09 (m, 2H, Ar-CH), 7.10-7.17 (m, 2H,
Ar-CH), 7.18-7.24 (m, 2H, Ar-CH), 7.28-7.33 (m, 1H, Ar-CH),
7.37-7.44 (m, 2H, Ar-CH). 13C NMR (100 MHz, CDCl3): δ =
14.9, 15.4, 50.3, 67.1, 115.3, 121.2, 127.0, 128.5, 129.6, 129.8,
131.5, 132.1, 141.9, 155.6, 158.9. 11B NMR (96 MHz, CDCl3): δ
= 3.00 (s). 19F NMR (282 MHz, CDCl3): δ = -173.92 (s). MS
(ESI): Calculated for [C27H27BN5OF]H+: 468.2365. Found:
468.2376. Calculated for [C27H27BN5OF]Na+: 490.2185. Found:
490.2195. Calculated for 2[C27H27BN5OF]Na+: 957.4478. Found:
957.4497. UV: λmax: 500 nm. FT: λmax,emission: 509 nm. QY: 27% ±
0.5% (acetonitrile).
pyrrolidineCHb), 3.56 (dd, 1H, 2J = 9.4 Hz, 3J = 3.8 Hz, pyrrolidineCHb),
3.68-3.77 (m, 1H, pyrrolidineCH), 4.42 (t, 2H, 3J = 4.9 Hz,
3
NtriazoleCH2), 4.79 (t, 2H, J = 4.9 Hz, OCH2), 5.08 (s, 2H,
NtsatineCH2), 5.85 (s, 2H, ArpyrroleCH), 6.94 – 7.03 (m, 2H, ArCH),
7.10-7.17 (m, 2H, ArCH), 7.18-7.24 (m, 2H, ArCH), 7.28-7.33
3
(m, 1H, ArCH), 7.37-7.44 (m, 2H, ArCH), 7.56 (d, 1H, J = 8.3
4
Hz, ArisatineCH), 7.92 (s, 1H, ArtriazoleCH), 8.02 (d, 1H, J = 1.8
Hz, ArisatineCH), 8.09 (dd, 1H, 3J = 8.3 Hz, 4J = 1.8 Hz,
ArisatineCH). 13C NMR (100 MHz, CDCl3): δ = 14.8, 15.9, 24.2,
28.9, 35.7, 49.4, 50.1, 59.2, 59.3, 66.2, 74.9, 112.1, 115.0, 117.4,
121.5, 124.6, 127.0, 129.4, 129.8, 130.0, 131.5, 132.0, 134.4,
137.7, 140.8, 141.3, 141.4, 153.0, 155.6, 157.7, 158.1, 181.8. 11
B
NMR (96 MHz, CDCl3): δ = 3.01 (s). 19F NMR (282 MHz,
4.2.3. General procedure A for copper-catalyzed [3+2]-
cycloadditons (“Click-reaction”)
CDCl3):
δ
=
-173.86 (s). MS (ESI): Calculated for
[C44H45BFN7O6S]Na+: 852.3121. Found: 852.3137. Calculated
for [C44H45BFN7O6SCH3OH]Na+: 884.3383. Found: 884.3400.
UV: λmax: 500 nm. FT: λmax,emission: 509 nm. QY: 12% ± 0.5%
(acetonitrile).
(S)-5-{[2-(Methoxymethyl)pyrrolidin-1-yl]sulfonyl}-1-(prop-
2-yn-1-yl)indoline-2,3-dione (ISA) (86 mg, 0.236 mmol) and
BODIPYs 8 or 9 were dissolved in dimethylformamide (40 mL)
under an inert atmosphere. A suspension of a solution of
copper(II)sulfate pentahydrate (429 mg, 1.72 mmol) in water
(1.5 mL) and sodium ascorbate (426 mg, 2.15 mmol) in water
(1.5 mL) was added after 5 min (suspension must be
orange/white). After stirring at RT for 24 h dichloromethane
(200 mL) was added. The organic layer was washed with water
(4  100 mL), EDTA-solution (0.1 mol/L, 50 mL) and again with
water (100 mL). The organic layer was dried over anhydrous
sodium sulfate and solvents were removed under reduced
pressure. The residue was purified by column chromatography
with neutral Alumina (40 g, 12 cm  2 cm, 10 mL fractions,
cyclohexane/ethyl acetate, 1:1, v/v moving to pure ethyl acetate)
to obtain compounds 11 or 1, respectively.
