Radiosynthesis of 1-(4-(2-[18F] fluoroethoxy)benzenesulfonyl)-3-butyl urea: A potential β-cell imaging agent

Article abstract of DOI:10.1002/jlcr.599

Tolbutamide (1) is a sulfonurea agent used to stimulate insulin secretion in type 2 diabetic patients. Its analogue 1-(4-(2-[¹⁸F]fluoroethoxy)benzenesulfonyl)-3-butyl urea (3) was synthesized in overall radiochemical yields of 45% as a potential β-cell imaging agent. Compound 3 was synthesized by ¹⁸F-fluoroalkylation of the corresponding hydroxy precursor (2) with 2-[¹⁸F]fluoroethyltosylate in DMF at 120°C for 10 min followed by purification with HPLC in a synthesis time of 50 min. Insulin secretion experiments of the authentic ¹⁹F-standard compound on rat islets showed that the compound has a similar stimulating effect on insulin secretion as that of tolbutamide (1). The partition coefficient of compound 3 between octanol/water was determined to be 1.3 × 0.3 (n = 5). Copyright

Full text of DOI:10.1002/jlcr.599

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 763–774.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.599
Research Article
Radiosynthesis of 1-(4-(2-[18F]
fluoroethoxy)benzenesulfonyl)-3-butyl urea:
a potential b-cell imaging agent
Ralf Schirrmacher*1, Michael Weber1, Alexander Schmitz2,
Chyng-Yann Shiue2, Abass A. Alavi2, Peter Feilen3,
3
3
1
.
Stefan Schneider , Peter Kann and Frank Rosch
1 Institute of Nuclear Chemistry, Johannes Gutenberg-University Mainz,
Fritz Strassmann-Weg 2, D-55128 Mainz, Germany
2 Department of Radiology, University of Pennsylvania, Philadelphia,
PA 19104, USA
3 1. Med. Clinic, Division of Endocrinology and Metabolic Disease, Johannes
Gutenberg-University Mainz, D-55131 Mainz, Germany
Summary
Tolbutamide (1) is a sulfonurea agent used to stimulate insulin secretion in type
2 diabetic patients. Its analogue 1-(4-(2-[18F]fluoroethoxy)benzenesulfonyl)-3-
butyl urea (3) was synthesized in overall radiochemical yields of 45%
as a potential b-cell imaging agent. Compound 3 was synthesized by
18F-fluoroalkylation of the corresponding hydroxy precursor (2) with
2-[18F]fluoroethyltosylate in DMF at 1208C for 10 min followed by purification
with HPLC in a synthesis time of 50 min. Insulin secretion experiments of the
authentic 19F-standard compound on rat islets showed that the compound has
a similar stimulating effect on insulin secretion as that of tolbutamide (1). The
partition coefficient of compound 3 between octanol/water was determined to
be 1.3 Æ 0.3 (n=5). Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: 18F-sulfonureas; b-cell imaging agents; diabetes; 18F-fluoro-
alkylation
*Correspondence to: R. Schirrmacher, Institute of Nuclear Chemistry, D-55128 Mainz, Germany.
E-mail: schirrmacher@mail.kernchemie.uni-mainz.de
Copyright # 2002 John Wiley & Sons, Ltd.
Received 9 October 2001
Revised 25 February 2002
Accepted 12 March 2002

