Comparison of in Silico, Electrochemical, in Vitro and in Vivo Metabolism of a Homologous Series of (Radio)fluorinated σ1Receptor Ligands Designed for Positron Emission Tomography

DOI: 10.1002/cmdc.201600366

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Comparison of in Silico, Electrochemical, in Vitro and

in Vivo Metabolism of a Homologous Series of

(Radio)fluorinated s₁Receptor Ligands Designed for

Positron Emission Tomography

Christian Wiese,^[a]Eva Große Maestrup,^[a]Fabian Galla,^[a]Dirk Schepmann,^[a]Achim Hiller,^[b]

Steffen Fischer,^[b]Friedrich-Alexander Ludwig,^[b]Winnie Deuther-Conrad,^[b]

Cornelius K. Donat,^[b]Peter Brust,^[b]Lars Bꢀter,^{[c, d]}Uwe Karst,^{[c, d]}and Bernhard Wꢀnsch*^{[a, d]}

The imaging of s₁receptors in the brain by fluorinated radio-

tracers will be used for the validation of s₁receptors as drug

targets as well as for differential diagnosis of diseases in the

central nervous system. The biotransformation of four homolo-

gous fluorinated PET tracers 1’-benzyl-3-(w-fluoromethyl to w-

about the presence of unchanged parent radiotracers and

their radiometabolites. Radiometabolites were not found in the

brain after application of [¹⁸F]2–4, but liver, plasma, and urine

samples contained several radiometabolites. Less than 2% of

the injected dose of [¹⁸F]4 reached the brain, rendering [¹⁸F]4

less appropriate as a PET tracer than [¹⁸F]2 and [¹⁸F]3. Com-

pounds [¹⁸F]2 and [¹⁸F]3 possess the most promising properties

for imaging of s₁receptors in the brain. High s₁affinity (K_i=

0.59 nm), low lipophilicity (logD_7.4=2.57), high brain penetra-

tion (4.6% of injected dose after 30 min), and the absence of

radiometabolites in the brain favor the fluoroethyl derivative

[¹⁸F]2 slightly over the fluoropropyl derivative [¹⁸F]3 for human

use.

fluorobutyl)-3H-spiro[2]benzofuran-1,4’-piperidine]

([¹⁸F]1–4)

was investigated. In silico studies using fast metabolizer (FAME)

software, electrochemical oxidations, in vitro studies with rat

liver microsomes, and in vivo metabolism studies after applica-

tion of the PET tracers [¹⁸F]1–4 to mice were performed. Com-

bined liquid chromatography and mass spectrometry (HPLC–

MS) analysis allowed structural identification of non-radioactive

metabolites. Radio-HPLC and radio-TLC provided information

Introduction

The s receptor was first postulated in 1976 by Martin et al.,

who found that various opioid analgesics exert different phar-

macological profiles.^[1]This observation led to the sub-differen-

tiation of the opioid receptor into three opioid receptor sub-

types, which were termed according to their prototypical li-

gands m (morphine), k (ketocyclazocine), and s receptors (SKF-

10,047).^[1,2]Because typical opioid antagonists such as nalox-

one and naltrexone are unable to inhibit the effects caused by

s ligands, the s receptor was removed from the class of opioid

receptors.^[3]Today s receptors represent a unique class of non-

opioid, non-NMDA, and non-dopamine receptors, which are

classified into s₁and s₂receptor subtypes.^[4,5]Herein we focus

on the s₁receptor subtype.

Cloning of the s₁receptor led to a protein with 223 amino

acids and a molecular weight of 25.3 kDa, which is not related

to any other mammalian protein. It shares a 30% homology

with the yeast enzyme sterol-D⁸/D⁷-isomerase.^[6,7]The s₁recep-

tor is composed of two transmembrane helices, an extracellu-

lar loop (50 residues), a short N-terminal domain (10 residues)

and a long C-terminal domain containing the ligand binding

site. Both the N- and C-terminal domains are located intracellu-

larly.^[8]Very recently a 3D homology model of the s₁receptor

was reported showing the 3D organization of the protein and

its interaction with ligands. A combined site-directed mutagen-

esis and theoretical analysis of the receptor protein provided

profound insight into the receptor–ligand interactions at the

molecular level.^[9,10]

[a] Dr. C. Wiese, Dr. E. Große Maestrup, Dr. F. Galla, Dr. D. Schepmann,

Prof. Dr. B. Wꢀnsch

Institut fꢀr Pharmazeutische und Medizinische Chemie, Westfꢁlische Wil-

helms-Universitꢁt Mꢀnster, Corrensstraße 48, 48149 Mꢀnster (Germany)

E-mail: wuensch@uni-muenster.de

[b] Dr. A. Hiller, Dr. S. Fischer, Dr. F.-A. Ludwig, Dr. W. Deuther-Conrad,

Dr. C. K. Donat, Prof. Dr. P. Brust

Helmholtz-Zentrum Dresden-Rossendorf, Institut fꢀr Radiopharmazeutische

Krebsforschung, Forschungsstelle Leipzig, Permoserstraße 15, 04318 Leipzig

(Germany)

[c] Dr. L. Bꢀter, Prof. Dr. U. Karst

Institut fꢀr Anorganische und Analytische Chemie, Westfꢁlische Wilhelms-

Universitꢁt Mꢀnster, Corrensstraße 30, 48149 Mꢀnster (Germany)

The s₁receptor is located within the membrane of the en-

doplasmic reticulum, but also within the cell membrane and

the membrane of mitochondria. It controls the Ca²⁺transfer

from the endoplasmic reticulum to mitochondria via interac-

tion with the Ca²⁺binding chaperone BiP (binding immuno-

globulin protein) and the inositol triphosphate receptor.^[11–13]

[d] Dr. L. Bꢀter, Prof. Dr. U. Karst, Prof. Dr. B. Wꢀnsch

Cells-in-Motion Cluster of Excellence (EXC 100–CiM), Westfꢁlische Wilhelms-

Universitꢁt Mꢀnster, 48149 Mꢀnster (Germany)

Supporting information for this article can be found under http://

dx.doi.org/10.1002/cmdc.201600366.

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Moreover, it has been shown that s₁receptors are able to reg-

ulate K⁺and Na⁺channels.^[14,15]In addition to the influence on

ion channels, s₁receptors modulate various neurotransmitter

systems including the cholinergic, dopaminergic and glutama-

tergic neurotransmission.^[16–18]

alkyl fluorides can be defluorinated by elimination, substitution

or oxidation losing the [¹⁸F]label. Moreover, radioactive metab-

olites penetrating the blood–brain barrier (BBB) will lead to dis-

torted images.

In this manuscript the biotransformation of the homologous

fluorinated s₁receptor ligands 1–4 is reported. After prediction

of metabolically labile positions using the software package

Fast Metabolizer (FAME), the products formed by electrochemi-

cal oxidation were analyzed. Then, the in vitro metabolism

during incubation with rat liver microsomes was investigated

and the formed metabolites were compared with the predict-

ed and electrochemically formed metabolites. Finally, the radio-

metabolites formed after injection of the [¹⁸F]-labeled tracers

[¹⁸F]1–[¹⁸F]4 in mice were characterized.

A high density of s₁receptors is not only found in the cen-

tral nervous system (CNS), but also in various tissues of the pe-

riphery, including heart, kidney, liver, endocrine organs, and

placenta. In the CNS, a high density of s₁receptors is found in

the frontal cortex, hypothalamus, hippocampus, and cerebel-

lum regions associated with memory and emotion as well as

motor and sensory control.^[19–22]

It has been shown that the s₁receptor is involved in several

psychiatric and neurodegenerative disorders including depres-

sion, psychosis, anxiety, neuropathic pain and Alzheimer’s and

Parkinson’s disease.^[22–25]The naphthyl substituted pyrazole de-

rivative S1RA is currently being investigated in phase II clinical

trials of neuropathic pain.^[26–28]Due to the important role of

the s₁receptor in the above mentioned psychiatric and neuro-

degenerative disorders, imaging of the s₁receptor in positron

emission tomography (PET) studies is of particular interest. A

PET tracer suitable for imaging of s₁receptors in the brain will

be used for target validation with the aim of developing novel

therapeutic concepts. Moreover, a correlation of s₁receptor

density and activity with a particular disease could be exploit-

ed for diagnosis and prognosis.^[29]

Results and Discussion

Prediction of metabolically labile positions by FAME

Data about phase 1 and phase 2 metabolites of a large

number of organic molecules are collected in FAME.^[39]Based

on this information, heavy atoms of unknown compounds are

ranked according to the probability of being involved in a bio-

transformation reaction. Values between 0 and 1 are attributed

to the heavy atoms; a value of 0 indicates a low and a value of

1 a high probability of the heavy atom being involved in a bio-

transformation reaction. In this study, heavy atoms showing

a value greater than 0.22 were considered. In Figure 2 the

heavy atoms of 1–4 predicted by FAME as potentially metabol-

ically labile positions are marked with arrows.

