A Novel 18F-Labeled Radioligand for Positron Emission Tomography Imaging of 11β-Hydroxysteroid Dehydrogenase (11β-HSD1): Synthesis and Preliminary Evaluation in Nonhuman Primates

Article abstract of DOI:10.1021/acschemneuro.8b00715

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the conversion of cortisone to cortisol and controls a key pathway in the regulation of stress. Studies have implicated 11β-HSD1 in metabolic diseases including type 2 diabetes and obesity, as well as stress-related disorders and neurodegenerative diseases, such as depression and Alzheimer's disease (AD). We have previously developed [¹¹C]AS2471907 as a PET radiotracer to image 11β-HSD1 in the brain of nonhuman primates and humans. However, the radiosynthesis of [¹¹C]AS2471907 was unreliable and low-yielding. Here, we report the development of the ¹⁸F-labeled version [¹⁸F]AS2471907, including the synthesis of two iodonium ylide precursors and the optimization of ¹⁸F-radiosynthesis. Preliminary PET experiments, composed of a baseline scan of [¹⁸F]AS2471907 and a blocking scan with the reversible 11β-HSD1 inhibitor ASP3662 (0.3 mg/kg), was also conducted in a rhesus monkey to verify the pharmacokinetics of [¹⁸F]AS2471907 and its specific binding in the brain. The iodonium ylide precursors were prepared in a seven-step synthetic route with an optimized overall yield of 7sim;2%. [¹⁸F]AS2471907 was synthesized in good radiochemical purity, with the ortho regioisomer of iodonium ylide providing greater radiochemical yield as compared with the para regioisomer. In monkey brain, [¹⁸F]AS2471907 displayed high uptake and heterogeneous distribution, while administration of the 11β-HSD1 inhibitor ASP3662 significantly reduced radiotracer uptake, thus demonstrating the binding specificity of [¹⁸F]AS2471907. Given the longer half-life of F-18 and feasibility for central production and distribution, [¹⁸F]AS2471907 holds great promise to be a valuable PET radiotracer to image 11β-HSD1 in the brain.

Full text of DOI:10.1021/acschemneuro.8b00715

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Article
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A Novel F-Labeled Radioligand for Positron Emission Tomography
Imaging of 11#-Hydroxysteroid Dehydrogenase (11#-HSD1):
Synthesis and Preliminary Evaluation in Nonhuman Primates
Evan Baum, Wenjie Zhang, Songye Li, Zhengxin Cai, Daniel Holden, and Yiyun Huang
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00715 • Publication Date (Web): 28 Jan 2019
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A Novel 18F-Labeled Radioligand for Positron Emission Tomography Imaging of 11-
Hydroxysteroid Dehydrogenase (11-HSD1): Synthesis and Preliminary Evaluation in
Nonhuman Primates
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Evan Baum1§, Wenjie Zhang2§, Songye Li1*, Zhengxin Cai1*, Daniel Holden1, Yiyun Huang1
1PET Center, Department of Radiology and Biomedical Imaging, Yale University School of
Medicine, 801 Howard Ave, New Haven, CT 06520-8048
2Department of Nuclear Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan
610041, China
§These two authors contribute equally to this work
*Corresponding authors
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ABSTRACT
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11-hydroxysteroid dehydrogenase type 1 (11-HSD1) catalyzes the conversion of
cortisone to cortisol and controls a key pathway in the regulation of stress. Studies have implicated
11-HSD1 in metabolic diseases including type 2 diabetes and obesity, as well as stress-related
disorders and neurodegenerative diseases, such as depression and Alzheimer’s disease (AD). We
have previously developed [11C]AS2471907 as a PET radiotracer to image 11-HSD1 in the brain
of nonhuman primates and humans. However, the radiosynthesis of [11C]AS2471907 was
unreliable and low-yielding. Here, we report the development of the 18F-labeled version
[18F]AS2471907, including the synthesis of two iodonium ylide precursors and the optimization
of 18F-radiosynthesis. Preliminary PET experiments, composed of a baseline scan of
[18F]AS2471907 and a blocking scan with the reversible 11β-HSD1 inhibitor ASP3662 (0.3
mg/kg), was also conducted in a rhesus monkey to verify the pharmacokinetics of [18F]AS2471907
and its specific binding in the brain. The iodonium ylide precursors were prepared in a 7-step
synthetic route with an optimized overall yield of ~2%. [18F]AS2471907 was synthesized in good
radiochemical purity, with the ortho regioisomer of iodonium ylide providing greater
radiochemical yield as compared to the para regioisomer. In monkey brain, [18F]AS2471907
displayed high uptake and heterogenous distribution, while administration of the 11β-HSD1
inhibitor ASP3662 significantly reduced uptake, thus demonstrating the binding specificity of
[18F]AS2471907. Given the longer half-life of F-18 and feasibility for central production and
distribution, [18F]AS2471907 holds great promise to be a valuable PET radiotracer to image 11-
HSD1 in the brain.
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Keywords:
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11-HSD1, 11-beta hydroxysteroid dehydrogenase type 1, positron emission tomography,
AS2471907, nonhuman primates, radiofluorination.