764
R. SCHIRRMACHER ET AL.
Introduction
Diabetes mellitus comprises a heterogeneous group of disorders
characterized by high blood glucose levels. Two major types of diabetes
mellitus have been defined: type 1 and type 2. Although hyperglycemia
is the common denominator of both type 1 and type 2, the etiology and
pathophysiology of these syndromes are distinct. Type 1 is a chronic
autoimmune disease characterized by the selective destruction of
insulin-producing b-cells of the islets of Langerhans. When autoimmune
destruction affects more than 90% of the b-cell mass, the resulting
insulin deficiency culminates into the development of overt hypergly-
cemia. In type 2, on the other hand, the pancreatic b-cells are initially
intact, and the disease is associated with insulin resistance and loss of b-
cell function, and eventual insulin dependency.1,2
Due to the ongoing efforts in transplanting b-cell mass, there is a
great medical interest in specific b-cell imaging agents to quantify the
acceptance of transplanted islets in human in vivo.6–8 Additionally, in
the context of type 1 diabetes mellitus the chronic and progressive loss
of b-cells due to autoimmune destruction has led to concerted efforts to
prevent further loss of b-cells by autoantigen-specific immunotherapy of
pre-diabetic patients. Furthermore, the monitoring of b-cell mass during
the silent phase of pre-diabetes in diabetes mellitus 2 patients is of
special interest. For all these purposes it is of great importance to have a
non-invasive method to quantify b-cell mass in vivo.
The aim of this study was to synthesize b-cell-specific positron
emitting radiolabeled sulfonureas such as tolbutamide (1) to image the
b-cell mass in vivo using positron emission tomography (PET).
Sulfonureas are antidiabetic agents which block pancreatic ATP-
sensitive potassium channels, located at the insulin producing b-cells
of the islets of Langerhans, either directly or via a plasma membrane
associated protein, resulting in an increase of intracellular calcium ion
concentration and consequent insulin secretion.3–5
Tolbutamide (1) is a sulfonurea agent used to stimulate insulin
secretion in type 2 diabetic patients.9–11 We hypothesize that in vitro
testing of this class of compounds would provide a strong basis to
proceed with in vivo studies in a non-human primate model, initially,
and in human experiments eventually. We intend to determine the
efficacy of these radiolabeled agents in visualizing and quantifying b-cell
concentrations in the pancreas of normal non-human primates by
PET.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 763–774

SYNTHESIS OF A b-CELL IMAGING AGENT
765
O
O
N
X
S
N
H
H
O
1. X=CH3
2. X=OH
Tolbutamide
1-(4-Hydroxybenzenesulfonyl)-3-butyl urea
3. X=O-(CH2)2[18F]F 1-(4-(2-[18F]fluoroethyl)benzenesulfonyl)-3-butyl urea
Figure 1. Structure of tolbutamide and its derivatives
The 18F-fluoro derivative of tolbutamide, 1-(4-[18F]fluorobenzenesul-
fonyl)-3-butyl urea, was synthesized recently in a low overall yield of
1–2%.12 This low yield makes it difficult to eventually use this
radiopharmaceutical routinely in nuclear medicine. To overcome this
problem partially, we have synthesized its 18F-fluoroethoxylated
derivative (3) (Figure 1) and tested its efficacy in insulin secretion.
Results and discussion
We have labeled the tolbutamide analogue (3) with fluorine-18 for PET
studies. The precursor 1-4-(hydroxybenzenesulfonyl)-3-butyl urea (2)
was synthesized in multiple steps (Scheme 1). Diazotization of 4-
aminobenzenesulfonamide (4) followed by hydroxylation gave
4-hydroxybenzenesulfonamide (5).13 Acetylation of compound 5 with
acetic anhydride gave compound 6 which prevents the formation of
1-(4-butylcarbamoylbenzenesulfonyl)-3-butyl urea14 as a by-product in
the subsequent step. Reaction of compound 6 with butyl isocyanate
in DMF with Cu(I)Cl as a catalyst15 yielded compound 11 which was
de-protected with sodium methoxide in methanol to give the final
product (2). The overall chemical yield was 58%.
The non-radioactive authentic compound 1-(4-(2-fluoroethoxy)ben-
zenesulfonyl)-3-butyl urea (8) was synthesized in a similar manner
(Scheme 1). Reaction of compound 5 with 1-bromo-2-fluoroethane in
DMF yielded 4-(2-fluoroethoxy)benzenesulfonamide (7). Coupling of
compound 7 with butyl isocyanate in triethylamine gave (8)16 in an
overall chemical yield of 40%. The identity of the compounds was
verified by 1H-NMR as well as elemental analysis and for some
compounds additionally by 13C-NMR and MS (FD).
