Radiofluorinated N-Octanoyl Dopamine ([18F]F-NOD) as a Tool to Study Tissue Distribution and Elimination of NOD in Vitro and in Vivo

Article abstract of DOI:10.1021/acs.jmedchem.6b01191

To mitigate pretransplantation injury in organs of potential donors, N-octanoyl dopamine (NOD) treatment might be considered as it does not affect hemodynamic parameters in braindead (BD) donors. To better assess optimal NOD concentrations for donor treat

Full text of DOI:10.1021/acs.jmedchem.6b01191

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Article
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Radiofluorinated N-Octanoyl Dopamine ([F]F-NOD) as tool to study
tissue distribution and elimination of NOD in vitro and in vivo
Marc Pretze, Prama Pallavi, Mareike Roscher, Sarah Klotz, Julio Caballero,
Uta Binzen, Wolfgang Greffrath, Rolf-Detlef Treede, Martin C. Harmsen,
Mathias Hafner, Benito Yard, Carmen Wängler, and Björn Wängler
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01191 • Publication Date (Web): 12 Oct 2016
Downloaded from http://pubs.acs.org on October 13, 2016
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Radiofluorinated N-Octanoyl Dopamine ([ F]F-
NOD) as tool to study tissue distribution and
elimination of NOD in vitro and in vivo.
1
*
2,5
1
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Marc Pretze ‡ , Prama Pallavi ‡, Mareike Roscher , Sarah Klotz , Julio Caballero ,Uta
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Binzen , Wolfgang Greffrath , Rolf-Detlef Treede , Martin C. Harmsen , Mathias Hafner ,
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1*
Benito Yard , Carmen Wängler , Björn Wängler
1:
Molecular Imaging and Radiochemistry, Department of Clinical Radiology and Nuclear
Medicine, Medical Faculty Mannheim of Heidelberg University, Theodor-Kutzer-Ufer 1-3,
2
:
68167 Mannheim, Germany; V. Department of Medicine, University Hospital Mannheim,
3
:
Medical Faculty Mannheim of Heidelberg University, Mannheim, Germany;
Center for
Bioinformatics and Molecular Simulations, Faculty of Engineering in Bioinformatics,
4
:
Universidad de Talca, Talca, Chile, Division of Neurophysiology, Centre of Biomedicine and
Medical Technology Mannheim (CBTM) , Medical Faculty Mannheim of Heidelberg University,
5:
Mannheim, Germany;
University of Groningen, University Medical Centre Groningen,
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:
Department of Pathology and Medical Biology, The Netherlands; Institute for Molecular and
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:
Cellular Biology, Mannheim University of Applied Sciences, Mannheim, Germany;
Biomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine, Medical
Faculty Mannheim of Heidelberg University.
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KEYWORDS (Dopamine, NOD, Fluor-18, radiolabeling)
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Abstract
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To mitigate pre-transplantation injury in organs of potential donors N-octanoyl dopamine (NOD)
treatment might be considered as it does not affect hemodynamic parameters in braindead (BD)
donors. To better assess optimal NOD concentrations for donor treatment we report on the fast
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and facile radiofluorination of the NOD-derivative [ F]F-NOD [ F]5 for in vivo assessment of
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NOD’s elimination kinetics by means of PET imaging. [ F]5 was synthesized with reproducible
high radiochemical yield and purity (>98%) as well as high specific activities (>20 GBq/µmol).
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Stability tests showed no decomposition of [ F]5 over a period of 120 minutes in rat plasma. In
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vitro, low cell association was found for [ F]5 indicating no active transport mechanism into
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cells. In vivo, [ F]5 exhibited a fast blood clearance and a predominant hepatobiliary
elimination. As these data suggest that also NOD might be cleared fast, further pharmacokinetic
evaluation is warranted.
Introduction
Current evidence suggests that low dose dopamine treatment of braindead (BD) donors
1
improves transplantation outcome in recipients of renal and heart allografts. Although the
salutary effect of donor dopamine treatment has not been completely delineated, it has been
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suggested that the hemodynamic properties of dopamine do not play an essential role herein. In
vitro studies have identified three putative mechanisms by which dopamine may convey its
protective properties in transplantation settings. Firstly, dopamine can protect cells against cold
3,4
inflicted injury as tested in vitro on endothelial cells, renal epithelial cells and cardiomyocytes.
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Secondly, dopamine is able to activate the transcription factor Nrf2 which regulates the
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expression of the tissue protective protein Heme oxygenase 1 (HO-1). Thirdly, dopamine has
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anti-inflammatory properties, reflected by down-regulation of adhesion molecules and
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chemokines.
Even though both prospective and retrospective studies have advocated the use of dopamine in
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BD donors as a meaningful modality to improve transplantation outcome
, it has not been
implemented in general guidelines for donor management. Currently dopamine is used in donor
management to stabilize blood pressure, albeit in clinical practice (nor)adrenaline is more
frequently used for blood pressure stabilization of BD donors. In approximately 12% of treated
donors dopamine treatment needs to be discontinued as a consequence of tachycardia or
hypertension. Moreover, the potential impact of inotropic agents on cardiomyocytes, i.e.
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increased cytosolic calcium concentration and opening of the mitochondrial permeability
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transition pore , may raise further concerns on the use of such agents in donor management.
This underscores the need for dopamine-like compounds that are devoid of hemodynamic action
and yet have a similar or higher efficacy to protect organ allografts from pre-transplantation
injury. By N-acylation of dopamine, which largely abrogates adrenoceptor and dopaminergic
receptor binding, a class of synthetic compounds was developed lacking hemodynamic actions
1c, 4b
while retaining the protective properties of dopamine.
As representative of N-acyl dopamines
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we have shown that N-octanoyl dopamine (NOD) is superior to dopamine in vitro
and is
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highly effective in vivo to protect rat kidneys from ischemia induced acute kidney injury (AKI).
The latter seems to be a consequence of the interaction with the transient receptor potential
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-/-
cation channel subfamily V member 1 (TRPV1) as NOD was not renoprotective in TRPV1
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rats. Recently it was also demonstrated that NOD treatment of BD donor rats reduced
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inflammation in the donor kidney and improved renal function when such grafts were
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transplanted into allogeneic recipients.