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INTRODUCTION
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The 11-hydroxysteroid dehydrogenases (11-HSD) catalyze the interconversion of active
glucocorticoids to inactive 11-keto forms, thus regulating glucocorticoid access to steroid
receptors. In humans, two isozymes, 11-hydroxysteroid dehydrogenase type 1 (11-HSD1) and
type 2 (11-HSD2), interconvert cortisone and cortisol through NADPH- and NAD+-dependent
reactions, respectively (Fig. 1).1 11-HSD2 is found primarily in tissues targeted by aldosterone,
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such as kidney, colon, salivary, and sweat glands. Genetic deficiencies of 11-HSD2 lead to an
excess of cortisol, which cross-reacts with non-selective mineralcorticoid receptors in the distal
nephron and causes hypertension, hypokalemia, and other symptoms of apparent mineralcorticoid
excess.1, 2 11-HSD1 is widely distributed in metabolically active organs such as the liver, adipose
tissue, skeletal muscle, and central nervous system (CNS), where it has high yet uneven expression
in the cerebellum, hippocampus, and cortex regions.1, 3 11-HSD1 overexpression in adipose tissue
of transgenic mice leads to obesity, insulin resistance, hyperlipidemia, and hypertension, while
targeted overexpression in the liver leads to these same symptoms of metabolic syndrome without
associated obesity.4-6 Compared to non-diabetic patients, obese diabetic patients have been found
to have elevated or sustained levels of 11-HSD1 in the liver, implicating the enzyme as a potential
target for treatment of type 2 diabetes mellitus and concurrent cardiovascular conditions.3, 7 In the
CNS, 11-HSD1 deletion or inhibition has been shown to mitigate the age-associated cognitive
decline in mice,8, 9 while administration of the 11-HSD1 inhibitor UE-2316 was found to improve
cognitive function in a mouse model of Alzheimer’s disease (AD) independent of A plaque
formation.10 Sandeep et al.11 reported that in both healthy elderly men and type 2 diabetic patients,
treatment with the 11-HSD1 inhibitor carbenoxolone improved verbal fluency and memory,
further implicating that local reduction of glucocorticoids in the brain improves cognition.
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Due to its association with diabetes, metabolic syndrome, age-related cognitive decline,
and AD, 11-HSD1 has thus become an attractive target for investigation of disease mechanisms
and therapeutic development. A Positron Emission Tomography (PET) radiotracer targeting the
11-HSD1 would allow for noninvasive imaging of the enzyme in vivo to better understand its
expression under normal and pathological states. PET imaging is also invaluable in evaluating the
target occupancy and efficacy of novel 11-HSD1 inhibitors in many clinical trials, such as ABT-
384,12, 13 UE-2343,14 PF-915275,15 MK0916,16 BMS-77076717 and ASP366218 (Fig. 2).
Molecules targeting 11-HSD1 employ several structural motifs, including triazole, amide,
and sulfonamide groups (Fig. 2). A highly specific triazole compound 3-(2-chlorophenyl)-4-
methyl-5-(2-(2,4,6-trifluorophenoxy)propan-2-yl)-4H-1,2,4-triazole (8, AS2471907, Fig. 2) was
recently reported19 , which displayed an IC50 of 5.6 nM for human 11-HSD1, >10,000 nM for
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human 11-HSD2, and no affinities or cross-activities for various receptors, ion channels,
transporters and other enzymes. The 11C-labeled form, [11C]AS2471907 ([11C]8), was shown to be
a promising radiotracer for imaging brain 11-HSD1 in monkeys20 and humans.21 However, the
radiosynthesis of [11C]AS2471907 was unreliable and low-yielding, as it produced three
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regioisomers with the desired radiotracer as the minor product (Scheme 1). Besides, C-labeled
radiotracers, in general, have limitations in their applications due to the short radioactive half-life
(t1/2 = 20.4 min) of the radionuclide and thus the requirement of an onsite cyclotron for their
production. The three aromatic fluorine atoms in AS2471907 (8) present the opportunity for 18F-
labeling. While aromatic rings without strongly electron-withdrawing substituents are difficult to
radiofluorinate,22, 23 recent developments in radiofluorination methodologies provided additional
avenues for radiofluorination. For example, using the recently developed iodonium ylides as
precursor for 18F-fluorination,24, 25 we have successfully prepared the -opioid receptor antagonist
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radiotracer [18F]LY2459989,26 which could not be accessed through conventional
radiofluorination method. We have since translated this chemistry to the preparation of 18F-labeled
AS2471907 ([18F]8).
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In this report we detail the chemical synthesis of two novel iodonium ylide precursors 8-
((4-((2-(5-(2-chlorophenyl)-4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)oxy)-3,5-
difluorophenyl)-λ3-iodaneylidene)-6,10-dioxaspiro[4.5]decane-7,9-dione (7a, Scheme 1) and 8-
((2-((2-(5-(2-chlorophenyl)-4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)oxy)-3,5-
difluorophenyl)-λ3-iodaneylidene)-6,10-dioxaspiro[4.5]decane-7,9-dione (7b, Scheme 1), and
discuss the optimization of the radiofluorination with the para-regioisomer 7a to produce p-[18F]8,
which was further refined with the ortho-regioisomer 7b to provide the most efficient radiolabeling
with the highest molar activity in the production of [18F]8. A preliminary in vivo evaluation of this
first 18F-labeled 11-HSD1 PET radiotracer [18F]8 in non-human primates is also described.
RESULTS AND DISCUSSION
Precursor synthesis
Based on the recent use of iodonium ylide precursors for 18F-labeling,26-28 a synthetic
strategy was devised to produce both the para- and ortho- iodonium ylide precursors 7a and 7b
with the 6,10-dioxaspiro[4.5]decane-7,9-dione (9, SPI-5) spirocyclic auxiliary attached to the
aromatic iodine (Scheme 2). 2,6-difluorophenol (1a) and 2,4-difluorophenol (1b) were first
iodinated, followed by a substitution reaction with ethyl 2-bromoisobutyrate to produce the ester
intermediates 2a and 2b. Hydrolysis, then coupling with 2-chlorobenzohydrazide yielded 4a and
4b, which were cyclized with 2-chloro-1,3-dimethylimidazolinium chloride (DMC) to afford the
oxidiazoles 5a and 5b. Conversion of 5a and 5b to the corresponding triazoles 6a and 6b proved
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to be the most challenging step, requiring fresh trifluoroacetic acid (TFA) for the in house
preparation of methylamine trifluoroacetate (MeNH2-TFA), followed by heating at 150 oC for 48
h over molecular sieves. The synthesis of SPI-5 (9) was achieved through the reaction of malonic
acid and cyclopentanone in the presence of acetic anhydride and concentrated sulfuric acid, as
described previously.26 The aromatic iodine in 6a and 6b was oxidized with oxone and TFA,
followed by addition of 9 under basic conditions to form the para- and ortho-iodonium ylides (7a
and 7b) as radiolabeling precursors. Both 7a and 7b were fully characterized by melting point,
1H and 13C NMR, and HRMS, with purity of > 98% based on HPLC analysis. The overall yield
for the synthetic pathway in Scheme 1 was ~2.2%.
