Fluorinated GluN2B Receptor Antagonists with a 3-Benzazepine Scaffold Designed for PET Studies

Accepted Article

Title: Fluorinated GluN2B receptor antagonists with a 3-benzazepine

scaffold designed for PET studies

Authors: Bernhard Wünsch, Marina Szermerski, Frederik Börgel, Dirk

Schepmann, Thomas Betzel, Simon Ametamey, and Ahmed

Haider

This manuscript has been accepted after peer review and appears as an

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content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.201700819

Link to VoR: http://dx.doi.org/10.1002/cmdc.201700819

A Journal of

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FULL PAPER

Fluorinated GluN2B receptor antagonists with a 3-benzazepine

scaffold designed for PET studies

Dr. Marina Szermerski,^[a]Frederik Börgel,^[a]Dr. Dirk Schepmann,^[a]Ahmed Haider,^[b]Dr. Thomas

Betzel,^[b]Prof. Dr. Simon M. Ametamey,^[b]Prof. Dr. Bernhard Wünsch*^[a,c]

[a]

Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Corrensstraße 48, D-48149 Münster, Germany Tel.: +49-251-8333311;

Fax: +49-251-8332144; E-mail: wuensch@uni-muenster.de

Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland

Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), Westfälische Wilhelms-Universität Münster, Germany

[b]

[c]

Abstract: In order to analyze the NMDA receptor distribution in the

central nervous system, fluorinated ligands selectively addressing the

ifenprodil binding site of GluN2B subunit containing NMDA receptors

were developed. Various strategies to introduce a fluorine atom into

the potent GluN2B ligand 2 (3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-

3-benzazepin-1,7-diol) were pursued including the replacement of the

benzylic OH moiety by a F-atom (13) and introduction of fluoroethoxy

moieties at different positions (14 (7-position), 17 (9-position) 18a-c

(1-position)). With respect to GluN2B affinity and selectivity over

related receptors, the fluoroethoxy derivatives 14 and 18a represent

the most promising ligands. Radiosynthesis of fluoroethoxy derivative

[¹⁸F]14 was performed by nucleophilic substitution of the phenol 2 with

2-[¹⁸F]fluoroethyl tosylate. On rat brain slices the fluorinated PET

tracer [¹⁸F]14 accumulated in regions with high density of NMDA

receptors containing GluN2B subunits. The bound radioactivity could

not be replaced by (S)-glutamate. However, the GluN2B ligands

eliprodil, Ro 25-6981 and the non-labeled 3-benzazepine 14 were

able to abolish the specific binding of [¹⁸F]14.

GluN1 subunit (GluN1a-h) are known, four and two genes encode

four GluN2A-D and two GluN3A-B subunits, respectively. In

particular the expression of different GluN2 subunits differs

considerably in various regions of the central nervous system. As

an example, the GluN2B subunit is predominantly expressed in

the hippocampus, striatum and cortex, but not in the cerebellum,

where the GluN2C subunit is found in high density. Thus, subunit-

selective NMDA receptor ligands will allow addressing only some

regions of the central nervous system, whereas other regions

without the particular subunit are not affected.^[7-10]

Very recently, the structure of the heterotetrameric NMDA

receptor containing two GluN1a splice variants and two GluN2B

subunits together with various small molecules (glutamate,

glycine, ifenprodil or Ro 25-6981) has been reported.^[11,12]The

structure shows the amino terminal domain (ATD) with the

ifenprodil binding site at the interface between the GluN2B and

GluN1a subunits, the ligand binding domain (LBD) with the

glutamate (GulN2) and the glycine binding sites (GluN1) and the

transmembrane domain (TMD) with the ion channel pore. Ligands

interacting with the ifenprodil binding site within the ATD

selectively modulate the opening state of only GluN2B subunit

containing NMDA receptors as this binding site is only present at

the interface between GluN2B and GluN1 subunits.

Introduction

The excitatory amino acid neurotransmitter (S)-glutamate

interacts with eight metabotropic receptors (i.e. G-protein coupled

receptors) and three ionotropic receptors (i.e. ligand gated ion

channels). Whereas all three ionotropic glutamate receptors

(NMDA, AMPA, kainate receptors) control the passage of Na⁺-

and K⁺-ions across the cell membrane, the opened NMDA (N-

methyl-D-aspartate) receptor allows additionally the influx of Ca²⁺-

ions into the cell. The increase of intracellular Ca²⁺-ion

concentration is associated with neuronal development and

neuronal growth, but also with excitotoxic effects. Therefore, the

NMDA receptor represents an interesting target for the

development of novel drugs for the treatment of various

neurological and neurodegenerative disorders (e.g. depression,

cerebral ischemia, stroke, Parkinson’s, Alzheimer’s and

Huntington’s disease.^[1-6]

The NMDA receptor ion channel is structured by association of

four proteins. These proteins belong to three types of subunits,

GluN1, GluN2 and GluN3. Whereas eight splice variants of the

Figure 1. Design of GluN2B selective ligands 3 bearing a 2-

fluoroethoxy moiety at various positions.

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Ifenprodil (1) represents the prototypical ligand giving name to the

ifenprodil binding site. (Figure 1) However, its selectivity is rather

low, as it interacts with some types of Ca²⁺-channels,1, 5-HT_1A,

5-HT₂, _and ₂receptors as well, which results in side effects

such as psychotomimetic effects, memory deficit and

hypertension.^[13-16]The selectivity could be considerably

increased by rearrangement of the characteristic features of

ifenprodil resulting in the 3-benzazepine 2. (Figure 1) Ifenprodil

and 3-benzazepine 2 display comparable GluN2B affinity, but 2

shows high selectivity against more than 100 relevant drug targets

including adrenergic, serotonergic and  receptors.^[17]

Scheme 1: Synthesis of central building blocks 9 and 10.

Reagents and reaction conditions: (a) TosNHCH₂CO₂CH₃, DIAD,

Ph₃P, THF, 0 °C → RT, 24 h, 64 %. (b) NaOH, CH₃OH, reflux, 3

h, 90 %. (c) SnCl₄, trifluoroacetic anhydride, CH₂Cl₂, -15 °C, 18 h,

39 % mixture of 7 and 8. (d) NaBH₄, CH₃OH, RT, 16 h, 48 % (9),

17 % (10).