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Although these pre-clinical findings are promising tissue distribution and pharmacokinetics of
NOD have not been addressed so far. As a first step we developed in the present study a NOD
based radiotracer to study the in vivo tissue distribution and elimination kinetics of NOD by
means of PET imaging.
In this work, we developed a fluorinated derivative of NOD, 2-fluoro-N-octanoyl dopamine 5
(
F-NOD) and compared the in vitro behavior of F-NOD with NOD in various assays in order to
prove whether F-NOD could act as an analogue to NOD. In brief, we compared the induction of
HO-1, redoxactivity, TRPV1 activation and EC50. Further we performed a catechol O-methyl
transferase (COMT) assay with NOD and F-NOD in order to investigate the cell intern
metabolism. We developed a precursor 10 for radiofluorination in order to obtain the radiotracer
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[ F]F-NOD. Next, we performed an in vitro cell assay with [ F]F-NOD at human umbilical
vein endothelial cells (HUVEC) for the determination of cell association. After this, we
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evaluated the in vivo behavior of radiolabeled [ F]F-NOD in three Lewis rats.
Results and discussion
Synthesis of F-NOD
F-NOD was synthesized from 3,4-dimethoxy-2-fluorobenzaldehyde 1 as outlined in Scheme
1
. It was successfully converted to the β-nitrostyrene derivative 2 by the method of Milhazes et
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al. To this end, nitromethane was used as solvent and ammonium acetate as base for generation
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of 2 (yield >81%), which was subsequently reduced with LiAlH to the corresponding amine 3.
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This was followed by introduction of an octanoyl group at the primary amine using octanoyl
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chloride in excess and selective removal of both methoxy groups with 5 eq. BBr at -12°C.
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Scheme 1. Synthesis pathway of the non-radioactive reference compound 5.
In vitro studies with F-NOD and NOD
Several in vitro studies were conducted to test if F-NOD 5 displayed similar relevant biological
properties as NOD 7, i.e. inhibition of TNFα mediated vascular cell adhesion molecule 1
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(
VCAM-1) expression, induction of HO-1, redox activity and the ability to activate TRPV1.
At higher concentrations both NOD 7 and F-NOD 5 inhibited VCAM-1 expression almost to a
similar extent, while at low concentrations F-NOD 5 seemed to be less effective. This was more
pronounced for induction of HO-1 (Figure 1A). We have previously demonstrated that the redox
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activity of NOD 7 is crucial for its ability to inhibit VCAM-1 expression. In essence this redox
activity is provided by the ortho dihydroxy moiety on the benzene structure. Since these
functional groups are also present in F-NOD 5, this largely explains its ability to inhibit VCAM-
1
expression and induce HO-1. We made use of peroxidase based luminol reaction, to assess if a
difference exists in the redox activity of the compounds Dose response experiments revealed
highly significant effects of concentration (F(15,559) = 194.42, p<0.001; two-way ANOVA,
fixed effects) as well as of substance F(1,559) = 26.78, p<0.001. Although both NOD 7 and F-
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NOD 5 were able to completely quench chemiluminescence, for NOD 7 (EC50 : 12.12 µM) it
was achieved at lower concentration as compared to F-NOD 5 (EC50 : 32.6 µM) (Figure 1B
Interaction: F(15,559) = 5.98, p<0.001 two-way ANOVA, fixed effects). This suggests a small
difference in redox activity between both compounds and might explain why F-NOD 5 is less
efficiently inhibiting VCAM-1 and inducing HO-1 expression at low concentrations. In
agreement with their redox activity it was found that both compounds were able to protect
endothelial cells against cold inflicted injury (Figure S1).
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Figure 1. In vitro F-NOD 5 shows similar biological activity as NOD 7: (A) Influence of F-NOD and NOD on
TNF mediated VCAM-1 expression and induction of HO-1. Cells were stimulated overnight with TNF 10 ng/ml
in the presence of different concentrations of F-NOD 5 or NOD (0–100 µM). Cell lysates were prepared and 15 µg
of total protein was loaded on a 10% SDS PAGE. The gel was blotted and stained for VCAM-1 and HO-1. Equal
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loading of protein was confirmed with ß-Actin. Note that at higher concentrations inhibition of VCAM-1 and
induction of HO-1 are comparable but not at lower concentrations to NOD in terms of its anti-inflammatory
properties. (B) Redox activity of NOD and F-NOD: F-NOD 5 and NOD 7 showed comparable ability to quench
luminescence in the luminol assay. Serial dilutions of NOD 7 or F-NOD 5 were added to 100 µL of reaction mixture
containing 0.005 U of horseradish peroxidase (HRP), 2.5 mM luminol and 0.9 mM p-coumaric acid.
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2 2
Chemiluminescence was measured directly after addition of 0.03% H O . The ability to quench peroxidase mediated
chemiluminescence was tested and results are expressed as relative to the control to which neither NOD nor F-NOD
were added along with ± SEM of all measurements. For each concentration and for each compound at least 3
independent measurements were performed. EC50 of NOD 7 was determined to be 12.12 µM and EC50 of F-NOD 5
32.60 µM indicated by dashed lines, *p<0.05, *** p<0.001 ANOVA with LSD post-hoc test.
The TRPV1 activating properties were assessed in a heterologous cell assay. F-NOD 5 like NOD
was able to activate TRPV1 (Figure 2A). Dose response experiments revealed highly
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significant effects of concentration (F (11,137) = 17.86, p<0.001; two-way ANOVA, fixed effects)
as well as of substance (F (1,137) = 12.38, p<0.001). Thus, both substances were able to activate
TRPV1 in a concentration dependent manner but NOD (EC = 7.10 µM [-5.15 ± 0.08]) was
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much more effective as compared to F-NOD (EC50 = 45.94 µM [-4.34 ± 0.12]) (Figure 2B).
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Figure 2. TRPV1 activation by F-NOD and NOD: (A) Representative images of a calcium imaging experiment
using rTRPV1 transfected HEK293 cells (Left panel). The ratio 340/380 at 510 nm – a measure proportional to free
intracellular calcium – is false color coded where warmer colors indicate increasing calcium values. Average time
course traces are shown on the right panel. Cells were first either stimulated with 10 µM NOD 7 or 10 µM of F-
NOD 5, followed by 10 µM capsaicin to select rTRPV1 transfected cells and to see maximum response after
washing away of the first substance; (B) rTRPV1 transfected HEK293 cells were stimulated with different
concentrations of NOD 7 or F-NOD 5: For each concentration and for each compound at least 3 independent
measurements were performed. The results are expressed as average ratio 340/380 at 510 nm ± SEM of all
measurements (n = 3 to 13). EC50 of F-NOD 5 was determined to be 45.94 µM and EC50 of NOD 7 7.10 µM; EC50
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values (NOD 7: 7.10 µM and F-NOD 5: 45.94 µM) indicated by dashed lines, *p<0.05, *** p<0.001 ANOVA with
LSD post-hoc test.
