Radiosynthesis and evaluation of an 18F-labeled positron emission tomography (PET) radioligand for brain histamine subtype-3 receptors based on a nonimidazole 2-aminoethylbenzofuran chemotype

Article abstract of DOI:10.1021/jm201690h

A known chemotype of H₃ receptor ligand was explored for development of a radioligand for imaging brain histamine subtype 3 (H ₃) receptors in vivo with positron emission tomography (PET), namely nonimidazole 2-aminoethylbenzofurans,

Full text of DOI:10.1021/jm201690h

Article
pubs.acs.org/jmc
18
Radiosynthesis and Evaluation of an F-Labeled Positron Emission
Tomography (PET) Radioligand for Brain Histamine Subtype-3
Receptors Based on a Nonimidazole 2-Aminoethylbenzofuran
Chemotype
Xiaofeng Bao, Shuiyu Lu, Jeih-San Liow, Sami S. Zoghbi, Kimberly J. Jenko, David T. Clark,
Robert L. Gladding, Robert B. Innis, and Victor W. Pike*
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Building 10, Room B3 C346A, 10
Center Drive, Bethesda, Maryland, 20892, United States
S
* Supporting Information
ABSTRACT: A known chemotype of H3 receptor ligand was
explored for development of a radioligand for imaging brain
histamine subtype 3 (H3) receptors in vivo with positron
emission tomography (PET), namely nonimidazole 2-amino-
ethylbenzofurans, represented by the compound (R)-(2-(2-(2-
methylpyrrolidin-1-yl)ethyl)benzofuran-5-yl)(4-fluorophenyl)-
methanone (9). Compound 9 was labeled with fluorine-18
(t1/2 = 109.7 min) in high specific activity by treating the
prepared nitro analogue (12) with cyclotron-produced [18F]-
fluoride ion. [18F]9 was studied with PET in mouse and in
monkey after intravenous injection. [18F]9 showed favorable
properties as a candidate PET radioligand, including
moderately high brain uptake with a high proportion of H3
receptor-specific signal in the absence of radiodefluorination. The nitro compound 12 was found to have even higher H3 receptor
affinity, indicating the potential of this chemotype for the development of further promising PET radioligands.
and [18F]8 have shown promise in animal experiments, and
INTRODUCTION
The histamine subtype 3 (H3) receptor is one of the four G-
■
only [11C]6 is known to have progressed to studies in human
subjects.14
protein-coupled receptors of the histamine receptor family.1−3
H3 receptors are widely expressed in the mammalian brain and
regulate the presynaptic release of histamine and other
neurotransmitters such as acetylcholine, noradrenalin, and
dopamine. Brain H3 receptors are attractive drug targets for
the treatment of cognitive and other disorders such as
narcolepsy, attention-deficit hyperactivity disorder, and pain.4,5
The use of selective H3 receptor radioligands with PET
(positron emission tomography) has potential (i) to elucidate
any changes in the distribution and density of H3 receptors in
living human brain during the progression of neuropsychiatric
disorders, and (ii) to determine the dose-dependence of extent
and duration of brain H3 receptor occupancy by candidate
drugs that may be directed at the treatment of such disorders.6
So far, few radioligands have been prepared and evaluated for
imaging brain H3 receptors with PET. These radioligands may
be structurally categorized as imidazoles, such as [18F]FUB 272
([18F]1),7 [18F]VUF 5000 ([18F]2),8,9 [18F]fluoroproxyfan
([18F]3),10,11 [11C]UCL 1829 ([11C]4),7 or nonimidazoles,
such as [11C]JNJ-10181457 ([11C]5),12 [11C]GSK189254
([11C]6),13,14 [11C]Merck 1b ([11C]7),15 and [18F]Merck 2b
([18F]8)15 (Chart 1). The imidazoles have not shown
encouraging results. Only the nonimidazoles [11C]6, [11C]7,
In our efforts to develop an 18F-labeled H3 receptor PET
ligand, we selected a new chemotype, the reported non-
imidazole 2-aminoethylbenzofuran-based H3 receptor antago-
nist/inverse agonist 9,16 for radiolabeling and evaluation in
animals. Ligand 9 appeared attractive as a candidate for
development as a PET radioligand because it was already
known to exhibit some of the properties recognized as being
desirable,17−20 including high affinity and selectivity for binding
to target human receptors, ability to cross the blood−brain
barrier, and potential to be labeled with no-carrier-added
(NCA) fluorine-18 (t1/2 = 109.7 min) at an aryl carbon.21 Here
we report the radiosynthesis of [18F]9 and an evaluation of its
radioligand behavior in mouse and monkey. Our findings show
[18F]9 is an effective H3 receptor radioligand.
RESULTS AND DISCUSSION
■
Chemistry. Reference ligand 9 was prepared from
commercially available (4-fluorophenyl)(4-hydroxyphenyl)-
methanone, as described previously.16 We prepared the nitro
Received: December 14, 2011
Published: February 7, 2012
This article not subject to U.S. Copyright.