Since 1990 more than 60 compounds have been radiolabeled for

in vivo imaging of the centrally located NMDA receptor. The

majority of these radiotracers suffer from severe problems

including (1) poor brain penetration, (2) extensive metabolism, (3)

high non-specific and/or low specific binding, (4) homogenous

brain distribution and/or (5) distribution inconsistent with known

distribution of NMDA receptors.^[18,19]Very recently, a 18-F-labeled

memantine derivative was reported addressing the phencyclidine

binding site within the channel pore with rather low affinity.^[20]

However, in case of open-channel blockers an additional problem

exists since the PCP binding site within the channel pore is only

accessible in the active state (open channel). Therefore, GluN2B

subunit containing NMDA receptors were addressed by the 11-C-

labeled dimethylaminopyridine [¹¹C]Ro-647312 showing high

affinity towards the ifenprodil binding site.^[21]A 11-C-labeled 3-

benzazepine showing promising in vivo labeling of the ifenprodil

binding site of GluN2B NMDA receptors was recently reported by

us.^[22]The 18-F-labeled piperidine derivative [¹⁸F]MK-0657

revealed promising in vitro data, but low uptake into the brain.^[23]

In in vivo studies with 18-F-labeled benzoxazolone-based

fluorophenyl derivative RGH-896 fast defluorination was

observed resulting in high radioactivity in bones.^[24]

After saponification of the methyl ester 5, various reaction

conditions were tried to perform the intramolecular Friedel-Crafts

acylation of carboxylic acid 6. Treatment of acid 6 with

trifluoroacetic anhydride for 30 min and subsequent addition of

SnCl₄afforded a mixture of 3-benzazepinones 7 and 8 in 39 %

yield. During formation of the regioisomer with adjacent carbonyl

and benzyloxy moieties, an unexpected cleavage of the benzyl

ether was observed leading to the phenol 8. After reduction of the

mixture of ketones 7 and 8 with NaBH₄, the alcohols 9 and 10

were separated by flash chromatography. The benzyl ether 9,

which is the key intermediate of this study, had already been

prepared by cleavage of the methoxy analog of benzyl ether 7

with AlCl₃and subsequent benzylation of the resulting phenol.^[17]

Altogether, a fluorinated PET tracer selectively labeling NMDA

receptors fulfilling all requirements of a PET tracer does not exist.

Therefore, we decided to use 3-benzazepine 2 as starting point

for the development of a fluorinated PET tracer for selective

labeling of the ifenprodil binding site of GluN2B subunit containing

NMDA receptors. As displayed in compound 3 fluoroethoxy

groups will be introduced at various positions of the aromatic and

aliphatic part of the 3-benzazepine moiety. Furthermore, an F-

atom will be introduced in 1-position. (Figure 1)

Scheme 2: Synthesis of fluorinated 3-benzazepine 13. Reagents

and reaction conditions: (a) Mg, CH₃OH, reflux, 6 h, 50 %. (b) 1-

chloro-4-phenylbutane, Bu₄NI, CH₃CN, K₂CO₃, reflux, 72 h, 92 %.

(c) Et₂NSF₃(DAST), CH₂Cl₂, -78 °C, 1 h, then rt, 16 h, 53 %.

Removal of the tosyl protective group of 9 was performed with Mg

in refluxing CH₃OH to yield the secondary amine 11.^[17]Upon

reaction with 1-chloro-4-phenylbutane, the benzyloxy derivative

11 was converted into 4-phenylbutyl derivative 12, which was

obtained in 92 % yield, a yield that is slightly higher than the

reported one.^[17]The F-atom was introduced by reaction of 12 with

diethylaminosulfur trifluoride (DAST)^[28]to convert the benzylic OH

moiety into a F-atom. The 1-fluoro derivative 13 was isolated in

53 % yield. Although 13 could be characterized spectroscopically,

it showed fast degradation upon standing in solution. Due to the

low stability of 13, it could not be included into the biological

studies. (Scheme 2)

Results and Discussion

Synthesis

In order to obtain 3-benzazepin-1-one 7 and 3-bezazepin-1-ol 9a

with a benzyl protected phenol efficiently, a similar synthetic

approach was followed as described for the synthesis of the

analogous methyl ethers. (Scheme 1).^[25]At first tosylated glycine

ester^[26]was alkylated with benzyloxy substituted 2-phenylethanol

4 under Mitsunobu conditions.^[27]

In addition to the F-atom in benzylic position, a fluoroethyl moiety

was considered as alternative F-containing substituent. This

strategy will finally allow a facile radiofluorination, by using 18-

fluorine labeled (2-fluoroethyl) tosylate as alkylating agent.

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substituent in 7-position. 18c with a phenolic OH moiety showed

a K_ivalue of approx. 1 µM, the corresponding OCH₃derivative

18b revealed a K_ivalue of 470 nM, but the BnO derivative 18a

was the most potent GluN2B ligand of this series showing a K_I

value of 71 nM. (Table 1)

The moderate GluN2B affinity of the fluoroethoxy derivative 14

was unexpected, since the corresponding methoxy derivative (R¹

= OH, R²= 7-OCH₃) showed very high GluN2B affinity (K_i= 5.4

nM) and high selectivity.^[25]We assume that the ifenprodil binding

pocket is rather limited around the alkoxy group: whereas the

methoxy moiety fits nicely into the binding pocket, the slightly

larger 2-fluoroethoxy moiety is too big to be well accepted by the

binding pocket.

Alkylation of the hydroxy group in 1-position with a 2-fluoroethyl

moiety also led to considerably reduced GluN2B affinity. This

effect can be nicely observed by the Ki values recorded for the 1-

OH compound 2 (K_i= 14 nM)^[17]and the corresponding 1-

FCH₂CH₂O compound 18c (K_i= 1020 nM). It is postulated that

the OH moiety in 1-position is required to establish H-bonds with

some important amino acid residues in the binding pocket (e.g.

Gln110, sSr132).