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It was further investigated via computational molecular modeling, whether F-NOD also
interacted with the TRPV1 binding pocket in similar fashion as NOD. No differences in putative
interactions with amino acids inside the TRPV1 binding pocket were found between NOD 7 and
F-NOD 5 (Figure S2). The 6-fold increase in EC value of F-NOD for TRPV1 activation may
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be a result of the insertion of fluorine as substituent in the aromatic ring. There, the fluorine
might exert an inductive (-I) effect, thereby reducing the negative charge density in the aromatic
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ring, which in turn may influence the ability of F-NOD to access the TRPV1 binding site.
Radiochemistry
For radiolabeling purposes we initially intended to synthesize the precursor 10 by introduction
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of a nitro group into NOD 7 which can be substituted by [ F]fluoride (Scheme 2). Although the
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synthesis of NOD 7 was successful , the subsequent nitration step to generate precursor 10
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failed under various conditions. This was most likely due to the acidic conditions during the
nitration, which caused the hydrolysis of the octanoyl group yielding only small amounts (<10%)
of nitro dopamine 8. Therefore, precursor 10 was synthesized from 5-nitrified dopamine 8, which
was subsequently reacted with an excess of octanoyl chloride in order to maximize the yield of 9
(
Scheme 2). The O-octanoyl groups of 9 were removed selectively by NaOH.
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Scheme 2. Synthesis pathway towards the precursor 10.
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The radiolabeling of precursor 10 (0.5–1.8 mg) was optimized and various reaction conditions
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were tested (reaction temperature of 20–120 C in CH
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CN, DMF or DMSO (0.5–1.0 mL) for 20–
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conditions, which were obtained with 1.5 mg 10 in 0.5 mL DMSO for 30 min reaction time at
20°C (radiochemical yield (RCY) = 45% (decay corrected – d. c.) (Figure 3A); specific activity
2 3
CO (Table 1)). Scheme 3 depicts the best radiolabeling
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21.9–34.5 GBq/µmol for [ F]F-NOD [ F]5). Purification was performed by SepPak C18 light
cartridges resulting in a radiochemical purity (RCP) of >99% and an absolute amount of
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radiotracer of 880 MBq after 90 min (starting from 3.43 GBq [ F]fluoride) (Figure 3B). The
identity of the product was checked by comparison of retention times on HPLC and TLC with
that of the reference compound 5.
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Scheme 3. Radiosynthesis scheme of radiotracer [ F]F-NOD ([ F]5).
Radiolabeling with acetonitrile as solvent resulted in a comparable RCY (38%, d. c.) but also in
the formation of a lipophilic side product (>29%). Removal of this side product was only
possible by semi-preparative HPLC which ended in prolonged overall synthesis time and lower
product activity. Radiolabeling utilizing DMF resulted in much lower RCY (6%, d. c.) (Table 1).
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reaction mixture and F-incorporation (t
R R
= 0.65 min) into [ F]F-NOD (t = 4.07 min) after 30 min at 120°C in
1
8
DMSO; (B) Representative analytical UV and radio-HPLC chromatograms of [ F]F-NOD (t
R
= 4.07 min) after
purification via a SepPak C18 light cartridge: γ-trace (thick line) is depicting the radiochemical purity >99%, UV-
trace at 210 nm (dashed line) is depicting the chemical purity >96%.
Table 1. Optimization of the radiolabeling of precursor 10 reaction time, temperature, solvent and concentration of
precursor.
RCP [%]
amount of
volume temperature
labeling
solvent
RCY [%]*
of crude
1
0 [mg]
[mL]
[°C]
time [min]
1
8
[
F]5
1.5
1.5
MeCN
MeCN
MeCN
MeCN
0.5
0.5
0.5
1.0
20
30
10
40
60
10
19
18
38
25
42
55
61
100
100
100
1
1
.8
.8
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.5
.5
.5
.5
DMF
0.5
0.5
0.5
0.5
100
110
100
120
40
40
40
40
6
65
83
58
69
DMSO
DMSO
DMSO
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33
45
10
11
12
13
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*
Unless otherwise stated RCYs are all corrected for decay
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Lipophilicity and in vitro stability tests of [ F]F-NOD
The lipophilicity of a pharmacologically active compound is a fundamental physicochemical
parameter. Hence, logP/logD determinations are of utmost importance and relevance to evaluate
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structure-activity relationships in medicinal chemistry as well as in radiopharmacy. The
18
lipophilicity (logD) of [ F]5 was determined at pH 7.5 to be 3.35 ± 0.04 (n = 3) using PBS and
2
4
18
n-octanol according to the shake-flask method (OECD guideline). Next, the stability of [ F]5
1
8
was evaluated in vitro. To this end, a solution of [ F]5 in PBS/3% EtOH (100 µl) was incubated
with rat plasma (200 µl) and shaken for at least 120 min at 37°C. Radio-TLC as well as radio-
HPLC analyses were performed after 30, 60 and 120 min, respectively, and showed no
1
8
decomposition or radiodefluorination of [ F]5 (RCP ≥ 98%) in the supernatant fraction, which
18
showed a recovery rate of [ F]5 of 77–85% after precipitation of the incubation solution
(
100 µl) with acetonitrile (100 µl) at 0°C. The precipitate contained 15–23% of the remained
activity.
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Cell uptake and stability studies
Cell uptake studies were performed with HUVEC to assess to what extent [ F]5 is taken-up by
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the cells, and if so whether it is further metabolized. As depicted in Figure 4A [ F]5 cell
associated activity was approximately 0.14–0.21% of the applied dose (ID) after 120 minutes of
incubation at 37°C. HPLC analysis of the cell lysate extracts revealed only one radioactive peak
1
8
18
R
at t = 4.07 min, corresponding to the intact [ F]5, indicating that [ F]5 was not metabolized
intracellularly within the time-frame of the experiment.