Published 2012 by the American Chemical
Society
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Chart 1. Previously Reported PET Radioligands for H3 Receptors (Imidazole-Based Radioligands Are Shown on the Left and
Nonimidazole-Based Radioligands on the Right)
a
Scheme 1. Synthesis of Nitro Precursor 12
a
Reagents, conditions and yields: (i) HBr, AcOH, reflux, 9 h; 87%; (ii) I2, KI, NH4OH, rt, 48 h; 26%; (iii) (a) 1-(3-butynyl)-2-(R)-
methylpyrrolidine, Pd(OAc)2, (p-tol)3P, CuI, rt, 10 min, (b) i-Pr2NH, MeCN, 60 °C,16 h, 28%.
analogue 12 to provide a precursor suitable for use in a single-
step labeling of 9 with cyclotron-produced NCA [18F]fluoride
ion because p-nitrophenylketones are well-known to be
susceptible to aromatic nucleophilic substitution with [18F]-
fluoride ion.21,22 The synthesis of 12 was analogous to that
known16 for the fluoro compound 9 (Scheme 1). An initial
attempt to demethylate the commercially available (4-
methoxyphenyl)(4-nitrophenyl)methanone with NaSMe in
DMF23,24 proved unsuccessful due to substitution of the nitro
group to form a thioether. This observation nevertheless
confirmed the susceptibility of the nitro group toward aromatic
nucleophilic substitution. Selective demethylation of (4-
methoxyphenyl)(4-nitrophenyl)methanone to the required
(4-hydroxyphenyl)(4-nitrophenyl)methanone (10) was readily
achieved by use of a non-nucleophilic Brønsted acid reagent,
HBr in AcOH.25,26 Treatment of 10 with 0.5 equiv of iodine
and potassium iodide selectively produced 11 having iodine in
ortho position to the hydroxy group in a moderate yield (30%)
Use of a substoichiometric amount of iodine reduced the
unwanted di-iodination that we observed from the use of a
stoichiometric amount of iodine. 1-(3-Butynyl)-2(R)-methyl-
pyrrolidine16 was used directly as a MeCN solution (∼0.1 M)
for ring closure with 11 to give the target nitro precursor 12 in
useful yield (28%).
Pharmacological Assays and Screening. Assay of
compound 9 for binding to human recombinant H3 receptors,
expressed in HEK293T cells, confirmed high affinity with Ki in
the nanomolar range (Table 1). The affinities of 9 for a wide
range of other human recombinant receptors and binding sites,
including the other subtypes of histamine receptor, were found
to be at least 200-fold lower. These results accord with the
previous report of the selectivity of 9 for binding to H3
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decomposition of [18F]9 became significant above 100 °C.
[18F]9 was separated with single-pass reverse phase HPLC in
high radiochemical purity (>99%). The pharmacologically
active precursor 12 eluted at 29.1 min and was well separated
from [18F]9 (tR = 36.0 min). No residual 12 or other chemical
impurity was detected in the formulated radioligand by
analytical HPLC. The specific radioactivity of [18F]9, when
finally formulated for intravenous injection at about 110 min
from the end of radionuclide production, was 1311 221 mCi/
μmol (n = 12). The average RCY of formulated [18F]9 was 9.3
5.8% (n = 12).
Table 1. Ki Values of Compounds 9 and 12 Determined from
in Vitro Competitive Binding Assays
Ki (nM)
receptor or binding site
9
12
H1
5440
1708
7055
923
H2
H3
1.9 0.23
>10000
432
0.4 0.04
>10000
>10000
>10000
5237
H4
5-HT1A
5-HT1B
5-HT1D
5-HT2A
5-HT2B
DAT
M1
>10000
6380
Measurement of LogD7.4 and Computation of cLogD
and cLogP. The lipophilicity of a candidate radioligand is an
important consideration17−20,29,30 because this property
strongly influences (i) ability of a radioligand to penetrate the
blood−brain barrier, (ii) nonspecific binding of the radioligand
in brain, (iii) the magnitude and measurability of the plasma
free fraction (f P), a parameter which may need to be known for
the application of certain types of PET data quantitative
analyses, and (iv) general susceptibility of a radioligand to
metabolism. Moderate lipophilicity is usually considered
desirable for achieving adequate blood−brain barrier pene-
tration without incurring unacceptable nonspecific binding and
for avoiding troublesome lipophilic brain-penetrant radio-
metabolites29 and low f P.30 The measured LogD7.4 of [18F]9
was 2.95 0.06 (n = 6). Thus, the lipophilicity of 9 was found
to be within the desirable range of LogD 1.5−3.5.19 Computed
cLogD (at pH = 7.4) values of 9 and 12 were 2.90 and 2.45,
respectively, and hence Pallas software appears quite accurate
for predicting lipophilicity for this type of structure. Computed
LogP values for 9 and 12 were 4.79 0.43 and 4.34 0.34.
PET Imaging of [18F]9 in Mice. Mouse brain is known to
contain relatively (i) high densities of H3 receptors in striatum,
parietal cortex, insular cortex, nucleus acumbens, globus
pallidus, and olfactory tubercle, (ii) moderate densities in
thalamus, hypothalamus, and hippocampus, and (iii) low
densities in cerebellum and brain stem.31,32 H3 receptor
concentrations in striatum have been reported to be 95 fmol/
mg tissue, which approximates to 95 nM.31 A practical
guideline19 is that to achieve successful imaging with PET
radioligands that are intended to bind reversibly to target
receptors, Bmax/Kd should exceed 10 in vivo. [18F]9 has a Ki
value of 1.0 nM for rodent (rat) H3 receptors16 and therefore,
the density of H3 receptors (Bmax, 95 nM) should be adequate
for PET imaging with this radioligand.
