F-18 labelled N,N-bis-haloethylamino-phenylsulfoxides - A new class of compounds for the imaging of hypoxic tissue

Article abstract of DOI:10.1002/jlcr.1129

Two N,N-bis-chloroethylamino-phenylsulfoxides have been synthesized and radiolabelled with F-18 via halogen exchange. The radiolabelling of both compounds proceeds smoothly with K[¹⁸F]F-kryptofix 2.2.2 complex at 100°C in DMSO. Decay-corrected

Full text of DOI:10.1002/jlcr.1129

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 1089–1103.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1129
Research Article
F-18 labelled N,N-bis-haloethylamino-phenylsulfoxides
} a new class of compounds for the imaging of
hypoxic tissue
Cheryl L. Falzon1,2,3, Uwe Ackermann2,3,*, Neil Spratt3,4,
Henri J. Tochon-Danguy2,3, Jonathon White1, David Howells3,4 and
Andrew M. Scott2,3,5
1 Department of Chemistry, The University of Melbourne, Parkville, Vic. 3010, Australia
2 Centre for PET, Austin Health, Austin Campus, Studley Road, Heidelberg, Vic. 3084,
Australia
3 Department of Medicine, The University of Melbourne (AHNH), Austin Campus,
Studley Road, Heidelberg, Vic. 3084, Australia
4 National Stroke Research Institute, Austin Health, Austin Campus, Studley Road,
Heidelberg, Vic. 3084, Australia
5 Ludwig Institute for Cancer Research, Melbourne Centre for Clinical Sciences,
Melbourne, Australia
Summary
Two N,N-bis-chloroethylamino-phenylsulfoxides have been synthesized and radi-
olabelled with F-18 via halogen exchange. The radiolabelling of both compounds
proceeds smoothly with K[18F]F-kryptofix 2.2.2 complex at 100 8C in DMSO. Decay-
corrected radiochemical yields were 25–35% for [18F]12 (SO201) and 35–45% for
[18F]11 (SO101), with an average specific activity of 1.8 Ci/mmol. Both compounds
remained 80% intact in plasma over a period of 2 h. In vivo binding in a stroke model
indicates that both markers are selectively retained in hypoxic tissue. Copyright #
2006 John Wiley & Sons, Ltd.
Received 27 July 2006; Revised 9 August 2006; Accepted 9 August 2006
Key Words: hypoxia; PET; radiolabelling; stroke
*Correspondence to: Uwe Ackermann, Centre for PET, Austin Health, Austin Campus, Studley Road,
Heidelberg, Vic 3084, Australia. E-mail: ackerman@petnm.unimelb.edu.au
Contract sponsor: NHMRC; contract/grant number: 251525
Contract/grant sponsor: Cancer council
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Introduction
Hypoxic tissue plays an important role in tumors as well as in ischemic stroke.
In oncology, hypoxic tumors are more resistant to chemo- and radiotherapy1,2
and may behave more aggressively than normoxic tumors.3–5 In acute stroke,
hypoxic tissue is functionally impaired but morphologically intact and at risk
of infarction.6 This tissue has the potential to recover completely if perfusion is
restored. In order to better understand tissue recovery as well as assessing the
efficiency of neuroprotective agents, imaging of hypoxic tissue is of great
importance. Positron emission tomography (PET) is the perfect tool for such
imaging studies due to its ability to quantify tissue uptake in vivo.7 So far, most
F-18-based compounds for PET imaging of hypoxic tissue in stroke are
nitroimidazole derivatives such as [18F]FMISO and [18F]FAZA (Figure 1).8–10
These compounds are believed to undergo bioreduction at the nitro group and
subsequently become trapped in the hypoxic cell by binding to intracellular
molecules.11 In normoxic cells the reduced intermediates are believed to be
reoxidized to the original molecule, which can diffuse freely across the cell
membrane and is therefore not taken up by the normoxic cell. Due to the lack of
metabolism, no radiotracer uptake is observed in necrotic tissue. However, the
optimum time for in vivo imaging with these nitroimidazole-based radiotracers is
2 h post tracer administration.12 This time frame renders the routine use of these
tracers in stroke difficult and justifies the need for the development of novel tracers.