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25
In order to investigate the stability of [ F]5 in more detail, a catechol O-methyl transferase
2
6
18
(
COMT) assay was performed to assess if [ F]5 is a genuine substrate for COMT. COMT is a
ubiquity intracellular enzyme which deactivates catechols by addition of a methyl group to one
27
of the hydroxyl groups. The assay was optimized using dopamine as gold standard, which is
converted to 3-methoxythyramine (3-MT) (11) (Scheme 4).
Scheme 4. Synthesis pathway towards the O-methylated metabolite 12 of NOD in COMT assay.
Because of the high lipophilicity of both NOD and F-NOD, 10 µL of Tween20 was added and
higher concentrations of the methyl donor S-adenosyl-L-methionine (SAM) and COMT were
used with prolonged reaction times (120–240 min) in order to allow the formation of 3-MNOD
2
12 and (OMe) -F-NOD 4. Figure 4 displays the HPLC chromatograms of combined injections of
different NOD and F-NOD derivatives (Figures 4 B,D) and the performed COMT assay (Figures
4 C,E) at different time points.
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The conversion to the methylated derivatives proceeded slower in comparison to dopamine
which was totally converted to 3-MT after 120 minutes using low concentration of SAM and
COMT. Methylation of NOD and F-NOD occurred at both hydroxy groups resulting in double
methylated derivatives. The rate of conversion to the methylated derivatives of F-NOD was
slightly faster in comparison to NOD (see chromatograms C and E at 180 minutes in Figure 4).
This might be explained by the strong negative inductive effect of the fluorine.
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Figure 4. (A) Uptake of [ F]5 by endothelial cells over a period of 120 min at 37°C (values represent means ± SD
of three independent experiments); Representative chromatograms of: (B) NOD and its derivatives; (C) COMT
assay for NOD, indicating a new more lipophilic peak (16%) at the same retention time like 3-O-methoxy N-
octanoyl dopamine 12; (D) F-NOD and its methylated derivative; (E) COMT assay for F-NOD; HPLC condition: 0–
5
0% MeCN + 0.1% TFA within 8 minutes.
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In vivo evaluation of [ F]5
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For assessment of the elimination kinetics of [ F]5 in three Lewis rats, different amounts of
activity (4.8, 5.9 and 13.2 MBq) were injected to find the optimal dose for PET imaging which
18
was found to be 13.2 MBq. Elimination of [ F]5 was characterized by a fast blood clearance and
substantial renal and hepatobiliary elimination (Figure 5A) being comparable with other
2
8
lipophilic radiotracers. The activity is flowing through the kidneys, peaking between 5 and 10
minutes (SUVbw = 8 ± 5 g/mL) and being subsequently reallocated from the renal pelvis into the
bladder within 30 min. Most of the injected activity accumulated in the liver as early as 8 min
1
8
p.i. (SUVbw = 21 ± 10 g/mL) possibly because of the lipophilic nature of [ F]5. It seems
29
18
probable that a substantial oxidative defluorination of [ F]5 occurs in the liver by rat specific
3
0
dehalogenase
as reflected by a gradual accumulation of activity in bones starting at 60
minutes. After 120 minutes, most of the activity was found in the bladder (SUVbw = 38.5 g/mL)
and bones (SUVbw = 9.8 g/mL). Time-activity curves for different organs are given in Figure 5B.
The activity remains at a certain level (SUVbw = 8 g/mL) within the renal pelvis and slowly
eliminates into the bladder after 30 minutes. The liver activity is higher but also shows a faster
elimination than from the renal pelvis without formation of a plateau.
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Figure 5. (A) PET images (MIP - maximal intensity projection) of Lewis rat injected with 13.2 MBq [ F]5; (B)
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Distribution of [ F]5 within 120 min (n = 3). Most of activity accumulates in liver. Some of the activity remains at a
certain level inside the renal pelvis for 30 min.
Conclusion
In summary, we compared the in vitro behavior of F-NOD 5 with NOD 7 in various assays in
order to prove, whether F-NOD 5 could act as an analogue to NOD 7. The results of F-NOD
compared to NOD were similar at induction of HO-1, redoxactivity, TRPV1 activation and EC50
.
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NOD and F-NOD are both substrates for COMT. At both derivatives O-methylation takes place
but in comparison to dopamine, NOD and F-NOD exhibit a higher stability. This might be due to
the higher lipophilicity of 5 and 7, which hinders the O-methylation by the enzyme.
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These data suggested that the radiolabeled [ F]F-NOD could act as a tool for further
18
determination of the in vivo behavior of NOD. In vitro cell assay with [ F]F-NOD at HUVEC
revealed low cell association (0.14–0.21%), which means that there is no active transport
mechanism into the cell. The cell experiment suggests, that the concentration of NOD must be at
least 500 times higher outside the cell in order to achieve a certain concentration inside the cell
after 2 h.
1
8
The in vivo experiments revealed a fast renal and hepatobiliary clearance of [ F]F-NOD. Since
the concentration of the radiotracer is very low, the metabolism of such small doses can be faster
in comparison to a higher concentration of non-radioactive NOD given therapeutically. Due to
the fast clearance it is recommended to use a high concentration of NOD as bolus injection or
semi-high concentration via continuous injection in order to reach a renoprotective level in the
kidneys for transplantation purposes.
In conclusion, we herein report on the fast and facile radiofluorination of the NOD-derivative
1
8
18
[
F]F-NOD [ F]5 for in vivo assessment of NOD’s elimination kinetics by means of PET
imaging. We demonstrated that F-NOD 5 and NOD 7 behave similarly in different in vitro
1
8
assays and that both are substrates for COMT. [ F]5 was synthesized with reproducibly high
radiochemical yield and purity (>98%) as well as high specific activities (>20 GBq/µmol). After
a synthesis time of approximately 90 minutes, the radiotracer was ready for injection. Stability
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8
tests show no decomposition of [ F]5 over a period of 120 minutes in rat plasma. Fast blood
1
8
clearance and a predominant elimination by liver and kidneys were found for [ F]5 in vivo.
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Although these data suggest that also NOD 7 might be subject to a fast clearance, further
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pharmacokinetic evaluation is warranted. However, [ F]5 is useful for studying
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pharmacokinetics via biodistribution within a period of up to 30 minutes, due to the fast renal
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and hepatobiliary elimination and degradation in vivo between 30–60 min. [ F]5 might not be
suitable for elongated pharmacokinetic and biodistribution studies in rodents as it likely underlies
an oxidative defluorination after 60 min.