Recently, a set of four homologous fluoroalkyl-substituted

spirocyclic s₁receptor ligands 1–4 has been synthesized and

pharmacologically evaluated (Figure 1). Due to the high s₁re-

ceptor affinity (K_i=0.59–1.4 nm) and selectivity against the s₂

subtype and other related receptors, the compounds 1–4 were

labeled with [¹⁸F]fluoride and investigated as PET tracers for

imaging of s₁receptors in the brain. With respect to organ dis-

tribution and pharmacokinetic properties the fluoroethyl deriv-

ative 2 (fluspidine) represents the most promising radioligand

and is currently used in clinical studies.^[30–38]

Figure 2. Metabolically labile positions predicted by FAME using the algo-

rithm for rat phase 1 reactions; black arrows indicate metabolically labile po-

sitions; the dashed arrow indicates a position which was only predicted for

the phenylbutyl derivative 4.

Figure 1. Homologous fluorinated spirocyclic s₁receptor ligands 1–4 devel-

oped for imaging of s₁receptors in the brain.

According to the predictions of FAME the N atom and the

adjacent benzylic C atom have the highest probability for met-

abolic reactions. Additionally, the para position of the benzyl

moiety was identified as metabolically labile. For all homologs,

C atoms of the side chain and the terminal fluorine atom were

marked as metabolically labile positions. Unexpectedly, the

benzene ring of the benzofuran was predicted only for the flu-

orobutyl derivative 4. Here, the C atom at position 5 was pre-

dicted to be biotransformed.

For studies with living organisms the biotransformation of

the respective ligand has to be considered. During the devel-

opment of a PET tracer the metabolism is of particular interest,

since the tracer should have a biological half-life longer than

the period of observation, usually at least 30–60 min. In gener-

al, aliphatic CÀF bonds are rather stable. Nevertheless, primary

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Electrochemical oxidation of 1–4

ence of oxidation products resulting from N-debenzylation and

hydroxylation indicates that hydroxylation probably has taken

place at the positions labeled in the respective structures

shown in Figure 4. For the homologous fluoroalkyl derivatives

1–4, very similar oxidation products were detected. However,

in the case of the fluoroethyl derivative 2, the hydroxylated

and N-debenzylated product 2i was not detected, indicating

that further dehydrogenation of 2i to form 2j or dehydrogena-

tion of the corresponding precursors 2a and 2g have already

taken place quantitatively.

The CYP-catalyzed oxidative biotransformation of compounds

1–4 can also be simulated by means of electrochemical oxida-

tion. Subsequent mass spectrometric detection and the ab-

sence of biological matrices allow the identification of reactive

and short-lived species such as radical cations as well as to

evaluate their binding tendency toward selected biomole-

cules.^[40–44]To investigate the oxidative biotransformation of

compounds 1–4 electrochemically, a potential ramp was ap-

plied to the electrochemical cell and the detected mass spectra

were plotted depending on the applied oxidation potential.

The obtained mass voltammograms allow an easy identifica-

tion of possible biotransformation products, since electro-

chemically generated oxidation products typically show in-

creasing signal intensities during the potential ramp, while the

signal intensity of the starting compound is decreasing.

A comparison of in silico predicted (FAME) and electrochemi-

cally generated metabolites shows that the N atom and the

adjacent benzylic C atom are the most labile positions. In con-

trast to the in silico prediction, electrochemical oxidation of

the fluoroalkyl side chain was not detected. However, the ben-

zene ring of all four homologs was oxidized electrochemically,

although this biotransformation reaction was predicted only

for the fluorobutyl derivative 4.

In Figure 3, the mass voltammogram of 1 is shown. The

signal intensity of 1 (m/z 312) starts to decrease at a potential

of ~1.7 V. At the same time, several signals appear with in-

creasing intensities. The signal of m/z 328, which can be traced

back to a hydroxylated oxidation product of 1, is already pres-

ent without applying a potential. This can be explained by the

fact that electrochemical oxidations can also occur during the

electrospray ionization process. However, this is the only oxida-

tion product present in the mass spectrum of the initial solu-

tion. Based on the detected exact masses of all indicated oxi-

dation products, sum formulae were determined and chemical

structures were deduced.

In vitro metabolism using rat liver microsomes

Analysis of metabolites formed upon incubation of 1–4 with

rat liver microsomes and NADPH

To analyze the metabolic labile positions, the fluoroalkyl-substi-

tuted s₁ligands 1–4 were incubated with rat liver microsomes

and Nicotinamide adenine dinucleotide phosphate (NADPH).

After precipitation of the proteins with acetonitrile, the super-

natant containing the metabolites was analyzed by HPLC–

HRMS methods.

In Figure 4, the suggested structures of the oxidation prod-

ucts of compounds 1–4 are shown. Hydroxylation of the ben-

zofuran moiety (e.g., 1g–4g), dehydrogenation of the piperi-

dine ring (e.g., 1k–4k), N-debenzylation (e.g., 1a–4a), and all

possible combinations of these reactions (e.g., 1h–4h, 1i–4i,

1m–4m) have taken place. The position of the hydroxy group

cannot be specified based on the exact masses, but the pres-

In general six metabolites of the homologous fluoroalkyl-

substituted derivatives 1–4 could be identified (Figure 5). The

secondary amines 1a–4a belong to the main metabolites.

Their exact mass indicates the removal of the N-benzyl group

by cytochrome P450 (CYP) catalyzed dealkylation. Moreover,

the structures of the fluoroethyl and fluoropropyl derivatives

Figure 3. Mass voltammogram obtained from the electrochemical oxidation of compound 1. The signal intensity of 1 (m/z 312) is decreasing, while signals

for oxidation products appear with increasing signal intensities. For clarity only normal mass values of oxidation products are shown.

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Figure 4. Possible oxidation pathway of compounds 1–4. Structural formulae were deduced based on the determined sum formulae, which were obtained

from the detected exact masses. (a) Electrochemical N-debenzylation.

Figure 5. Metabolites formed by incubation of 1–4 with rat liver microsomes and NADPH. Metabolites of 2 and 3 have already been reported.^{[33, 36]}

2a and 3a were confirmed unequivocally by comparison of

the retention times and mass spectra with independently syn-

thesized compounds.^[32,36]

Three monooxygenated products were formed by CYP cata-

lyzed oxidation, respectively. Compounds 1b,c–4b,c bearing

the additional oxygen atom in the fluoroalkyl side chain or the

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benzyl moiety represent important metabolites. In the MS

analyses of the hydroxybenzyl metabolites 1c–4c fragments

with a mass decreased by 106 Da were detected. It is assumed

that the M+H⁺À106 fragment results from the cleavage of

the hydroxybenzyl moiety. Additionally, the fragments M+H⁺

À106 of the metabolites 1c–4c are identical with the frag-

ments M+H⁺À91 of the parent compounds 1–4 and produce

the same fragmentation pattern. The 4-hydroxybenzyl substi-

tuted compound 2c had been synthesized to prove the struc-

ture of this metabolite and furthermore the position of the OH

moiety in the para position.^[34]

the four homologous fluoroalkyl derivatives 1–4 after incuba-

tion with rat liver microsomes and electrochemical oxidation.

In addition to N-debenzylation, rat liver microsomes oxidized

compounds 1–4 at the fluoroalkyl side chain (1b–4b), the

benzyl moiety (1c–4c), and the N atom (1d–4d). These posi-

tions were also predicted by FAME. In contrast, the electro-

chemical oxidation did not modify the fluoroalkyl side chain

and the phenyl ring of the benzyl moiety. Nevertheless, dehy-

drogenation of the piperidine ring (1k–4k) was observed

during electrochemical oxidation, which could be the first step

in oxidative transformations around the tertiary amine. The

predicted and detected (electrochemical oxidation) products

with a hydroxy moiety in the benzofuran ring (1g–4g) could

not be found after incubation with rat liver microsomes.

The fragmentation pattern of the metabolites 1b–4b ex-

cludes hydroxylation in the benzyl moiety and the piperidine

ring. The stepwise fragmentation of the metabolite 2b showed

elimination of the benzyl moiety (M+H⁺À91) and subsequent

loss of two molecules of water (M+H⁺À91À18 and M+H⁺

À91À18À18). The removal of two molecules of water is only

possible if the OH moiety is in an aliphatic position, that is, in

the fluoroalkyl side chain. The first H₂O molecule resulted from

removal of the additional hydroxy moiety, the second one

stems from the benzofuran subunit. Moreover, in the chroma-

togram, the metabolite 2b shows two peaks close together

with the same exact mass and the same fragmentation pat-

tern. These peaks could result from two diastereomeric metab-

olites, since hydroxylation in the fluoroalkyl side chain led to

an additional center of chirality.

Kinetics of metabolic degradation

Next, the stability of the four homologous fluoroalkyl substitut-

ed spirocyclic s₁ligands 1–4 during incubation with rat liver

microsomes and NADPH was determined. For this purpose, in-

cubations were stopped after 5, 10, 30, 40, 60 and 90 min, by

addition of cold (À208C) acetonitrile. The amount of remaining

parent compound 1–4 was determined by HPLC. A matrix

matched calibration was performed without addition of

NADPH using the compound concentration in the assay as

highest calibration concentration.