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Radiochemistry
Radiolabeling test-run and optimization were first performed using the para-iodonium
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ylide precursor 7a. Mossine et al.29 detailed the Cu-mediated F-fluorination of boronic acids
using potassium trifluoromethanesulfonate (KOTf) and potassium carbonate (K2CO3) to elute the
18Ffluoride from the ion exchange cartridge, followed by drying and aliquoting small amounts of
the resulting base and [18F]KF mixture to perform radiolabeling tests. They described the optimal
conditions as having 0.09 mol of KOTf and 0.012 mol of K2CO3 with 4.0 mol of precursor in
0.10 mL of solvent. These low base aliquot conditions were assessed with 2.0-2.5 mg of precursor
7a to screen the solvents for radiolabeling, as shown in Table 1, entry 1-3. Acetonitrile (MeCN),
N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) were tested at various
temperatures, with the radiochemical conversion (RCC) of 7a to p-[18F]8 determined by analytical
HPLC. The radiolabeling proceeded with maximal conversion of 8% at 200 oC in DMF, while no
product was formed when MeCN or DMSO was used as solvent. These results were in agreement
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with past reports, which found DMF to be the preferred solvent for radiofluorination of a variety
of iodonium ylide substrates,24 as well as diaryliodonium salts.30, 31 All subsequent reactions were
thus performed in DMF.
The method of aliquoting small amounts of dried [18F]KF to achieve low concentrations of
base, however, is not practical for routine production of a PET radiotracer, where larger amount
of the radioactive product is desired for imaging studies. Furthermore, eluting with such low
concentrations of base may result in poor elution efficiency,29 or necessitate backwashing of
18Ffluoride trapping cartridge,32 which cannot be easily translated to an automated synthesis
module. For these reasons, a series of bases and additives in different amounts were tested in
reactions at different temperatures (Table 1, entry 4-13). Addition of Kryptofix 222 or 18-crown-
6 with KOTf and K2CO3 resulted in low conversion to product. RCC increased to a maximum of
12.4% with 5.0 mg (26.5 mol, 6.6 eq.) of KOTf and increasing amounts of tetraethylammonium
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bicarbonate (TEAB) up to 2.0 mg (10.5 mol, 2.6 eq.), which also provided good (> 90%) elution
efficiency. These optimized conditions (Table 1, entry 12) for precursor 7a were translated to a
GE TRACERlab FXN Pro automated synthesis module. Radiolabeling, purification, and
formulation of 7a produced p-[18F]8 with > 99% radiochemical purity by analytical HPLC.
Average product activities after formulation were sufficient for monkey imaging studies (0.41 ±
0.31 GBq, n = 11), while molar activities (Am) at end of synthesis (EOS) were moderate (28.5 ±
9.9 GBq/mol, n = 11). The radiochemical yield (RCY), calculated as isolated product
activity/trapped activity, was low, with an average of 0.5%. The total synthesis time was 88 ± 4
min (n = 11).
The optimized conditions for para-iodonium ylide precursor 7a were tested and further
refined on the ortho-iodonium ylide precursor 7b, leading to improved RCC and RCY. The KOTf
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and TEAB condition was tried alongside with other bases in DMF at 150 or 160oC (Table 2). RCC
increased to 22% with 7b (entry 1, Table 2) when heating at 150 oC for 20 min with KOTf (5 mg)
and TEAB (2 mg), as compared to 12.4% with 7a under the same conditions. Use of
tetrabutylammonium trifluoromethanesulfonate (TBAOTf) with or without cesium carbonate
(Cs2CO3) resulted in low RCC. Meanwhile, addition of 2 mg TEAB alone provided an RCC of
34%, and increasing amounts of TEAB up to 16 mg did not increase the conversion rate. The use
of 2-10 mg of TEAB in 0.2-0.5 mL of DMF has been shown to be ideal for radiofluorination of
iodonium ylides in past studies24, 26 as well, while greater amounts of base may lead to
decomposition of the precursors, therefore decrease RCC. The optimal condition for 7b (Table 2,
entry 4) was adopted to the FXN Pro module to produce o-[18F]8 for PET scans in nonhuman
primates. Use of the ortho-precursor 7b afforded o-[18F]8 in > 99% radiochemical purity by
analytical HPLC. Average isolated product activity after formulation were 1.02 ± 0.68 GBq (n =
18) with greatly improved EOS Am of 121.5 ± 101.4 GBq/mol (n = 18), four times higher than
that of radiofluorination with 7a. RCY increased by six-fold at 3.2 ± 2.1% (n = 18). Total synthesis
time on the FXN Pro module was 84 ± 5 min (n = 14). Based on higher RCC, RCY, and Am, the
automated, optimized synthesis using the ortho-precursor 7b will be used for subsequent studies
of [18F]8 in nonhuman primates and translation to clinical studies in human.