In oncology, sulfoxide containing nitrogen mustards have shown great
promise as targeted therapeutics for hypoxic tumors.13,14 It is believed that this
class of compounds undergoes bioreduction to the corresponding thioether in
hypoxic cells, which activates the nitrogen mustard moiety.15 This process
results in the formation of an aziridinium ion intermediate, which binds in an
irreversible manner to reductases in the cell and is responsible for the cytotoxic
effect of these compounds. In normoxic tissue, the sulfoxide moiety is believed
to be further oxidized to the sulfone which has no cytotoxic effect, can diffuse
freely across the cell membrane, and therefore does not accumulate in the
normoxic cell (Figure 2).16
Our aim was to radiolabel compounds 1 and 2 with the positron emitting
isotope F-18 (t12 = 110 min) by halogen exchange, determine the stability of
N
18
F
NO
OH
N
2
O
HO
NO
2
N
OH
18
N
F
18
18
[
F]FAZA
[
F]FMISO
Figure 1. Chemical structures of [18F]FMISO and [18F]FAZA
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NEW CLASS OF COMPOUNDS FOR IMAGING HYPOXIC TISSUE
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Cl
O
R
S
N
O
Cl
Cl
Cl
O
R
S
N
Cl-
Cl
Cl
1: R = Me
2: R = p-NO2C6H4
S
N
S
N
+
R
R
Cl
Figure 2. Mechanism of action of nitrogen mustards
these compounds in saline and human plasma, and determine whether they are
selectively retained in hypoxic tissue in vivo.
Results and discussion
Chemistry
Synthesis of labelling precursors and F-19 standards
Reaction of 4 equivalents of 2-chloroethyltriflate with 1 equivalent of either
4-methylthioaniline (3) or 4-(p-nitrophenylthio) aniline (4) in the presence of
potassium carbonate yielded the dialkylated products 5 and 6. The dialkylated
products 5 and 6 were oxidized with in situ generated trifluoroperacetic acid12
to form the sulfoxide precursors 1 and 2 required for the labelling (Scheme 1).
The monoalkylated intermediates 7 and 8 were synthesized by reacting
either 4-methylthioaniline (3) or 4(p-nitrophenyl)-thioaniline (4) with 0.5
equivalent of 2-chloroethyltriflate in the presence of potassium carbonate to
minimize the formation of the dialkylated products, which were difficult to
separate. 7 and 8 were then reacted with fluoroethyltriflate using 2,6-lutidine
as base to give the thioethers 9 and 10. Oxidation of these compounds with
in situ generated trifluoroperacetic acid gave the F-19 standards 11 and 12
(Scheme 2).
Radiochemistry
Radiolabelling of the sulfoxide precursors was achieved by halogen exchange
using no-carrier added, dried K[18F]F kryptofix 2.2.2 complex (Scheme 3).
Labelling efficiency at various temperatures and time points was measured
and found to be optimum at 1008C for 15 min for both compounds. Optimized
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Cl
Cl
Cl
i
O
R
ii
S
R
NH2
S
N
S
N
R
Cl
3: R = Me
4: R = p-NO2C6H4
5: R = Me
6: R = p-NO2C6H4
1: R = Me
2: R = p-NO2C6H4
Reagents and conditions:(i): 2-chloroethyltriflate (4 equiv.), K2CO3 (1 equiv.), CH2Cl2, reflux, 18 h
(ii): H2O2 (1 equiv.), TFA, -15˚C, 1 h
Scheme 1. Synthesis of labelling precursors
F
F
O
i
iii
ii
S
N
S
NH
S
N
S
NH2
R
R
R
R
Cl
Cl
Cl
11: R = Me
12: R = p-NO2C6H4
9: R = Me
10: R = p-NO2C6H4
3: R = Me
4: R = p-NO2C6H4
7: R = Me
8: R = p-NO2C6H4
Reagents and conditions: (i): 2-chloroethyltriflate (0.5 equiv), K2CO3 (1 equiv.), CH2Cl2, reflux, 18 h
(ii): 2-fluoroethyltriflate (0.5 equiv), 2,6-lutidine (1 equiv.), CH2Cl2, reflux, 18 h
(iii): H2O2 (1 equiv), TFA, -15˚C, 1 h
Scheme 2. Synthesis of labelling standards
18F
Cl
O
O
R
S
N
S
N
R
Cl
Cl
18F]11: R = Me
18F]12: R = p-NO2C6H4
1: R = Me
2: R = p-NO2C6H4
[
[
Reagents and conditions: K[18F]F kryptofix 2.2.2, 100˚C, 15 min
Scheme 3. Synthesis of [18F]11 and [18F]12
yields for [18F]11 were 35–45%, and 25–35% for compound [18F]12. Average
specific activity was 1.8 Ci/mmol in both cases. The F-18 labelled compounds
showed HPLC profiles that were identical to the F-19 standards. Separation of
the radioactive products from their respective precursor molecules was
achieved by reversed phase semi-preparative HPLC. After collection of the
desired radioactive peak, the ligands were reformulated in 10% ethanol using
the Sep Pak method.17 The synthesis time including HPLC purification and
reformulation was 1 h.