The retention times during RP-HPLC of the third monooxy-

genated metabolites 1d–4d were longer than those of the

parent compounds 1–4 indicating higher lipophilicity of the

metabolites. This observation together with the fragmentation

pattern points to the corresponding N-oxides 1d–4d. The N-

oxides 2d and 3d derived from the fluoroethyl and fluoro-

propyl derivatives 2 and 3 had been prepared independently

confirming the structures 1d–4d.^[34,37]

The metabolic degradation of the ligands 1–4 is shown in

Figure 6. It can be seen that the fluoropropyl and fluorobutyl

derivatives 3 and 4 with the elongated side chains are more

stable than the corresponding fluoroethyl and fluoromethyl

derivatives 2 and 1 with shorter side chains. After an incuba-

tion period of 90 min, ~25–30% of the parent compounds 3

and 4 were still detected, but 90% of 1 and almost 100% of 2

were metabolized by the microsome preparation.

The secondary amines 1e–4e with a hydroxy moiety in the

fluoroalkyl side chain represent the most polar metabolites.

After elimination of water (aliphatic OH group) in MSⁿfrag-

mentation experiments, the further fragmentation patterns

were very similar to the fragmentation patterns of the metabo-

lites 1a–4a. Additionally, the twin peaks in the chromatogram

indicate the existence of diastereomers originating from the

additional OH group in the fluoroalkyl side chain.

The amount of unchanged parent compound after an incu-

bation period of 30 min is of particular interest, since PET stud-

ies are usually performed during this period of time. The fluo-

ropropyl derivative 3 represents the most stable compound of

this series of fluorinated ligands with 46% of the parent com-

In addition to monooxygenated metabolites 1b,c,d–4b,c,d,

incubation with rat liver microsomes produced one dihydroxy-

lated metabolite 1 f–4 f, respectively. The mass of the main

fragment after cleavage of the hydroxybenzyl moiety (M+H⁺

À106) was identical to the mass of the metabolites 1e–4e.

Moreover, the fragmentation pattern of the main fragments

and the metabolites 1e–4e were very similar pointing to the

metabolites 1 f–4 f bearing an N-(hydroxybenzyl) moiety and

an additional OH moiety in the fluoroalkyl side chain. This

comparative study leads to the conclusion that incubation of

the four homologous fluoroalkyl substituted spirocyclic s₁li-

gands 1–4 with rat liver microsomes produced very similar pat-

terns of metabolites.

As predicted by FAME, the secondary amines 1a–4a formed

by oxidative debenzylation represent the main metabolites of

Figure 6. Degradation of the four homologous fluoroalkyl derivatives 1–4

over time during incubation with rat liver microsomes and NADPH.

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pound remaining unchanged after 30 min. The longer fluoro-

butyl derivative 4 showed almost the same stability (36% un-

changed after 30 min). However, the metabolic stability of the

fluoromethyl and fluoroethyl derivatives 1 and 2 was consider-

ably lower than the stability of 3 and 4, since only 20% and

13% of the parent compounds remained unchanged after an

incubation period of 30 min.

parent compounds from biological matrices was investigated.

In the majority of radiometabolite experiments, brain and liver

homogenates (tissues) as well as blood plasma and urine (bio-

fluids) were analyzed. Investigation of radiometabolites in the

brain is of major interest, since it represents the target organ

of the tracer. Analysis of plasma samples is also crucial for

studying metabolic pathways and for modelling of PET data.

The liver is the central organ responsible for the biotransforma-

tion of exogenous compounds. Polar metabolites are preferen-

tially excreted via the kidney and urine and, therefore, the radi-

ometabolites in the urine were determined.

It can be concluded that the fluoropropyl derivative 3 is

metabolically the most stable and the fluoroethyl derivative 2

is metabolically the least stable ligand of this series of fluori-

nated compounds. However, these results have to be interpret-

ed very carefully, since the rate of degradation in vivo depends

also on parameters such as lipophilicity, plasma protein bind-

ing, penetration into the tissue, etc. In order to image s₁re-

ceptors in the brain the penetration of the ligands into the

brain and the absence of radiometabolites in the brain are im-

portant features.

The first essential step in studying radiometabolites in tis-

sues and biofluids is the separation of low molecular weight

analytes from the biological matrix components in particular

macromolecular components such as proteins. Generally more

than 85% of the activity of the parent radiotracer and its me-

tabolites should be extracted. Due to the complexity of matrix

components, radiotracers and radiometabolites as well as their

interactions, a general method for separation does not exist.

However, protein precipitation is the most commonly used

method.^[45,46]

In vivo metabolism of [¹⁸F]1–[¹⁸F]4 in mice during PET

studies

Despite their strong precipitation efficiency, strong acids

such as HClO₄, and trichloroacetic acid are not suitable, since

they are chemically too aggressive and can lead to decomposi-

tion of acid labile metabolites. Organic solvents (CH₃CN,

CH₃OH, EtOH, acetone) and solvent mixtures (CH₃CN/CH₃OH,

CH₃OH/CH₂Cl₂, CH₃OH/EtOH, CH₃CN/H₂O, CH₃OH/CH₃CN/ace-

tone) are preferred precipitation agents.

Radiosynthesis

The fluorinated PET tracers [¹⁸F]1–[¹⁸F]4 were synthesized as

previously described.^{[30,33,36,38]}In brief the tosylates 5–8 were

reacted with [¹⁸F]fluoride in the presence of the cryptand Kryp-

tofix K2.2.2 (Figure 7). Heating of the acetonitrile solutions of

6–8 for 15 min provided the fluorinated PET tracers [¹⁸F]2–

[¹⁸F]4 in reproducible high labeling yields of ~55–70%. Howev-

er, the synthesis of the fluoromethyl derivative [¹⁸F]1 required

heating of the components in DMSO (1508C). The sterically de-

manding 2-benzofuran system close to the reaction center is

the reason for the sluggish reaction of the tosylate 5. In gener-

al radiochemical yields of 35–40%, radiochemical purity of

ꢀ99%, high chemical purity (free from non-radioactive com-

pounds) and a specific activity of ꢀ200 GBqmmol^À1within

a total synthesis time of 90–120 min were obtained for all radi-

otracers.

To investigate the recovery of fluorinated PET tracers from

complex biological matrices, [¹⁸F]2 (fluspidine) was selected as

model compound. A defined amount of [¹⁸F]2 was added to

freshly prepared biomaterials obtained from different sources

and various extracting agents were used. Plasma (rabbit), brain

homogenates (mouse) and liver homogenates (pig) were em-

ployed as biomaterials and CH₃CN (100%, 90% and 80%) as

well as CH₃OH (100%, 90% and 80%) were used for the pre-

cipitation of proteins and further matrix components. The first

precipitation step already provided ꢀ90% of the total ¹⁸F ac-

tivity in the supernatant (radio-HPLC). A second extraction step

increased the extracted activity less than 5% indicating that

the first extracting step was already very efficient.

Extraction of brain, plasma and liver samples with different

precipitation media resulted in very high amounts of the intact

parent radiotracer [¹⁸F]2 (96–99%) as monitored by radio-TLC.

These results indicate very low protein binding of the PET

tracer and negligible matrix effects. However, the UV signals

(254 nm) of the HPLC chromatograms of samples prepared

using different extraction media showed significant differences.

Whereas CH₃OH as well as aqueous CH₃OH and CH₃CN re-

vealed some UV-active impurities, samples extracted with pure

CH₃CN provided very clean chromatograms. Therefore, CH₃CN

was chosen as the preferred precipitating agent in further ex-

periments.

Figure 7. Radiosynthesis of [¹⁸F]1–[¹⁸F]4: (a) K[¹⁸F]F, K2.2.2, DMSO, 1508C,

25 min, for [¹⁸F]1; (b) CH₃CN, 858C, 15 min for [¹⁸F]2–[¹⁸F]4.

Recovery of PET tracer [¹⁸F]2 from complex biological

matrices

Additionally to these experiments, bovine serum albumin

was incubated with [¹⁸F]2 and subsequently the sample was

precipitated with cold CH₃CN. The recovery of the PET tracer

was >98%. According to the method of Bradford residual pro-

To analyze the metabolites formed in vivo after application of

the fluorinated tracers [¹⁸F]1–[¹⁸F]4 to mice, the recovery of the

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tein components were not found in the extract indicating

complete precipitation of the proteins. The ratio of biomaterial

and precipitating agent was crucial. Whereas a ratio of bioma-

terial: precipitating agent of 1:2 was somewhat critical to give

a good pellet, a ratio of 1:3 or 1:4 led to formation of good

pellets.

experiments. The recovery was lower than expected (60–90%).

As the amount of intact radiotracer was always >90% (excep-

tion [¹⁸F]F1 after 60 min), it is difficult to determine whether

the extraction of the parent compounds or their radiometabo-

lites is incomplete.

The relative activity in mouse brain at 30 min and at 60 min

after injection derived from organ distribution studies of

[¹⁸F]1–4 is depicted in Figure 8. The relative amount of the

metabolically more stable radiotracers [¹⁸F]2–4 decreased in

the order [¹⁸F]2>[¹⁸F]3>[¹⁸F]4. The logD values at pH 7.4 in-

crease in the same order indicating that the uptake of the

intact radiotracer decreases with increasing lipophilicity. It is as-

sumed that the more lipophilic fluorobutyl derivative [¹⁸F]4 is

trapped partly within membranes.^[38]

In summary, cold CH₃CN used in three- to four-fold excess

over the biomaterial represents the preferred procedure to ex-

tract the fluorinated PET tracers [¹⁸F]1–[¹⁸F]4 and their metabo-

lites almost quantitatively from the biomaterial.