Scheme 3: Synthesis of fluoroethoxy derivatives. Reagents and

reaction conditions: (a) CH₃CN, K₂CO₃, reflux, 16 h, 31 %. (b)

CH₃CN, K₂CO₃, reflux, 72 h, 78 %. (c) Mg, CH₃OH, reflux, 16 h,

60 %. (d) 1-chloro-4-phenylbutane, TBAI, CH₃CN, K₂CO₃, reflux,

72 h, 46 %. (e) NaH, DMF, 24-36 h, 13 % (18a), 34 % (18b). (f)

H₂, 1 bar, Pd/C, CH₃OH, RT, 30 min, 79 %.

At first, phenol 13c was transformed into the fluoroethoxy

derivative 14 by alkylation with (2-fluoroethyl) tosylate. The

regioisomeric fluoroethoxy derivative 17 was prepared starting

with the side product 10 formed during the intramolecular Friedel-

Crafts acylation. Reaction of phenol 10 with (2-fluoroethyl)

tosylate gave the fluoroethyl ether 15. Removal of the tosyl group

upon treatment with Mg in CH₃OH afforded the secondary amine

16, which was alkylated with 1-chloro-4-phenylbutane to provide

17 in 46 % yield. (Scheme 3)

Selectivity against related binding sites and receptors

In order to characterize the fluoroethoxy derivatives 14, 17 and 18

in more detail, the affinity towards the phencyclidine (PCP)

binding site within the channel pore of the NMDA receptor^[30,31]

and the affinity towards both  receptor subtypes^[32-34]were

determined in radioligand receptor binding studies. Up to a

concentration of 1 µM the test compounds did not compete with

the radioligand [³H]MK-801 for the PCP binding site indicating

very low PCP affinity and thus high selectivity for the ifenprodil

binding site. (Table 1)

Next, it was investigated, whether the ifenprodil binding pocket

tolerates a fluoroethyl moiety attached at the benzylic OH moiety

of 12. For this purpose, 12 and 19 were deprotonated with NaH

and subsequently reacted with (2-fluoroethyl) tosylate to provide

the fluoroethyl ethers 18a and 18b. The benzyl group of the

benzyl ether 18a was removed hydrogenolytically to give the

phenol 18c.

Due to similar pharmacophores^[35-38]of  receptor ligands and

ligands binding at the ifenprodil binding site, the affinity towards

_and _receptors was included into this study. Promising

selectivity over both  receptor subtypes was found for the most

potent GluN2B ligands 14 and 18a. Both compounds showed 7-

and 10-fold selectivity for the ifenprodil binding site of NMDA

receptors over _and _receptors, respectively.

Pharmacological evaluation

GluN2B affinity

The K_ivalues for interaction of 14 with _and _receptors are

higher than 1 µM indicating very low affinity towards these

receptors. The low _and _affinity of 14 is comparable with the

low affinities of the lead compound 2^[17]and its corresponding

methyl ether.^[25]However, the GluN2B/₁and GluN2B/₂

selectivity of 14 is considerably lower due to the reduced GluN2B

affinity. Nevertheless the 7-10-fold selectivity stimulated the first

radiosynthetic experiments.

The GluN2B affinity of the fluoro derivative 13 and the

fluoroethoxy derivatives 14, 17 and 18 was recorded in

competitive receptor binding studies. In brief, the receptor

material was obtained from L(tk-) cells stably transfected with a

vector encoding for the GluN1a and GluN2B subunits of the

NMDA receptor. In the assay, [³H]-labeled ifenprodil and the test

compounds were competing for a limited number of receptors

containing the ifenprodil binding site.^[29]

Unfortunately, the benzyl fluoride 13 could not be included into

the pharmacological study due to its low stability. In particular the

donor substituent in 7-position induces a fast fluoride elimination.

Whereas a fluoroethoxy substituent in 9-position (17) led to

negligible competition with the radioligand, the regioisomeric 7-

fluoroethoxy derivative 14 revealed promising GluN2B affinity of

162 nM. The GluN2B affinity of 3-benzazepines with the

fluoroethoxy moiety in 1-position was strongly dependent on the

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Table 1. Receptor affinity of fluoro derivative 13 and fluoroethoxy derivatives 14, 17 and 18 compared with reference compounds.

K_i± SEM [nM] (n = 3)^[a]

compd.

R¹

F

R²

7-OBn

GluN2B

PCP

σ₁

σ₂

[b]

13

14

OH

7-(FCH₂CH₂O)

9-(FCH₂CH₂O)

7-OBn

162 ± 22

21 %

71 ± 29

470

9 %

1040

5900

1700

113

17

OH

0 %

18a

18b

18c

FCH₂CH₂O

0 %

469

773

7-OMe

0 %

214 ± 63

5700

4 %

7-OH

1020

10 ± 0.7

13 ± 2.0

-

0 %

1000

98 ± 34

-

ifenprodil

-

125 ± 24

-

eliprodil

-

dexoxadrol

haloperidol

di-o-tolylguanidine

32 ± 7.4

-

6.3 ± 1.6

78 ± 2.3

-

89 ± 29

57 ± 18

^[a]The K_ivalues of potent compounds were recorded three times (n = 3). For low-affinity or very low-affinity compounds the competition

curves were recorded only once (single value) or the inhibition (in %) of the radioligand binding at a test compound concentration of

1 µM is given.

^[b]Due to its high reactivity (low stability) the benzyl fluoride 13 could not be tested pharmacologically

Radiosynthesis of fluoroethoxy derivative [¹⁸F]14

compound [¹⁸F]14 in non-decay corrected radiochemical yields of

4.4 – 16.7 % (1.3 – 4.1 GBq at the end of the synthesis).

Radiochemical purity was greater than 98 % and specific molar

radioactivity was between 85.6 and 179.9 GBq/µmol. (Scheme 4)

The remarkable selectivity over the PCP binding site, ₁and₂

receptors stimulated the radiosynthesis of 14, although its K_ivalue

for interaction with the ifenprodil binding site of GluN2B subunit

containing NMDA receptors was only moderate.