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Rotstein et al.25 have proposed a mechanism for the radiofluorination of spirocyclic
iodonium ylides that involves regioselective reductive elimination. More sterically hindered
compounds such as 7a and 7b are typically poor substrates for reductive elimination. Reductive
elimination of unsymmetrical diaryliodonium salts, however, demonstrates a preference for ortho-
substituted arenes, including those with an ortho methoxy group.33 A similar ortho preference was
observed in the radiolabeling of ortho- vs. para-benzyloxyphenyliodonium ylides.25 Furthermore,
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computer modeling of ortho- vs. para-methoxyphenyliodonium ylides shows a 5 kcal/mol lower
G‡ for the ortho reductive elimination transition state, indicating that iodonium ylides ortho to
an alkoxy moiety may undergo radiofluorination with lower activation energies.25 With less steric
destabilization, para-iodonium ylides adopt a planar configuration that requires additional energy
to rotate the arene to an out-of-plane transition state for reductive elimination.25 These mechanistic
considerations support the observations in this study, that the ortho-iodonium ylide 7b
demonstrated more efficient radiolabeling than the para compound 7a, perhaps due to a lower
energy barrier to the transition state.
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PET Imaging in Nonhuman Primates
The ortho-iodonium ylide precursor 7b was used to produce [18F]8 in preliminary PET
imaging studies in one rhesus monkey. Metabolism of [18F]8 was extremely slow, with >90 % of
intact radiotracer up to 90 min after injection. PET images and regional time-activity curves
(TACs) of [18F]8 from the baseline and blocking scans are shown in Figure 3 and Figure 4. Baseline
scan showed high uptake of the radiotracer, particularly in cortical regions (Figure 3, middle).
Regional concentrations of the radiotracer reached peak levels within 20 min after injection,
followed by a moderate rate of washout over time (Figure 4A). Pre-treatment of the animal with
the 11-HSD1 inhibitor ASP3662 (0.3 mg/kg) reduced activity uptake in all brain regions (Figure
3, bottom), indicating the binding specificity of the radiotracer in vivo (Figure 4B).
Regional TACs were processed with the one tissue compartment (1TC) model34 to generate
the regional volume of distribution (VT, Table 3). In the blocking study, VT was significantly
reduced in all regions. Receptor occupancy was calculated to be 98% from the occupancy plot,
with non-displaceable distribution volume (VND) of 3.5 mL/cm-3. Regional BPND values calculated
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from 1TC VT in the baseline and VND from the blocking scan are shown in Table 3. The rank order
of BPND in various regions are as follows: occipital cortex > cingulate cortex ≈ temporal cotex >
pons > cerebellum > putamen > insula > brainstem > frontal cortex > caudate > thalamus. Levels
of specific binding were high for [18F]8, consisting of up to 77% of the signals in high binding
regions.
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CONCLUSIONS
The enzyme 11-HSD1 is an important target for therapeutic development. We aimed to
develop a PET imaging agent for 11-HSD1 based on the potent and selective inhibitor
AS2471907. Recently developed iodonium chemistry was explored to prepare two iodonium ylide
precursors (7a and 7b) for radiofluorination. Optimization of radiolabeling conditions indicated
that DMF was the solvent of choice, and combination of TEAB/KOTf or TEAB alone gave the
best radiochemical yield for 18F-fluorination of precursors 7a and 7b, respectively, with the ortho-
iodonium ylide precursor 7b giving higher radiolabeling yield and molar activity in the synthesis
of radiotracer [18F]8. Preliminary PET imaging experiments in rhesus monkeys indicate that [18F]8
is a highly specific radiotracer with suitable properties for imaging 11-HSD1 in the primate brain.
Further evaluation of this novel radiotracer in non-human primates and humans is currently
underway.
EXPERIMENTAL SECTION
Chemistry
General. All reagents and solvents were purchased from commercial sources (e.g., Sigma-
Aldrich) and used without further purification unless noted otherwise. 2,6-Difluorophenol (1a) and
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2,4-difluorophenol (1b) were obtained from FisherSci. Proton (1H, 400 MHz or 600 MHz) and
carbon (13C, 151 MHz) nuclear magnetic resonance (NMR) spectra were recorded on an Agilent
400 MHz (A400a) or 600 MHz (A600a) spectrometer. Chemical shifts are reported in parts per
million, with the solvent resonance as the internal standard (1H NMR, CDCl3: 7.26 ppm; DMSO-
d6: 2.49 ppm). Melting point was determined on an Electralthermo MelTemp instrument. High-
resolution mass spectrometry (HRMS) was obtained on a Thermo LTQ Orbitrap spectrometer.
AS2471907 (8) and ASP3662 were provided by Astellas Pharma, and their synthesis has
been reported previously.19, 20, 35
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Ethyl 2-(2,6-difluoro-4-iodophenoxy)-2-methylpropanoate (2a): To a solution of 1a (6.8
g, 52.35 mmol), iodine (20.0 g, 78.80 mmol) and potassium iodide (13.0 g, 78.31 mmol) in de-
ionized (DI) water (100 mL) was added dropwise a solution of sodium hydroxide (4.3 g, 105
mmol) in DI water (25 mL) at 0 oC. Then the reaction mixture was warmed to room temperature
and kept stirring for 2 h. The reaction was neutralized with a solution of ammonium chloride in DI
water followed by a solution of sodium thiosulfate in DI water until de-colorization. This mixture
was then extracted with t-butyl methyl ether. The combined organic extracts were dried over
MgSO4 and concentrated in vacuo. To a solution of this crude product and ethyl 2-
bromoisobutyrate (14.0 mL, 95.39 mmol) in DMF (100 mL) was added K2CO3 (22.0 g, 159.18
mmol) under argon. The reaction mixture was stirred at 80 ˚C for 3 h. After cooling to room
temperature, the mixture was poured into ice cold H2O (200 mL) and extracted with EtOAc (50
mL × 3). The combined organic phase was dried over MgSO4 and concentrated in vacuo. The
crude product was purified on a silica gel column, eluting with gradient 0 - 10% EtOAc/hexane to
afford compound 2a as a clear oil (10.0 g, 52%). 1H NMR (CDCl3, 400 MHz): δ 7.23 (d, J = 7.18
Hz, 2H), 4.23 (q, J = 7.14 Hz, 2H), 1.53 (s, 6H), 1.29 (t, J = 7.14 Hz, 3H).