Experimental Section
General procedures. All reagents and solvents were purchased from commercial suppliers
and were used without further purification unless otherwise specified. NMR spectra were
recorded on a 300 MHz Varian Mercury Plus and a 500 MHz Varian NMR System spectrometer
(
Palo Alto, CA). Chemical shifts (δ) are given in ppm and are referenced to the residual solvent
1
13
19
resonance signals relative to (CH
3
)
4
3
Si ( H, C) and the internal standard CFCl ( F). Mass
TM
spectra were obtained on a Bruker Daltonics microflex
MALDI-TOF mass spectrometer
(
Bremen, Germany). Preparative column chromatography was performed on Merck silica gel 60.
Reactions were monitored by thin-layer chromatography (TLC) on Merck silica gel F254
aluminum plates, with visualization under UV (λ = 254 nm) or by ninhydrin staining. If
necessary, the purity was determined by high performance liquid chromatography (HPLC).
Purity of all final compounds was 95% or higher. Analytical (radio-)HPLC was performed on a
Dionex UltiMate 3000 HPLC system (Thermo Scientific, Dreieich, Germany), equipped with a
reverse phase column (Merck Chromolith RP-18e; 100x4.6 mm plus a guard column
1
0x4.6mm), a UV-diode array detector (210 nm, 254 nm) and a scintillation radiodetector Gabi
Star (Raytest, Straubenhardt, Germany). The solvent system used was a gradient of
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acetonitrile:water (containing 0.1% TFA) (0–8 min: 0–100% MeCN) at a flow rate of 4 mL/min
unless otherwise stated. All radioactive compounds were identified using analytical radio-HPLC
by comparison of the retention time of the reference compound and also by co-injection with the
reference substance. A difference of 0.11 min between UV and -trace occurs as a function of the
HPLC detector setup. Decay-corrected RCYs were quantified by calculation from QMA-eluted
activity against C18-eluted activity with respect to the purity of the final product in radio-HPLC
and radio-TLC using a radio-TLC scanner (BAS-2500, Raytest). Cartridge purification was
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performed with Waters C18 light cartridges. [ F]Fluoride was purchased from ZAG Zyklotron
AG (Karlsruhe, Germany).
Cell culture
HUVEC were isolated from the freshly available umbilical cord. Cell were grown in basal
endothelial cell growth medium (Provitro GmbH, Berlin, Germany), supplemented with 2% fetal
2
bovine serum and antibiotics. Cultures were maintained at 37ºC in a 5% CO humidified
atmosphere and experiments were conducted on cells at approximately 80–90% confluence.
2
HEK293 cells were cultured in T25cm flask with DMEM supplemented with 10% FCS, 1%
2
Penicillin Streptomycin at 37°C with 5% CO humidified atmosphere.
Calcium Imaging and data analysis
HEK293 cells were seeded on 15 mm round glass coverslips in 12 well plates. 24 hours post
plating, cells were transfected with rTRPV1 using Metafectane (metafectene pro - Biontex
Laboratories GmbH, Germany). 48 hours post transfection, cells were transferred into
extracellular solution containing 137.6 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl, 1.8 mM CaCl , 5
2
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mM glucose, and 10 mM HEPES (Roth, Karlsruhe, Germany), loaded with the fluorescent dye
FURA-2AM (1 µM; Biotrend, Cologne, Germany). Fluorescence was measured using an
inverted microscope (IX-81 with Cell^R, Olympus, Hamburg, Germany) and an ORCA-R2 CCD
camera (Hamamatsu Corp., Bridgewater, NJ, USA). After alternating excitation with light of 340
nm and 380 nm wavelength, the ratio of the fluorescence emission intensities at 510 nm (340
nm/380 nm [510 nm]) was calculated and digitized at 0.5 Hertz. This fluorescence ratio is a
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relative measure of intracellular calcium concentration . Analysis was done using cell^R
software (Olympus). All the cells that responded to Capsaicin were taken as region of interest
and each coverslip was taken as an independent experiment. To determine the absolute change in
ratio, the baseline value was subtracted from the peak. The data was analyzed using 2-way
ANOVA with fixed effects for substance and concentration (STATISTICA Vs. 4.5, StatSoft,
Inc.), LSD post-hoc test was used to identify substance differences at a given concentration.
Protein isolation and Western Blotting
HUVEC cell lysates were generated by lysing in lysis buffer consisting of 10 mM Tris-HCl,
150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 1 µM dithiothreitol
(
DTT), proteinase inhibitor cocktail and phosphatase inhibitor. Protein concentration was
measured using Coomassie-Reagent (Pierce, Rockford, USA). Samples (15 μg protein extract)
were heated to 95°C for 5 minutes, loaded and separated on 10% SDS-polyacrylamide gels
followed by semi-dry blotted onto PVDF membranes (Roche, Mannheim, Germany). The
membranes were incubated with 5% w/v non-fat dry milk in TBS/Tween 0.5% to block
unspecific background staining and hereafter incubated overnight at 4°C with anti-VCAM-1-
(AF643, R&D Systems, Wiesbaden, Germany) or anti-HO-1-antibodies (ADI-SPA-895, Enzo,
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Biochem Inc, USA). Subsequently, the membranes were thoroughly washed with TBS-Tween
0
.1% and incubated with the appropriate horseradish peroxidase conjugated secondary antibody,
followed by five wash steps in TBS/Tween 0.1%. Proteins were visualized using enhanced
chemiluminescence technology, according to the manufacturer’s instructions (Pierce, Rockford,
IL, USA). To confirm equal protein loading, membranes were stripped and re-probed with
monoclonal anti-β-actin antibody (clone AC-74, ascites fluid, Sigma Aldrich, USA).
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Luminol Assay
The redox activity of the NOD and F-NOD was measured using luminol assay. Serial dilutions
of the compounds were prepared in distilled water and added to Luminol reaction mix (luminol
2
.5 mM, p-Coumaric acid 0.9 mM and 0.3% H O ). HRP (0.1 µg/µl) was added to the reaction
2 2
mix and quenching of chemiluminescence was measured immediately using infinite 200 PRO –
Tecan microplate reader. The measurements were performed in triplicates.