Fluorine-18 labeled compound 3 was synthesized in a two-step
synthesis by direct nucleophilic substitution of ethylene glycol-1,2-dito-
sylate (9) with K[18F]/Kryptofix 2.2.2 to yield the 18F-fluoroethylating
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R. SCHIRRMACHER ET AL.
O
S
O
O
S
O
NH2
HO
S
NH2
H2N
NH2
O
O
O
4
6
5
O
O
O
O
O
OCN
Cu(I)Cl
NaOCH3, CH3OH
N
H
O
S
N
H
O
11
2
O
S
N
H
N
H
HO
O
O
O
F
O
S
NH2
HO
S
NH2
O
O
7
8
5
O
O
S
OCN
F
O
N
N
H
H
NEt3
O
Scheme 1. Syntheses of the labeling precursor 1-(4-hydroxybenzene)-3-butyl
urea (2) and the authentic 19F-standard compound 8
agent 2-[18F]fluoroethyl tosylate (10),17 and subsequent reaction with
the labeling precursor (2) at 1208C in DMF (Scheme 2). The overall
radiochemical yield after HPLC purification was 45% within a total
preparation time of 50 min.
In comparison to the recently described compound 1-(4-[18F]fluoro-
benzenesulfonyl)-3-butyl urea12 the new compound 3 could be obtained
in much higher yields. Due to the nature of nucleophilic aromatic
substitution with [18F]fluoride the aromatic system must be activated for
obtaining reasonable radiochemical yields. In the case of the previously
described two-step synthesis of 1-(4-[18F]fluorobenzenesulfonyl)-3-butyl
urea, radioactive labeling was limited by the nucleophilic substitution of
the nitro group of 4-nitrobenzenesulfonamide. Reaction with K[18F]/
Kryptofix 2.2.2 gave the corresponding product 4-[18F]fluorobenzene-
sulfonamide in 3–7% yield. These relatively low yields indicate that the
aromatic system is probably not well enough activated or the
sulfonamide moiety with its two H-atoms decreases the nucleophilicity
of the [18F]fluoride via complexation. All these limitations have no
influence on the 18F-fluoroalkylation of phenolic hydroxy groups with
2-[18F]fluoroethyl tosylate because the critical radioactive labeling step
has already been performed. Besides, the nucleophilicity of a
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 763–774

SYNTHESIS OF A b-CELL IMAGING AGENT
767
K[18F]/K2.2.2
OTos
OTos
TosO
18F
acetonitrile, 80°C
9
10
O
O
HO
S N
N
H
H
O
2
OTos
18F
NaOH/DMF
10
O
O
18F
O
S N
N
H
H
O
3
Scheme 2. Radiosynthesis of 1-(4-(2-[18F]fluoroethyl)benzenesulfonyl)-3-butyl
urea (3)
deprotonated phenolic hydroxy function is high and thus guarantees a
good radiochemical yield of the corresponding 2-[18F]fluoroethyl phenyl
ether.18
The identity of compound 3 was verified by both TLC and HPLC
compared to the non-radioactive authentic samples. In vitro experiments
showed that compound 8 has a similar stimulating effect on insulin
secretion as that of tolbutamide (1) itself. The increase of insulin
secretion after stimulation with compound 8 was determined to be
183 Æ 50% (n=6) and was in the same range as that of the original
tolbutamide 1 with 175 Æ 19% (n=6). These results indicate that the
derivatization of tolbutamide 1 does not affect the binding to the
sulfonylurea receptor significantly. The octanol/water partition coeffi-
cient of compound 3 was 1.3 Æ 0.3 (n=5) which is lower than that of
the recently reported 1-(4-[18F]fluorobenzenesulfonyl)-3-butyl urea
(3.13 Æ 0.28)
and
18F-fluoroethylated
glyburide
derivative
(124 Æ 21.61).12 The high lipophilicity of these tracers might result in
a higher liver uptake and will lead to a difficulty in differentiation
between pancreatic and liver radioactivity uptake. A less lipophilic
compound such as compound 3 decreases the liver uptake and
enhances the imaging quality of b-cell islets in pancreas. The evaluation
of compound 8 was performed before the labeling experiments were
done.
