No-carrier-added, 18F-labelling of a cholesterol derivative, used in detection of adrenal malignancies

Article abstract of DOI:10.1002/jlcr.496

[¹⁸F]Cholesteryl 4-(fluoromethyl)benzoate 6b was prepared from 3-cholesteryl [(4-nitrobenzenesulfonyl)oxymethyl]benzoate in one step using K⁺-Kryptofix 2.2.2, as counter ion and acetonitrile as the solvent at 40-60°C. The product was

Full text of DOI:10.1002/jlcr.496

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 747–755.
DOI: 10.1002/jlcr.496
Research Article
18
No-carrier-added, F-labelling of a
cholesterol derivative, used in detection of
adrenal malignancies
2
1
Amir Reza Jalilian1, , Maria Bineshmarvasti , Hamid Rafii ,
*
Hossein Afarideh1 and Abbas Shafiee2
1 Cyclotron Department, Nuclear Research Center for Agriculture & Medicine,
Karaj, Iran, P.O. Box: 31585-4395 Karaj
2 Faculty of Pharmacy of Tehran University of Medical Sciences, Tehran, Iran,
P.O. Box: 14155-6451, Tehran
Summary
[18F]Cholesteryl 4-(fluoromethyl)benzoate 6b was prepared from 3-
cholesteryl [(4-nitrobenzenesulfonyl)oxymethyl]benzoate in one step
using K+-Kryptofix 2.2.2, as counter ion and acetonitrile as the solvent
at 40–608C. The product was isolated by HPLC in 75% (decay
corrected) radiochemical yield with a radiochemical purity of more than
90% in 30 min (specific radioactivity 2000–2500 Ci/mmol). Biodistribu-
tion studies in mice showed that a high target/non-target ratio of
radioactivity was obtained in adrenal and ovaries. Copyright # 2001
John Wiley & Sons, Ltd.
Key Words: fluorine-18; PET; cholesteryl esters; adrenal malignancies;
[18F]cholesteryl 4-(fluoromethyl)benzoate ([18F]CFMB)
*Correspondence to: A. R. Jalilian, Cyclotron Department, Nuclear Research Center for
Agriculture & Medicine, Karaj, Iran, P.O. Box 31585-4395, Karaj, Iran. E-mail: ajalili@seai.
neda.net.ir
Copyright # 2001 John Wiley & Sons, Ltd.
Received 5 March 2001
Revised 24 April 2001
Accepted 21 May 2001

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A. R. JALILIAN ET AL.
Introduction
Cholesterol derivatives accumulate in steroid secreting tissues because
these organs use cholesterol as a precursor in steroid synthesis. Gamma
emitting isotopes of iodine (131I; t1=2 ¼ 8 day or 123I; t1=2¼ 13:2 h) have
been used to label cholesterol and its derivatives for administration to
human subjects. The labelled compounds were selectively absorbed in
adrenal and ovaries.1,2 Thus, cholesteryl esters can be used as adrenal
imaging agents to diagnose various adrenal diseases.3 Previously, a
fluorine-18-labelled cholesteryl ester, [18F]cholesteryl 4-fluorobenzoate
([18F]-CFB) had been prepared by a fluoro for nitro exchange reaction
and tested as a prospective radioligand for positron emission
tomography (PET).4,5 In this paper, a new radiolabelled cholesterol
ester, [18F]cholesteryl 4-(fluoromethyl)benzoate ([18F]CFMB), has been
developed. In view of the low efficacy of fluoro for nitro exchange used
in the previous labelling procedure, it was our intention to produce a
new cholesteryl aromatic ester that could be labelled in higher yields and
under mild conditions.
Results and discussion
Highly sterically hindered cholesteryl esters, when suitably radiolabelled
have been shown to be good diagnosticagents, based on their slow
rate of hydrolysis and high accumulation rate in steroid secreting
tissues.2,6
Aliphaticnulceophilicsubstitution is one of the best and most
efficient way to synthesize 18F-labelled ligands. Many research groups
have shown that sulfonyl esters are the best candidates for fluoro
exchange.7 The radiolabelled target molecule 6b, was prepared
according to Scheme 1. In the first step, cholesteryl 4-bromomethyl-
benzoate was prepared in a rapid procedure with high purity by
condensation of related acid 2 and cholesterol 1 using DCC in
anhydrous dichloromethane. The labile 4-nitrobenzenesulfonate group
had been shown to be the best leaving group for nucleophilic fluoride
anion in the presence of reactive aminopolyether, Kryptofix 222, in
acetonitrile as solvent. In the next step, in order to obtain a better
leaving group than bromo, freshly prepared silver 4-nitrobenzene
sulfonate salt 4, was treated with 3 in acetonitrile at room temperature
and darkness according to previous methods.8
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NO-CARRIER-ADDED, 18F
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Scheme 1. Preparation of 6a and 6b
The product, compound 5, was repeatedly recrystallized to ensure the
removal of silver ion, since trace silver impurities are susceptible to light
and temperature. The pure compound can be easily stored for several
months. The final solution was injected into mice intravenously.