2718
1355
3152
>10000
137
>10000
3266
931
M2
462
681
M3
204
512
M4
351
415
M5
437
336
a
all others
>10000
>10000
a
5-HT1E,2C,3,5A,6 and 7, α1A,1B,1D,2A,2B and 2C, β1−3, D1−5, σ1,2, SERT, and
NET
receptors among a wide battery of receptors (H1−4, D1,2s,2,4, 5-
HT1−3, adrenergic, and muscarinic) and transporters (NET,
DAT, SERT) binding sites from unidentified species.16
Therefore, 9 exhibited the necessary high human H3 receptor
affinity and selectivity for consideration as a candidate PET
radioligand. The nitro precursor 12 was similarly screened and
was found to have almost 5-fold higher affinity for human H3
receptors and also greater than 200-fold selectivity for all other
tested receptors and binding sites (Table 1).
Intrinsic activity may influence the utility of a PET
radioligand for imaging G-protein coupled neuroreceptors.
For example, antagonists are expected to bind to the full
population of target receptors whether present in the G-protein
coupled state or not, whereas agonists might be expected to
bind only to the subpopulation of receptors in the G-protein
coupled state. Ligand 9 was reported to be a competitive H3
receptor antagonist in a variety of assays, including a Ca2+ flux
assay, and also found to be a potent H3 receptor inverse agonist
for GTP-γ-S binding in H3 receptor-transfected cells.16 Both 9
and 12 were found to be inverse agonists in a GTP-γ-S assay
performed by the Psychoactive Drug Screening Program.
Radiochemistry. [18F]9 was produced automatically within
a lead-shielded hot-cell from cyclotron-produced [18F]fluoride
ion (200−300 mCi) in a Synthia device27 equipped with a
microwave heater (Scheme 2).28 In trial experiments, reaction
of 12 with [18F]fluoride ion in DMF gave a higher decay-
corrected radiochemical yield (RCY) of [18F]9 (34%) than in
MeCN (2%) or in DMSO (6%). In these experiments,
microwave power was set between 50 and 60 W so that
reaction temperature did not exceed 90 °C because
Brain time−activity curves of [18F]9 in mice were acquired at
baseline, and after treatment of mice with the selective high-
affinity H3 inverse agonist ciproxifan33 (2.0 mg/kg, iv), nitro
precursor 12 (2.0 mg/kg, iv), or ligand 9 itself (1.0 mg/kg, iv)
(Figure 1). At baseline, radioactivity entered brain quickly after
iv injection of [18F]9, with peak whole brain uptake reaching a
quite high level of 3.36 SUV at about 6.5 min. Brain
radioactivity concentration then slowly declined to about 2.3
SUV by 90 min. In mice pretreated with ciproxifan, brain
Scheme 2. Radiosynthesis of [18F]9
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that [18F]9 gives a strong H3-receptor specific signal in mice,
encouraged our further evaluation of [18F]9 in nonhuman
primate.
PET Imaging of [18F]9 in Monkey. The distribution of H3
receptors in rhesus monkey brain is similar to that in human
brain.15,34 As in rat, H3 receptors are enriched in basal ganglia
and also present in hippocampus and cortical areas, whereas
cerebellum has lower levels of H3 receptors.
Brain time−activity curves of [18F]9 in male rhesus monkey
were acquired at baseline (Figure 2A) and after treatment with
ciproxifan (2.0 mg/kg, iv) or 9 (1.0 mg/kg, iv) (Figure 2B).
After injection of [18F]9 at baseline, radioactivity entered brain
well with peak uptake in H3 receptor-rich regions, such as
striatum (4.45 SUV) and frontal cortex (3.86 SUV), occurring
at about 27.5 and 47.5 min, respectively. Peak radioactivity
concentration in other regions was lower but still higher than in
cerebellum (2.76 SUV). The concentration of radioactivity in
all regions slowly and continuously decreased to the end of the
PET experiment (180 min).
Figure 1. Whole brain time−activity curves after [18F]9 was injected in
mice under baseline and pretreatment conditions. Data points are
means with one-sided error bars showing SD.
radioactivity concentration quickly peaked at a lower level of
about 1.98 SUV at 4.5 min after radioligand injection and then
gradually reduced to 1.09 SUV at 90 min, a value much lower
than seen at the same time after radioligand injection in the
baseline experiment. When mice were pretreated with either
the nitro precursor 12 or the ligand 9 itself, brain radioactivity
peaked at 3.5 min and at higher values of 4.34 or 4.07 SUV,
respectively. Brain radioactivity then decreased quickly to <1.20
SUV at 90 min, a level similar to that in the ciproxifan
pretreatment experiment. Together, these data indicate that at
baseline a high proportion of radioactivity in whole brain
represents specific binding of [18F]9 to H3-receptors.
Summed PET images of monkey brain at baseline showed a
distribution of radioactivity reflecting the expected distribution
of H3 receptors (Figure 3), with no evidence of radioactivity
uptake into skull.
The reasons for the differences in brain radioactivity uptake
between the pretreatment experiments with ciproxifan and
those using the structural congeners 12 and 9 are unclear. All
three ligands appear to be highly selective for H3 receptors.
One possibility is that at their administered doses 12 and 9
displace the structurally similar [18F]9 from plasma proteins,
giving a higher plasma free fraction (fP) and greater entry of
radioligand into brain than in the baseline experiment. Another
possibility is that 12 or 9, but not ciproxifan, blocks [18F]9 from
binding to other unknown sites in periphery, thereby increasing
availability of radioligand for brain entry. However, the
cumulative in vitro data on the strong selectivity of both 12
and 9 for binding to H3 receptors argue against this possibility.