Copyright # 2002 John Wiley & Sons, Ltd.
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R. SCHIRRMACHER ET AL.
Experimental
4-Aminobenzenesulfonamide, ethyleneglycol-1,2-ditosylate and butyl
isocyanate were purchased from Aldrich Chemical Company
(Germany) and used without further purification. Tolbutamide was
purchased from Sigma (Germany). Solid phase columns were purchased
from Merck (Lichrolut EN). Analytical thin layer chromatography
(TLC) was performed using plates from Merck (Silicagel 60 F254). High-
performance liquid chromatography (HPLC) was performed with a
Sycam S1100 system, UV detection was obtained using an UV detector
Sycam S3200. NMR spectra were recorded using a Bruker 200-MHz-
FT-NMR spectrometer AC 200. Chemical shifts are quoted in d (ppm)
downfield from tetramethylsilane (TMS) as an internal standard. MS
spectra were obtained on a Finnigan MAT90 spectrometer. Elemental
analyses were performed with an EL2 system (Elementar vario).
Melting points were determined on an Electrothermal 9100 apparatus
and are uncorrected. Detection of radioactivity was performed using an
Instant Imager (Packard Canberra) for radio-TLC and detection of
radio-HPLC was performed using an NaI-radiodetector (Packard
Canberra). [18F]fluoride as produced via the 18O(p,n)18F reaction was
purchased from different institutions. Purification of the labeling
precursor 2-[18F]fluoroethyl tosylate was accomplished using an HPLC
column (Lichrosphere RP18-EC5, 250 Â 10 mm). Purification of the
labeled compound was achieved using an HPLC column (Lichrosphere
100 RP 18-5 mEC 250 Â 4.6 mm).
Syntheses of precursors and authentic samples
4-hydroxybenzenesulfonamide (5)
Sulfanilamide 4 (5 g, 29 mmol), dissolved in a mixture of concentrated
sulfuric acid (20 ml) and water (40 ml), was diazotized at 08C with
sodium nitrite (2 g, 29 mmol) in water (20 ml) and then heated on a
water-bath until the evolution of nitrogen ceased. After the mixture had
been kept overnight at 48C, the crystals of 4-hydroxybenzenesulfona-
mide (5) were collected. A further quantity of the compound was
obtained by exhaustively extracting the filtrate with ether. On
recrystallization from water, the sulfonamide was obtained in the form
of large yellow needles (4 g, 23.6 mmol, 80%), mp 1758C (1768C13).
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF A b-CELL IMAGING AGENT
769
1H-NMR (d6-DMSO): (2 H, d, 6.85 ppm), (2 H, s, 7.1 ppm), (2 H, d,
7.6 ppm), (1 H, s, 10.2 ppm); Anal. (C5H7NO3S) C, H, N.
4-acetyloxybenzenesulfonamide (6)
To 4-hydroxybenzenesulfonamide (5) (2 g, 11.8 mmol) dissolved in a
sodium carbonate solution (pH 8), acetic anhydride (2.55 g, 25 mmol)
was added with cooling. After a short while 4-acetyloxybenzene
sulfonamide (6) was separated and recrystallized from 50% aqueous
1
alcohol (2.1 g, 9.7 mmol, 86%), mp 1568C (1588C13). H-NMR (d6-
DMSO): (3 H, s, 2.3 ppm), (2 H, d, 7.3 ppm), (2 H, s, 7.4 ppm), (2 H, d,
7.85 ppm); Anal. (C8H9NO4S) C, H, N.
1-(4-acetyloxybenzenesulfonyl)-3-butyl urea (11)
4-Acetyloxybenzenesulfonamide (6) (0.6 g, 2.8 mmol) was dissolved in
DMF (2.5 ml). Butylisocyanate (0.55 g, 5.6 mmol) and copper (I)
chloride (20 mg) were added under an argon atmosphere and stirred
for 24 h. The crude mixture was poured into ice water (70 ml) under
vigorous stirring and hydrochloric acid (2 N) was added until a white
solid was precipitating. After drying under vacuum the product was
obtained as a white solid (0.8 g, 2.5 mmol, 91%), 1568C (decomposition).