Copyright # 2006 John Wiley & Sons, Ltd.
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SO201 standard
SO101 standard
70000
60000
50000
40000
30000
20000
10000
0
120000
100000
80000
60000
40000
20000
0
0
2
4
6
8
10 12 14 16 18 20
0
2
4
6
8
10 12 14 16 18 20
time (min)
time (min)
F-18 SO201
F-18 SO101
1000
800
600
400
200
0
600
500
400
300
200
100
0
0
2
4
6
8
10 12 14 16 18 20
0
2
4
6
8
10 12 14 16 18 20
time (min)
time (min)
Figure 3. HPLC chromatograms of the independent quality control of SO101
and SO201
Independent quality control was performed on a Develosil UG (C-30, 5 m,
10 ꢀ 250 mm, 20 ml injection loop) HPLC column at a flow rate of 2 ml/min
with 0.1 M NH4HCO2/ ethanol : 40/60 (v/v) as mobile phase (Figure 3).
Stability in saline and plasma
The in vitro stability of compounds [18F]11 and [18F]12 was assessed by
incubating 100 mCi of activity at 37 8C in 4 ml of saline or plasma over various
time points. After the end of each timepoint, radioactivity was extracted from
the plasma by addition of 1 ml of acetonitrile. The sample was spun down and
the supernatant analyzed by radio thin layer chromatography (TLC) and
HPLC. In both cases 80% of the tracer is still intact after 2 h. [18F]11 and
[18F]12 were found to be stable in saline over a period of 2 h.
Uptake in ischemic tissue
For all experimental animals for each compound there was increased signal
within the normal distribution of the occluded middle cerebral artery (MCA)
(Figure 4). Average hypoxic:normoxic ratio was 2.07 ꢁ 0.27 ([18F]11) and
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Figure 4. [18F]11 (A) and [18F]12 (B) autoradiographs of representative brains
after 2 h focal ischemia. For each animal 6 ꢀ 2 mm coronal sections are shown.
Increased radioactivity is seen within the normal distribution of the occluded
middle cerebral artery in each case
2.26 ꢁ 0.20 ([18F]12). The area of increased binding was 479 ꢁ 170 mm3
([18F]11) and 526 ꢁ 94 mm3 ([18F]12).
The results of the labelling studies have demonstrated that halogen
exchange is a simple and convenient way to synthesize compounds [18F]11
and [18F]12. The difference in HPLC retention time between the precursors 1
and 2 and their [18F]labelled analogues [18F]11 and [18F]12 are 6.16 and 8 min,
respectively, which is sufficiently large to ensure complete separation on a
semi-preparative HPLC column. It is noteworthy that both compounds could
be synthesized in good radiochemical yields, despite the need to displace the
relatively weak leaving group Clꢂ. These yields are sufficient for animal and
potential human studies.
We decided to target compounds [18F]11 and [18F]12 because of the large
difference in hypoxic selectivity between the parent molecules 1 and 2.
Clonogenic assays with V-79 cells have shown that under normoxic conditions
the IC90 value of 2 is 3680 and 182 mM under hypoxic conditions.14 The value
for hypoxic selectivity, which is defined as the ratio between the normoxic and
hypoxic IC90 values, is 20 for compound 2. In the same set of experiments a
Copyright # 2006 John Wiley & Sons, Ltd.
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NEW CLASS OF COMPOUNDS FOR IMAGING HYPOXIC TISSUE
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hypoxic selectivity of 3 was measured for compound 1. This difference in
hypoxic selectivity of the parent molecules could result in higher uptake of
[18F]12 in hypoxic tissue compared to [18F]11.
Since uptake of these putative tracers depends on active metabolism, it can
be expected that both [18F]11 and [18F]12 will not be taken up by necrotic cells.
In vivo binding of both radiotracers indicates successful retention within
hypoxic tissue, consistent with potential applications as hypoxic imaging
agents. Further characterization of time-course of binding and comparison
with established hypoxic tracers in stroke18 and oncology19 is being under-
taken.
Experimental
General
No-carrier-added [18F] fluoride was produced by the 18O(p, n)18F nuclear
reaction with a 10 MeV proton beam generated by the IBA Cyclone 10/5
cyclotron in a titanium target using [18O]H2O at Austin Health, Centre for
PET. Typical irradiation parameters were 20 mA/h for 30 min, which produced
5.4–8.1 GBq (146–219 mCi) of [18F]fluoride. Isolation of the [18F]Fꢂ ion from
[18O]H2O was achieved by trapping on a QMA ion exchange column. Elution
of the column with a solution containing 3.5 mg of potassium carbonate in
0.2 ml of water and 20 mg of kryptofix 2.2.2 in 0.4 ml of acetonitrile followed
by repeated azeotropic distillation with acetonitrile then evaporation to
dryness gave the anhydrous fluoride ion used in the labelling experiments.