4.3. Radiosyntheses
4.3.1. 8-[4-(2-Azidoethoxy)phenyl]-4-[18F]fluoro-4-phenyl-
1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene ([18F]9)
A solution of compound 8 (580 μg, 1.25 μmol) was dissolved
in
acetonitrile
(400 μL)
and
trimethylsilyl
trifluoromethanesulfonate (5.5 μL, 30 μmol, 24 equiv) was
added. The solution was shaken at RT for 15 min and added to
previously dried [18F]fluoride (540–4924 MBq) in
a
TRACERLab FxFDG synthesizer. The mixture was stirred for
5 min at 60 °C and purified on HPLC-system B using a
PHENOMENEX Gemini 5 µm C18 column (Rt: 19.8 min). The
product was obtained in radiochemical yields (rcy) of 21% ±
2.2% (d.c.) in 84 ± 13 min (n = 4). To verify high purity, the
isolated compound [18F]9 was injected to analytical HPLC-
system A (Rt = 8.9 min). The gradient was 10% to 90% CH3CN
in water (0.1% TFA) over 9 min, followed by a linear gradient
from 90% to 10% CH3CN in water (0.1% TFA) over 6 min with
λ = 254 nm and a flow rate of 1 mL·min-1. The radiochemical
purity was determined to be > 98% in all cases. To exclude the
possibility of [18F]fluoride contamination, additional TLC quality
control (QC) was performed (see SI). Usually to determine the
specific activity (AS) of a radiolabeled compound a UV
calibration curve of the corresponding non-radioactive
isotopomer is generated with defined concentrations using
analytical radio-HPLC. Then the concentration can be calculated
after HPLC quality control using the solution of the radiolabeled
compound, which also contains the corresponding carrier (the
19F-counterpart). With a known concentration the AS can be
determined. In the case of compound [18F]9 the corresponding
isotopomer 9 showed a detection limit of 0.8 μg/mL in the UV-
channel. Since UV-signals belonging to 9 were not occurring
during QC of the final preparation of [18F]9, the AS was
calculated to ≥ 514 GBq/μmol using the concentration of the
detection limit.
4.2.3.1. BODIPY-OH-labeled isatin 11
Applying the general procedure (4.2.3) compound 8 (100 mg,
0.215 mmol) was transformed to compound 11, which was
1
obtained as an orange solid. Yield: 60 mg (34%). H NMR (400
MHz, CDCl3): δ = 1.45 (s, 6H, Ar-CH3) 1.56-1.73 (m, 2H,
pyrrolidineCHa), 1.80-1.94 (m, 2H, pyrrolidineCHb), 2.22 (s, 6H, Ar-
CH3), 3.04-3.15 (m, 1H, pyrrolidineCHa), 3.34 (s, 3H, OCH3), 3.35
(dd, 1H, 2J = 9.4 Hz, 3J = 7.6 Hz, pyrrolidineCHa), 3.36-3.45 (m, 1H,
pyrrolidineCHb), 3.56 (dd, 1H, 2J = 9.4 Hz, 3J = 3.8 Hz, pyrrolidineCHb),
3.68-3.77 (m, 1H, pyrrolidineCH), 4.42 (t, 2H, 3J = 4.9 Hz,
3
NtriazoleCH2), 4.79 (t, 2H, J = 4.9 Hz, OCH2), 5.08 (s, 2H,
NisatineCH2), 5.85 (s, 2H, ArpyrroleCH), 6.94-7.03 (m, 2H, ArCH),
7.10-7.17 (m, 2H, ArCH), 7.18-7.24 (m, 2H, ArCH), 7.28-7.33
3
(m, 1H, ArCH), 7.37-7.44 (m, 2H, ArCH), 7.56 (d, 1H, J = 8.3