1H-NMR (d6-DMSO): (3 H, s, 0.8 ppm), (4 H, m, 1.1–1.3 ppm),
(3 H, s, 2.3 ppm), (2 H, q, 2.9 ppm), (1 H, t, 6.5 ppm), (2 H, d, 7.35 ppm),
(2 H, d, 7.9 ppm), (1 H, s, 10.6 ppm). Elemental analysis (C13H18N2O5S)
C, H, N.
1-(4-hydroxybenzenesulfonyl)-3-butyl urea (2)
1-(4-Acetyloxybenzenesulfonyl)-3-butyl urea (11) (0.8 g, 2.5 mmol) was
dissolved in a small amount of methanol (3 ml) and a pH of 8.5 was
adjusted using a 1% NaOMe/MeOH solution and was stirred overnight
at room temperature. The mixture was added slowly under stirring to a
hydrochloric acid/diethyl ether mixture (5%) (10 ml) and the resulting
precipitate was filtered, washed with water and dried under vacuum to
yield the product nearly quantitatively (0.65 g, 2.3 mmol, 94%), mp
2808C (decomposition).
1H-NMR (d6-DMSO): (3 H, t, 0.8 ppm), (4 H, m, 1.1–1.3 ppm), (2 H,
q, 2.8 ppm), (1 H, t, 5.9 ppm), (2 H, d, 6.7 ppm), (2 H, d, 7.5 ppm), (1 H,
s, 10.6 ppm); 13C-NMR (d6-DMSO) d [ppm]: 14, 20, 32, 39, 115, 129,
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R. SCHIRRMACHER ET AL.
132, 151, 162; MS (FD): m/z (% relative intensity) 272.6 (100.0, [M]+);
Anal. (C11H16N2O4S) C, H, N.
4-(2-fluoroethoxy)benzenesulfonamide (7)
Elemental sodium (0.132 g, 5.7 mmol) was carefully added to methanol
(30 ml) and refluxed until no sodium was left. The sodium methanolate
solution was cooled to room temperature and 4-hydroxybenzene
sulfonamide (5) (1 g, 5.7 mmol) was added and stirred for 30 min. The
methanol was evaporated under vacuum and the remaining sodium
phenolate was dissolved in DMF (15 ml). Sodium iodide (10 mg) and 1-
bromo-2-fluoroethane were added and the mixture was kept at 708C
overnight. The DMF was removed under vacuum (10À3 mbar) and the
crude product was purified via column chromatography (Si-60, chloro-
form/methanol/acetone (6/1/1)) to yield (7) as a white solid (0.75 g,
3.4 mmol, 59%), mp 132–1348C.
1H-NMR (d6-DMSO): (1 H, t, 4.2 ppm), (1 H, t, 4.4 ppm), (1 H, t,
4.6 ppm), (1 H, t, 4.9 ppm), (2 H, d, 7.1 ppm), (2 H, s, 7.2 ppm), (2 H, d.
7.75 ppm); Anal. (C7H10FNO3S) C, H, N.
1-(4-(2-fluoroethoxy)benzenesulfonyl)-3-butyl urea (8)
4-(2-Fluoroethoxy)benzenesulfonamide (7) (0.25 g, 1.1 mmol) and bu-
tylisocyanate (0.55 g, 5.6 mmol) were added to triethylamine (10 ml) and
stirred for 15 h at 908C. The reaction mixture was cooled to room
temperature and poured into DMF (40 ml). Hydrochloric acid (4 ml
water/20 ml HClconc.) was added, the precipitating product was filtered
and washed with water to give compound 8 as a white solid (0.27 g,
0.85 mmol, 75%), mp 148–1498C.