Analysis of radiometabolites of [¹⁸F]1–[¹⁸F]4 in different

tissues and biofluids

To analyze radiometabolites in different tissues and biofluids,

brain and liver samples were homogenized and urine and

plasma samples were used directly. The proteins of the sam-

ples were precipitated by cold CH₃CN.

Radiometabolites of [¹⁸F]1–4 found in mouse brain

In brain, more than 95% of intact radiotracers [¹⁸F]2–4 were

present at 30 min and 60 min after injection (Table 1). At these

time points, radiometabolites could not be detected indicating

that radiometabolites were not formed in the brain and radio-

metabolites potentially formed in the periphery could not pass

the BBB.

Figure 8. Relative amounts of activity in mouse brain 30 min and 60 min

after injection of radiotracers [¹⁸F]1–4. Doses were standardized related to

wet weight; data are mean values Æstandard deviation; number of experi-

ments n=3–5; distribution coefficients at pH 7.4 (logD_7.4) were recorded by

distribution of the radiotracers between n-octanol and phosphate buffer

pH 7.4 and subsequent quantification of activity.^{[30,33,36, 38]}

Table 1. Amount of parent compounds [¹⁸F]1–4 and recovery in mouse

brain samples 30 and 60 min after injection (extraction agent: CH₃CN).

Compd

30 min^[a]

60 min^[a]

intact

recovery [%]^[c]

intact

recovery [%]^[c]

radiotracer [%]^[b]

A similar trend was reported for N-([¹⁸F]fluoroalkyl)spiperone

derivatives, where increasing lipophilicity led to decreased

amounts of radiotracers in the brain. However, the logD_7.4

values of fluorinated spiperone derivatives are higher than the

logD_7.4values of the radiotracers [¹⁸F]1–4 and differ more from

each other. Within this series of fluoroalkyl spiperone deriva-

tives the preference was given to metabolically more stable

compounds.^[47]

[¹⁸F]1

[¹⁸F]2

[¹⁸F]3

[¹⁸F]4

91Æ2.5

97Æ1.1

96Æ1.5

96Æ1.0

75–80

50–60

85–90

80–85

81Æ2.0

98Æ0.5

98Æ1.4

95Æ2.7

60–68

65–70

80–85

85–95

[a] Time after radiotracer injection. [b] Mean valuesÆSD (n=3) from radio-

TLC and radio-HPLC experiments. [c] Amount of activity in the CH₃CN ex-

tract relative to the total activity in the respective brain sample.

The absence of radiometabolites in the brain together with

the high relative amount of radiotracer in the target tissue

brain (4.6% of injected dose) render the fluoroethyl derivative

[¹⁸F]2 the favored radiotracer of this series of homologs.

Notably, the fluoromethyl compound [¹⁸F]1 differed signifi-

cantly from [¹⁸F]2–4 (Table 1, and Supporting Information (SI)

Figure SI1). HPLC analysis resulted in two radioactive com-

pounds at shorter retention times of 35 min (compound 1-A)

and 26 min (compound 1-B) indicating higher polarity of these

radiometabolites than the parent compound [¹⁸F]1. The

amount of both radiometabolites increased over the time. At

30 min after injection of [¹⁸F]1, the amount of radiometabolites

was 6% (1-A) and 4% (1-B), whereas at 60 min, 9% and 12%

of the radiometabolites 1-A and 1-B were found, respectively

(Figure SI1). In parallel, the amount of intact radiotracer [¹⁸F]1

decreased from 90% (30 min) to 79% (60 min). HPLC and TLC

data were in agreement (Æ3%). The reproducibility of the re-

sults was excellent, considering the natural variability of animal

Radiometabolites of [¹⁸F]1–4 found in mouse liver

Because the liver plays a central role in the biotransformation

of xenobiotics and their excretion, the presence of radiotracers

[¹⁸F]1–4 and their radiometabolites in mouse liver homoge-

nates was analyzed; 30 min after injection, the amounts of un-

changed radiotracers [¹⁸F]2–4 in the liver sample were rather

similar (57–69%), but the amount of intact fluoromethyl deriv-

ative [¹⁸F]1 was only 27% (Table SI1). Clearly, the biotransfor-

mation of the fluoromethyl derivative [¹⁸F]1 is faster than the

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biotransformation of the longer chained homologs [¹⁸F]2–4.

Surprisingly, the amounts of [¹⁸F]1, [¹⁸F]3 and [¹⁸F]4 remained

constant between 30 and 60 min after injection, whereas the

fluoroethyl derivative [¹⁸F]2 was further biotransformed.

All detected radiometabolites were more polar than the cor-

responding parent compounds [¹⁸F]1–4. However, neither in

radio-TLC nor in radio-HPLC was a signal for [¹⁸F]fluoride ob-

served. At 30 min after injection, radio-HPLC of liver samples

showed three major metabolites 1-A, 1-B and 1-D for the fluo-

romethyl derivative [¹⁸F]1. At the same time, the chromato-

grams of liver samples of mice treated with the homologous

fluoroalkyl derivatives [¹⁸F]2–4 were very similar considering

the increasing retention times of homologs with longer alkyl

chains (Figure 9). The corresponding radio-HPLC chromato-

grams of liver samples at 60 min after injection of the radio-

tracers [¹⁸F]1-4 were very similar to the chromatograms ob-

tained at 30 min.

Figure 10. Radiochromatograms of mouse liver samples using different ex-

tracting agents. Top: Precipitation with CH₃CN; bottom: Precipitation with

CH₃OH; insets: chromatograms with UV detection (254 nm). Liver homoge-

nates were obtained at 30 min after injection of the radiotracer [¹⁸F]1; the

additional radiometabolite is marked in the second chromatogram; reten-

tion times of parent compound [¹⁸F]1 and its radiometabolites: [¹⁸F]1:

t_R=44.5 min; 1-A: t_R~35 min; 1-B: t_R~27 min; 1-C: t_R~24.1 min (additional

radiometabolite); 1-D: t_R~22 min.

or aqueous Ch₃CN (90% CH₃CN_aq) resulted in lower recovery of

activity than extraction with CH₃CN. However, two very polar

radiometabolites with very short retention times (t_R<4 min)

were found after precipitation with aqueous CH₃CN and aque-

ous CH₃OH. Extraction of liver samples containing the higher

homologous fluorobutyl radiotracer [¹⁸F]4 with CH₃OH led to

~10% higher amounts of activity.

Figure 9. Chromatograms of radioactive metabolites of tracers [¹⁸F]1–4 de-

tected by radio-HPLC in samples of mouse liver at 30 min after injection (ex-

traction agent CH₃CN). Retention times of parent compounds: [¹⁸F]1:

t_R=44.5 min; [¹⁸F]2: t_R=49.9 min; [¹⁸F]3: t_R=53.7 min; [¹⁸F]4: t_R=54.7 min.

Radiometabolites of [¹⁸F]1–4 found in mouse plasma

At 30 min after injection, more than 84% of the activity of

plasma samples was represented by the unchanged radiotrac-

ers [¹⁸F]2–4. Only for the fluoromethyl derivative [¹⁸F]1 was

a decreased amount (75%) of intact radiotracer detected.

Treatment of the mice for 60 min led to reduced amounts of

unchanged radiotracers in the plasma, which can be attributed

to increased amounts of radioactive metabolites in the plasma.

In the radio-HPLC chromatograms displayed in Figure 11

(30 min samples) only two small peaks were observed in addi-

tion to the unchanged fluorobutyl derivative [¹⁸F]4 (parent ra-

diotracer 84%). These peaks appear at retention times of

In Figure SI2 the chromatograms of liver samples at 30 min

after injection of the fluoromethyl and fluoroethyl radiotracers

[¹⁸F]1 and [¹⁸F]2 are compared. For the fluoroethyl derivative

[¹⁸F]2 a few trace radiometabolites and two main radiometabo-

lites 2-A and 2-B were detected. On the contrary, three major

radiometabolites 1-A, 1-B and 1-D in considerably higher

amounts were observed for the fluoromethyl derivative [¹⁸F]1.

The number of minor radiometabolites, however, increased at

60 min after injection. In liver samples of mice treated with flu-

oropropyl and fluorobutyl homologs [¹⁸F]3 and [¹⁸F]4, several

radiometabolites (>10) were detected by radio-HPLC. Two of

these radiometabolites can be considered as major metabo-

lites.

As documented in Table SI1 the recovery of activity after ex-

traction of liver homogenates with CH₃CN was generally rather

low. In some experiments, other extracting agents were investi-

gated. Precipitating liver samples containing the fluoromethyl

radiotracer [¹⁸F]1 with CH₃OH, led to the same amount of un-

changed radiotracer [¹⁸F]1 as found with CH₃CN. However, an

additional radiometabolite 1-C at a retention time of 24.1 min

was detected after extraction with CH₃OH, which was not visi-

ble after extraction with CH₃CN (Figure 10).

Figure 11. Radiochromatograms of plasma samples 30 min after injection of

the radiotracers [¹⁸F]1–4 (extraction agent CH₃CN). The peaks of the parent

For the fluoroethyl radiotracer [¹⁸F]2, extraction with more

polar solvents such as CH₃OH, aqueous CH₃OH (90% CH₃OH_aq)

radiotracers are marked with . The peak for radiometabolite 2-B of tracer

*

[¹⁸F]2 is increased by a factor of 3.