Autoradiography of fluoroethoxy derivative [¹⁸F]14

Fluoroethoxy derivative [¹⁸F]14 was evaluated in autoradiography

studies using rat brain slices. (Figure 2) High accumulation of

radioactivity (fluoroethoxy derivative [¹⁸F]14) was observed in the

cortex, hippocampus, striatum and the hypothalamus, regions

known to have high concentration of NMDA receptors with

GluN2B subunit. As expected, treatment with (S)-glutamate did

not lead to displacement of radioactivity on the slices indicating

that (S)-glutamate had no effect on radioligand binding. The

GluN2B antagonists eliprodil and Ro 25-6981 as well as the non-

radioactive compound 14 abolished the binding of the radioligand

suggesting in vitro specific binding of the radioligand [¹⁸F]14 to

GluN2B subunit containing NMDA receptors.

Scheme 4: Radiosynthesis of 18-F-labeled fluoroethoxy

derivative [¹⁸F]14. Reagents and reaction conditions: (a) CH₃CN,

Bu₄NOH, 90 °C, 4 min. (b) DMSO, NaOH, 12 min, 120 °C.

Fluoroethoxy derivative [¹⁸F]14 was obtained in a two-step

radiosynthesis, starting from ¹⁸F-labeling of ethylene ditosylate to

give 2-[¹⁸F]fluoroethyl tosylate. In the second reaction step, 2-

[¹⁸F]fluoroethyl tosylate was reacted with phenol 2, to afford

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Figure 2: Rat brain slices incubated with Fluoroethoxy derivative [¹⁸F]14. A: Incubation with PET tracer [¹⁸F]14 only; B – E: In addition

to incubation with [¹⁸F]14, glutamate (B), eliprodil (C), Ro 25-6981 (D) and 14 (E) were added.

Experimental Section

Conclusions

Chemistry, General Methods

The potent and subtype selective NMDA receptor antagonist 2 (K_i

= 14 nM) represents the starting point for the development of

fluorinated PET tracers deigned for imaging of NMDA receptors

with GluN2B subunit in the central nervous system. Since

introduction of the F-atom in benzylic position (13) resulted in a

rather unstable compound, fluoroethoxy moieties were introduced

in 1- (18), 7- (14) and 9-position (17). Promising GluN2B affinity

and selectivity over  receptors were found the 1-and 7-

fluoroethoxy derivatives 18a and 14. Radiosynthesis of [¹⁸F]14

succeeded by alkylation of phenol 2 with 2-[¹⁸F]fluoroethyl

tosylate to give the PET tracer [¹⁸F]14 in good radiochemical

yields and radiochemical purity. [¹⁸F]14 was able to label

selectively NMDA receptors with GluN2B subunits of rat brain

slices. The radioactivity could be blocked by co-incubation with

the GluN2B ligands eliprodil, Ro 25-6981 and non-labeled 14.

According to these results, we conclude that the class of 3-

benzazepines is well suited for the development of a fluorinated

PET tracer for imaging of GluN2B subunit containing NMDA

receptors. However, before in vivo evaluation we aim to increase

the GluN2B affinity and selectivity over related receptors.

Oxygen and moisture sensitive reactions were carried out under

nitrogen, dried with silica gel with moisture indicator (orange gel,

VWR, Darmstadt, Germany) and in dry glassware (Schlenk flask

or Schlenk tube). All solvents were of analytical or technical grade

quality. Demineralized water was used. CH₂Cl₂was distilled from

CaH₂; THF was distilled from sodium/benzophenone; MeOH was

distilled

from

magnesium

methanolate.

Thin

layer

chromatography (tlc): tlc silica gel 60 F₂₅₄on aluminum sheets

(VWR). Flash chromatography (fc): Silica gel 60, 40–63 µm

(VWR); parentheses include: diameter of the column (∅), length

of the stationary phase (l), fraction size (v) and eluent. Automated

flash chromatography: Isolera^TMSpektra One (Biotage^®);

parentheses include: cartridge size, flow rate, eluent, fraction size

was always 20 mL. Melting point: Melting point system MP50

(Mettler Toledo, Gießen, Germany), open capillary, uncorrected.

MS: MicroTOFQII mass spectrometer (Bruker Daltonics, Bremen,

Germany); deviations of the found exact masses from the

calculated exact masses were 5 ppm or less; the data were

analyzed with DataAnalysis^®(Bruker Daltonics). NMR: NMR

spectra were recorded in deuterated solvents on Agilent DD2 400

MHz and 600 MHz spectrometers (Agilent, Santa Clara CA, USA);

chemical shifts (δ) are reported in parts per million (ppm) against

the reference substance tetramethylsilane and calculated using

the solvent residual peak of the undeuterated solvent; coupling

constants are given with 0.5 Hz resolution; assignment of ¹H and

According

to

our

philosophy,

pharmacological

and

physicochemical properties of ligands have to be optimized in

vitro in order to reduce the number of animal experiments.

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¹³C NMR signals was supported by 2-D NMR techniques were

necessary.IR: FT/IR IR Affinity^®-1 spectrometer (Shimadzu,

Düsseldorf, Germany) using ATR technique.

brine (3 x 100 mL) and dried over Na₂SO₄. The solution was

concentrated in vacuo giving the crude product, which was

purified by flash column chromatography (d = 4 cm, h = 15 cm, V

= 20 mL, cyclohexane:ethyl acetate = 2:1 + 2 % AcOH) to yield 6.

Colorless solid, mp 132 °C, yield 2.58 g (90 %). C₂₄H₂₅NO₅S

(439.5). R_f= 0.13 (cyclohexane:ethyl acetate = 2:1 + 2 % AcOH).