1H-NMR (d6-DMSO): (3 H, t, 0.8 ppm), (4 H, m, 1.1–1.3 ppm), (2 H,
q, 2.9 ppm), (1 H, t, 4.2 ppm), (1 H, t, 4.4 ppm), (1 H, t, 4.6 ppm), (1 H, t,
4.9 ppm), (1 H, t, 6.4 ppm), (2 H, d, 7.15 ppm), (2 H, d, 7.8 ppm), (1 H, s,
10.4 ppm); 13C-NMR (d6-DMSO) d [ppm]: 14, 20, 32, 39, 68, 82, 115,
129, 132, 151, 162; MS (FD): m/z (% relative intensity) 318.6 (100.0,
[M]+); Anal. (C13H19FN2O4S) C, H, N.
2-[18F]fluoroethyltosylate (10)
No-carrier-added (NCA) aqueous [18F]fluoride (540–1100 MBq) pre-
pared by the 18O(p,n)18F nuclear reaction on an enriched water (95%)
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF A b-CELL IMAGING AGENT
771
target was added to a solution of K2CO3 (15 ml (1 N)) and Kryptofix
2.2.2. (10–15 mg) in CH3CN (800 ml). The water was removed using a
stream of nitrogen at 808C and coevaporated to dryness with CH3CN
(2 Â 1 ml).
To the dried Kryptofix 2.2.2/[18F]fluoride (380–750 MBq) complex in
acetonitrile (1 ml) ethylenglycol-1,2-ditosylate (9) (8–10 mg, 20–25 mmol)
was added and heated under stirring in a sealed vial for 3 min.
Purification of the crude product was accomplished using HPLC
(acetonitrile/water, 50:50, flow rate: 5 ml/min, Rt: 8 min). After diluting
the HPLC fraction containing the 2-[18F]fluoroethyltosylate with water,
the product was loaded on an 18C-Sepac column, dried with nitrogen
gas and eluted with 1 ml of tempered (40–508C) DMF to yield a product
activity of 300–600 MBq 2-[18F]fluoroethyltosylate (10).
1-(4-(2-[18F]fluoroethoxy)benzenesulfonyl)-3-butyl urea (3)
To 1-(4-hydroxybenzenesulfonyl)-3-butyl urea (2) (5 mg, 18.4 mmol)
dissolved in DMF (0.6 ml), sodium hydroxide solution (1 N, 18.4 ml) was
added and the solution was heated at 808C for at least 3 min. A solution
of 2-[18F]fluoroethyltosylate (300–600 MBq) in DMF (300–500 ml) was
added and stirred in a sealed reaction vessel at 1208C for 10 min. The
product was purified with HPLC (acetonitrile/water 1:1, flow rate 1 ml/
min, tr: 5.9 min). After diluting the HPLC fraction containing the
product with water, it was loaded on a solid phase column (LiChrolut
EN, Merck), dried with nitrogen and eluted with 1 ml ethanol to yield
220–440 MBq of the product (3). HPLC analysis showed a radio-
chemical purity of >98%. Radio-TLC analysis confirmed the results
(chloroform/methanol/acetone 6:1:1, Rf=0.5). The specific activity of 3
was between 40 and 48.1 GBq/mmol as determined via a UV-calibration
curve.
Determination of partition coefficient of compound 3
The lipid/water partition coefficient of compound 3 was measured by
adding 5 ml of the compound in saline solution to a 5 ml vial containing
1 ml each of 1-octanol and pH 7.0 phosphate buffer. The vial was
capped and vortexed vigorously for 10 min at room temperature.
After reaching equilibrium, the organic phase was pipetted out and
each phase was analyzed via radio-TLC at an Instant Imager
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R. SCHIRRMACHER ET AL.
(Packard Canberra). The partition coefficient was calculated as (cpm in
1-octanol)/(cpm in pH 7.0 phosphate buffer).
Insulin secretion experiments of compound 8 on rat islets
For testing the in vitro function of the tolbutamide derivative (8) and the
original tolbutamide (1) a standardized batch stimulation was
performed. Adult rat islets were isolated by collagenase digestion and
purified by a density gradient.