All chemicals and solvents were purchased from Sigma-Aldrich and used as
received. 2-Chloroethyltriflate and 2-fluoroethyltriflate were synthesized as
described and used without further purification. Flash chromatography was
1
carried out with silica gel 60 (0.040–0.063 mm, Merck). H Nuclear magnetic
resonance spectra were recorded at 400 MHz and 19F spectra were recorded at
375 MHz using a JOEL GX-400 spectrometer. 13C nuclear magnetic resonance
spectra were recorded at 100 MHz using a JOEL GX-400 spectrometer.
Melting points were determined with a Gallenkamp Capillary melting point
apparatus and are uncorrected.
The labelling procedure including semi-preparative HPLC purification,
formulation and sterile filtration was carried out using a modified IBA
automated synthesis module.
Semi-preparative HPLC was performed using a Shimadzu LC-10AS
isocratic pump equipped with a 1.2 ml injection loop and a reversed phase
column (Apollo C-18, 5 m, 10 ꢀ 250 mm). For SO101 the mobile phase
consisted of 0.1 M NH4HCO2/ acetonitrile : 40/60 (v/v) at a flow rate of 4 ml/
min. 0.1 M NH4HCO2/ acetonitrile : 50/50 (v/v) at a flow rate of 4 ml/min was
used as mobile phase for SO201. Detection of chemical compounds was
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achieved with a Shimadzu SPD-6AV UV detector (254 nm) and a Geiger-
Muller tube as radiodetector.
¨
Radioactive analytical TLC was carried out on aluminum backed silica
gel 60 (0.25 mm plates, Alltech) with CH3CN/H2O : 95/5 (v/v) as mobile
phase.
For both compounds, independent quality control was performed on a
Develosil UG (C-30, 5 m, 10 ꢀ 250 mm, 20 ml injection loop) HPLC column at a
flow rate of 2 ml/min with 0.1 M NH4HCO2/ ethanol : 40/60 (v/v) as mobile
phase. Detection of chemical compounds was achieved with a Shimadzu SPD-
6AV UV detector (254 nm) and a Berthold LB506 C-1 monitor for
radiochemical detection.
Chemistry
2-chloroethyltriflate
Trifluoromethanesulfonic anhydride (5 g, 3 ml, 17.7 mmol) was added to a
stirred solution of 4-(polyvinyl) pyridine (5.70 g, 51.75 mmol) in dichloro-
methane (100 ml). 2-Chloroethanol (1.29 g, 1.08 ml, 16.02 mmol) was then
added dropwise over 1 min. The reaction was left to stir at room temperature
for 30 min, and then filtered under gravity. The 4-(polyvinyl) pyridinium
precipitate was washed with 5 ml of dichloromethane. The organic phases were
combined and washed with NaHCO3 (3 ꢀ 50 ml) and water (3 ꢀ 50 ml)
respectively, dried (MgSO4), and concentrated under reduced pressure to give
2-chloroethyltriflate (2.105 g, 9.9 mmol, 61.8%) as colorless oil. This product
was used without further purification.
1H NMR (CDCl3): 3.80 (t, 2H, CH2-Cl), 4.65 (t, 2H CH2-OTf)
13C NMR (CDCl3): 40.32 (CH2-Cl), 75.07 (CH2-OTf), 118.35 (q, J=319 Hz)
IR: 1144.4, 1215.4 (O=S=O).
2-fluoroethyltriflate
Trifluoromethanesulfonic anhydride (8.57 g, 5.13 ml, 30.37 mmol) was added
to a stirred solution of 4-(polyvinyl) pyridine (6.68 g, 60.78 mmol) in
dichloromethane (100 ml). 2-Fluoroethanol (1.77 g, 1.62 ml, 27.63 mmol) was
then added drop wise over 1 min. The reaction was left to stir at room
temperature for 30 min, and then filtered under gravity. The 4-(polyvinyl)
pyridinium precipitate was washed with 5 ml of dichloromethane. The organic
phases were combined and washed with NaHCO3 (3 ꢀ 50 ml) and water
(3 ꢀ 50 ml) respectively, dried (MgSO4), and concentrated under reduced
pressure to give 2-fluoroethyltriflate (5.10 g, 26 mmol, 94.1%) as colorless oil.
The product was used without further purification.
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1H NMR (CDCl3): 4.66 (m, 2H, CH2-F), 4.65 (m, 2H CH2-OTf)
13C NMR (CDCl3): 74.50 (d, CH2-F, J=20 Hz), 79.86 (d, CH2-OTf,
J=175 Hz), 118.47 (q, CF3, J=320 Hz)
IR: 1156.3, 1217.8 (O=S=O).