HPLC: 99.2 %, t_R= 22.71 min. ¹H NMR (600 MHz, CDCl₃): δ [ppm]

= 2.40 (s, 3H Ph-CH₃), 2.83 (t, J = 7.7 Hz, 2H, CH₂CH₂NTs), 3.46

(t, J = 7.7 Hz, 2H, CH₂CH₂NTs), 3.97 (s, 2H, CH₂COOH), 5.03 (s,

2H, O-CH₂-Ph), 6.71 – 6.85 (m, 3H, 2-H_arom, 4-H_arom.and 6-H_arom.),

7.18 (t, J = 7.8 Hz, 1H, 5-H_arom.), 7.25 – 7.44 (m, 7H, 3-H_tosyland

HPLC method for the determination of the purity

Equipment 1: Pump: L-7100, degasser: L-7614, autosampler: L-

7200, UV detector: L-7400, interface: D-7000, data transfer: D-

line, data acquisition: HSM-Software (all from Merck Hitachi,

Darmstadt, Germany); Equipment 2: Pump: LPG-3400SD,

degasser: DG-1210, autosampler: ACC-3000T, UV-detector:

VWD-3400RS, interface: DIONEX UltiMate 3000, data

acquisition: Chromeleon

7

(equipment and software from

5-H_tosyl, benzyl), 7.69 (d, J = 7.9 Hz, 2H, 2-H_tosyland 6-H_tosyl). ¹³

C

Thermo Fisher Scientific, Lauenstadt, Germany); column:

LiChrospher^®60 RP-select B (5 µm), LiChroCART^®250-4 mm

cartridge; flow rate: 1.0 mL/min; injection volume: 5.0 µL;

detection at λ = 210 nm; solvents: A: demineralized water with

0.05 % (V/V) trifluoroacetic acid, B: CH₃CN with 0.05 % (V/V)

trifluoroacetic acid; gradient elution (% A): 0 - 4 min: 90 %;

4 - 29 min: gradient from 90 % to 0 %; 29 - 31 min: 0 %;

31 - 31.5min: gradient from 0 % to 90 %; 31.5 - 40 min: 90 %.

NMR (151 MHz; CDCl₃): δ [ppm] = 21.7 (1C, Ph-CH₃), 35.2 (1C,

CH₂CH₂NTs), 48.7 (1C, CH₂COOH), 50.3 (1C, CH₂CH₂NTs), 70.1

(1C, O-CH₂-Ph), 113.1 (1C, C-4_arom.), 115.6 (1C, C-2_arom.), 121.4

(1C, C-6_arom.), 127.5 (2C, C-2_tosyland C-6_tosyl), 127.6 (2C, C-2_benzyl

and C-6_benzyl), 128.1 (1C, C-4_benzyl), 128.7 (2C, C-3_benzyland C-

5_benzyl), 129.8 (2C, C-3_tosyland C-5_tosyl), 129.9 (1C, C-5_arom.), 136.5

(1C, C-1_tosyl), 137.0 (1C, C-1_benzyl), 139.7 (1C, C-1_arom.), 143.9 (1C,

C-4_tosyl), 159.1 (1C, C-3_arom.), 173.8 (1C, COOH). IR: 푣̃ [cm^-1] =

3032 (=C-H), 2932 2874 (CH_aliph.), 1717 (C=O), 1597 (C=C_arom.),

1346, 1157 (SO₂). Exact Mass (APCI): m/z = 440.1550 (calcd.

440.1526 for C₂₄H₂₆NO₅S [MH]⁺).

Synthetic procedures

Methyl N-[3-(benzyloxy)phenylethyl]-N-tosylglycinate (5)

2-[3-(Benzyloxy)phenyl]ethan-1-ol (4, 4.48 g, 19.6 mmol, 1.0 eq)

was dissolved in abs. THF (250 mL) and the solution was cooled

to 0 °C. PPh₃(15.4 g, 58.8 mmol, 3.0 eq) was added. After

addition of methyl (tosylamino)acetate (5.25 g, 21.6 mol, 1.1 eq),

DIAD (11.6 mL, 58.8 mmol, 3.0 eq) was added dropwise and the

mixture was stirred at 0 °C for 1 h. The mixture was warmed to

room temperature and stirred for 24 h. After filtration, the solvent

was removed under reduced pressure and the residue was

purified by flash column chromatography (d = 10 cm, h 15 cm, V

= 65 mL, cyclohexane:ethyl acetate = 4:1) to give 5. Yellow oil,

7-(Benzyloxy)-3-tosyl-2,3,4,5-tetrahydro-3-benzazepin-1-one

(7) and 9-hydroxy-3-tosyl-2,3,4,5-tetrahydro-3-benzazepin-1-

one (8)

The acid 6 (1.0 g, 2.28 mmol, 1.0 eq) was dissolved in CH₂Cl₂(80

mL) and the solution was cooled to -15 °C. Trifluoroacetic

anhydride (1.20 mL, 8.55 mmol, 3.75 eq) was added and the

solution was stirred for 30 min. Afterwards SnCl₄(1.0 mL, 8.55

mmol, 3.75 eq) was slowly added and the solution was stirred for

18 h at -15 °C. Water (30 mL) was added before the solution was

warmed to room temperature and neutralized with NaOH. After

dilution with brine, the aqueous layer was extracted with CH₂Cl₂

(3 x 100 mL) and the combined organic layers were dried over

Na₂SO₄. The solvent was removed in vacuo and the residue was

purified by flash column chromatography (d = 4 cm, h = 15 cm, V

= 20 mL, cyclohexane:ethyl acetate = 4:1), yielding a mixture of 7

and 8. Colorless foam, yield 377 mg (39 %). C₂₄H₂₃NO₄S (421.5).

R_f= 0.35 (cyclohexane:ethyl acetate = 2:1). The NMR data were

generated with another sample. Ratio 7:8 = 55:45. The signals for

yield 5.67

g

(64 %). C₂₅H₂₇NO₅S (453.6). R_f

= 0.80

(cyclohexane:ethyl acetate = 2:1). HPLC: 93.3 %, t_R= 23.99 min.