COMT assay (for hydrophilic substrate - dopamine) The assay is performed with
manufacturer specifications. In brief, the assay solution consists of 1 mM SAM in 100 µL of 20
mM tracepure HCl mixed with 100 µL of 10 mM MgCl
dithiothreitol in tracepure water and 200 µL of 0.5 M Tris buffer (pH 8.0 at 37°C). The substrate
10 mM dopamine) is added in 200 µL tracepure water. 100 µL of this assay solution were
2
in tracepure water and 100 µL of 5 mM
(
mixed with 100 µl freshly prepared COMT-solution (10 units) in 1% BSA (bovine serum
albumin) and incubated at 37°C for 1 h. Afterwards, 100 µl of HCl conc. were added and the
mixture was cooled on ice for 5 minutes, centrifuged and the supernatant was analyzed with
analytical HPLC.
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COMT assay (for hydrophobic substrate). The assay solution consists of 100 µL of 10 mM
2
MgCl in tracepure water and 100 µL of 5 mM dithiothreitol in tracepure water and 500 µL of
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5
6
7
8
9
0
0.5 M Tris buffer (pH 8.0 at 37°C). 100 µL of this assay solution were mixed with 10 µl
Tween20 and 20 µl of 14.6 mM SAM (300 nmol). 3 µL (300 nmol) of the substrate (100 mM
NOD or F-NOD in EtOH) is added and finally 100 µL freshly prepared COMT-solution (75
units) in 1% BSA (bovine serum albumin) is added. The assay mixture is incubated at 37°C for
2
–4 h. Afterwards, the mixture was cooled on ice for 5 minutes, centrifuged and the supernatant
was extracted 3x with CHCl . The organic solvent is removed in vacuum and the residue is
3
dissolved in 1:1 MeCN/water and analyzed with analytical HPLC.
Cell uptake assay
18
Cells were incubated with ~450 kBq [ F]5 per well on a six-well plate for different time
1
8
points at 37°C under normal atmosphere. For this purpose, 9.11 MBq [ F]5 were added to 20
mL cell medium, mixed and 1 mL from that solution was added to every well containing cells.
After incubation, the supernatant was collected, the cells were washed three times with 1 mL
PBS and the third washing solution was collected and finally 50 µL lysis buffer was added to the
cells and cells were scratched from the bottom of the well and collected for gamma counting.
Animal experiments and data analysis
Lewis rats (LEW/Crl, Charles River Laboratories, Sulzfeld, Germany), aged 10 weeks and
with a weight of 270–280 g, were employed for the initial PET experiments. The rats were
anesthetized by isoflurane (2–2.5% delivered at 3.5 L/min) and a catheter was implanted in the
V. femoralis for administration of the radiotracer. First experiments were set-up as terminal
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experiments in order to assess the biodistribution after injection of different doses of [ F]F-NOD
(
4.8, 5.9 and 13.2 MBq, in 0.9% NaCl solution, 150 µL, respectively).
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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4
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4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
All experiments were performed in compliance with the National Guidelines for Animal
Protection, Germany, and the approval of the animal care committee. Small-animal PET
experiments were conducted on the Albira PET System (Bruker Biospin MRI GmbH, Ettlingen,
Germany). On injection of the radiotracer a 120-min dynamic scan was initiated. The protocol
comprised 31 frames (10x 60, 10x 120, 5x 300, and 6x 600 sec). Reconstruction was performed
using maximum likelihood expectation maximization (MLEM) algorithm with a matrix size of
20x20 and a pixel size of 0.5 mm (12 iterations) with the Albira Suite Reconstructor (Bruker
Biospin MRI GmbH, Ettlingen, Germany) with the data output in kBq/cc. For data analysis
volumes of interests (VOI) were drawn in PMOD (version 3.608) to quantify the injected dose
(PMOD Technologies Ltd, Zurich, Switzerland).
Materials.
(
E)-1-Fluoro-4,5-dimethoxy-2-(2-nitrovinyl)benzene
(2).
3,4-Dimethoxy-2-
fluorobenzaldehyde (1, 552 mg, 3.00 mmol) and ammonium acetate (58 mg, 0.75 mmol) were
dissolved in nitromethane (5 mL) and the mixture was stirred at 80°C for 6 h. After cooling to
ambient temperature, excess nitromethane was removed and substituted with Et
2
O. This solution
was washed with water (2x 10 mL), dried with MgSO and the yellow product (555 mg, 81%)
4
was crystallized from a solution of Et
2
O/petroleum ether (PE) 1:1. The purity of 2 was >97% and
1
the product was used without further purification. R
F
= 0.47 (Et
2
O:PE 1:1). H NMR (500 MHz,
3
3
CDCl
3
): δ = 8.04 (d, JH,H = 13.7 Hz, 1H, -HC=CH-NO
2
), 7.64 (d, JH,H = 13.7 Hz,
4
3
1H, -HC=CH-NO
2
), 6.87 (d, JH,F = 6.7 Hz, 1H, H-6), 6.71 (d, JH,F = 11.7 Hz, 1H, H-3), 3.93 (s,
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
1
3
2
H, 4-OCH
3
), 3.90 ppm (s, 3H, 5-OCH
3
). C NMR (126 MHz, CDCl
3
): δ = 157.6 (d, JC,F
=
3
4
3
51.9 Hz, C-1), 153.7 (d, JC,F = 10.5 Hz, C-4), 146.0 (d, JC,F = 2.2 Hz, C-5), 137.1 (d, JC,F
=
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
4
10.5 Hz, C-3), 132.6 (d, JC,F = 1.0 Hz, C=C-NO
2
), 111.1 (d, JC,F = 4.4 Hz, C=C-NO
2
), 100.5 (d,
2
19
JC,F = 27.9 Hz, C-6), 56.5 ppm (2xOCH
3
). F NMR (282 MHz, CDCl
3
): δ = -114.6 ppm. MS
+
+
(
MALDI-TOF): m/z (%) 228 (30) [M + H] , 250 (30) [M + Na] . See Figure S3 for NMR signal
assignment.
2
-(2-Fluoro-4,5-dimethoxyphenyl)ethan-1-amine (3). To a solution of 2 (2.43 g, 10.69
mmol) in anhydrous THF (40 mL) under an N atmosphere a fresh solution of LiAlH (1.02 g,
2
,
4
2
6.9 mmol) in THF (40 mL) was slowly added at 0°C. The mixture was stirred for 15 min and
the reaction was quenched with ice-cold water. THF was removed under reduced pressure at
0°C, brine was added and the aqueous phase was extracted with ethyl acetate (EA) (3x 50 mL).