4
Hz, ArisatineCH), 7.92 (s, 1H, ArtriazoleCH), 8.02 (d, 1H, J = 1.8
Hz, ArisatineCH), 8.09 (dd, 1H, 3J = 8.3 Hz, 4J = 1.8 Hz,
ArisatineCH). 13C NMR (100 MHz, CDCl3): δ = 14.8, 15.6, 24.2,
28.9, 35.7, 49.4, 50.1, 59.2, 59.3, 66.2, 74.9, 112.1, 115.0, 117.4,
121.5, 124.6, 127.0, 129.4, 129.8, 130.0, 131.5, 132.0, 134.4,
137.7, 140.8, 141.3, 141.4, 153.0, 155.6, 157.7, 158.1, 181.8. 11
B
NMR (96 MHz, CDCl3): δ = 1.17 (s). MS (ESI): Calculated for
[C44H46BN7O7S]Na+: 850.3165. Found: 850.3144. Calculated for
[C44H46BN7O7SCH3OH]Na+: 882.3427. Found: 882.3394. UV:
λmax: 499 nm. FT: λmax,emission: 509 nm. QY: 6% ± 0.5%
(acetonitrile).
4.3.2. BODIPY-18F-labeled isatin ([18F]1)
A solution of compound 11 (490 μg, 0.59 μmol) was
dissolved in acetonitrile (400 μL) and trimethylsilyl
trifluoromethanesulfonate (5.5 μL, 30 μmol, 51 equiv) was
added. The solution was shaken at RT for 15 min and added to
previously dried [18F]fluoride (366–4835 MBq) in the automated
synthesizer. The mixture was stirred for 10 min at 60 °C and
purified on HPLC-System B using the a ACE 5 AQ column (Rt:
17.8 min). The product was obtained in radiochemical yields
(rcy) of 11% ± 6.1% (d.c.) in 91 ± 6 min ( n = 4). To verify high
purity the isolated compound [18F]1 was injected to analytical
4.2.3.2. BODIPY-F-labeled isatin 1
Applying the general procedure (4.2.3) compound 9 (98 mg,
0.210 mmol) was transformed to compound 1, which was
obtained as a red solid. Yield: 66 mg (38%). 1H NMR (400 MHz,
CDCl3): δ = 1.42 (s, 6H, Ar-CH3), 1.55-1.73 (m, 2H,
pyrrolidineCHa), 1.81-1.93 (m, 2H, pyrrolidineCHb), 2.23 (s, 6H, Ar-
CH3), 3.04-3.15 (m, 1H, pyrrolidineCHa), 3.34 (s, 3H, OCH3), 3.35
(dd, 1H, 2J = 9.4 Hz, 3J = 7.6 Hz, pyrrolidineCHa), 3.36-3.45 (m, 1H,

HPLC-system A (Rt = 12.0 min). The gradient was 10% to 90%
CH3CN in water (0.1% TFA) over 9 min, followed by a linear
gradient from 90% to 10% CH3CN in water (0.1% TFA) over
6 min with λ = 254 nm and a flow rate of 1 mL·min-1. The
radiochemical purity was determined to be 97% ± 2%. To
exclude the possibility of [18F]fluoride contamination, additional
TLC quality control was performed (see SI). The test of serum
stability was also performed on this system. In human blood
serum [18F]1 was stable for at least 120 min at 37 °C. The logDexp
value was determined to be 1.57. Specific activity had to be
calculated similar to preparations of [18F]9 due to concentrations
below the detection limit. In the case of compound [18F]1 the
corresponding isotopomer 1 showed a detection limit of
0.8 μg/mL in the UV-channel at different wavelengths. Since
UV-signals belonging to 1 were not occurring during QC of the
final preparation of [18F]1, the AS was calculated to
≥ 166 GBq/μmol using the concentration of the detection limit.