H3 receptors also exist in peripheral organs such as lung and
gastrointestinal tract.1 The administered doses of the agents 9
and 12 may be more effective than that of ciproxifan at blocking
the binding of [18F]9 to peripheral H3 receptors, thereby
increasing the free concentration of [18F]9 in plasma and hence
uptake of radioactivity into brain. The imaging results, showing
Figure 3. Axial, coronal, and sagittal PET images of brain through the
striatum, after intravenous injection of a monkey with [18F]9 at
baseline (top panel) and after treatment with 9 (1.0 mg/kg, iv)
(bottom panel). Images were acquired for 180 min immediately after
each radioligand injection.
Pretreatment of monkeys with ciproxifan reduced the peak
radioactivity concentration in H3 receptor-rich regions such as
Figure 2. Brain region time−activity curves after [18F]9 was injected intravenously into monkey at baseline (A). (B) The effect of pretreatment with
either ciproxifan (2.0 mg/kg, iv) or 9 (1 mg/kg, iv) on the time−activity curve for striatum. The mean curve for cerebellum from the three
experiments is shown for comparison. Error bars represent mean SD.
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Figure 4. Time course of plasma concentration (SUV) of total radioactivity and unchanged [18F]9 after intravenous injection of [18F]9 into a rhesus
monkey, under baseline, ciproxifan pretreatment, and 9-pretreatment conditions..
frontal cortex and striatum but not in cerebellum. Subsequent
washout of radioactivity from all H3 receptor containing regions
was slow. Pretreatment with 9 reduced peak radioactivity
concentrations in all brain regions, and thereafter all brain
region concentrations declined to a common low level at 180
min. The summed PET images from pretreatment experiments
with 9 show a uniform low distribution of radioactivity across
brain, again with no evidence of radioactivity uptake in skull.
Thus, pretreatment with 9 appeared more effective than
pretreatment with ciproxifan in showing specific binding of
[18F]9 in monkey brain.
Figure 5. Percentage of radioactivity in plasma represented by
unchanged radioligand after injection of a monkey with [18F]9 at
baseline, after treatment with 9, and after treatment with ciproxifan.
The remainder of radioactivity was composed of radiometabolites
[18F]A−C.
Ciproxifan is known to show species differences in H3
binding affinity, with over 100-fold lower affinity for human
H3 receptors (Ki = 63 nM) than for rat H3 receptors (Ki = 0.51
nM). The binding affinity of ciproxifan for monkey H3
receptors is unknown. There is a possibility that the affinity
of ciproxifan for monkey H3 receptors is similar to that for
human H3 receptors, and this may account for incomplete
blockade of the brain H3 receptors at the administered dose.
Stability of [18F]9 in Buffer, Whole Blood, and Plasma
in Vitro. [18F]9 was found to be 98.8
0.2% (n = 6)
unchanged after 2.5 h in sodium phosphate buffer (pH 7.4) at
room temperature and was also stable in monkey whole blood
(98.4% 0.07%, n = 6) and monkey plasma (99.2% 0.1%, n
= 6) at room temperature in vitro for 2.5 h. Therefore, accurate
measurement of unchanged radioligand and radiometabolites in
plasma was feasible by radio-HPLC for the ultimate purpose of
generating radioligand arterial input functions.
Emergence of Radiometabolites of [18F]9 in Monkey
Plasma in Vivo. In the analyses of all studied monkey plasma
samples, extractions of radioactivity from plasma with
acetonitrile for radio-HPLC analysis were very effective (95.3
7.44%, n = 115). After administration of [18F]9 into monkey
at baseline, radioactivity cleared rapidly from plasma until about
50 min, when the low level of decay-corrected plasma
radioactivity concentration became almost constant (Figure
4). Similar plasma time−radioactivity curves were seen in
monkey that had been pretreated with ciproxifan or 9 (Figure
4). HPLC analyses of plasma showed that the concentration of
unchanged radioligand declined continuously (Figure 4) while
three radiometabolites [18F]A−C emerged (Figure 5). These
radiometabolites appeared to be less lipophilic than [18F]9 (tR =
5.63 min) according to their shorter retention times on reverse
phase HPLC (Figure 6). Radiometabolites emerged similarly in
plasma of monkeys pretreated with ciproxifan (Figure 5). By
contrast, in monkeys pretreated with 9, parent radioligand more
quickly decreased to become the minor component in plasma.
Figure 6. Reverse phase HPLC radiochromatogram of plasma sampled
from monkey at 75 min after intravenous injection of [18F]9 (3.87
mCi). See text for details of analysis.
In these experiments, the least lipophilic radiometabolite [18F]
A became the major component in plasma. The ability of the
radiometabolites to penetrate the blood−brain barrier is
presently unknown. Nonetheless, because of their lower
lipophilicities, these radiometabolites might be expected to
enter brain less readily than parent radioligand. They would not
therefore be expected to be troublesome to quantification of
radioligand binding to H3 receptors.