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Ethyl 2-(2,4-difluoro-6-iodophenoxy)-2-methylpropanoate (2b): Compound 2b was
prepared in procedures similar to those described above for compound 2a. Yield: 52%. 1H NMR
(CDCl3, 400 MHz): δ 7.35-7.27 (m, 1H), 6.88-6.78 (m, 1H), 4.26 (q, J = 7.12 Hz, 2H), 1.53 (s,
6H), 1.29 (t, J = 7.12 Hz, 3H).
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2-(2,6-difluoro-4-iodophenoxy)-2-methylpropanoic acid (3a): To a solution of compound
2a (6.0 g, 16.21 mmol) in EtOH (30 mL) was added dropwise a 3M solution of NaOH in DI water
(11 mL, 33.00 mmol) at 0 °C and the mixture was stirred at room temperature for 3 h. The mixture
was then poured into ice H2O (50 mL) and washed with diisopropyl ether/heptane (v/v, 1/1, 20 mL
× 2). The aqueous layer was separated, acidified with concentrated HCl to pH = 4, and then the
cloudy solution was extracted with ethyl acetate (30 mL × 3). The combined organic phase was
dried over MgSO4, and concentrated in vacuo to afford compound 3a as a light brown solid (4.75
g, 86%). The product was used in the next step without further purification. 1H NMR (CDCl3, 400
MHz): δ 7.30 (d, J = 6.83 Hz, 2H), 1.58 (s, 6H).
2-(2,4-difluoro-6-iodophenoxy)-2-methylpropanoic acid (3b): Compound 3b was prepared
in procedures similar to those described above for compound 3a. Yield: 86%. 1H NMR (CDCl3,
400 MHz): δ 7.34 (dt, J = 7.22, 2.17 Hz, 1H), 6.88 (td, J = 8.06, 2.92 Hz, 1H), 1.63 (s, 6H).
2-chloro-N'-(2-(2,6-difluoro-4-iodophenoxy)-2-methylpropanoyl)benzohydrazide (4a): To
a solution of compound 3a (4.74 g, 13.87 mmol) in anhydrous CH2Cl2 (30 mL) under argon was
added 1,1’-carbonyldiimidazole (CDI, 2.40 g, 14.36 mmol). The reaction mixture was stirred at
room temperature for 1 h and 2-chlorobenzohydrazide (2.50 g, 14.36 mmol) was added. After
stirring for 16 h, the reaction was quenched with DI water (50 mL) and extracted with CH2Cl2 (30
mL × 3). The combined organic phase was dried over MgSO4, and concentrated in vacuo. The
crude product was purified on a silica gel column, eluting with gradient 20 - 40% EtOAc/hexane
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to afford compound 4a as an off-white solid (3.88 g, 57%). 1H NMR (CDCl3, 400 MHz): δ 9.71
(d, J = 6.40 Hz, 1H), 9.15 (d, J = 6.49 Hz, 1H), 7.85 (d, J = 7.51 Hz, 1H), 7.44 (q, J = 7.82 Hz,
2H), 7.37 (t, J = 7.61 Hz, 1H), 7.32 (d, J = 6.81 Hz, 2H), 1.57 (s, 6H).
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2-chloro-N'-(2-(2,4-difluoro-6-iodophenoxy)-2-methylpropanoyl)benzohydrazide
(4b):
Compound 4b was prepared in procedures similar to those described above for compound 4a.
Yield: 57%. 1H NMR (CDCl3, 400 MHz): δ 9.63 (d, J = 6.42 Hz, 1H), 9.22-9.09 (m, 1H), 7.84 (d,
J = 7.65 Hz, 1H), 7.42 (q, J = 7.76 Hz, 2H), 7.37 (t, J = 7.02 Hz, 2H), 6.90 (dt, J = 7.97, 2.94 Hz,
1H), 1.64 (s, 6H).
2-(2-chlorophenyl)-5-(2-(2,6-difluoro-4-iodophenoxy)propan-2-yl)-1,3,4-oxadiazole (5a):
To a solution of compound 4a (3.88 g, 7.84 mmol) and 2-chloro-1,3-dimethylimidazolinium
chloride (DMC, 2.20 g, 11.71 mmol) in anhydrous CH2Cl2 (70 mL) was slowly added
triethylamine (TEA, 3.3 mL, 23.68 mmol) at 0 oC. After stirring at 0 oC for 1 h, the reaction was
quenched with DI water (50 mL) and extracted with CH2Cl2 (25 mL × 2). The combined organic
phase was dried over MgSO4, and concentrated in vacuo. The crude product was purified on a
silica gel column, eluting with gradient 0-20% EtOAc/hexane to afford compound 5a as a colorless
oil (2 g, 53%). 1H NMR (CDCl3, 400 MHz): δ 7.99 (d, J = 7.72 Hz, 1H), 7.55 (d, J = 7.98 Hz, 1H),
7.47 (t, J = 7.70 Hz, 1H), 7.41 (t, J = 7.51 Hz, 1H), 7.20 (d, J = 6.95 Hz, 1H overlap with m, 1H),
1.90 (s, 6H).