General procedure for the alkylation of anilines (procedure A)
The aniline and 1 equivalent of the respective base were added to a stirred
solution of the crude alkylating agent in dichloromethane (100 ml). The
reaction was refluxed (80 8C) overnight, then cooled and washed with
NaHCO3 (3 ꢀ 50 ml) and water (3 ꢀ 50 ml), respectively. The organic layers
were combined, dried (MgSO4) and evaporated under reduced pressure. The
crude material was then purified by flash column chromatography on silica gel.
N,N-bis-(2-chloroethyl)-(4-methylthio) aniline 5
Compound 5 was synthesized following procedure A starting from 4-
(methylthio) aniline (3) (0.38 g, 2.73 mmol), anhydrous potassium carbonate
as base and 2-chloroethyltriflate (2.32 g, 10.91 mmol) as alkylating agent.
Purification by flash column chromatography (eluent 5% ether in hexane)
gave 0.48 g (1.82 mmol, 66.7%) of product as brown oil. TLC Rf0.76 in ether/
hexane (1:1).
1H NMR (CDCl3): 2.43 (s, 3H), 3.62 (t, 4H, CH2, J=7.2 Hz), 3.74 (t, 4H, CH2,
J=7.2 Hz), 6.64 (d, 2H, J=8.4 Hz), 7.28 (d, 2H, J=8.4 Hz)
13C NMR (CDCl3): 18.83 (CH3), 43.30 (CH2-Cl), 45.38 (CH2-N), 113.79 (CH-
Ar), 125.34 (C-S, Ar), 131.24 (CH-Ar), 145.78 (C-N, Ar).
N-(2-chloroethyl)-(4-methylthio) aniline 7
Compound 7 was obtained following procedure A, starting from starting from
4-(methylthio) aniline (3) (8.89 g, 63.85 mmol), anhydrous potassium carbo-
nate as base and 2-chloroethyltriflate (6.79 g, 31.93 mmol) as alkylating agent.
Purification by flash column chromatography (eluent 5% ether in hexane)
gave 2.23 g (11.05 mmol, 34.6%) of product as brown oil. TLC Rf 0.61 in
ether/hexane (1:1)
1H NMR (CDCl3): 2.43 (s, 3H), 3.48 (t, 2H, CH2, J=4.8 Hz), 3.69 (t, 2H, CH2,
J=4.8 Hz), 6.58 (d, 2H, CH, J=6.4 Hz), 7.24 (d, 2H, CH, J=6.4 Hz).
13C NMR (CDCl3): 18.79 (CH3), 43.23 (CH2-Cl), 45.43 (CH2-N), 113.86 (CH-
Ar), 125.46 (C-S, Ar), 131.16 (CH-Ar), 145.61 (C-N, Ar).
Accurate mass (ESI): calculated (M+H) 202.0457. Found 202.0455.
N-(2-chloroethyl)-N-(2-fluoroethyl)-(4-methylthio) aniline 9
Compound 9 was obtained from N-(2-chloroethyl)-(4-methylthio) aniline (7)
(8.89 g, 44.07 mmol), 2,6-lutidine as base and fluoroethyltriflate (4.32 g,
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22.03 mmol) as alkylating agent following procedure A. The crude material
was purified by flash column chromatography (eluent 5% ether in hexane) to
give 2.23 g (9.0 mmol, 40.9%) of product as brown oil. TLC Rf 0.61 in ether/
hexane (1:1).
1H NMR (CDCl3): 2.42 (s, 3H), 3.59–3.69 (m, 6H), 4.58 (dt, 2H, F-CH2,
J=47.5, 5.0 Hz), 6.63 (d, 2H, CH, J=8.0 Hz), 7.27 (d, 2H, CH, J=8.0 Hz).
13C NMR (CDCl3): 18.79 (CH3), 40.02 (CH2-Cl), 51.72 (d, F-CH2-CH2-N,
J=21.5 Hz), 53.53 (CH2-N), 81.38 (d, F-CH2-CH2-N, J=169.7 Hz), 112.70
(CH-Ar), 124.79 (CH-Ar), 131.39 (C-S, Ar), 145.20 (C-N, Ar).
Accurate mass (ESI): calculated (M+H) 248.0676. Found 248.0673.