pump controlled saline flush (300 µL/min). After 90 min of PET
scanning, the scanning bed was transferred to the computed
tomography (CT) scanner (Inveon, Siemens Medical Solutions,
U.S.) and a CT acquisition with a spatial resolution of 80 µm was
performed for each mouse. Reconstructed image data sets were
co-registered based on extrinsic markers attached to the
multimodal scanning bed and the in-house developed image
analysis software MEDgical. Three-dimensional volumes of
interest (VOIs) were defined over the respective organs in CT
data sets, transferred to the co-registered PET data and analyzed
quantitatively. Regional uptake was calculated as percentage of
injected dose by dividing counts per milliliter in the ROI by total
counts in the mouse multiplied by 100 (% ID/mL).
After PET imaging, mice were sacrificed by cervical
dislocation and organs of interest were harvested, weighted and
counted using a Wizard2 automatic γ-counter (PERKIN ELMER).
The percentage of tracer uptake stated as percentage injected
dose per gram of tissue (% ID/g) was calculated as the activity
bound to tissue per actual injected dose per organ weight, decay-
corrected to the start time of counting.
4.4. In vitro enzyme inhibition assay
An in vitro inhibition assay was performed to determine the
effect of the BODIPY-labeling on both inhibition potency and
selectivity. The inhibitory activity of the lead compound ISA and
compound 1 was evaluated as IC50 values towards caspases-1, -3,
-6, and -7. Recombinant human caspases and their substrates
were purchased from ALEXIS BIOMEDICALS (Switzerland). As
described previously,47 reaction rates showing the inhibitory
activity were determined by measuring the accumulation of the
cleaved fluorogenic product AMC (7-amino-4-methylcoumarin)
with a TriStar2 Multimode Reader (BERTHOLD TECHNOLOGIES) at
excitation and emission wavelengths of 360 and 460 nm,
respectively. All assays were performed in a volume of 200 µL at
37°C in reaction buffer containing the non-radioactive
compounds in DMSO each in single doses (500 µM, 50 µM, 5
µM, 500 nM, 50 nM, 5 nM, 500 pM, 50 pM, or 5 pM).
Recombinant caspases were diluted into the appropriate buffer to
a concentration of 0.5 units per assay (= 500 pmol substrate
conversion after 60 min). After incubation for 10 min, the peptide
substrate (10µM) was added and reacted for further 10 min. The
IC50 values were determined by nonlinear regression analysis
using the XMGRACE program (Linux software).
4.7. Fluorescence reflectance imaging (FRI)
For Fluorescence Reflectance Imaging (FRI) mice were
anesthetized by isoflurane/O2, and the BODIPY-labeled
compound 1 (2 nmol) was injected into the tail vein. Two hours
after injection mice (n = 3) were sacrificed and organs were
rapidly removed and cut on a Vibrating Blade Microtome
VT1200S (LEICA) at 500 μm thickness and placed on a petri dish.
FRI was performed using the IVIS Spectrum small-animal
imaging system (PERKIN ELMER). Excitation was achieved at 465
nm, and emitted light was filtered by a bandpass filter (540 nm).
The following settings were chosen: binning: 1, f-stop: 2, field of
view: B (6.6 cm), exposure time: 120 sec. Images were analyzed
with the software Living Image 4.2.0 (PERKIN ELMER). Grayscale
photographic images and fluorescence color images were
superimposed. Regions of interest (ROIs) were drawn over the
tissue samples using the photographic images. Fluorescence
emission was measured by fluorescence emission radiance per
incident excitation irradiance (p/sec/cm2/sr/μW/cm2).