¹H NMR (600 MHz, CDCl₃): δ [ppm] = 2.41 (s, 3H, Ph-CH₃), 2.84

(t, J = 7.7 Hz, 2H, CH₂CH₂NTs), 3.46 (t, J = 7.8 Hz, 2H,

CH₂CH₂NTs), 3.62 (s, 3H, OCH₃), 3.98 (s, 2H,CH₂COOCH₃), 5.03

(s, 2H, O-CH₂-Ph), 6.73 - 6.85 (m, 3H, 2-H_arom., 4-H_arom.and 6-

H_arom.), 7.19 (t, J = 7.8 Hz, 1H, 5-H_arom.), 7.27 (d, J = 8.1 Hz, 2H,

3-H_tosyland 5-H_tosyl), 7.31 - 7.44 (m, 5H, benzyl), 7.70 (d, J = 7.9

Hz, 2H, 2-H_tosyland 6-H_tosyl). ¹³C NMR (151 MHz, CDCl₃): δ [ppm]

= 21.7 (1C, Ph-CH₃), 35.3 (1C, CH₂CH₂NTs), 48.7 (1C,

1

7 are marked with letter a. Letter b indicates signals from 8. H

CH₂COOCH₃), 50.1 (1C, CH₂CH₂NTs), 52.3 (1C, OCH₃), 70.1 (1C, NMR (400 MHz, CDCl₃): δ [ppm] = 2.33 (s, 3H, Ph-CH₃), 2.90 –

O-CH₂-Ph), 113.0 (1C, C-4_arom.), 115.6 (1C, C-2_arom.), 121.5 (1C,

C-6_arom.), 127.5 (2C, C-2_tosyland C-6_tosyl), 127.6 (2C, C-2_benzyland

C-6_benzyl), 128.1 (1C, C-4_benzyl), 128.7 (2C, C-3_benzyl, C-5_benzyl),

129.7 (2C, C-3_tosyland C-5_tosyl), 129.8 (1C, C-5_arom.), 136.8 (1C, C-

1_tosyl), 137.1 (1C, C-1_benzyl), 140.0 (1C, C-1_arom.), 143.6 (1C, C-

4_tosyl), 159.1 (1C, C-3_arom.), 169.5 (1C, COOCH₃). IR: 푣̃ [cm^-1] =

3032 (=C-H), 2951, 2870 (C-H_aliph.), 1751 (C=O), 1597, 1489

(C=C_arom.), 1450 (CH₂deform.), 1153 (SO₂), 737 (=C-H deform.).

Exact Mass (ESI): m/z = 476.1497 (calcd. 476.1502 for

C₂₅H₂₇NO₅SNa [M+Na]⁺).

3.00 (m, 2H, 5-CH₂), 3.67 (t, J = 6.6 Hz, 2  0.55H, 4-CH₂, a), 3.73

(t, J = 6.6 Hz, 2  0.45H, 4-CH₂, b), 4.17 (s, 2  0.55H, 2-H, a), 4.33

(s, 2  0.45H, 2-CH₂, b), 5.10 (s, 2  0.55H, O-CH₂-Ph, a), 6.62 (d,

J = 7.2 Hz, 0.45H, 6-H, b), 6.69 – 6.75 (m, 1H, 6-H and 8-H), 6.80

(dd, J = 8.7/2.4 Hz, 0.55H, 8-H, a), 7.02 – 7.12 (m, 2H, 3-H_tosyland

5-H_tosyl), 7.23 – 7.28 (m, 0.45H, 7-H, b), 7.33 – 7.48 (m, 2H, 2-

H_tosyl, 6-H_tosyl; 0.55 H, 9-H, a; 5  0.55H, benzyl, a), 11.28 (s, 1H,

OH, b). ¹³C NMR (101 MHz, CDCl₃): δ [ppm] = 21.57, 21.59 (1C,

Ph-CH₃and Ph-CH₃), 33.3 (0.55C, C-5, a), 34.1 (0.45C, C-5, b),

46.6 (0.55C, C-4, a), 46.9 (0.45C, C-4, b), 54.7 (0.55C, C-2, a),

55.0 (0.45C, C-2, b), 70.3 (0.55C, O-CH₂-Ph, a), 113.4 (0.55C, C-

8, a), 115.8 (0.55C, C-6, a), 117.5 (0.45C, C-8, b), 118.9 (0.45C,

C-9a, b), 120.9 (0.45C, C-6, b), 126.4 (2C, C-2_tosyland C-6_tosyl),

126.9, 128.5, 128.9 (5  0.55C, benzyl, a), 129.7 (2 0.55C, C-3_tosyl

and C-5_tosyl, a), 129.8 (2 0.45C, C-3_tosyland C-5_tosyl, b), 130.0

(0.55C, C-9a, a) 131.7 (0.55C, C-9, a), 136.09 (1C, C-1_tosyl),

136.10 (1C, C-1_benzyl), 140.0 (0.55C, C-5a, a), 140.3 (0.45C, C-5a,

N-[3-(Benzyloxy)phenylethyl]-N-tosylglycine (6)

5 (2.85 g, 6.29 mmol, 1.0 eq) was dissolved in CH₃OH (10 mL), 2

M NaOH (35 mL) was added and the mixture was heated to reflux

for 3 h. At room temperature, HCl was added to adjust pH 2. After

addition of brine (20 mL), the solution was extracted with CH₂Cl₂

(3 x 100 mL).The combined organic layers were washed with

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b), 143.4 (0.55C, C-4_tosyl, a), 143.6 (0.45C, C-4_tosl, b), 162.7

(0.55C, C-7, a; 0.45C C-9, b) , 200.3 (0.55C, C-1, a), 205.1 (0.45C,

C-1, b). IR: 푣̃ [cm^-1] = 3032 (=C-H), 2924, 2882 (C-H_aliph.), 1674

(C=O), 1597 (C=C_arom.), 1339, 1153 (SO₂). Exact Mass (ESI): m/z

= 444.1245 (calcd. 444.1240 for C₂₄H₂₃NO₄SNa [M+Na]⁺, 7),

354.0782 (calcd. 354.0770 for C₁₇H₁₇NO₄SNa [M+Na]⁺, 8).

7-(Benzyloxy)-2,3,4,5-tetrahydro-1H-3-benzazepin-1-ol (11)

[17]

9 (84.2 mg, 0.20 mmol, 1 eq) and Mg (106 mg, 4.37 mmol, 22 eq)

were suspended in abs. CH₃OH (20 mL) and the suspension was

heated to reflux for 6 h. At 0 °C, conc. H₂SO₄(0.5 mL) was added

until no reaction of Mg was observed. 2 M NaOH was added to

adjust pH 9 and the suspension was filtered. The filtrate was

extracted with CH₂Cl₂(4 x 20 mL). The combined organic layers

were dried over Na₂SO₄and the solvent was removed in vacuo.