N,N-bis-(2-chloroethyl)-[4-(4-nitrophenylthio)] aniline 6
Compound 6 was synthesized from (4-nitrophenylthio) aniline (4) (0.87 g,
3.53 mmol), anhydrous potassium carbonate as base and 2-chloroethyltriflate
(3.00 g, 14.13 mmol) as alkylating agent, following procedure A. The crude
material was purified by flash column chromatography (eluent 5% ether in
hexane) to give 0.57 g (1.54 mmol, 43.5%) of 6 as yellow solid. TLC Rf 0.60 in
ether/hexane (1:1).
1H NMR (CDCl3): 3.68 (t, 4H, CH2, J=7.2 Hz), 3.80 (t, 4H, CH2, J=7.2 Hz),
6.74 (d, 2H, J=8.4 Hz), 7.09 (d, 2H, J=8.4 Hz), 7.43 (d, 2H, J=8.4 Hz), 8.04
(d, 2H, J=8.4 Hz).
13C NMR (CDCl3): 40.12 (CH2-Cl), 53.30 (CH2-N), 112.96 (CH-Ar), 115.75
(C-S, Ar), 123.92 (CH-Ar), 125.29 (CH-Ar), 137.43 (CH-Ar), 144.84 (C-S, Ar),
147.47 (C-N, Ar), 150.64 (C-N, Ar).
MP 113.6–114.98C.
N-(2-chloroethyl)[4-(4-nitrophenylthio)] aniline 8
Compound 8 was obtained from (4-nitrophenylthio) aniline (4) (0.56 g,
2.27 mmol), anhydrous potassium carbonate as base and 2-chloroethyltriflate
(0.24 g, 1.13 mmol) as alkylating agent following procedure A. The crude
material was purified by flash column chromatography (eluent 5% ether in
hexane) to give 0.32 g (1.04 mmol, 92.0%) of product as yellow solid. TLC Rf
0.61 in ether/hexane (1:1).
1H NMR (CDCl3): 3.57 (t, 2H, CH2, J=5.9 Hz), 3.76 (t, 2H, CH2, J=5.9 Hz),
6.70 (d, 2H, J=8.7 Hz), 7.09 (d, 2H, J=8.7 Hz), 7.37 (d, 2H, J=8.7 Hz), 8.03
(d, 2H, J=8.7 Hz).
13C NMR (CDCl3): 43.10 (CH2-Cl), 44.92 (CH2-N), 114.06 (CH-Ar), 115.83
(C-S, Ar), 123.89 (CH-Ar), 125.15 (CH-Ar), 137.27 (CH-Ar), 144.72 (C-S, Ar),
148.64 (C-N, Ar), 151.01 (C-N, Ar).
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Accurate mass (ESI): calculated (M+Na) 331.0284. Found 331.0278.
MP 102.5–1048C.
N-(2-chloroethyl)-N-(2-fluoroethyl)-[4-(4-nitrophenylthio)] aniline 10
Compound 10 was prepared from N-(2-chloroethyl)-[4-(4-nitrophenylthio)]
aniline (8) (0.19 g, 0.62 mmol), 2,6-lutidine as base and 2-fluoroethyltriflate
(0.06 g, 0.31 mmol) as alkylating agent. The crude material was purified by
flash column chromatography (eluent 5% ether in hexane) to give 62.5 mg
(0.18 mmol, 56.8%) of product as a yellow solid. TLC Rf 0.50 in ether/hexane
(1:1).
1H NMR (CDCl3): 3.70 (t, 2H, CH2-Cl, J=7.3 Hz), 3.76–3.83 (m, 4H), 4.64
(dt, 2H, F-CH2, J = 47.8, 4.7 Hz), 6.75 (d, 2H, J=8.8 Hz), 7.09 (d, 2H,
J=8.8 Hz), 7.42 (d, 2H, J=8.8 Hz), 8.64 (d, 2H, J=8.8 Hz).
13C NMR (CDCl3): 39.79 (CH2-Cl), 51.61 (d, F-CH2-CH2-N, J=21.4 Hz),
53.34 (CH2-N), 81.23 (d, F-CH2-CH2-N, J=169.4 Hz), 112.98 (CH-Ar),
115.40 (C-S, Ar), 123.91 (CH-Ar), 125.26 (CH-Ar), 137.36 (CH-Ar), 144.70
(C-S, Ar), 147.88 (C-N, Ar), 150.76 (C-N, Ar).
19F NMR (CDCl3): ꢂ146.4 (m).
Accurate mass (ESI): calculated (M+Na) 377.0503. Found 377.0502.
MP 116.3–117.88C.
General procedure for the oxidation of thioethers to sulfoxides (procedure B)
A solution of 30% H2O2 (0.033 g, 0.030 ml, 0.29 mmol) in TFA (0.970 ml) was
added to a solution of 0.29 mmol of the respective thioether in TFA (5 ml)
while stirring at ꢂ158C. The reaction was allowed to proceed for 1 h at ꢂ158C.