4.8. Metabolic stability
4.5. Animals
Preparation of DMSO stock solutions
Eleven- to fourteen-week-old C57/BL6 mice (21-30 g body
weight) were used for all experiments. Animal studies were
approved by the office for environment, nature and municipal
affairs of the land of North Rhine-Westphalia, Germany.
A small amount (1.5-5 mg) of each compound was weighed
in an Eppendorf cup on a XP26 Delta Range® (METTLER
TOLEDO) weighing machine. An exact amount of DMSO was
added to prepare the DMSO stock solutions with a defined
concentration. The concentrations of the solutions were 10 mM,
if not stated otherwise.
4.6. Small Animal PET/CT Scanning
For PET/CT studies anaesthesia was induced by placing the
mice (n = 3) in an anaesthesia induction chamber filled with 3%
isoflurane in air until the animal was completely anaesthetized.
Then, the mice were transferred to a heated PET scanner bed,
isoflurane anaesthesia was maintained (1.5 - 2% in oxygen) and
biosignals (respiration, ECG, body temperature) were monitored.
A catheter (27G diameter) was placed in one of the tail veins,
flushed with saline (50 µL) and connected to the injection line. In
order to prevent the eyes of running dry they were moistened
with eye salve. The setup for radiotracer injection consists of a
tail vein catheter, a 100 µL reservoir filled with the radiotracer
[18F]1 (9.2-9.9 MBq) and an injection pump holding a syringe
filled with saline. For PET imaging (32 module quadHIDAC,
Oxford Positron Systems Ltd., Oxford, UK) the scanner bed was
automatically positioned in the centre of the camera field-of-view
and PET list mode acquisition was started. Simultaneously the
radiotracer reservoir was injected into the mouse by an injection
Preparation of aliquots
Aliquots of the cofactors NADPH and UDPGA were
prepared by weighing an exact amount of each cofactor in an
EPPENDORF cup on a XP26 Delta Range® (METTLER TOLEDO)
weighing machine. They were stored at -20 °C prior to use and
solved in an exact amount of PBS (0.1 M, pH 7.4) to give the
desired concentration.
Preparation of mouse liver microsomes
Deep Frozen livers (-80 °C) of C57/BL6 mice were obtained
from the European Institute for Molecular Imaging (Münster,
Germany). Livers (30 g) were thawed in 1.15 % (m/v) potassium
chloride solution at 4 °C and cut in slices and homogenized in an
Elvehjem-Potter (10 strokes, 3 s) with cold phosphate buffer
(30 mL, pH 7.4, 0.1 M) containing sodium EDTA (0.5 mM).
Cold sodium phosphate buffer (90 mL, pH 7.4, 0.1 M) was added

and the resulting suspension was centrifuged at 9,000 g for 20
min at 4 °C. The supernatant was centrifuged at 45,000 g for
90 min. The resulting microsome pellet was suspended in sodium
phosphate buffer (pH 7.4, 0.1 M). Aliquots were stored at -80 °C
prior to use.
volume of 100 µL sample online dilution was used. Therefore a
second pump with an aqueous solvent was added to the method,
to dilute the solvent of the first pump in a ratio of 1:4. For online
dilution, the calibration window was set at 2.0 – 2.2 min.
Detailed LC/MS gradients are described in the supporting
information.
Determination of the protein concentration
The determination of the protein concentration of the mice
liver microsomes was done according to Bradford.[73]
Coomassie® Brilliant Blue G 250 (5 mg) was dissolved in abs.
ethanol (2.5 mL). Dist. water (10 mL) and phosphoric acid
(5 mL) were added. The solution was diluted with dist. water to
50 mL. The resulting solution was stored under darkness and at 4
°C overnight. Before the experiment, the solution was filtered
twice through paper filters. A stock solution of BSA in dist.
water (10 mg/mL) was prepared. A multi-point calibration curve
(0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL,
0.5 mg/mL) was performed by dilution of the stock solution with
dist. water. The samples were diluted 20-fold (50 μL microsome
solution, 200 μL 1 M NaOH, 750 μL dist. water) and 50-fold
(20 μL microsome solution, 200 μL 1 M NaOH, 780 μL dist.
water). The measurements were performed in a 96-well plate. To
10 μL of diluted sample or 10 μL of calibration solution 190 μL
Bradford solution were added. The plate was shaken for 5 min
and the absorption was recorded at 595 nm. Sample and
calibration solutions were prepared in triplicate and measured
once.