The residue was purified by flash column chromatography (d = 2

cm, h = 15 cm, V = 10 mL, CH₂Cl₂: CH₃OH = 95:5 + 2 % NH₃) to

afford 11. Colorless solid, mp 138 °C, yield 26.9 mg (50 %).

C₁₇H₁₉NO₂(269.3). R_f= 0.14 (CH₂Cl₂:CH₃OH = 9:1 + 2 % NH₃).

HPLC: 95.4 %, t_R= 15.23 min. ¹H NMR (400 MHz, CDCl₃):δ [ppm]

= 2.65 (dd, J = 15.0/5.7 Hz, 1H, 5-H), 2.78 (t, J = 12.4 Hz, 1H, 4-

H), 2.88 (d, J = 12.7 Hz, 2H, 2-H), 3.21 – 3.34 (m, 3H, 2-H, 4-H

and 5-H), 4.60 (d, J = 6.1 Hz, 1H, 1-H), 5.04 (s, 2H, O-CH₂-Ph),

6.70 – 6.75 (m, 2H, 6-H and 8-H), 7.12 (d, J = 8.0 Hz, 1H, 9-H),

7.30 – 7.43 (m, 5H, benzyl). Signals for OH and NH protons are

not seen in the spectrum. ¹³C NMR (101 MHz, CDCl₃): δ [ppm] =

39.0 (1C, C-5), 49.5 (1C, C-4), 53.7 (1C, C-2), 70.1 (1C, O-CH₂-

Ph), 74.1 (1C, C-1), 111.2 (1C, C-8), 118.0 (1C, C-6), 127.6,

128.1, 128.7 (5C, benzyl), 130.2 (1C, C-9), 135.9 (1C C-9a),137.2

(1C, C_benzyl-1), 141.9 (1C, C-5a), 158.3 (1C, C-7). IR: 푣̃ [cm^-1] =

3294 (N-H), 3032 (=C-H), 2905, 2851 (C-H_aliph.), 1612, 1578, 1493

(C=C_arom.), 1258 (C-OH). Exact Mass (APCI): m/z = 270.1531

(calcd. 270.1498 for C₁₇H₂₀NO₂[MH]⁺).

7-(Benzyloxy)-3-(N-tosyl)-2,3,4,5-tetrahydro-1H-3-

benzazepin-1-ol (9) and 3-(N-tosyl)-2,3,4,5-tetrahydro-1H-3-

benzazepine-1,9-diol (10)

NaBH₄(153 mg, 3.94 mmol, 6 eq) was added to a suspension of

7/8 (277 mg, 1 eq) in abs. CH₃OH (20 mL) and the mixture was

stirred 16 h at room temperature. Water (20 mL) and CHCl₃(30

mL) were added and the layers were separated. The aqueous

layer was extracted with CHCl₃(3 x 20 mL) and the combined

organic layers were washed with H₂O (3 x 30 mL) and dried over

Na₂SO₄. The solvent was removed in vacuo and the residue was

purified by flash column chromatography (d = 2 cm, h = 15 cm, V

= 10 mL, cyclohexane:ethyl acetate = 2:1) yielding the separated

compounds 9 and 10 in a 3:1 ratio.

9 (R_f= 0.37 (cyclohexane:ethyl acetate = 2:1)): Colorless solid,

mp 121 °C, yield 133 mg (48 %). C₂₄H₂₅NO₄S (423.5). HPLC:

96.4 %, t_R= 22.85 min. ¹H NMR (600 MHz, CDCl₃): δ [ppm] = 2.40

(s, 3H, Ph-CH₃), 2.83 (dd, J = 15.3/7.5 Hz, 1H, 5-H), 3.08 (dd, J =

12.9/9.7 Hz, 1H, 4-H), 3.23 (d, J = 13.2 Hz, 1H, 2-H), 3.25 - 3.32

(m, 1H, 5-H), 3.62 (dd, J = 12.9/7.6 Hz, 1H, 4-H), 3.70 (dd, J =

13.2/7.1 Hz, 1H, 2-H), 4.83 (d, J = 6.8 Hz, 1H, 1-H), 5.02 (s, 2H,

O-CH₂-Ph), 6.70 - 6.77 (m, 2H, 6-H, 8-H), 7.21 (d, J = 8.4 Hz, 1H,

9-H), 7.28 (d, J = 8.1 Hz, 2H, 3-H_tosyland 5-H_tosyl), 7.30 - 7.40 (m,

5H, benzyl), 7.66 (d, J = 8.1 Hz, 2H, 2-H_tosyland 6-H_tosyl). A signal

for the OH proton is not seen in the spectrum. ¹³C NMR (151 MHz,

CDCl₃): δ [ppm] = 21.6 (1C, Ph-CH₃), 36.8 (1C, C-5), 48.6 (1C, C-

4), 53.8 (1C, C-2), 70.1 (1C, O-CH₂-Ph), 72.9 (1C, C-1), 111.9

(1C, C-8), 117.6 (1C, C-6), 127.3 (2C, C-2_tosyland C-6_tosyl), 127.5,

128.1, 128.7 (5C, benzyl), 129.4 (1C, C-9), 129.9 (2C, C-3_tosyland

C-5_tosyl), 134.0 (1C, C-9a), 135.5 (1C, C-1_tosyl), 136.9 (1C, C-

1_benzyl), 140.1 (1C, C-5a), 143.7 (1C, C-4_tosyl), 158.5 (1C, C-7). IR:

푣̃ [cm^-1]: = 3341 (O-H), 3063, 3032 (=C-H), 2920, 2862 (C-H_aliph.),

1613, 1578, 1493 (C=C_arom.), 1331, 1153 (SO₂), 1096 (C-OH).

Exact Mass (APCI): m/z = 422.1421 (calcd. 422.1432 for

C₂₄H₂₄NO₄S [MH]^-).