The dry ice bath was removed and the reaction was left to warm to room
temperature. TFA was evaporated and the residual product dissolved in
dichloromethane (30 ml) and washed with NaHCO3 (3 ꢀ 30 ml) and water
(3 ꢀ 30 ml) respectively. The organic layers were combined, dried (MgSO4) and
evaporated under reduced pressure and the crude material was purified by
flash column chromatography on silica gel.
N,N-bis-(2-chloroethyl)-(4-methylsulfinyl) aniline 1
This compound was prepared from N,N-bis-(2-chloroethyl)-(4-methylthio)
aniline (5) (76 mg, 0.29 mmol) following procedure B. The crude material was
purified by flash column chromatography (eluent 80% ether in hexane) giving
58 mg (0.21 mmol, 71%) of 1 as a brown solid. TLC Rf 0.31 in ether hexane
(7:1).
1H NMR (CDCl3): 2.68 (s, 3H), 3.64 (t, 4H, CH2, J=6.9 Hz), 3.78 (t, 4H, CH2,
J=6.9 Hz), 6.77 (d, 2H, CH, J=7.2 Hz), 7.54 (d, 2H, CH, J=7.2 Hz).
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13C NMR (CDCl3): 40.04 (CH2-Cl), 43.70 (CH3), 53.26 (CH2CH2N), 112.06
(CH-Ar), 125.91 (CH-Ar), 132.52 (C-S, Ar), 148.50 (C-N, Ar).
N-(2-chloroethyl)-N-(2-fluoroethyl)-(4-methylsulfinyl) aniline 11
This compound was prepared from N-(2-chloroethyl)-N-(2-fluoroethyl)-
(4-methylthio) aniline (9) (72 mg, 0.29 mmol) following procedure B. The
crude material was purified by flash column chromatography (eluent 80%
ether in hexane) to give 65 mg (0.25 mmol, 85%) of pure product as a brown
oil. TLC Rf 0.31 in ether hexane (7:1).
1H NMR (CDCl3): 2.73 (s, 3H), 3.61–3.82 (m, 6H), 4.58 (dt, 2H, F-CH2,
J = 47.1, 4.8 Hz), 6.77 (d, 2H, CH, J=9.0 Hz), 7.54 (d, 2H, CH, J=9.0 Hz).
13C NMR (CDCl3): 39.71 (CH3), 43.63 (CH2-Cl), 51.63 (d, F-CH2-CH2-N,
J=21 Hz), 53.32 (CH2-N), 81.15 (d, F-CH2-CH2-N, J=169.4 Hz), 112.09
(CH-Ar), 125.93 (CH-Ar), 132.08 (C-S, Ar), 148.96 (C-N, Ar).
Accurate MASS (ESI): calculated (M+Na) 286.0445. Found 286.0443.
N,N-bis-(2-chloroethyl)-[4-(4-nitrophenylsulfinyl)] aniline 2
This compound was prepared from N,N-bis-(2-chloroethyl)-[4-(4-nitrophe-
nylthio)] aniline (6) (107 mg, 0.29 mmol) following procedure B. The crude
material was purified by flash column chromatography (eluent 80% ether in
hexane) to give 110 mg (0.28 mmol, 98%) of pure product as a yellow solid.
TLC Rf 0.30 in ether hexane (7:1).
1H NMR (CDCl3): 3.62 (t, 4H, CH2, J=6.8 Hz), 3.77 (t, 4H, CH2, J=6.8 Hz),
6.71 (d, 2H, J=9.0 Hz), 7.50 (d, 2H, J=9.0 Hz), 7.78 (d, 2H, J=8.8 Hz), 8.30
(d, 2H, J=8.8 Hz).
13C NMR (CDCl3): 39.89 (CH2-Cl), 53.22 (CH2N), 112.26 (CH-Ar), 124.22
(CH-Ar), 125.27 (CH-Ar), 128.27 (CH-Ar), 131.29 (C-S, Ar), 148.97 (C-S, Ar),
149.26 (C-N, Ar), 153.37 (C-N, Ar).
MP 106-107.58C.
N-(2-chloroethyl)-N-(2-fluoroethyl)-[4-(4-nitrophenylsulfinyl)] aniline 12
This compound was prepared from N-(2-chloroethyl)-N-(2-fluoroethyl)-[4-
(4-nitrophenylthio)] aniline (9) (103 mg, 0.29 mmol) following procedure B.
The crude material was purified by flash column chromatography (eluent 80%
ether in hexane) to give 91 mg (0.25 mmol, 85%) of pure product as a yellow
oil. TLC Rf 0.35 in ether hexane (7:1).