The routes of radioligand metabolism and the identities of
the radiometabolites are also unknown. However, a close
analogue of 9 having a nitrile (CN) group in place of the fluoro
(F) group has been found to be a substrate for CYPs 3A4, 1A2,
and 2D6 as well as flavin monooxygenases FMO-1 and FMO-
3.16 The lack of radioactivity uptake in skull (Figure 3)
indicates that none of the radiometabolites of [18F]9 is
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[18F]fluoride ion and that radiodefluorination does not occur
for this radioligand. Radioligands that radiodefluorinate may
give high radioactivity uptake in skull which may compromise
PET measurements in nearby brain through “partial volume
effects”.
Plasma Free Fraction of [18F]9. The plasma free fraction
(f P) of [18F]9 in monkey plasma was accurately measurable and
found to be 2.08 0.14% at baseline and 1.4 0.1% during
pretreatment with 9.
Biomathematical Analysis of PET Data Acquired with
[18F]9. Time−activity curves in baseline monkey experiments
with [18F]9 fitted quite well to both one-tissue (1TC) and two-
tissue (2TC) compartmental models. An F test showed that the
2TC model gave the best fit to acquired data (Figure 7).
Figure 8. Time stability of normalized total volume of distribution
(VT) determined in three regions in one monkey with [18F]9. Each VT
value was estimated from only the PET data collected up to that time
point.
position which may be susceptible to radiodefluorination in
human subjects in vivo to give high radioactivity uptake in bone
including skull. Skull uptake of radioactivity is known to occur
for other PET radioligands labeled with fluorine-18 in a
35
fluoromethoxy position, including (S,S)-[18F]FMeNER-d2
and [18F]FMEPEP-d2.36 In fact the incorporation of deuterium
into these radioligands is intended to counter radiodefluorina-
tion in vivo. We found that [18F]9 shows no radio-
defluorination in monkey. [18F]9 would not be expected to
be radiodefluorinated in human subjects because aryl
carbon−18F bonds are usually stable in vivo. The radioligand
[11C]7 has higher peak brain uptake (∼2.3 SUV) than [18F]8,
but this is still lower than that of [18F]9. However, [11C]7
appears superior in giving a very high proportion (∼83%) of H3
receptor-specific binding. The radioligand [11C]6 gave very
high peak uptake in pig brain and a very high proportion
(>90%) of H3 receptor-specific signal.13 So far, this is the only
H3 receptor radioligand studied in human subjects.14 In
baseline human experiments, this radioligand demonstrates
progressively increasing radioactivity uptake in H3 receptor-rich
regions, apparently due to a slow off rate from the receptor.
Although this high-affinity radioligand can be used to measure
drug receptor occupancy, care has to be taken to avoid mass
effects of coadministered carrier in the use of this radioligand,
and in particular undesirably significant and prolonged
occupancy of H3 receptors by carrier.
Figure 7. Fitting to two-tissue compartmental models of time−activity
curves acquired in three brain regions after iv injection of monkey with
[18F]9.
Application of 2TC modeling of the data from the two
monkeys showed that on average ciproxifan reduced the VT
26−34% whereas 9 reduced VT around 49−58% (Table 2).
These data indicate that the majority of radioactivity in H3
receptor-rich regions of brain represents specific binding of
[18F]9 to H3 receptors.
By truncating acquired PET data, we obtained VT values for
different time periods after radioligand injection. Reasonably
stable VT values were obtained from data acquired between 130
and 180 min (Figure 8), suggesting that for this radioligand in
monkey, the ingress of troublesome radiometabolites into brain
is not greatly problematic.
Comparison of [18F]9 with Other H3 Receptor Radio-
ligands. The only other 18F-labeled radioligand for brain H3
receptors that has been reported to give a sizable receptor-
specific signal in vivo is [18F]8 (Chart 1).15 In rhesus monkey,
this radioligand gives much lower peak brain radioactivity
uptake (<1.3 SUV) in H3 receptor-rich regions than [18F]9
(∼4.5 SUV; Figure 2), although a somewhat higher proportion
(∼75%) of this lower radioactivity uptake appears to be
receptor-specific binding. [18F]8 is labeled in a fluoromethoxy
CONCLUSIONS
■
[18F]9 demonstrates favorable properties as a PET radioligand
for brain H3 receptors in monkey, including moderately high
brain uptake and sizable receptor-specific signal in the absence
of radiodefluorination. Moreover, this study shows that higher
affinity ligands, such as 12, exist among this chemotype,16
which may thus serve as a platform for developing further
Table 2. Estimation of VT from 2TC Model in Two Monkeys Injected with [18F]9 at Baseline, After Treatment with Ciproxifan,
And after Treatment with 9
monkey 1 Vt
monkey 2 Vt
mean Vt decrease (%)
region
baseline ciproxifan pretreatment 9 pretreatment baseline ciproxifan pretreatment 9 pretreatment ciproxifan 9 pretreatment
frontal cortex
striatum
64.1
54.7
60.4
53.7
42.0
43.5
42.7
45.3
40.8
33.5
29.5
29.1
31.2
29.9
24.8
42.1
45.3
50.8
49.9
28.5
26.7
31.6
30.4
29.4
19.6
16.4
19.7
20.8
19.4
12.0
−34
−26
−33
−33
−26
−58
−52
−54
−53
−49
hippocampus
thalamus
cerebellum
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129.48, 124.19, 115.03, 85.61 ppm. GC-MS: found 368.77 (M+); calcd
for C13H8INO4, 368.95.
improved 11C-labeled and 18F-labeled H3 receptor PET
radioligands.