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27.1 min (4-D, 14.3%) and 3.5 min (4-E, 3.6%). A few further

metabolites were detected in traces. A very similar radiochro-

matogram was obtained for the fluoropropyl derivative [¹⁸F]3

(86% parent) showing two major metabolites at retention

times of 25.6 min (3-D, 10.3%) and 3.5 min (3-E, 3.8%). For the

smallest homolog, the fluoromethyl derivative [¹⁸F]1, several

small and two main radioactive metabolites were detected,

which correlate nicely with the metabolites 1-A (37.1 min,

9.5%) and 1-D (21.7 min, 12.0%) found in tissue samples (only

1-A). The plasma sample of the fluoroethyl derivative [¹⁸F]2

(89% parent) however reveals a large peak for the radiometa-

bolite 2-D at a retention time of 28.2 min (5.6%) and a smaller

peak at 3.5 min (2-E, 5.4%). The formation of the metabolite 2-

D is the reason for decreased amounts of unchanged radio-

tracer in plasma (Table 2).

decreased to ~50% of the activity obtained at 30 min. Clearly

the more polar radiotracers [¹⁸F]1–3 are distributed faster into

tissues and organs, whereas the more lipophilic fluorobutyl ra-

diotracer [¹⁸F]4 remains in the plasma for a longer time, which

could be due to higher plasma protein binding.

Metabolites of [¹⁸F]1–4 found in mouse urine

Since urine samples were only available in some experiments,

the analysis of radiometabolites was not as exhaustive as for

tissue and plasma samples. In Table SI2 the amounts of un-

changed radiotracers in urine samples at 30 min and at 60 min

after injection of [¹⁸F]1–4 is summarized. In the urine samples,

only a small amount (<4%) of parent radiotracers was found.

Clearly, the intact radiotracers were not eliminated via the

kidney and urine.

Table 2. Amount of recovered parent compounds [¹⁸F]1–4 and recovery in

mouse plasma 30 min and 60 min after injection (extraction agent:

CH₃CN).

On the contrary, up to 10 radiometabolites were detected

by analytical HPLC. The majority of radiometabolites were

found in trace amounts only, but a few radiometabolites oc-

curred in large amounts (Figure 13, Table 3). For all tracers

[¹⁸F]1–4, rather polar radiometabolites 1-E to 4-E with a reten-

tion time of 3.5 min were detected, which represent major me-

tabolites, in the case of fluoroethyl and fluorobutyl derivatives

[¹⁸F]2 and [¹⁸F]4. The metabolites 1-E to 4-E appeared directly

after the exclusion volume of the radio-HPLC. It is possible that

this peak is caused by [¹⁸F]fluoride. However, separate analyses

of femur, that is, activity in bone versus marrow, did not indi-

cate any defluorination of the tracers [¹⁸F]1–4. The short reten-

tion time in the HPLC analysis indicates the presence of

[¹⁸F]fluoroethanol or [¹⁸F]fluoroacetic acid.

Compd

30 min^[a]

60 min^[a]

intact

recovery [%]^[c]

intact

recovery [%]^[c]

radiotracer [%]^[b]

[¹⁸F]1

[¹⁸F]2

[¹⁸F]3

[¹⁸F]4

75Æ10

89Æ3

86Æ2

84Æ9

76–80

60–75

55–65

60–70

52Æ7

75Æ8

83Æ5

86Æ5

60–65

50–60

60–68

67–75

[a] Time after radiotracer injection. [b] Data are mean valuesÆSD (n=3–5)

from radio-TLC and radio-HPLC experiments. [c] Amount of activity in the

CH₃CN extract relative to the total activity in the respective plasma

sample.

The main metabolites for the four homologous fluoroalkyl

derivatives [¹⁸F]1–4 are the metabolites 1-C to 4-C with reten-

tion times of 22–29 min. The increasing retention times from

1-C to 4-C are due to increasing chain length. Interestingly, the

radiometabolite pattern in the urine of mice treated with [¹⁸F]1

differs considerably from the pattern of the homologous trac-

ers [¹⁸F]2–4. Although the urine is already a solution of the ra-

diometabolites, extraction of the solution with CH₃CN and sub-

sequent analysis of activity led to a recovery of 81% for [¹⁸F]1,

66% for [¹⁸F]2, 59% for [¹⁸F]3 and 60% for [¹⁸F]4.

Figure 12 shows the amount of activity in CH₃CN extracts of

mouse plasma related to the injected dose obtained during

organ distribution studies.^{[30,33,36,38]}The following trend was ob-

served for plasma samples obtained at 30 min: [¹⁸F]3>[¹⁸F]4>

[¹⁸F]1~[¹⁸F]2. The most lipophilic fluorobutyl derivative [¹⁸F]4

revealed the smallest difference between the amount of activi-

ty recorded at 30 min and at 60 min as both values are almost

identical. On the contrary, the activity amount determined for

the more polar radiotracers [¹⁸F]1–3 60 min after injection was

Figure 12. Amount of activity in mouse plasma in relation to the injected

dose at 30 min and at 60 min after injection of the radiotracers [¹⁸F]1–4; wet

weight normalized dose; data are mean values Æstandard deviation, n=3–

5.

Figure 13. Radiochromatograms of urine samples 30 min after injection of

the radiotracers [¹⁸F]1–4 (extraction agent CH₃CN). The peaks of the parent

radiotracers are marked with

.

*

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Table 3. Relative amounts of major radiometabolites 60 min after injection.^[a]

Compd

Metab.

t_R[min]

Amount [%]

Metab.

t_R[min]

Amount [%]

Metab.

t_R[min]

Amount [%]

[¹⁸F]1

[¹⁸F]2

[¹⁸F]3

[¹⁸F]4

1-E

2-E

3-E

4-E

3.5

3.6

2–9

31–48

4–6

1-D

2-D

3-D

4-D

22.6

24.1

24.3

14–29

5–7

6–10

0.10

1-C

2-C

3-C

4-C

24.2

26.3

26.9

28.6

47–64

42–59

74–89

23–34

41–61

[a] Values are the mean (n=2) from radio-TLC and radio-HPLC experiments.

Comparison of metabolite spectra from brain, liver, plasma

and urine using [¹⁸F]2 as example

in vivo studies with mice were correlated. The most lipophilic

metabolites formed as major metabolites with rat liver micro-

somes are the hydroxybenzyl derivatives 1c–4c. In vivo, radio-

metabolites 1-A to 4-A represent the most lipophilic radiome-

tabolites, which are also formed as major radiometabolites (see

Figure 10–12). Therefore, it can be speculated that the radio-

metabolites 1-A to 4-A formed in vivo bear a hydroxy moiety

in the benzyl ring as shown for metabolites 1c–4c.

Debenzylation of the tertiary amines (1a–4a) represents the

second important metabolic pathway catalyzed by CYP en-

zymes of rat liver microsomes and NADPH. The secondary

amines 1a–4a were also detected as major products after elec-

trochemical oxidation and predicted by FAME. As the secon-

dary amines 1a–4a are considerably more polar than metabo-

lites bearing a hydroxy moiety in the phenyl ring (1c–4c) or in

the fluoroalkyl side chain (1b–4b), we speculate that the radi-

ometabolites 1-D to 4-D are the secondary amines [¹⁸F]1a–4a.

According to this hypothesis, the less polar radiometabolites

marked with 1-B to 4-B could bear the hydroxy moiety in the

fluoroalkyl side chain corresponding to metabolites 1b–4b.

After analyzing the organ-specific radiometabolites separately,

the radiometabolite spectra of the homologous tracers [¹⁸F]1–

4 were compared. Here the fluoroethyl derivative [¹⁸F]2 is dis-

cussed. In Figure 14, the radiochromatograms recorded from

tissue samples and body fluids at 30 min after application of

[¹⁸F]2 are displayed. In the chromatogram of brain samples,

only one peak for the unchanged parent compound [¹⁸F]2 is

shown indicating that radiometabolites are not formed in the

brain and radiometabolites are not able to pass the BBB. In

contrast, the urine sample does not contain the parent com-

pound [¹⁸F]2, but two major very polar radiometabolites 2-D

and 2-E with retention times of 26.4 min and 3.5 min, respec-

tively. Due to their high polarity these radiometabolites are

eliminated via kidney and urine. In the liver and plasma sam-

ples the parent compound [¹⁸F]2 represents the major compo-

nent indicating high metabolic stability. In both samples only

trace amounts of the radiometabolites 2-A (t_R=38.2 min) and

2-B (t_R=31.0 min) were detected.

The highly polar radiometabolite 2-E was present predomi-

nantly in urine and in low amounts in plasma. Its structure has

not yet been identified, but the fast renal elimination inhibits

the penetration into various tissues and in particular the CNS.