1H NMR (CDCl3): 3.73 (t, 2H, CH2CH2-Cl, J=7.2 Hz), 3.70–3.78 (m, 4H),
4.57 (dt, 2H, F-CH2, J=47.2, 4.8 Hz), 6.69 (d, 2H, J=8.8 Hz), 7.47 (d, 2H,
J=8.8 Hz), 7.76 (d, 2H, J=8.8 Hz), 8.28 (d, 2H, J=8.8 Hz).
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13C NMR (CDCl3): 39.51 (CH2-Cl), 51.50 (d, F-CH2-CH2-N, J =21.4 Hz),
53.14 (CH2-N), 81.00 (d, F-CH2-CH2-N, J=170.2 Hz), 112.15 (CH-Ar),
124.14 (C-S, Ar), 125.24 (CH-Ar), 128.20 (CH-Ar), 130.75 (CH-Ar), 148.86
(C-S, Ar), 149.64 (C-N, Ar), 153.36 (C-N, Ar).
Accurate mass (ESI): calculated (M+Na) 393.0452. Found 393.0450.
General procedure for the radiosynthesis of [18F]11 and [18F]12
The precursor (2 mg) in DMSO (1 ml) was added and to the dried K[18F]F
kryptofix complex and heated at 1008C for 15 min. After the labelling, 10 ml of
water was added to the reaction vessel and the crude reaction mixture was
subsequently pushed through a C-18 Sep Pak for trapping of the crude
product. The Sep Pak was rinsed with 10 ml of water before elution with 1 ml
of ethanol. The ethanol fraction was injected into the semi-preparative HPLC
(Apollo C-18, 5 m, 10 ꢀ 250 mm) using conditions as described below.
HPLC conditions for SO101
0.1 M NH4HCO2/ acetonitrile : 40/60 (v/v) at a flow rate of 4 ml/min was used
as mobile phase. The radioactive peak at 12.16 min was collected and
reformulated in 10% ethanol using the Sep Pak method. Retention time of the
precursor was 18.32 min under these conditions.
Independent quality control (Develosil UG C-30, 5 m, 10 ꢀ 250 mm, 20 ml
injection loop) gave a single radioactive peak at 8.5 min, which was identical to
the F-19 standard.
HPLC conditions for SO201
0.1 M NH4HCO2/ acetonitrile : 50/50 (v/v) at a flow rate of 4 ml/min was used
as mobile phase. The radioactive peak at 11.5 min was collected and
reformulated in 10% ethanol using the Sep Pak method. Retention time of
the precursor was at 19.5 min under these conditions.
Independent quality control (Develosil UG C-30, 5 m, 10 ꢀ 250 mm, 20 ml
injection loop) gave a single radioactive peak at 11.7 min, which was identical
to the F-19 standard.
In vivo binding
Animal experimentation was approved by the Austin Health animal ethics
committee and was performed in accordance to guidelines of the NHMRC.
Focal cerebral ischemia was induced in Sprague-Dawley rats (n ¼ 8).20 The
middle cerebral artery was permanently occluded with a silicone-coated nylon
monofilament. 150 mCi of the radiotracer was injected into the tail vein 10 min
after induction of ischemia, and animals were decapitated 2 h later (n ¼ 4 each
compound). Brains were removed and immediately cut into 2 mm coronal
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sections using a rat brain template. These sections were covered with cling-
film, then placed on phosphor-imaging plates for 20 min in the dark. Plates
were read in a BAS-5000 plate reader (Fuji, Japan). Image analysis was
performed using the MCID program (Imaging Research, Canada). Images
were smoothed using a 15 ꢀ 15 smoothing kernel. Area of increased uptake
was defined by thresholding at mean + 3 SD. above the contralateral
hemisphere optical density.
Conclusion
In conclusion, we have been able to radiolabel two sulfoxide containing bis-
haloethyl anilines (SO101 and SO201) with F-18 via halogen exchange. The
optimum decay corrected labelling yields were 35–45% for [18F]11 (SO101)
and 25–35% for [18F]12 (SO201) at 1008C for 15 min for both compounds. The
putative tracers [18F]11 and [18F]12 were found to be stable in saline and 80%
intact in plasma over a period of 2 h. Uptake by hypoxic tissues in animals
suggest that further investigation of [18F]11 and [18F]12 as PET imaging agents
for hypoxic tissue are warranted.
Acknowledgements
N.S. is supported by an NHMRC postgraduate medical scholarship, and has
received a CVL grant (Pfizer Australia). This work was supported in part by
NHMRC program grant #251525. The authors would also like to thank the
Cancer Council for support and Dr Rachel Mulligan for helpful discussions.
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