The organic phase was dried with MgSO , concentrated and the crude product 3 was obtained as
3
4
1
4
yellow oil (2.11 g, 99%). R
F
= 0.16 (EE). H NMR (300 MHz, CDCl
3
): δ = 6.68 (d, JH,F = 7.1
3
Hz, 1H, H-6), 6.61 (d, JH,F = 11.0 Hz, 1H, H-3), 3.85 (s, 3H, 5-OCH
3
), 3.84 (s, 3H, 4-OCH ),
3
3
3
13
2
.98 (t, J = 6.1 Hz, 2H, NCH
2
), 2.77 (t, J = 6.9 Hz, 2H, CCH
2
2
), 2.33 ppm (br s, 2H, NH ). C
1
3
NMR (75 MHz, CDCl
3
): δ = 155.2 (d, JC,F = 237.8 Hz, C-1), 148.4 (d, JC,F = 9.9 Hz, C-4),
4
2
3
1
45.1 (d, JC,F = 2.6 Hz, C-5), 116.2 (d, JC,F = 17.5 Hz, C-1), 113.3 (d, JC,F = 6.5 Hz, C-6),
2
100.2 (d, JC,F = 28.5 Hz, C-3), 56.5 (OCH
3 3 2 2
), 56.1 (OCH ), 42.0 (NCH ), 31.9 ppm (CCH ). MS
+
(
MALDI-TOF): m/z (%) 223 (100) [M + Na] .
N-(2-Fluoro-4,5-dimethoxyphenethyl)octanamide (4). To a solution of 3 (2.07 g, 10.40
mmol) in CH Cl (20 mL), a solution of octanoyl chloride (1.6 mL, 9.36 mmol) in CH Cl (25
mL) was slowly added at 0°C. To the resulting dark orange suspension Et N (2.2 mL, 15.6
2
2
2
2
3
mmol) was added and the mixture was stirred for 15 min at 0°C. The resulting pale yellow
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solution was washed with water and brine and dried with MgSO . The solvent was removed and
the crude product was purified by column chromatography (PE  PE:EA 10:1  5:1  4:1 
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1:1) to obtain compound 4 as pale yellow solid (1.41 g, 42%). R
F
= 0.33 (PE:EA 1:2). Hot
): δ = 6.66 (t,
1
petroleum ether was used for recrystallization of 4. H NMR (500 MHz, DMSO-d
6
4
3
J
H,F = 7.1 Hz, 1H, H-6), 6.61 (d, JH,F =11.0 Hz, 1H, H-3), 5.49 (s, 1H, NH), 3.85 (s, 3H,
3
3
3
OCH
3
), 3.84 (s, 3H, OCH
3
2
), 3.48 (dt, JH,H = 6.8 Hz, JNH,H = 6.1 Hz, 2H, NCH ), 2.79 (t, J =
3
6
.9 Hz, 2H, CCH
2
), 2.13 (t, J = 7.6 Hz, 2H, H-α), 1.62–1.56 (m, 2H, H-β), 1.31–1.23 (m, 8H,
13
H-γ,δ,ε,ζ), 0.89–0.86 ppm (m, 3H, CH
3
). C NMR (126 MHz, DMSO-d
6
): δ = 173.2 (C=O),
1
3
4
1
55.1 (d, JC,F = 237.7 Hz, C-2), 148.4 (d, JC,F = 9.9 Hz, C-4), 145.2 (d, JC,F = 2.7 Hz, C-5),
2
3
2
116.1 (d, JC,F = 17.4 Hz, C-1), 113.0 (d, JC,F = 6.4 Hz, C-6), 100.1 (d, JC,F = 28.5 Hz, C-3),
6.4 (OCH ), 56.4 (OCH ), 39.7 (NCH ), 36.9 (C-α) 31.7 (C-ε), 29.2 (C-γ), 29.0 (C-δ), 28.8
3
CCH ), 25.7 (C-β), 22.6 (C-ζ), 14.1 ppm (CH ). F NMR (282 MHz, CDCl ): δ = -126.1 ppm.
5
3
3
2
1
9
(
2
3
+
MS (MALDI-TOF): m/z (%) 326 (100) [M + H] . Analytical HPLC: t
R
= 4.54 min.
N-(2-Fluoro-4,5-dihydroxyphenethyl)octanamide (5). To a solution of 4 (734 mg, 2.26
mmol) in CH Cl (100 mL), a solution of BBr (11.3 mL, 11.3 mmol, 1 M) in CH Cl was slowly
2
2
3
2
2
added at -12°C within 5 min. The mixture was stirred for 135 min at -10°C and was allowed to
reach ambient temperature for 60 min. Water (30 mL) was added for quenching the reaction and
2 2
the mixture was extracted with CH Cl (1x) and EA (2x). The combined organic phases were
dried with MgSO , concentrated under reduced pressure and the crude product was purified by
4
column chromatography (PE  PE:EA 1:1) to obtain compound 5 as pale yellow solid (602 mg,
1
90%). R
F
= 0.44 (PE:EA 1:3), 0.85 (methanol). H NMR (300 MHz, DMSO-d
6
): δ = 9.09 (s, 1H,
3
4
OH), 8.72 (s, 1H, OH), 7.81 (t, J = 5.7 Hz, 1H, NH), 6.54 (d, JH,F = 7.8 Hz, 1H, H-6), 6.48 (d,
3
3
3
J
2 2
H,F = 10.9 Hz, 1H, H-3), 3.18–3.11 (m, 2H, NCH ), 2.52 (t, J = 7.3 Hz, 2H, CCH ), 2.01 (t, J
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2
2
2
2
2
2
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4
4
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5
5
5
5
5
5
5
5
5
6
=
7.2 Hz, 2H, H-α), 1.48–1.43 (m, 2H, H-β), 1.27–1.23 (m, 8H, H-γ,δ,ε,ζ), 0.88–0.83 ppm (m,
13
1
3H, CH
3
6
). C NMR (75 MHz, DMSO-d ): δ = 171.9 (C=O), 153.3 (d, JC,F = 233.2 Hz, C-2),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
3
144.2 (d, JC,F = 11.2 Hz, C-4), 141.3 (d, JC,F = 2.3 Hz, C-5), 116.5 (d, JC,F = 6.0 Hz, C-6),
2
2
1
15.0 (d, JC,F = 17.2 Hz, C-1), 102.8 (d, JC,F = 26.4 Hz, C-3), 39.0 (NCH
ε), 28.5 (C-γ), 28.4 (C-δ), 28.1 (CCH
MHz, DMSO-d
HPLC: t = 3.96 min.