Conclusions

Different levels of animal experiments provided complementa-

ry information about the biotransformation of homologous flu-

oroalkyl substituted s₁receptor ligands 1–4. Theoretical analy-

sis of metabolites using databases (e.g., FAME) does not re-

quire any lab experiments. Electrochemical oxidation can be

performed without any animal material and provides valuable

ideas about oxidatively labile positions in the s₁ligands. Al-

though structural information can be derived from these ex-

periments and computational studies, the biological relevance

remains to be elucidated. In vitro metabolism studies using rat

liver microsomes require liver from rats as native biological ma-

terial. The degradation rate and the structure of metabolites

formed by oxidation with CYP enzymes can be analyzed with

this artificial system. However, studies with mice resemble best

the fate of a PET tracer in a living organism. Disadvantages of

in vivo studies are the requirement of animals and the missing

structural information about radiometabolites due to the very

low amount of PET tracers used in animal experiments. These

drawbacks are compensated by in vitro studies with liver mi-

crosomes and electrochemical oxidation.

Figure 14. Comparison of radio HPLC chromatograms of samples obtained

from different tissues and biofluids at 30 min after injection of the fluoroeth-

yl derivative [¹⁸F]2 (extraction agent CH₃CN); major metabolites are marked

*

with 2-A, 2-B, 2-D and 2-E; minor metabolites are marked with

.

Proposed structures of radiometabolites

For in vivo studies, receptor affinity and selectivity as well as

radiochemical availability represent important features of PET

tracers. However, pharmacokinetic parameters, such as logD_7.4

value determining the passage of membranes, penetration

In order to speculate about the nature of the in vivo formed

radiometabolites, the information gained in in vitro studies

with rat liver microsomes, electrochemical oxidations and

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ferred into an ultracentrifuge (Beckmann L8M with rotor Ti 70.1)

and centrifuged for 60 min at 100000 g and 48C. The resulting su-

pernatant (cytosol) was discarded, the pellet carefully washed with

buffer, resuspended and the centrifugation repeated. Finally the

supernatant was removed, the pellet resuspended in a small

amount of phosphate buffer (pH 7.4) and the microsome suspen-

sion was stored at À808C. The protein concentration was deter-

mined according to the method of Bradford^[48]using bovine serum

albumin as standard.

into the CNS and biotransformation are also important proper-

ties of PET tracers. This study showed that the database

(FAME), electrochemical oxidation experiments, in vitro bio-

transformation with rat liver microsomes and in vivo metabo-

lism studies with mice provided complementary information

supporting the final decision, which compound of this series

of homologous fluoroalkyl derivatives should be further devel-

oped.

In the case of the fluoroalkyl substituted s₁ligands [¹⁸F]1–

[¹⁸F]4, the fluoroethyl and fluoropropyl derivatives [¹⁸F]2 and

[¹⁸F]3 revealed a good brain uptake, while their metabolites

could not be detected in the brain. Clearly, the radiometabo-

lites detected in liver, plasma and urine are not able to pass

the BBB. Considering the high amount in the brain (4.6% of in-

jected dose), the high s₁affinity (K_i=0.59 nm) and the low

logD_7.4value (2.57), [¹⁸F]2 is the favored radioligand to be fur-

ther developed as PET tracer.

Incubation of 1–4 with rat liver microsomes: The incubation was

carried out in phosphate buffer (pH 7.4) at room temperature in

a circular shaker (IKA vibrax VXR, Staufen, Germany) and contained

rat liver microsomes (1.5 mgmL^À1protein), MgCl₂(0.86 mm) and

NADPH (2.6 mm). The concentration of 1–4 was 260 mm in a final

volume of 0.9 mL. Usually, the incubation was stopped after

90 min by addition of cold acetonitrile (À208C). The samples were

stored for 10 min on ice to complete protein precipitation. After

thawing, the samples were centrifuged for 8 min (10000 g in Het-

tich Mikro 20 benchtop centrifuge). The supernatant was decanted,

filtered with a 0.45 mm pore size syringe filter made from regener-

ated cellulose and analyzed.

Experimental Section

Structure elucidation by HPLC–MS: The LC–MS system consisted

of a LTQ Orbitrapꢂ XL with Accelaꢂ HPLC pump and autosampler

(Thermo Fisher). The prepared incubation solutions (20 mL) were in-

jected onto a LiChrospherꢂ RP Select B column (5 mm, 250ꢃ

4.6 mm) with guard column LiChroCARTꢂ RP Select B (4ꢃ4.0 mm)

at a flow rate of 1.0 mLmin^À1. The mobile phase was composed of

(A) 15% CH₃CN in water and (B) 60% CH₃CN in water; both com-

ponents contained formic acid (0.1%). The following gradient was

applied (A%): 0–20 min: gradient from 100% to 0%, 20–23 min:

0%, 23–24 min: gradient from 0% to 100%, 24–30 min: 100%. For

MS analysis the eluent was reduced to 25% by a post-column split-

ter (Acurate, LC Packings, Dionex). The MS parameters were as fol-

lows: ion spray voltage: 4 kV in positive mode, sheath gas flow: 40

arbitrary units, aux gas flow: 15 arbitrary units, sweep gas flow: 10

arbitrary units, capillary temperature: 2758C, capillary: voltage

40.5 V. First a total ion current (TIC) spectrum was recorded. In

order to elucidate the structures of the metabolites, collision in-

duced dissociation (CID), fragmentation experiments were carried

out. The Orbitrap MS system generated data with fragmentations

up to MS³.

In silico prediction of metabolically labile positions

ChemDraw was used to draw the basic amines 1–4 as protonated

species as they exist at pH 7.4. Then, sdf files were created. These

files were used by FAME for the prediction of metabolically labile

positions. The applied algorithm included phase 1 reactions from

rats (“ratPhase1”).

Electrochemical oxidation of 1–4

Compounds 1–4^{[30,32,36,38]}were dissolved in ammonium formate

(50/50 (v/v) 10 mm, pH 7.4, adjusted with aqueous ammonia) and

CH₃CN to a final concentration of 10 mm. The solutions were con-

tinuously pumped through an amperometric electrochemical thin-

layer cell (ReactorCell, Antec Leyden, Netherlands) equipped with

a boron-doped diamond working electrode, a graphite-doped

Teflon counter electrode and a Pd/H₂reference electrode, to which

all potentials mentioned in this work refer. The flow rate was set to

10 mLmin^À1, and a potential ramp between 0 and 2.5 V with a scan

rate of 10 mVs^À1was applied. The effluent of the electrochemical

cell was directly analyzed by means of ESI-HRMS using an Orbitrap

mass analyzer (Exactive, Thermo Fisher Scientific, Bremen, Germa-

ny). Mass spectra were recorded in the positive ionization mode

with the following MS parameters: ESI(+); resolution, high; AGC

target, balanced; sheath gas flow (N₂), 10 au; capillary temperature,

2758C; spray voltage, 4.0 kV; capillary voltage, 95 V; tube lens volt-

age, 120 V; skimmer voltage, 38 V; maximum injection time,

100 ms; m/z range, 100–1000.

Metabolic stability: Six incubations of the compounds were car-

ried out in phosphate buffer (pH 7.4) at room temperature in a cir-

cular shaker containing rat liver microsomes (1.5 mgmL^À1protein),

MgCl₂(0.86 mm) and NADPH (2.6 mm). The concentration of com-

pounds 1–4 was 320 mm in a final volume of 900 mL. After periods

of 5, 10, 30, 40, 60 and 90 min, the incubations were stopped by

addition of cold CH₃CN (500 mL, À208C). Then praziquantel solu-

tion (200 mL, 0.6 mgmL^À1in CH₃CN) was added as internal standard

resulting in a total volume of 1.6 mL. All incubations were carried

out as duplicates. The samples were kept at À208C for 30 min to

complete protein precipitation. After thawing, the samples were

centrifuged at 10000 g for 10 min, then the supernatant was deca-

nted, filtered with a 0.45 mm pore size syringe filter made from re-

generated cellulose (Macherey–Nagel, Dꢀren, Germany) and ana-

lyzed. A calibration was carried out with the same microsomal

matrix except NADPH. All calibration standards were treated in the

same way (90 min on the shaker, protein precipitation with aceto-

nitrile, centrifugation, etc.). The HPLC analysis was carried out with

a Merck Hitachi equipment consisting of UV detector: L-7400; au-

tosampler: L-7200; pump: L-7150; interface: D-7000. Twenty micro-

liters of the prepared incubation solutions were injected onto

In vitro metabolism using rat liver microsomes

Preparation of rat liver microsomes: Frozen rat livers from Wistar

rats were thawed in phosphate buffer (pH 7.4) with sucrose

(0.25m) and EDTA (5 mm), cut into pieces and homogenized with

a Potter (Elvehjem Potter, B. Braun Biotech International, Melsun-

gen, Germany) at 48C. The resulting suspension was centrifuged

(high-speed cooling centrifuge model Sorvall, RC-5C plus, Thermo

Finnigan, Langerwehe, Germany) at 10000 g for 15 min at 48C. Fat

was removed by absorption with cellulose. The pellet was resus-

pended in buffer, the mixture centrifuged again and afterward

both supernatants were combined. This suspension was trans-

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a Phenomenex Geminiꢂ C₁₈column (5 mm, 250ꢃ4.6 mm) at a flow

rate of 1.0 mLmin^À1and UV detection wavelength of 210 nm. The

mobile phase was composed of (A) 15% CH₃CN in water and

(B) 60% CH₃CN in water. Trifluoroacetic acid (0.05%) was added to

both components. The following gradient was applied (A%): 0–

20 min: gradient from 100% to 0%, 20–23 min: 0%, 23–24 min:

gradient from 0% to 100%, 24–30 min: 100%.