Lewis rats (Central Animal Facility, University of Mainz), 6–8 weeks
old, body weight 250–270 g, were used as islet donors. Rats were
anesthetized by intraperitoneal pentobarbital administration (60 mg/
kg); a midline abdominal incision was performed; and the pancreas was
exposed and injected via the pancreatic duct with Hanks’ balanced salt
solution (HBSS; Gibco BRL, Long Island, NY) containing 1.7 mg/ml
collagenase (Serva PanPlus, Heidelberg, Germany). After the death of
the animal, the pancreatic tissue was surgically removed and incubated
for 10 min at 378C in the collagenase solution. Mechanical disruption of
the digested pancreatic tissue was achieved by further incubation at
378C for 10 min in collagenase solution, interrupted every 2 min by
shaking for 30 s. The digestion process was stopped by addition of
cooled HBSS plus 10% fetal calf serum (48C). Islet purification was
achieved using a discontinuous three-phase Ficoll density gradient
(densities: 1.090, 1.077 and 1.040). Islets were cultured in RPMI medium
(Biochrom KG, Berlin, Germany) at 378C. The medium contained
5.1 mmol/l (1 g/l) d-glucose, 25 mmol/l HEPES, 10% fetal calf serum
(Greiner Laboratories, Frickenhausen, Germany), 0.2 g/l Glutamax
(GibcoBRL, Paisley, Scotland), penicillin, streptomycin (GibcoBRL,
Paisley, Scotland) and Ciprobay (Hoechst, Frankfurt, Germany).
For each sample ten islets were picked (equal in size and shape) in a
culture insert with a membrane of 12 mm pore size (Millicell PCF,
Millipore, France) and incubated in a 24-well culture plate (Falcon
Multiwell, Becton Dickinson, USA). First the basal insulin secretion
was tested by incubating the islets with normo-glycemic culture media
(RPMI 1640+d-glucose 1 g/l+10% FCS+P/S) for 1 h at 378C. After
the culture period the media were collected and stored at À208C. The
inserts with islets were transferred to normo-glycemic culture media
with several concentrations (2.5, 25 and 250 mg/ml) of the tolbutamide
(1) and the 19F-derivative (8) and incubated for a stimulation period
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SYNTHESIS OF A b-CELL IMAGING AGENT
773
Table 1. Insulin secretion of 8, 1 and D-glucose at different concentrations
Compound
Stimulation effect (% Æ SD%) at different concentrations
2.5 mg/ml
25 mg/ml
250 mg/ml
3 g/l
1
8
118 Æ 13
127 Æ 15
}
175 Æ 19
183 Æ 50
}
120 Æ 8
108 Æ 16
}
}
}
297 Æ 30
d-Glucose
of 1 h. As a positive control several inserts with islets were cultured with
a hyperglycemic culture media (RPMI 1640 d-glucose 3 g/l+10%
FCS+P/S) only. For the negative control normo-glycemic culture
media (RPMI 1640 d-glucose 1 g/l+10% FCS+P/S) with a diluted
solution but with neither compound 1 nor compound 8 was used. The
insulin content of each probe was quantified by a rat-insulin ELISA
(Mercodia, Sweden). The stimulation effect (in %) was calculated as
stimulated insulin secretion divided by basal insulin secretion  100
(Table 1).
Conclusion
We have synthesized 1-(4-(2-[18F]fluoroethoxy)benzenesulfonyl)-3-butyl
urea (3) in an attempt to use it as a b-cell imaging agent for the non-
invasive determination of the progressive loss of b-cell mass during the
silent phase of pre-diabetes or transplanted islet mass. The overall yield
of the radiosynthesis was 45% within 50 min and the specific activity
was determined to be in the range of 40–48 GBq/mmol. In comparison to
the recently published fluorine-18 labeled tolbutamide and glyburide
derivatives, compound 3 is easier to synthesize and gives a better yield.
In addition, compound 3 is less lipophilic than fluorine-18 labeled
tolbutamide and glyburide which may decrease the liver uptake and
could lead to a better resolution for the endocrine pancreatic tissue. The
feasibility of using sulfonylurea receptor ligands as b-cell imaging agents
is under investigation.
References
1. Taylor SI. Cell 1999; 97: 9–12.
2. DeFronzo R. Diabetes Rev 1997; 5: 177–269.
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