Enhanced radioligand uptake was observed 30 min after ethynyl
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750
A. R. JALILIAN ET AL.
esteradiol injection as previously reported in the literature.9–11 After 1 h,
the subjects were dissected and the radioactivity of different organs was
measured using a radiometer. Previous studies had shown that
lipoprotein uptake in adrenal regions was enhanced after ethinyl
esteradiol pretreatment.12 Due to cholesterol derivative incorporation
into lipoproteins, their absorption is increased using ethynyl esteradiol.
In this study, ethynyl esteradiol pretreatment effectively increased the
adrenal uptake 5 fold and spleen uptake by greater than 16 fold. Uptake
of radioactivity by bone was low in the control and in ethinyl esteradiol
pretreated groups due to low defluorination of 18F-CFMB.
Experimental
Cholesterol, Kryptofix 222 (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo
[8,8,8] hexacosane) and other chemicals were purchased from the
1
Aldrich Chemical Co., UK. H NMR spectra were obtained on a
Bruker FT-80 (80 MHz) or a Varian (400 MHz) instrument with
tetramethylsilane as the internal standard. Infrared spectra were taken
on a Perkin-Elmer 781 spectrometer (KBr disks). Mass spectra were
recorded using a Finnigan Mat TSQ-70 spectrometer. Thin-layer
chromatography (TLC) of non-radioactive products was performed
on silica gel polymer backed (F 1500/LS 254, 20 ꢀ 20 cm2, TLC Ready
Foils Schleicher & SchuellTM) or glass plates (25 ꢀ 35 cm2, E-Merck).
Acetonitrile, used for the labelling step was of ‘Sure-Seal’TM grade
supplied by Aldrich. Analytical HPLC was performed to determine
specific radioactivity, with a Shimadzu LC-10AT instrument, equipped
with two detector systems, that included a flow scintillation analyzer
(with Packard-150TR) and UV-visible (Shimadzu) detector using on Si
Kromasil 100, 5 mm 250 ꢀ 4.6 mm2 (M & W), InchromTM column. The
specific radioactivity of 6b was calculated using a standard curve
prepared for 6a. Radiochromatography was performed using a rotary
motor coupled to a CanberraTM germanium detector (model GC1020-
7500SL) using polymer-backed silica gel paper. Purification of 6b was
performed using a Silica Kromasil preparative column purchased from
WatersTM. Melting points were determined on a Reichert-JungTM
microscope and are not corrected. Elemental microanalyses were within
ꢁ 0.4% of theoretical values for C, H, F, and N.
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NO-CARRIER-ADDED, 18F
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Preparation of [18F]-potassium fluoride from [18O]-water
[18F]fluoride was prepared by bombardment of an enriched 18O-water
sample (1.7 ml, >95 at%, CortecTM, France) held in an all-silver target
with 18 MeV protons from a 30 MeV cyclotron at the Nuclear Research
Center for Agriculture and Medicine (NRCAM) Karaj-Iran. After the
18
recovery of O-water over an anion exchange membrane (Bio-RadTM
AG 1-X8 Anion exchange, carbonate form 47 mm), fluoride-18 anion
was eluted by a 1% potassium carbonate solution (1%, 100 ml). The
eluted solution was directly used in the labelling reaction.