Fragmentation of the metabolites of the fluoropropyl deriva-

tive 3

Metabolite 3a: HPLC: t_R=12.6 min. MS¹(ESI+ exact mass): m/z

calcd for [C₁₅H₂₀FNO+H] 250.1602, found 250.1602. MS²(ESI+

exact mass): m/z calcd for [C₁₅H₁₈FN+H] 232.1496, found 232.1493

[MÀH₂O].

Metabolite 3b: HPLC: t_R=15.1 min and 16.9 min (diastereomers).

MS¹(ESI+ exact mass): m/z calcd for [C₂₂H₂₆FNO₂+H] 356.2020,

found 356.2016. MS²(ESI+ exact mass): m/z calcd for [C₁₂H₁₃FO₂+

H] 209.0972, found 209.0969 [MÀCH₂C₆H₅ÀN(CH₂)₃].

Data of the metabolites: Exact masses of metabolites and corre-

sponding fragments were determined using LTQ Orbitrapꢂ XL. The

exact masses confirm the elemental composition of the postulated

metabolites.

Metabolite 3c: HPLC: t_R=18.3 min and 18.6 min (regioisomers).

MS¹(ESI+ exact mass): m/z calcd for [C₂₂H₂₆FNO₂+H] 356.2020,

found 356.2016. MS²(ESI+ exact mass): m/z calcd for [C₁₅H₂₀FNO+

H] 250.1602, found 250.1598 [MÀCH₂C₆H₄OH].

Fragmentation of the metabolites of the fluoromethyl deriva-

tive 1

Metabolite 1a: HPLC: t_R=8.3 min. MS¹(ESI+ exact mass): m/z

calcd for [C₁₃H₁₆FNO+H] 222.1294, found 222.1291. MS²(ESI+

exact mass): m/z calcd for [C₁₂H₁₄FN+H] 204.1189, found 204.1183

[MÀH₂O].

Metabolite 1b: HPLC: t_R=12.0 min and 12.7 min (diastereomers).

MS¹(ESI+ exact mass): m/z calcd for [C₂₀H₂₂FNO₂+H] 328.1713,

found 328.1711. MS²(ESI+ exact mass): m/z calcd for [C₁₀H₉FO₂+

H] 181.0665, found 181.0659 [MÀCH₂C₆H₅ÀN(CH₂)₃].

Metabolite 3e: HPLC: t_R=8.1 min. MS¹(ESI+ exact mass): m/z

calcd for [C₁₅H₂₀FNO₂+H] 266.1551, found 266.1551.

Metabolite 3 f: HPLC: t_R=12.6 min. MS¹(ESI+ exact mass): m/z

calcd for [C₂₂H₂₆FNO₃+H] 372.1969, found 372.1969. MS²(ESI+

exact mass): m/z calcd for [C₂₂H₂₄FNO₂+H] 354.1864, found

354.1863 [MÀH₂O]. MS³(ESI+ exact mass): m/z calcd for

[C₁₅H₁₈FNO+H] 248.1445, found 248.1445 [MÀH₂OÀCH₂C₆H₄OH].

Metabolite 1c: HPLC: t_R=13.5 min. MS¹(ESI+ exact mass): m/z

calcd for [C₂₀H₂₂FNO₂+H] 328.1713, found 328.1711. MS²(ESI+

exact mass): m/z calcd for [C₁₃H₁₆FNO+H] 222.1294, found

222.1290 [MÀCH₂C₆H₄OH].

Fragmentation of the metabolites of the fluorobutyl deriva-

tive 4

Metabolite 4a: HPLC: t_R=14.9 min. MS¹(ESI+ exact mass): m/z

calcd for [C₁₆H₂₂FNO+H] 264.1764, found 264.1758. MS²(ESI+

exact mass): m/z calcd for [C₁₆H₂₀FN+H] 246.1658, found 246.1654

[MÀH₂O].

Metabolite 4b: HPLC: t_R=20.2 min and 20.6 min (diastereomers).

MS¹(ESI+ exact mass): m/z calcd for [C₂₃H₂₈FNO₂+H] 370.2182,

found 370.2176. MS²(ESI+ exact mass): m/z calcd for [C₁₂H₁₃FO₂+

H] 223.1134, found 223.1129 [MÀCH₂C₆H₅ÀN(CH₂)₃].

Metabolite 4c: HPLC: t_R=14.6 min and 18.8 min (regioisomers).

MS¹(ESI+ exact mass): m/z calcd for [C₂₃H₂₈FNO₂+H] 370.2182,

found 370.2176. MS²(ESI+ exact mass): m/z calcd for [C₁₅H₂₀FNO+

H] 264.1764, found 264.1758 [MÀCH₂C₆H₄OH].

Metabolite 4e: HPLC: t_R=9.8 min. MS¹(ESI+ exact mass): m/z

calcd for [C₁₆H₂₂FNO₂+H] 280.1713, found 280.1709.

Metabolite 1e: HPLC: t_R=6.0 min. MS¹(ESI+ exact mass): m/z

calcd for [C₁₃H₁₆FNO₂+H] 238.1234, found 238.1238.

Metabolite 1 f: HPLC: t_R=9.4 min and 9.87 min (isomers). MS¹

(ESI+ exact mass): m/z calcd for [C₂₀H₂₂FNO₃+H] 344.1662, found

344.1658. MS²(ESI+ exact mass): m/z calcd for [C₂₀H₂₀FNO₂+H]

326.1556, found 326.1551 [MÀH₂O]. MS³(ESI+ exact mass): m/z

calcd

for

[C₁₃H₁₄FNO+H]

220.1138,

found

220.1133

[MÀH₂OÀCH₂C₆H₄OH].

Fragmentation of the metabolites of the fluoroethyl derivative

2

Metabolite 2a: HPLC: t_R=11.1 min. MS¹(ESI+ exact mass): m/z

calcd for [C₁₄H₁₈FNO+H] 236.1451, found 236.1444. MS²(ESI+

exact mass): m/z calcd for [C₁₄H₁₆FN+H] 218.1345, found 218.1338

[MÀH₂O].

Metabolite 2b: HPLC: t_R=13.4 min and 15.4 min (diastereomers).

MS¹(ESI+ exact Mass): m/z calcd for [C₂₁H₂₄FNO₂+H] 342.1869,

found 342.1865. MS²(ESI+ exact mass): m/z calcd for [C₁₁H₁₁FO₂+

H] 195.0821, found 195.0813 [MÀCH₂C₆H₅ÀN(CH₂)₃].

Metabolite 2c: HPLC: t_R=16.6 min and 17.0 min (regioisomers).

MS¹(ESI+ exact mass): m/z calcd for [C₂₁H₂₄FNO₂+H] 342.1869,

found 342.1865. MS²(ESI+ exact mass): m/z calcd for [C₁₄H₁₈FNO+

H] 236.1451, found 236.1444 [MÀCH₂C₆H₄OH].

Metabolite 2e: HPLC: t_R=6.6 min. MS¹(ESI+ exact mass): m/z

calcd for [C₁₄H₁₈FNO₂+H] 252.1400, found 252.1392.

Metabolite 4 f: HPLC: t_R=14.5 min. MS¹(ESI+ exact mass): m/z

calcd for [C₂₃H₂₈FNO₃+H] 386.2131, found 386.2128 MS²(ESI+

exact mass): m/z calcd for [C₂₃H₂₆FNO₂+H] 368.2026, found

368.2023 [MÀH₂O]. MS³(ESI+ exact mass): m/z calcd for

[C₁₆H₂₀FNO+H] 262.1607, found 262.1604 [MÀH₂OÀCH₂C₆H₄OH].

In vivo metabolism of radiofluorinated s₁ligands [¹⁸F]1–4 in

mice

Animal experiments: All procedures involving animals were car-

ried out according to the Principles of Laboratory Animal Care (NIH

publication No. 86-23, revised 1985) and following the national

laws on the protection of animals. For the HZDR group, the studies

were performed based on an authorized approval (Landesdirektion

Leipzig, No. 24-9168, 11/12/23; TVV 22/10).

Metabolite 2 f: HPLC: t_R=10.7 min. MS¹(ESI+ exact mass): m/z

calcd for [C₂₁H₂₄FNO₃+H] 358.1818, found 358.1810. MS²(ESI+

exact mass): m/z calcd for [C₂₁H₂₂FNO₂+H] 340.1713, found

340.1704 [MÀH₂O]. MS³(ESI+ exact mass): m/z calcd for

[C₁₄H₁₆FNO+H] 234.1284, found 234.1284 [MÀH₂OÀCH₂C₆H₄OH].

Radiosynthesis: Aqueous [¹⁸F]fluoride solution was transformed

into the reactive K[¹⁸F]F–K2.2.2–carbonate complex using K₂CO₃

(1.8 mg, 12.9 mmol) and K2.2.2 (Kryptofix, 11.2 mg, 29.7 mmol). This

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complex was reacted with tosylates 5–8 (2.5–3 mg) dissolved in an-

hydrous CH₃CN (1 mL). The mixture was heated at 858C for 15 min.