(R)-(2-(2-(2-Methylpyrrolidin-1-yl)ethyl)benzofuran-5-yl)(4-
nitrophenyl)methanone (12). Compound 11 (0.500 g, 1.36 mmol)
and a solution of 1-(3-butynyl)-2-(R)-methylpyrrolidine (0.1M) in
MeCN (16.9 mL) were mixed in a round-bottomed flask (100-mL).
Pd(OAc)2 (9.0 mg, 0.04 mmol), tri-p-tolylphosphine (24.3 mg, 0.08
mmol), and CuI (77 mg, 0.40 mmol) were added. The resultant
mixture was stirred at 25 °C for 10 min. i-Pr2NH (1.9 mL, 13.6 mmol)
was then added and the mixture heated at 60 °C for 16 h. The reaction
mixture was allowed to cool and then filtered through a plug of Celite.
The filtrate was concentrated under reduced pressure. Flash
chromatography (silica gel; CH2Cl2−MeOH−NH4OH, 90:9.9:0.1 by
vol) of the residue gave 12 as a dark-brown semisolid (143 mg, 28%).
1H NMR (CDCl3): δ 8.35 (d, 2H, J = 6.8 Hz), 7.94 (m, 3H), 7.75 (d,
1H, J = 8.4 Hz), 7.53 (d, 1H, J = 8.4 Hz), 6.56 (s, 1H), 3.27 (m, 2H),
3.06 (t, 2H, J = 8.4 Hz), 2.54 (m, 1H), 2.44 (m, 1H), 2.26 (m, 1H),
1.98 (m, 1H), 1.80 (m, 2H), 1.48 (m, 1H), 1.16 (d, 3H, J = 6.0 Hz)
ppm. 13C NMR (CDCl3): δ 194.61, 159.80, 157.43, 149.54, 143.77,
131.25, 130.55, 129.09, 125.97, 123.68, 123.44, 111.08, 103.14, 60.34,
53.76, 51.60, 32.58, 27.90, 21.66, 18.70 ppm. LC-MS (M+ + 1) 379.1.
HRMS (M+ + 1) found, 379.1668; calcd for C22H23N2O4, 379.1658.
HPLC: tR = 12.7 min, purity >99%.
EXPERIMENTAL SECTION
■
Animal Procedures. All animal experiments were performed in
accordance with the Guide for Care and Use of Laboratory Animals37
and were approved by the National Institute of Mental Health Animal
Care and Use Committee.
Materials and General Methods. All reagents and solvents were
ACS grade or higher and used without further purification. Unless
otherwise noted, all chemicals were purchased from Sigma-Aldrich
(Milwaukee, WI). Reactions were performed under argon atmosphere
with standard Schlenk techniques. 1-(3-Butynyl)-2-(R)-methylpyrroli-
dine was synthesized with a published method16 from (R)-2-
methylpyrrolidine and 3-butynyl-4-toluenesulfonate; this reagent was
used as a ∼0.1 M solution in MeCN without purification. Ligand 9 was
prepared from commercially available (4-flourophenyl)(4-
hydroxyphenyl)methanone, as described previously,16 and obtained
as a brown oil in >99% chemical purity.
1H (400 MHz), 13C NMR (100 MHz), and 19F NMR (376 MHz)
spectra were recorded on an Avance 400 spectrometer (Bruker;
Billerica, MA). Chemical shifts are reported in δ units (ppm)
downfield relative to the chemical shift for tetramethylsilane.
Abbreviations br, s, d, t, and m denote broad, singlet, doublet, triplet,
and multiplet, respectively. GC-MS spectra were obtained on a Polaris-
Q GC-MS instrument (Thermo Fisher Scientific Corp., Waltham,
MA). LC-MS was performed on a LCQ Deca instrument (Thermo
Fisher Scientific Corp.) equipped with a reverse-phase HPLC column
(Luna C18, 3 μm, 50 mm × 2 mm; Phenomenex, Torrance, CA),
eluted at 200 μL/min with a mixture of A (H2O−MeOH−AcOH,
90:10:0.5 v/v) and B (MeOH−AcOH, 100:0.5 v/v), initially
composed of 20% B and linearly reaching 80% B in 3 min). High
resolution mass spectra (HRMS) were acquired at the Mass
Spectrometry Laboratory, University of Illinois at Urbana
Champaign (Urbana, IL) under electron ionization conditions with a
double-focusing high resolution instrument (Autospec; Micromass
Inc.). Thin layer chromatography was performed on silica gel layers
(type 60 F254; EMD Chemicals, Gibbstown, NJ), and compounds
were visualized under UV light (λ = 254 nm). Prepared compounds
were analyzed by HPLC on a Prodigy column (10 μm, 4.6 mm × 250
mm; Phenomenex) eluted with 85%B at 2 mL/min with eluate
monitored for absorbance at 243 nm (Gold 168 detector; Beckman)
and were of >95% purity.