Preparation of 4-fluoromethylbenzoic acid 8
Kryptofix 222 (140 mg, 0.38 mmol), methyl 4-bromomethyl benzoate8
(150 mg, 6.6 mmol) and potassium fluoride (60 mg, 1.05 mmol) were
refluxed in anhydrous acetonitrile (10 ml) at 508C for 15 h. The reaction
was monitored by TLC, using hexane as the mobile phase. The reaction
mixture was cooled to room temperature and filtered. The filtrate
was concentrated in vacuum and purified by TLC over silica gel
coated glass plates using hexane as the mobile phase. The fraction
at Rf ¼ 0:3 was separated and extracted by chloroform. Then the
extract was concentrated in vacuum. The physicochemical data
were as reported previously.13 The product can be used in the next
step directly. 4-Fluoromethyl benzoic acid 8 was prepared by equimolar
hydrolysis of related ester with 1 N sodium hydroxide solution and
subsequent acidification. The physicochemical data were as reported
previously.13
Preparation of cholesteryl 4-bromomethylbenzoate 3
A mixture of 4-bromomethylbenzoicacid 2 (215 mg, 1 mmol), choles-
terol 1 (386 mg, 1 mmol) and dicyclohexyl carbodiimide (DCC) (206 mg,
1 mmol) was stirred vigorously in anhydrous dichloromethane (15 ml)
for 24 h at room temperature. The mixture was filtered and the filtrate
evaporated to dryness. The residue was crystallized from ethyl acetate–
hexane mixture (7 : 3, v/v) to give a white light powder (70%). m.p.:
177–1808C. 1H NMR (CDCl3) d (ppm) 7.48–8.17 (ABq, 4 H, aromatic),
5.30–5.34 (m, 1 H, C6-H), 4.52 (s, 2 H, CH2), 3.33–3.37 (m, 1 H, C3-H),
0.67–2.35 (m, 43 H, aliphatic). IR (KBr) nmax 2967, 1640, 1243, 640, 729.
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A. R. JALILIAN ET AL.
Preparation of cholesteryl 4-fluoromethylbenzoate 6a
Compound 6a was prepared according to the procedure discussed above
for 3, using equimolar portions of acid 8, cholesterol 1 and DCC in
1
anhydrous dichloromethane and led to a yellow oil (60%). H NMR
(CDCl3) d (ppm) 7.49–8.27 (ABq, 4 H, aromatic), 5.50 (s, 1 H, FCH2),
5.33–5.37 (m, 1 H, C6-H), 5.29 (s, 1 H, FCH2), 3.48–3.53 (m, 1 H, C3-H),
0.61–2.31 (m, 43 H, aliphatic). IR (KBr) nmax 3050, 1642, 1245.
Preparation of cholesteryl 4-[(4-nitrobenzene sulfonyl)-
oxymethyl] benzoate 5
Compound 5 was synthesized using silver 4-nitrobenzene sulfonate salts
4 and 3 in acetonitrile at room temperature. Freshly prepared silver
4-nitrobenzenesulfonate (0.85 g, 2.7 mmol) was dissolved in Sure-SealTM
grade acetonitrile (10 ml) and the mixture was added to 3 (560 mg,
0.96 mmol) in a capped vial and stirred vigorously at room temperature
for 15 h. Then the mixture was filtered and the solvent was evaporated
in vacuum. the residue was redissolved in hot ethyl acetate and passed
through a small silica column. The column extract was concentrated in
vacuum and crystallized from dichloromethane–hexane mixture (2 : 3,
v/v). White fine crystals were obtained. m.p.: 108–1118C (43%).
1H NMR (CDCl3) d (ppm) 8.12–8.43 (ABq, 4 H, aromatic), 7.45–7.53
(ABq, 4 H, aromatic), 5.27–5.30 (m, 1 H, C6-H), 5.21 (s, 2 H, CH2),
3.45–3.49 (m, 1 H, C3-H), 0.65–2.23 (m, 43 H, aliphatic). IR (KBr) nmax
3114, 1699, 1354, 1541, 1150, 1400.
Fluorination of cholesteryl 4-[(4-nitrobenzenesulfonyl)oxymethyl]benzo-
ate 5 with potassium fluoride and Kryptofix 2.2.2
Anhydrous acetonitrile (5 ml) was added to a vial containing potassium
fluoride (40 mg, 0.7 mmol) in water (0.5 ml) and Kryptofix 2.2.2 (200 mg,
0.54 mmol) in anhydrous acetonitrile (1 ml). The mixture was stirred
and then dried by the pressure of an argon stream. Acetonitrile (5 ml)
was added and dried again. The vial was cooled and 5 (150 mg,
0.22 mmol) in acetonitrile (5 ml) was added to the dried mixture. The
vial was capped and heated at 40–608C for 8 h. After cooling, the
mixture was purified by preparative TLC on silica gel and eluted with
chloroform–ethanol (9 : 1, v/v). The fluoride compound was separated
(Rf ¼ 0:35). The identity of the product was confirmed by comparison
with an authenticsample of 6a prepared previously.