2-(2-chlorophenyl)-5-(2-(2,4-difluoro-6-iodophenoxy)propan-2-yl)-1,3,4-oxadiazole (5b):
Compound 5b was prepared in procedures similar to those described above for compound 5a.
Yield: 53%. 1H NMR (CDCl3, 400 MHz): δ 8.01 (d, J = 7.70 Hz, 1H), 7.56 (d, J = 8.03 Hz, 1H),
7.48 (t, J = 7.66 Hz, 1H), 7.41 (t, J = 7.53 Hz, 1H), 7.31 (d, J = 7.33 Hz, 1H), 6.79 (t, J = 10.05
Hz, 1H), 1.95 (s, 6H).
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3-(2-chlorophenyl)-5-(2-(2,6-difluoro-4-iodophenoxy)propan-2-yl)-4-methyl-4H-1,2,4-
triazole (6a): A mixture of compound 5a (0.70 g, 1.47 mmol) and methylamine trifluoroacetate
(3.20 g, 22.06 mmol, prepared by combining equal stoichiometry ratio of methylamine and
trifluoroacetic acid in methanol followed by removal of solvent in vacuo) and molecular sieve
powder (0.70 g) were suspended in methylamine (2M MeOH solution, 15.0 mL, 30.00 mmol). The
reaction mixture was heated in a sealed tube at 150 oC for 48 h. After the reaction was cooled to
room temperature, molecular sieves were removed via filtration and the filtrate was concentrated
in vacuo. The crude compound was purified on a silica gel column, eluting with gradient 0-10%
EtOH/EtOAc to afford compound 6a as a light brown solid (0.2 g, 28%). 1H NMR (CDCl3, 400
MHz): δ 7.58-7.43 (m, 3H), 7.41 (t, J = 7.32 Hz, 1H), 7.23 (s, 2H overlap with CHCl3 solvent
peak), 3.75 (s, 3H), 1.87 (s, 6H).
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3-(2-chlorophenyl)-5-(2-(2,4-difluoro-6-iodophenoxy)propan-2-yl)-4-methyl-4H-1,2,4-
triazole (6b): Compound 6b was prepared in procedures similar to those described above for
compound 6a. Yield: 28%. 1H NMR (CDCl3, 400 MHz): δ 7.56-7.42 (m, 3H), 7.44-7.34 (m, 1H),
7.36-7.26 (m, 1H), 6.85-6.73 (m, 1H), 3.74 (s, 3H), 1.91 (s, 6H).
8-((4-((2-(5-(2-chlorophenyl)-4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)oxy)-3,5-
difluorophenyl)-λ3-iodaneylidene)-6,10-dioxaspiro4.5decane-7,9-dione (7a): To a solution of
compound 6a (100 mg, 0.20 mmol) in CHCl3 (3.0 mL) was added trifluoroacetic acid (0.48 mL,
6.23 mmol). Oxone (300 mg, 0.98 mmol) was added and the reaction mixture was stirred at room
temperature for 2 h. Volatile contents were then removed in vacuo. The dried residue was
suspended in EtOH (2 mL) and 6,10-dioxaspiro4.5decane-7,9-dione (9, 44 mg, 0.31 mmol) was
added followed by 10% Na2CO3 (aq) until pH = 10. The reaction mixture was stirred for 3 h,
diluted with water, and extracted with CH2Cl2 (3 mL × 3). The combined organic phase was dried
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over MgSO4 and concentrated in vacuo. The crude product was purified on a silica gel column,
eluting with gradient 0-10% EtOH/EtOAc to afford compound 7a as a white solid (78 mg, 58%).
This compound can be further purified via trituration with a solvent mixture of EtOAc and hexane.
M. P. 124 - 128 oC; 1H NMR (600 MHz, DMSO-d6): δ 7.72 (d, J = 7.85 Hz, 1H), 7.67-7.62 (m,
1H), 7.68-7.45 (m, 4H), 3.67 (s, 3H), 1.97-1.91 (m, 4H), 1.80 (s, 6H), 1.71-1.62 (m, 4H);13C NMR
(151 MHz, DMSO-d6): δ 164.32 (2C), 155.76, 155.21, 134.47, 132.58 (2C), 132.13 (2C), 129.95
(2C), 121.34 (2C), 126.51, 118.06, 117.87, 114.38, 81.94, 57.48, 37.42 (2C), 32.41, 26.09 (2C),
23.39 (2C). HRMS: calculated for C26H23ClF2IN3O5 (M + H+): 658.0418; found: 658.0412.
8-((2-((2-(5-(2-chlorophenyl)-4-methyl-4H-1,2,4-triazol-3-yl)propan-2-yl)oxy)-3,5-
difluorophenyl)-λ3-iodaneylidene)-6,10-dioxaspiro4.5decane-7,9-dione (7b): Compound 7b was
prepared in procedures similar to those described above for compound 7a. Yield: 58%. M. P. 125
- 128 oC; 1H NMR (600 MHz, ppm, DMSO-d6): δ 7.68 (d, J = 7.94 Hz, 1H), 7.64-7.60 (m, 1H),
7.58-7.54 (m, 1H), 7.53-7.48 (m, 2H overlap), 7.48-7.46 (m, 1H), 3.60 (s, 3H), 1.92 (t, J = 7.30
Hz, 4H), 1.88 (s, 6H), 1.64 (t, J = 7.36 Hz, 4H); 13C NMR (151 MHz, ppm, DMSO-d6): δ 163.89
(2C), 156.73, 154.20, 133.87 (2C overlap), 133.10 (2C overlap), 132.80, 130.21 (2C overlap),
128.12 (2C overlap), 127.10 (2C overlap), 112.77 (2C), 82.06, 59.81, 37.12 (2C), 32.37, 26.91
(2C), 23.15 (2C). HRMS: calculated for C26H23ClF2IN3O5 (M + Na+): 680.0231; found: 680.0276.