4.9. Microsomal stability
LC/MS method
UPLC-UV/MS (Agilent Technologies): Precolumn: Zorbax
Eclipse Plus-C18 (2.1  12.5 mm, 5 μm particle size), Main
column: Zorbax SB-C18 (2.1  50 mm, 1.8 μm particle size),
Degasser: 1260 HiP (G4225A), Pump: 1260 Bin Pump
(G1212B), Autosampler: 1260 HiP ALS (G1367E), Column
Oven: 1290 TCC (G1316C), MS-Detector: 6120 Quadrupol
LC/MS (G1978B). MS Source: Multimode source.
The alignments of the capillaries from the column oven were
changed, so that the six-port-valve which normally switches
between two columns was used as a divert valve to protect the
mass spectrometer from salts of the sample. After 2.0 min the
valve was switched from “waste” to “MS-source”. At the end of
a single run the valve was switched to “waste”. The Multimode
source was running only in the ESI mode. The quadrupole was
running in the SIM mode. The calibration of the quadrupole was
achieved by injection of APCI/APPI Tuning Mix (G2432A,
AGILENT TECHNOLOGIES) once a week. Detailed LC/MS
gradients and further information are described in the supporting
information.
Incubation procedure for 1 with mouse liver microsome
suspension
Optimization of fragmentor voltage and gas temperature:
FIA was used to determine the optimum value of the gas
temperature and the fragmentor voltage for each compound. For
this purpose, solutions consisting of 990 μL of bidist.
H2O/CH3CN 1:1 and 10 μL of the 10 mM DMSO stock solutions
were prepared. 1.0 μL of each solution was injected varying the
values for the fragmentor voltage or the drying gas temperature.
The highest area under the curve (AUC, SIM mode) for each
analyte indicated the optimum value.
1 μL of the DMSO stock solution (10 mM) of the respective
compound was added to 73 μL of PBS (pH 7.4, 0.1 M), 50 μL of
magnesium chloride solution (50 mM), 26 μL of microsome
suspension (mouse liver, 7.8 mg/mL protein), 50 µL NADPH
solution (2 mg/mL in PBS) and 50 µL UDPGA solution
(2 mg/mL in PBS). The suspension was mixed vigorously and
incubated (90 min, 900 rpm, 37 °C). The incubation was stopped
by addition of 400 μL of acetonitrile:methanol (1:1). The
EPPENDORF cups were cooled down to 0 °C for 10 min using a
water/ice bath. The precipitated proteins were separated via
centrifugation (15 min, 16000 rpm, 4 °C) and the supernatant was
analyzed with the LC/MS method 2. With the same procedure,
the empty value (without stock solution) and the blank value
(without NADPH and UDPGA) were prepared.
MS parameter:
ESI SIM mode: m/z 874 (1) and m/z 816 (12), Vaporizer
temperature: 200 °C, drying gas: 12 L/min, nebulizer pressure:
35 psi, VCap: 4000 V, fragmentor voltage: 150 V, drying gas
temperature: 180 °C.
LC/MS – method
For the determination of exact masses and MS/MS
experiments an LC system was coupled with a qTOF.