(4-Hydroxyphenyl)(4-nitrophenyl)methanone (10). (4-
Methoxyphenyl)(4-nitrophenyl)methanone (150 mg, 0.58 mmol)
was suspended in hydrobromic acid (5 mL, 48% w/w)25,26 and glacial
acetic acid (5 mL). The mixture was refluxed for 9 h and then taken to
dryness under vacuum. The residue was dissolved in EtOAc and
extracted with H2O. The organic layer was dried with MgSO4. After
removal of solvent, flash chromatography (silica gel; EtOAc−hexane,
20:80, v/v) of the residue gave 10 as a gray solid (122 mg, 87%); mp
198−200 °C. 1H NMR (CD3OD): δ 8.24 (d, 2H, J = 8.8 Hz), 7.77 (d,
2H, J = 8.8 Hz), 7.62 (d, 2H, J = 8.8 Hz), 6.79 (d, 2H, J = 8.8 Hz). 13C
NMR (CD3OD): δ 195.39, 164.44, 150.94, 145.42, 134.14, 131.38,
128.96, 124.50, 116.49 ppm. GC-MS: found 243.00 (M+); calcd for
C13H9NO4, 243.05.
Production of NCA [18F]fluoride Ion Reagent. NCA [18F]-
fluoride ion was produced by irradiating 18O-enriched water (98 atom
%) with a beam (20 μA) of 16.5 MeV protons38 from a PETrace
cyclotron (GE, Uppsala, Sweden) for 120 min. At the end of
irradiation, the aqueous solution (20−200 μL) of [18F]fluoride ion
(20−200 mCi) was transferred in a glass V-vial (1 mL) to a lead-
shielded hot-cell and placed in the cavity of a model 521 instrument
for accelerated microwave chemistry (Resonance Instruments Inc.,
Skokie, IL). The latter is integrated with a Synthia MKII radio-
chemistry platform inside the same hot-cell, as described previously.28
A solution of K2CO3/K 2.2.2 (100 μL stock solution of 0.5 mg K2CO3
and 5.0 mg K 2.2.2 in 9:1 MeCN and H2O mixture) and MeCN (600
μL) were added to the V-vial, which was then placed under N2 gas
flow (200 mL/min) and irradiated with microwaves (90 W in 2 × 2
min pulses). The addition of acetonitrile (600 μL) followed by
microwave irradiation was repeated three times to give dry NCA
[18F]fluoride ion-K+−K 2.2.2 reagent.
Radiosynthesis of [18F]9. Precursor 12 (1.0 mg) in DMF (0.3
mL) was introduced into the vial containing anhydrous [18F]fluoride
ion-K+−K 2.2.2 and irradiated with microwaves (50−55 W) in 3 × 2
min pulses. The reaction temperature was carefully held between 80
and 90 °C during the irradiation. The reaction mixture was then
diluted with water (0.7 mL) and injected onto a reverse phase column
(Prodigy, 10 μm, 10 mm × 250 mm; Phenomenex) eluted at 6 mL/
min with a mixture of aq NH4OH (0.025%, pH 8.5) (A, 70%) and
MeCN (B, 30%). B was kept at 30% for 5 min, linearly increased to
70% over 3 min, and then held at 70% for 45 min. The collected
fraction of [18F]9 (tR, 34−36 min) was transferred to a pear-shaped
flask and the solvent removed under vacuum. The residue was diluted
with sterile saline for injection (10 mL) containing EtOH (10%; USP
grade) and passed through a sterile filter (2.5 μm, Millex MP,
Millipore, Bedford, MA). The pH of the final dose was in the range 6−
8.
RCY was calculated from the radioactivity of formulated [18F]9.
Chemical and radiochemical purities, and specific radioactivity were
determined by reverse phase HPLC on a Prodigy column (10 μm, 4.6
mm × 250 mm; Phenomenex) eluted with 20% A and 80%B at 2 mL/
min with eluate monitored for absorbance at 243 nm (Gold 166
detector, Beckman) and for radioactivity (PMT, HC-003; Bioscan Inc.,
Washington, DC). [18F]9 (tR = 17.9 min) was identified by, (i)
coelution with nonradioactive 9 in the aforementioned analytical
HPLC method, and (ii) LC-MS analysis of associated carrier for
comparison with that of ref 9 (m/z = 352 (M+ + 1)).
(4-Hydroxy-3-iodophenyl)(4-nitrophenyl)methanone (11). A
solution of 10 (500 mg, 2.05 mmol) in NH4OH (1 M, 17.1 mL) was
stirred at 25 °C for 15 min and then treated with an aqueous solution
(2.05 mL) of KI (1.69 g, 10.25 mmol) and I2 (259 mg, 1.03 mmol)
with stirring at 25 °C for 48 h. Solids were filtered off, dissolved in
ethyl acetate, and then washed with H2O and brine. The organic layer
was dried with MgSO4. After removal of solvent, flash chromatography
(silica gel; EtOAc−hexane, 50:50 v/v) of the residue gave 11 as pale-
1
yellow solid (190 mg, 26%); mp 248−250 °C. H NMR (DMSO-d6/
Computation of cLogP and cLogD and Measurement of
LogD. cLogP and cLogD (at pH = 7.4) values for 9 were computed
with the program Pallas 3.0 for Windows (CompuDrug; San
Francisco, CA). The LogD value of [18F]9 was measured as the log
CD3OD): δ 8.31 (d, 2H, J = 8.4 Hz), 8.09 (s, 1H), 7.84 (d, 2H, J = 7.6
Hz), 7.62 (d, 1H, J = 8.4 Hz), 6.94 (d, 1H, J = 8.4 Hz). 13C NMR
(DMSO-d6): δ 192.37, 162.25, 149.74, 143.75, 141.81, 133.00, 130.89,
2412
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Journal of Medicinal Chemistry
Article
of its distribution coefficient between n-octanol and sodium phosphate
buffer (0.15 M, pH 7.4), as described previously.39,40 [18F]9 was
shown to be stable to buffer by radio-HPLC analysis. The radioactivity
in the organic phase and that in the aqueous phase were counted in a
γ-counter. Counting errors were <0.3 0.1% (n = 6) at one standard
deviation.
the total distribution volume (VT) under the three different conditions
(baseline, ciproxifan pretreatment, and 9 pretreatment). Time−activity
curve generation and nonlinear parameter fitting was carried out with
PMOD 3.0.