6,10-dioxaspiro4.5decane-7,9-dione (9, SPI-5). Compound 9 was prepared in procedures
described by Cai et al.26 1H NMR (CDCl3, 400 MHz): δ 3.62 (s, 2H), 2.26-2.15 (m, 4H), 1.96-1.80
(m, 4H).
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Radiochemistry
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[18O]H2O was obtained from Huayi Isotopes (Toronto, Canada). Anion exchange
Chromafix cartridges (PS-HCO3) were purchased from Macherey-Nagel (Dueringen, Germany).
Solid-phase extraction (SPE) SepPak cartridges were purchased from Waters Associates (Milford,
MA, USA). The HPLC system used for purification of crude product included a Shimadzu LC-
20A pump, a Knauer K200 UV detector, and a Bioscan -flow detector, with a Prodigy C18
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ODS(3) semi-preparative column (10 m, 10  250 mm, Phenomenex, Torrance, CA). The HPLC
system used for quality control tests was composed of a Shimadzu LC-20A pump, a Shimadzu
SPD-M20A PDA or SPD-20A UV detector, a Bioscan -flow detector, with a Luna C18(2) column
(5 m, 4.6 × 250 mm, Phenomenex, Torrance, CA, USA).
Optimized
radiosynthesis
of
3-(2-chlorophenyl)-5-(2-(2,6-difluoro-4-(fluoro-
18F)phenoxy)propan-2-yl)-4-methyl-4H-1,2,4-triazole (p-[18F]8). 18F-Fluoride was produced via
the 18O(p, n)18F nuclear reaction in a 16.5-MeV GE PETtrace cyclotron (Uppsala, Sweden). The
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cyclotron produced F-fluoride solution in [18O]H2O was transferred to a lead-shielded hot cell
with a GE TRACERlab FXN Pro module for automated synthesis or with a collection vial for
manual synthesis. The radioactivity was trapped on a Chromafix (PS-HCO3, Macherey-Nagel,
Dueringen, Germany) separation cartridge activated with 10 mL ethanol, followed by 10 mL 90
mg/mL KOTf solution in sterile H2O, and finally 10 mL sterile H2O. The 18F-fluoride was eluted
off the cartridge into a v-vial with a solution of TEAB (2 mg, 10.5 mol, 1.8-3.5 equiv) and KOTf
(5 mg, 26.5 mol, 4.4-8.8 equiv) in 1 mL of MeCN/water (7:3, v/v). The solvent was removed
under argon at 110 ºC, and dried azeotropically with the addition of 2 × 1 mL of MeCN. Precursor
7a (2-2.5 mg, 3-6 mol) in DMF (0.5 mL) was added to the reaction vessel, and the resulting
solution was heated at 150 ºC for 10 min. After cooling, the crude product was pre-purified by
solid phase extraction on a SepPak cartridge, eluted with 1 mL of EtOH, diluted with 3 mL of
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hydrochloric acid (0.04 N), and then loaded onto a semi-preparative Prodigy C18 ODS(3) HPLC
column for purification. The column was eluted with a mixture of 45% MeCN and 55% 0.04 N
HCl (v/v) at a flow rate of 5 mL/min. The eluent was monitored by a UV detector and a
radioactivity detector. The fraction containing p-[18F]8 was collected, diluted with 50 mL of DI
water and passed through a Waters Classic C18 SepPak cartridge. The cartridge was rinsed with
10 mL of 0.001 N HCl and air dried. The radioactive product, trapped on the SepPak, was
recovered by elution with 1 mL of absolute EtOH (USP), followed by 3 mL of USP saline into a
10 mL syringe barrel. The resulting EtOH-saline solution was then passed through a sterile
membrane filter (0.22 m) for terminal sterilization and collected in a sterile vial pre-charged with
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7 mL of USP saline and 20 L 8.4% USP NaHCO3, affording a formulated I.V. solution ready for
dispensing and injection.
Optimized
radiosynthesis
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18F)phenoxy)propan-2-yl)-4-methyl-4H-1,2,4-triazole (o-[18F]8). Radiosynthesis, formulation,
and characterization were the same as for p-[18F]8, except for the following: The Chromafix (PS-
HCO3) separation cartridge was activated with 10 mL ethanol followed by 10 mL sterile H2O. The
18F-fluoride was eluted off the cartridge into a v-vial with a solution of TEAB (2 mg, 10.5 mol,
1.8-3.5 equiv) in 1 mL of MeCN/water (7:3, v/v).
Quality Control and Analytical HPLC Conditions for [18F]8. Quality control of the
chemical purity, radiochemical purity, and molar activity of [18F]8 was determined by analytical
HPLC analysis of the final product solution. The identity of [18F]8 was confirmed by co-injection
of the product with the unlabeled standard (8) and detection of a single UV peak at 230 nm on the
chromatogram. Chemical and radiochemical purity > 99% by analytical HPLC eluting with a
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mobile phase of 52% MeCN and 48% 0.1 M ammonium formate solution (pH 4.2) with 0.5%
acetic acid at aflow rate of 2.0 mL/min.
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PET Procedures.
Imaging in Nonhuman Primates. Experiments were performed in rhesus monkeys (Macaca
mulatta) according to a protocol approved by the Yale University Institutional Animal Care and
Use Committee. The animals were fasted overnight prior to imaging. The animals were
immobilized with ketamine (10 mg/kg intramuscularly) and anesthetized with 1-2% isoflurane.