Materials:
1.5 mL tubes (EPPENDORF), phosphate buffered saline tabs
(SIGMA
ALDRICH),
rat liver microsomes, methanol
HPLC-DAD: Solvent rack (SRD 3600), Pump (DGP-3600RS),
Autosampler (WPS-3000RS), Column oven (TCC-3000RS),
DAD-detector (DAD-3000RS) operating at 230 and 250 nm. The
LC system was coupled with a microOTOF-Q II (BRUKER
DALTONICS). The ESI-qTOF was operated in positive and
negative ion polarity in the full scan mode (m/z = 70 – 700) with
the following settings: capillary 4500 V; end plate offset -500 V;
collision cell RF 300.0 Vpp; nebulizer 2.0 bar; dry heater 200 °C;
dry gas 9.0 L/min. In case of MS/MS experiments the isolation
window of the first quadrupole was set to 10 m/z units. The
collision energy of the second quadrupole was 35 eV. Data
handling and control of the system were realized with the
software DataAnalysis and Hystar (BRUKER DALTONICS). The
calibration of the TOF spectra was achieved by injection of 10
mM lithium formate (isopropanol:bidest. water 1:1) via a 20 uL
sample loop within each LC run at 1 – 1.2 min for calibration in
the range of m/z = 100 – 500. For the range of m/z = 500 – 1000
sodium formate was used as the calibrant. For an injection
(Chromasolv® LC-MS grade, FLUKA), acetonitrile (LC-MS
grade, FISHER SCIENTIFIC), DMSO (FISHER SCIENTIFIC),
centrifuge 5427 R and thermomixer comfort (EPPENDORF),
Incubation procedure of 1 for microsomal stability:
72.5 µL PBS (pH 7.4, 0.1 M), 50 µL MgCl2 solution
(0.05 M) were added to an EPPENDORF cup. After adding 50 µL
of the cofactor NADPH (2 mg/mL in PBS) and 1.5 µL of the
10 mM DMSO stock solution of 1 was added, the mouse liver
microsomes (26 µL, 7.8 mg protein/mL) were pipetted into the
cup. The suspension was mixed vigorously and incubated for
different periods of time (t = 5, 10, 15, 30, 60, 90 and 120 min,
900 rpm, 37°C). The incubation was stopped by addition of 400
μL of acetonitrile/methanol (1:1) containing 12 as internal
standard (2 µM). The EPPENDORF cups were cooled down to 0°C
for 10 min. The precipitated proteins were separated via
centrifugation (15 min, 16000 rpm, 0 °C) and the supernatant was
analyzed via LC/MS. With the same procedure, the empty value

(without stock solution) and the blank value (without NADPH)
were prepared.
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The calibration curve was obtained by the same procedure as
described above, but instead of the incubation the calibration
samples were quenched by addition of 400 μL of
acetonitrile/methanol (1:1) containing internal standard 12
(2 µM) directly after the addition of the rat liver microsomes.
Different concentrations were achieved by adding 1.5 µL of a
DMSO solution, which was prediluted of the 10 mM DMSO
stock solution. In this way, the final DMSO concentration and the
rest of the matrix of the calibration points match with the
samples. The calibration point, with a final concentration of
10 µM (40% parent) was used as the quality control (QC), with
the mistake being 1.69%.
The MS was used in SIM mode with traces m/z 874 for 1 and
m/z 816 for 12. For each incubation time and each calibration
point three samples were prepared. The injected volume was
15 µL and each sample and calibration point were measured in
triplicate.
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Acknowledgements
Financial support by the Deutsche Forschungsgemeinschaft
(DFG, collaborative research center 656 ‘Molecular Cardio-
vascular Imaging’, projects B01 and Z05) is gratefully
acknowledged. Compound ISA was prepared by Dr. Panupun
Limpachayaporn and Dr. Andreas Faust and was allocated for the
use in the present investigation.
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Supplementary data
Supplementary data (experimental procedures, spectroscopic
data for compounds 3, 4, 6, 7, 10 and 12 and copies of the NMR
spectra of all new compounds can be found, in online version, at
http://dx.doi.org/10.1016/j.bmc.2016 ...
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