ASSOCIATED CONTENT
■
PET Imaging of [18F]9 in Mouse. Wild type FVB mice (Taconic
Farm, Germantown, NY) were anesthetized with 1.5% isoflurane in
oxygen, and body temperatures were maintained at 36.5−37.0 °C with
a heating lamp. Intravenous injections were performed via poly-
ethylene cannulae (PE-10; Becton Dickinson, Franklin Lakes, NJ) in
the tail vein. The cannulae were secured with tissue adhesive
S
* Supporting Information
Chromatograms for the separation and analysis of [18F]9. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(Vetbond; 3M, St. Paul, MN). Thirteen mice (27.6
3.8 g) were
AUTHOR INFORMATION
Corresponding Author
*Phone: (301) 594 5986. Fax: (301) 480 5112. E-mail: pikev@
mail.nih.gov.
■
scanned either at baseline (n = 4) or after treatment with ciproxifan
(2.0 mg/kg, iv; n = 3), 9 (1.0 mg/kg, iv; n = 3), or 12 (1.0 mg/kg, iv; n
= 3) 30 min before injection of [18F]9.
Serial dynamic scans were acquired with a Focus 120 microPET
scanner (Siemens Medical Solutions, Knoxville, TN) started at the
time of injection of NCA [18F]9 (73 29 μCi) and were continued
for 120 min with increasing frame durations from 20 s to 20 min.
Images were reconstructed by a Fourier rebinning/2D ordered-subset
expectation maximization algorithm. No attenuation or scatter
correction was applied. Whole brain decay-corrected time−activity
curves were generated using PMOD 3.0 (PMOD Technologies,
Zurich, Switzerland). Brain uptake of radioactivity was expressed as
standardized uptake value (SUV) where SUV = (% injected dose per
cm3 brain) × (g body weight).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by the Intramural Research Program
of the National Institutes of Health (NIMH). We thank the
NIH Clinical PET Department (Chief Dr. P. Herscovitch) for
fluorine-18 production and PMOD Technologies for providing
the image analysis software. Receptor binding assays were
performed by the National Institute of Mental Health’s
Psychoactive Drug Screening Program, contract no. HHSN-
271-2008-00025-C (NIMH PDSP). The NIMH PDSP is
directed by Dr. Bryan L. Roth, MD Ph.D., at the University of
North Carolina at Chapel Hill and Project Officer Jamie
Driscoll at NIMH, Bethesda, MD, USA. We also thank Cheryl
L. Morse (NIMH) for assistance in radiochemistry.
PET Imaging of [18F]9 in Monkey. Four male rhesus monkeys
(7.3
0.8 kg) were used for PET scans of the brain. Two of the
monkeys had femoral artery indwelling catheters for blood sampling.
All four monkeys were studied at baseline (n = 4), three at 30 min
after treatment with ciproxifan (2.0 mg/kg, iv) and two at 30 min after
treatment with 9 (1.0 mg/kg, iv). All monkeys were immobilized with
ketamine (10 mg/kg, im) and maintained in anesthesia with 1.5%
isoflurane in O2 via an endotracheal tube. NCA [18F]9 (3.91 0.46
mCi, n = 9) was injected intravenously. All PET scans were acquired
with a Focus 220 microPET scanner (Siemens Medical Solutions,
Knoxville, TN) for 180 min except one scan for 120 min. Each scan
consisted of 45 frames of increasing duration, from 30 s to 5 min.
Images were reconstructed with a Fourier rebinning/2D filtered back
projection algorithm with scatter and attenuation correction. All data
were decay-corrected to the time of radioligand injection.
With a view to analyzing monkey plasma for radiometabolites and
thereby measuring an arterial-input function of unchanged radioligand,
the stability of [18F]9 for 2.5 h in whole monkey blood and plasma in
vitro was first confirmed by the radio-HPLC method to be used for
radiometabolite analysis. This method used a reverse phase column
(Novapak C18, 4 μm, 100 mm × 8 mm; Waters Corp.) within a radial
compression module (RCM-100) that was eluted at 2.0 mL/min with
MeOH−H2O−Et3N (85:15:0.1 by vol). Upon radioligand injection in
each of the PET scans in two of the monkeys, arterial blood was
sampled into heparin-treated syringes at every 15 s for 2 min, followed
by further sampling at 3, 5, 10, 30, 60, 75, 90, 120, 150, and 180 min
from injection. Plasma [18F]9 was then quantified in each sample with
radio-HPLC, as previously described.41
ABBREVIATIONS USED
■
5-HT, serotonin; DAT, dopamine transporter; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide; f P, plasma free
fraction; GTP, guanosine triphosphate; H3, histamine subtype-
3; HPLC, high performance liquid chromatography; HRMS,
high resolution mass spectrometry; NCA, no-carrier-added;
NET, noradrenalin transporter; PET, positron emission
tomography; RCY, decay-corrected radiochemical yield;
SERT, serotonin transporter; SUV, standardized uptake value
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