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NO-CARRIER-ADDED, 18F
753
Preparation of [18F]cholesteryl 4-fluoromethyl benzoate 6b
Compound 6b was prepared from 5 using a procedure similar to that
used above for 6a. The target solution (1.44 ml) was eluted with
potassium carbonate solution (1%, 100 ml), containing [18F]fluoride
(6 mCi) of activity and transferred to a ReactivialTM (2 ml, volume)
containing Kryptofix 222 (10 mg, 0.027 mmol) and anhydrous acetoni-
trile (0.5 ml). The mixture was then evaporated to dryness by slight heat
and the pressure of an argon stream. Drying was repeated after addition
of two more portions of anhydrous acetonitrile (2 ꢀ 0.5 ml). A mixture
of 5 (6 mg, 0.01 mmol) in Sure-SealTM grade acetonitrile (0.25 ml) was
added to the above dried sample and heated at 40–608C for 20 min.
After cooling, the mixture was injected into the HPLC (Si Kromasil)
instrument using a mixture of dichloromethane–acetonitrile–acetic acid
(14 : 85 : 1) as the mobile phase (flow rate: 2 ml/min). The fraction eluted
at 6.7 min was recovered. The active solution was checked for
radiochemical purity by TLC on a polymer-backed silica gel layer
eluted with chloroform (Rf ¼ 0:35) using a Canberra spectroscopy unit
armed with a step-motor. Radio thin layer chromatography showed a
high purity (greater than 95%) in the form of [18F]-CFMB. The final
mixture was then dried by the pressure of an argon stream and slight
heat prior to use in animal tests. The fraction eluted at 6.7 min was
evaporated to dryness and reconstituted in borate buffer (0.2 ml, pH=8)
followed by passing through a 0.22 mm filter.
Table 1. Effect of ethynyl esteradiol injection on biodistribution of 6b in
mature female mice 1 h after administration. Tissue uptake is represented as
%1D/g ꢁ SD with a dose between 30 ꢁ 2 lCi (n ¼ 5)
Pretreated test
Control
Tissue
21.7 ꢁ 7.95
10.2 ꢁ 1.34
543 ꢁ 167
4.23 ꢁ 0.34
1.23 ꢁ 0.16
36.1 ꢁ 11.2
3.26 ꢁ 0.45
4.94 ꢁ 0.23
0.49 ꢁ 0.04
0.19 ꢁ 0.05
0.77 ꢁ 0.18
4.98 ꢁ 0.18
0.05 ꢁ 0.01
0.98 ꢁ 0.24
Adrenal
Adrenal/blood
Adrenal/muscle
Ovaries
Blood
1.86 ꢁ 0.51
2.29 ꢁ 0.55
0.51 ꢁ 0.07
0.18 ꢁ 0.05
0.76 ꢁ 0.11
7.14 ꢁ 0.78
0.04 ꢁ 0.003
23.5 ꢁ 3.32
Bone
Fat
Kidney
Liver
Muscle
Spleen
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A. R. JALILIAN ET AL.
Administration of [18F]cholesteryl 4-(fluoromethyl) benzoate to mice
Mature female NMRI mice (Pasteur Institute, Iran) weighing 20–25 g
(n ¼ 10) were used in experiments. The animals were kept in groups of
ten, in cages under constant temperature (248C) and 12 h light/dark
schedule. They had free access to standard mouse diet and water except
during the experiment. On the day of the experiment, animals were
randomly transferred to individual cages and allowed to acclimatize for
30 min before injection of radioligand. Ethynyl esteradiol was
suspended in a mixture of Tween 80 solution (0.5%) and carboxymethyl
cellulose solution (1%) and the mixture was agitated vigorously. The
animals were divided into two groups. In the first group of ten mice,
ethynyl esteradiol was injected subcutaneusly under their thigh skins 2 h
before 18F-CFMB injection. The second group of ten (control animals)
was given only 18F-CFMB as shown in Table 1.
The dried radioactive residue obtained in the last part was dissolved
in borate buffer (0.5 ml, pH=8) with agitation and warming for half a
minute and injected to the mice intravenously.
Conclusion
Total synthesis and purification of the 18F-labelled cholesterol
derivative, [18F]CFMB, took about 45 min with 75% yield (decay
corrected) with a specific radioactivity of 2000–2200 Ci/mmol. Pre-
liminary biodistribution studies on mice demonstrated that 18F-CFMB
is a good candidate for adrenal and ovaries tomography as a result of its
good target to non-target ratio.
Acknowledgements
This work was financially supported by the Atomic Energy Organisa-
tion of Iran (AEOI). The authors would like to express their thanks to
the Cyclotron Group of NRCAM.
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