An indwelling port was surgically placed in a femoral artery for arterial blood sampling for
metabolite analysis.36 A water-jacket heating pad was used to maintain body temperature. The
animal was attached to a physiological monitor, and vital signs (pulse rate, blood pressure,
respirations, EKG, ETCO2, and body temperature) were continuously monitored. Baseline and
blocking scans were performed on a Siemens FOCUS 220 camera. Before radiotracer injection, a
9-minute transmission scan was obtained for attenuation correction. 18F-8 was injected
intravenously over 3 min as a bolus (~185 MBq/10 mL). PET scans were acquired over 2 h. In the
blocking scan the 11-HSD1 inhibitor ASP3662 (0.3 mg/kg) was administered over 5 min starting
at 15 min before radiotracer injection.
Image Reconstruction and Analysis. Procedures for PET image reconstruction and
definition of regions of interest (ROIs) have been described previously.36 Emission data were
attenuation corrected using the transmission scan, and dynamic images (33 frames over 120 min)
were reconstructed using a filtered back-projected algorithm with a Shepp-Logan filter. ROIs were
defined from a single representative anatomic rhesus MR image registered to a template image.
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For each PET scan, radiotracer concentrations over time were measured in the ROIs to generate
the regional time-activity curves (TACs).
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Regional TACs were analyzed to calculate regional volume of distribution (VT, mL/cm-3)
using the one-tissue compartmental model (1TC) 34. Target occupancy by the blocking drug was
obtained from occupancy plot using the regional VT from the baseline scan and VT difference
between baseline and blocking scans according to the method of Cunningham et al. 37.
Regional non-displaceable binding potential (BPND) was calculated using the 1TC VT
values from the baseline scan and the non-displaceable distribution volume (VND) derived from the
occupancy plot to assess levels of specific binding, with BPND = (VT ROI – VND)/VND.
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AUTHOR INFORMATION
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Corresponding Author: *E-mail: songye.li@yale.edu Telephone: 203-785-3605 Fax: 203-785-
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3107 and jason.cai@yale.edu, Telephone: 203-785-7691.
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Author Contributions: E.B. and W.Z. contributed equally to this work. All authors contributed
to and approved the final version of this manuscript.
ACKNOWLEDGEMENT
The authors thank the staff at the Yale PET Center for their expert technical assistance.
ABBREVIATIONS USED
11-HSD1, 11-beta hydroxysteroid dehydrogenase type 1; PET, positron emission tomography;
CDI, 1,1′-Carbonyldiimidazole; DMC, 2-chloro-1,3-dimethylimidazolinium chloride; MeCN,
acetonitrile; KOTf, potassium trifluoromethanesulfonate; TBAOTf, tetrabutylammonium
trifluoromethanesulfonate; K2CO3, potassium carbonate; CsCO3, cesium carbonate; TEAB,
tetraethylammonium bicarbonate; K222, Kryptofix® 222; RCC, radiochemical conversion; RCY,
radiochemical yield; MA EOS, molar activity at end of synthesis.
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TABLES AND FIGURES
5
6
7
8
N
N
F
N
N
F
O
O
F
O
I
N
O
F
N
O
Cl
Cl
18F
9
O
p-[18F]
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
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38
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40
41
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45
46
47
48
7a
8
Table 1. Optimization on radiolabeling of precursor 7a
RCCa (%)
No reaction
No reaction
2, 4.8, 8
Temp. (⁰C)
Entry
1b
2b
3b
4b
Base
Time (min)
Solvent
MeCN
DMSO
DMF
KOTf (170 µg), K2CO3 (1.7 µg)
KOTf (170 µg), K2CO3 (1.7 µg)
KOTf (170 µg), K2CO3 (1.7 µg)
KOTf (0.5 mg), K2CO3 (5 µg)
120
10
10
10
10
120, 160, 200
120, 160, 200
120, 160, 200
DMF
3.4, 3.8, 6.7
5
6
7
8
KOTf (5 mg), K2CO3 (50 µg)
160
10
10
10
10
DMF
DMF
DMF
DMF
1.9
KOTf (5 mg), K2CO3 (50 µg), K222 (0.5 mg)
KOTf (5 mg), K2CO3 (50 µg), 18-Crown-6 (0.35 mg)
KOTf (5 mg), TEAB (0.25 mg)
120, 160
120, 160
120, 160
0.5, 0.8
0.8, 0.8
0.9, 1.7
9
KOTf (5 mg), TEAB (0.5 mg)
KOTf (5 mg), TEAB (1 mg)
KOTf (5 mg), TEAB (1.5 mg)
KOTf (5 mg), TEAB (2 mg)
TEAB (2 mg)
160
160
160
160
160
10
10
10
10
10
DMF
DMF
DMF
DMF
DMF
6.9
6.7
9.9
12.4
6.5
10
11
12
13
aRadiochemical conversion (RCC) is calculated as integration of the product peak on analytical HPLC.
bElution of 18F-fluoride with KOTf (5 mg) and K2CO3 (50 g) followed by aliquoting to achieve the stated amount of reagents
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2
3
4
5
6
O
O
I
18F
N
N
O
N
O
F
N
N
O
O
F
Cl
N
7
F
Cl
8
9
F
o-[18F]
7b
8
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
Table 2. Base effect on radiolabeling of precursor 7b in DMF
RCCa (%)
Temp. (⁰C)
150
Entry
Base
Time (min)
22
1
2
KOTf (5 mg), TEAB (2 mg)
20
20
TBAOTf (5 mg), Cs2CO3 (5 µg)
160
2.5
2.3
34
16
28
16
TBAOTf (5 mg)
TEAB (2 mg)
TEAB (4 mg)
TEAB (8 mg)
TEAB (16 mg)
160
3
4
20
10
150
5
6
7
150
10
10
10
150
150
a Radiochemical conversion (RCC) was calculated as integration of the product peak on analytical HPLC.
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