For the synthesis of [¹⁸F]1 the transformation was performed in

DMSO (1 mL) and the mixture was heated at 1508C for 25 min. The

resulting mixture was diluted with water (3 mL) and directly ap-

plied to an isocratic semi-preparative HPLC for purification (Multis-

pherꢂ 120 RP18 AQ column, 150ꢃ10 mm, particle size 5 mm with

guard column, 50ꢃ10 mm, 5 mm); eluent: CH₃CN (55%) with

NH₄OAc (20 mm), flow rate 4.0 mLmin^À1. The fractions containing

the respective radiotracer were combined, diluted with water,

passed through a pre-conditioned Sep-Pak C₁₈cartridge, and the

purified radiotracer was eluted with CH₃OH (~1.5 mL). For in vivo

experiments, the solvent was evaporated and the radiotracer was

re-dissolved in saline.

again at 23500 g for 20 min at 48C. This procedure was repeated

twice. The final pellet was resuspended in 5–6 volumes of buffer.

The precipitation of proteins in brain, liver, plasma and urine sam-

ples was performed by addition of four volumes of ice-cold aceto-

nitrile. The precipitate was removed by centrifugation and the su-

pernatant was concentrated (608C, air flow).

Recovery of PET tracer [¹⁸F]2 from complex biological matrices:

Plastic tubes were filled with extracting agent (500 mL). Then, the

biological sample was added in a ratio of about 4:1 to 7:1 (extract-

ing agent/biological sample). The samples were vortexed and kept

in an ice bath for 15 min. After centrifugation, the supernatants of

all aliquots were combined and concentrated with argon gas and

gentle warming (~608C) up to a final residual volume of ~150 mL.

The activity of the extract was recorded and related to the activity

of the biological sample. Recovery (%) in this article is defined as

the activity of the extract divided by the activity of the original bio-

logical sample multiplied by 100%. The percentages of parent radi-

otracer and radiometabolites were analyzed by radio-HPLC and

radio-TLC. Data obtained for all radiotracers are based on repetitive

analyses (n=2–5). In general, good agreement was achieved be-

tween radio-HPLC and radio-TLC data (deviations for brain and

urine ~1%, for plasma ꢁ5–8% and liver ꢁ5%). Here, both inter-in-

dividual variability of mice, methodical discrepancies and the ana-

lytical error have to be considered.

Radio-thin-layer chromatography (radio-TLC):

Radio-thin-layer chromatography (radio-TLC) was performed using

SIL G/UV₂₅₄precoated plates using different mixtures of ethyl ace-

tate/petroleum ether/NH₃; (v/v/v) as optimized standard procedure.

All data are based on duplicate values. Spots were visualized under

UV light (254 nm) and by radioluminescence recording (BAS-1800 II

Bioimaging Analyzer, Fuji film, Tokyo, Japan), and quantitatively

evaluated with AIDA 2.31 software (raytest, Straubenhardt, Germa-

ny). Alternative eluents were applied: ethyl acetate/CH₃OH varying

between 95:5 to 80:20 (v/v) and CH₂Cl₂/CH₃OH/NEt₃=8:2:0.1. R_f

values of the reference compounds of the corresponding radiotrac-

ers: 1: R_f=0.74 (ethyl acetate/petroleum ether/NH₃5:5:0.1); 2: R_f=

0.54 (ethyl acetate/petroleum ether/NH₃3:7:0.1); 3: R_f=0.61 (ethyl

acetate/petroleum ether/NH₃3:7:0.1); 4: R_f=0.52 (ethyl acetate/pe-

troleum ether/NH₃3:7:0.1).

In general acetonitrile was used as standard precipitating and ex-

tracting agent. In experiments regarding the recovery of activity,

additional precipitating agents such as pure methanol, aqueous

methanol (90% CH₃OH_aq) and aqueous acetonitrile (90% CH₃CN_aq)

were investigated.

For comparative metabolite studies, non-native biological samples

preserved as Tris buffer solutions and stored in a deep freezer or

obtained from fresh ex vivo samples were applied. They included

plasma (rabbit), brain homogenate (mouse), and liver (pig). For this

purpose, tissue material (50–75 mL) or biofluids was mixed with ra-

diotracer in saline (~10 mL, ~1–5 MBq) and extractive agent

(500 mL) and processed as described before for in vivo metabolite

experiments.

Radio-HPLC

Method 1: Agilent device (HPLC 1100 ChemStation; quaternary

pump, degasser, multiwavelength detector), coupled with a radio-

activity-HPLC flow monitor (FAP 100k/HV, bte, Germany); Multos-

pher 120 RP18 AQ 250ꢃ4.6 mm, 5 mm with guard column (10ꢃ

4.6 mm, 5 mm) (CS Chromatographie Service, Germany); gradient:

5–80% CH₃CN/20 mmol NH₄OAc; 1.0 mLmin^À1; run time: ~60–

70 min; solution A: 5% CH₃CN+20 mm NH₄OAc; solution B: 80%

CH₃CN+20 mm NH₄OAc; gradient: 0–10 min (100% A) 10–50 min

(solution B increasing from 0% to 100%), 50–60 min. In few cases

an isocratic method using 42.5% CH₃CN+20 mm NH₄OAc as

mobile phase was used.

g-counting: The extraction efficiency of the total ¹⁸F activity (“activ-

ity balance” recovery) was monitored permanently for each step

and aliquots were measured by gamma counting of the superna-

tants and precipitates using a calibrated gamma counter (Wallac

WIZARD 3, PerkinElmer, USA).

Protein determination according to the method of Bradford:

The amount of residual proteins in supernatants was determined

by photometric analysis with the BCA method^[48]using a bovine

serum albumin standard (PerkinElmer) and a microplate reader

(BIORAD Model 680 microplate reader, Bio-Rad Laboratories, Ger-

many); Windows based Microplate Manager software; 96-well mi-

crotiter plate; Instruction Thermo Scientific Pierce BCA Protein

Assay Kit [23225/23227] (Pierce Biotechnology, Rockford, USA).

Method 2: Jasco HPLC Serie 2000 (interface LC-NET II ADC; pumps:

binary p., quaternary HP gradient system p.; 4-line-degassers; auto-

sampler; UV/Vis detectors; coupled with radioactivity-HPLC-low-

monitors (Gabi Star, raytest GmbH, Germany), software Chrompass;

Reprosil-Pur C₁₈-AQ 250ꢃ4.6 mm, 5 mm with guard column (Dr.

Maisch GmbH, Ammerbuch-Entringen, Germany); gradient 10–90%

CH₃CN/20 mm NH₄OAc.

Preparation of tissue samples: For the investigation of radiometa-

bolites, radiotracers [¹⁸F]1–[¹⁸F]4 (~200–300 MBq) were adminis-

tered to female CD-1 mice by tail vein injection. After 30 or 60 min,

brain and liver samples were homogenized and plasma and urine

were directly used. Mouse brain or liver was homogenized with

the Potter (500–800 rpm, 10 up-and-down strokes) in six volumes

of cold sucrose (0.3m). The suspension was centrifuged at 1000 g

for 10 min at 48C. The supernatant was separated and centrifuged

at 23500 g for 20 min at 48C. The pellet was resuspended in 5–6

volumes of buffer (500 mm Tris buffer, pH 7.4) and centrifuged

Author contributions

C.W., D.S., P.B., U.K., and B.W. participated in the research design;

C.W., E.G.M., A.H., S.F., F.A.L., W.D.-C., C.D., and L.B. contributed ex-

periments; F.G. contributed to the reagents or analytical tools; F.G.

and C.D. performed data analysis; A.H., S.F., U.K., and B.W. wrote or

contributed to the writing of the manuscript. The authors declare

no conflicts of interest.

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Revised: August 29, 2016

Published online on && &&, 0000

&

ChemMedChem 2016, 11, 1 – 15

www.chemmedchem.org

14

ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

FULL PAPERS

C. Wiese, E. Große Maestrup, F. Galla,

D. Schepmann, A. Hiller, S. Fischer,

F.-A. Ludwig, W. Deuther-Conrad,

C. K. Donat, P. Brust, L. Bꢀter, U. Karst,

B. Wꢀnsch*

&& – &&

Taking a close look: The biotransforma-

tion of a homologous series of fluo-

roalkyl-substituted s₁receptor ligands

designed as PET tracers was investigat-

ed at different levels: in silico using

FAME software, electrochemically, in

vitro, and in vivo. Electrochemical oxida-

tion and in vitro studies with rat liver

microsomes combined with LC–MS

analysis allowed identification of the

metabolite structure. The nature and

amount of radiometabolites in various

tissues were analyzed by radio-HPLC

and radio-TLC after application of the

[¹⁸F]-labeled PET tracers to mice.

Comparison of in Silico,

Electrochemical, in Vitro and in Vivo

Metabolism of a Homologous Series of

(Radio)fluorinated s₁Receptor Ligands

Designed for Positron Emission

Tomography

ChemMedChem 2016, 11, 1 – 15

www.chemmedchem.org

15

ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

&

These are not the final page numbers! ÞÞ

Article Doi

Comparison of in Silico, Electrochemical, in Vitro and in Vivo Metabolism of a Homologous Series of (Radio)fluorinated σ1Receptor Ligands Designed for Positron Emission Tomography

DOI: 10.1002/cmdc.201600366

Source and publish data:

Authors:

Article abstract of DOI:10.1002/cmdc.201600366

Full text of DOI:10.1002/cmdc.201600366

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