-(2-Aminoethyl)-5-nitrobenzene-1,2-diol (8). 6-Nitrodopamine 8 was prepared according to
2
), 35.3 (C-α) 31.1 (C-
19
2
), 25.2 (C-β), 22.0 (C-ζ), 13.9 ppm (CH
3
). F NMR (282
+
6
): δ = -129.7 ppm. MS (MALDI-TOF): m/z (%) 298 (100) [M + H] . Analytical
R
4
2
2d
the literature. In brief, to a solution of dopamine hydrochloride (1.50 g, 7.91 mmol) in water
(45 mL) being cooled to 0°C, NaNO (1.89 g, 27.39 mmol) was added and the mixture was
stirred for 15 min. To the pale yellow solution, an ice-cold solution of 20% H SO (3.5 mL) was
2
2
4
slowly added. The yellow product starts to precipitate from the brown solution. The product was
filtered, washed with cold water and methanol and dried in high vacuum to obtain 8 as yellow
2
2d
solid (1.21 g, 77%). NMR and MS analyses were consistent with the literature.
-Nitro-5-(2-octanamidoethyl)-1,2-phenylene dioctanoate (9). To a solution of 8 (500 mg,
.52 mmol) in CH Cl (10 mL), a solution of octanoyl chloride (1.19 mL, 6.98 mmol) in CH Cl
3 mL) was slowly added at 0°C. Et N (1.23 mL, 8.82 mmol) was added and the mixture was
stirred at ambient temperature for 16 h. CH Cl was removed and the residue was redissolved in
EA (20 mL) and washed with brine. The organic phase was dried with Na SO , concentrated
4
2
2
2
2
2
(
3
2
2
2
4
under reduced pressure and the crude product was purified by column chromatography (PE 
PE:EA 5:1  3:1) to obtain compound 9 as pale yellow solid (431 mg, 33%). R
F
= 0.59 (PE:EA
): δ = 7.89
1
1
:1). Hot acetonitrile was used for recrystallization of 9. H NMR (500 MHz, CDCl
3
3
3
(s, 1H, H-3), 7.21 (s, 1H, H-6), 5.79 (s, 1H, NH), 3.61–3.57 (dt, JH,H = 6.8 Hz, JNH,H = 6.4 Hz,
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H, NCH
7.5 Hz, 2H, 1-O(CO)CH
O(CO)CH CH ), 1.56–1.50 (m, 2H, N(CO)CH
.85 ppm (m, 9H, 3xCH
70.2 (1-OC=O), 146.0 (C-1), 145.8 (C-2), 140.9 (C-4), 133.2 (C-5), 127.5 (C-6), 120.9 (C-3),
9.7 (NCH ), 36.6 (N-C-α), 34.0 (2-O-C-α), 33.9 (1-O-C-α), 32.8 (CCH ), 31.7 (N-C-ε), 31.6
2
), 3.12 (t, J = 6.8 Hz, 2H, CCH
2
), 2.55 (t, J = 7.5 Hz, 2H, 2-O(CO)CH
2
), 2.54 (t, J
), 1.70–1.63 (m, 4H,
), 1.44–1.22 (m, 24H, 3xH-γ,δ,ε,ζ), 0.92–
). C NMR (126 MHz, CDCl ): δ = 173.7 (NC=O), 170.5 (2-OC=O),
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
=
2
), 2.17 (t, J = 7.7 Hz, 2H, N(CO)CH
CH
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
2
13
0
1
3
3
3
2
2
(
(
3
2xO-C-ε), 29.0 (6xC-γ,δ), 25.6 (N-C-β), 24.8 (2xO-C-β), 22.6 (3xC-ζ), 14.0 ppm (3xCH ). MS
+
MALDI-TOF): m/z (%) 577 (60) [M + H] . Analytical HPLC: t
R
= 7.75 min.
N-(4,5-Dihydroxy-2-nitrophenethyl)octanamide (10). 9 (150 mg, 0.26 mmol) was dissolved
in acetonitrile (25 mL) at 30°C. The mixture was cooled to 0°C and a solution of 0.1 M NaOH
2.6 mL, 0.26 mmol) was added dropwise. The mixture was then stirred for 4 h at 50°C. After
this, water (20 mL) was added and the mixture was extracted with EA (2x). The organic phase
was dried with MgSO , concentrated under reduced pressure and the crude product was purified
(
4
by column chromatography (PE  PE:EA 1:1) to obtain compound 10 as yellow solid (83 mg,
1
99%). R
F
= 0.20 (PE:EA 1:1). H NMR (500 MHz, DMSO-d
6
): δ = 10.33 (s, 1H, OH), 9.81 (s,
3
1
H, OH), 7.84 (t, J = 5.7 Hz, 1H, NH), 7.48 (s, 1H, H-6), 6.68 (s, 1H, H-3), 3.27–3.23 (m, 2H,
3
3
NCH
2
), 2.88 (t, J = 7.1 Hz, 2H, CCH
2
), 1.99 (t, J = 7.5 Hz, 2H, H-α), 1.47–1.41 (m, 2H, H-β),
13
1
.28–1.17 (m, 8H, H-γ,δ,ε,ζ), 0.87–0.84 ppm (m, 3H, CH
3
). C NMR (126 MHz, DMSO-d
6
): δ
=
171.9 (C=O), 151.7 (C-5), 143.9 (C-4), 139.0 (C-2), 128.1 (C-1), 118.1 (C-6), 111.9 (C-3),
39.7 (NCH
3.8 ppm (CH
min.
2
), 35.3 (C-α) 32.8 (C-ε), 31.0 (CCH
2
), 28.5 (C-γ), 28.3 (C-δ), 25.1 (C-β), 22.0 (C-ζ),
+
1
3
). MS (MALDI-TOF): m/z (%) 325 (100) [M + H] . Analytical HPLC: t
R
= 4.06
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