7-(Benzyloxy)-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-

benzazepin-1-ol (12)^[17]

11 (150 mg, 0.56 mmol, 1 eq) was dissolved in CH₃CN (20 mL).

After addition of 1-chloro-4-phenylbutane (0.14 mL, 0.85 mmol,

1.5 eq), K₂CO₃(619 mg, 4.48 mmol, 8 eq) and TBAI (207 mg, 0.56

mmol, 1 eq), the suspension was heated to reflux for 72 h. After

cooling to room temperature, K₂CO₃was filtered off and the

solvent was removed under reduced pressure. The residue was

purified by flash column chromatography (d = 2 cm, h = 15 cm, V

= 10 mL, cyclohexane:ethyl acetate = 2:1 + 1 % N,N-

dimethylethanamine) to yield 12. Colorless oil, yield 207 mg

(92 %). C₂₇H₃₁NO₂(401.6). R_f= 0.44 (cyclohexane:ethyl acetate

= 2:1 + 1 % N,N-dimethylethanamine). HPLC: 97.6 %, t_R= 20.92

1

min. H NMR (600 MHz, CDCl₃): δ [ppm] = 1.57 - 1.70 (m, 4H,

10 (R_f= 0.19 (cyclohexane:ethyl acetate = 1:1)): Colorless solid,

mp 76 °C, yield 37.5 mg (17 %). C₁₇H₁₉NO₄S (333.40). HPLC:

94.4 %, t_R= 18.68 min. ¹H NMR (600 MHz, CDCl₃): δ [ppm] = 2.41

(s, 3H, Ph-CH₃), 2.93 (dd, J = 7.3/4.0 Hz, 2H, 5-H₂), 3.12 (dt, J =

12.4/5.9 Hz, 1H, 4-H), 3.38 (dd, J = 13.9/6.9 Hz, 1H, 2-H), 3.60

(dt, J = 12.1/4.7 Hz, 1H, 4-H), 3.77 - 3.81 (m, 1H, 2-H), 5.36 (dd,

J = 6.8/2.2 Hz, 1H, 1-H), 6.59 (d, J = 7.5 Hz, 1H, 6-H), 6.75 (dd, J

= 8.1/1.2 Hz, 1H, 8-H), 7.04 (t, J = 7.8 Hz, 1H, 7-H), 7.30 (d, J =

8.0 Hz, 2H, 3-H_tosyland 5-H_tosyl), 7.66 (d, J = 8.1 Hz, 2H, 2-H_tosyl

and 6-H_tosyl), 8.49 (br s, 2H, OH_arom.and benzylic OH). ¹³C NMR

(151 MHz, CDCl₃): δ [ppm] =21.7 (1C, CH₃), 35.2 (1C, C-5), 48.0

(1C, C-4), 53.0 (1C, C-2), 72.3 (C-1), 116.7 (1C, C-8), 122.0 (1C,

C-6), 124.6 (1C, C-9a), 127.3 (2C, C-2_tosyland C-6_tosyl), 129.1 (1C,

C-7), 130.0 (2C, C-3_tosyland C-5_tosyl), 134.9 (1C, C-1_tosyl), 137.9

(1C, C-5a), 144.0 (1C, C-4_tosyl), 156.3 (1C, C-9). IR: 푣̃ [cm^-1] =

3422 (O-H), 2978, 2920, 2855 (C-H_aliph.), 1589 (C=C_arom.), 1466

(CH₂deform.), 1327, 1153 (SO₂), 1096 (C-OH). Exact Mass (ESI):

m/z = 356.0928 (calcd. 356.0927 for C₁₇H₁₉NO₄SNa [M+Na]⁺).

NCH₂CH₂CH₂CH₂Ph), 2.45 - 2.54 (m, 1H, 4-H), 2.57 - 2.74 (m,

6H, NCH₂CH₂CH₂CH₂Ph, 2-H and 5-H), 3.02 (t, J = 8.9 Hz, 1H, 4-

H), 3.18 (d, J = 10.5 Hz, 1H, 2-H), 3.24 - 3.32 (m, 1H, 5-H), 4.63

- 4.70 (m, 1H, 1-H), 5.03 (s, 2H, O-CH₂-Ph), 6.71 - 6.76 (m, 2H,

6-H and 8-H), 7.12 (d, J = 7.9 Hz, 1H, 9H), 7.16 - 7.43 (m, 10H,

phenyl and benzyl). A signal for the OH proton is not seen in the

spectrum. ¹³C NMR (151 MHz, CDCl₃): δ [ppm] = 26.5 (1C,

NCH₂CH₂CH₂CH₂Ph), 29.2 (1C, NCH₂CH₂CH₂CH₂Ph), 35.9 (1C,

NCH₂CH₂CH₂CH₂Ph), 36.6 (1C, C-5), 56.1 (1C, C-4), 59.7 (1C,

NCH₂CH₂CH₂CH₂Ph), 60.8 (1C, C-2), 70.1 (1C, O-CH₂-Ph), 72.2

(1C, C-1), 111.4 (1C, C-8), 117.6 (1C, C-6), 125.9, 127.6, 128.49,

128.52, 128.7 (10C, arom.), 129.7 (1C, C-9), 135.7 (1C, C-9a),

137.2 (1C, C-1_benzyl), 141.1 (1C, C-5a), 142.3 (1C, C-1_phenyl), 158.3

(1C, C-7). IR: 푣̃ [cm^-1] = 3348 (-OH), 3059, 3024 (=C-H), 2931,

2858, 2820 (C-H_aliph.), 1678, 1605, 1582, 1497 (C=C_arom.), 1242

(C-O). Exact Mass (APCI): m/z = 402.2405 (calcd. 402.2428 for

C₂₇H₃₂NO₂[MH]⁺).

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7-(Benzyloxy)-1-fluoro-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-

1H-3-benzazepine (13)

9-(2-Fluoroethoxy)-3-tosyl-2,3,4,5-tetrahydro-1H-3-

benzazepin-1-ol (15)

DAST (0.08 mL, 0.61 mmol, 4.2 eq) was dissolved under N₂

atmosphere in abs. CH₂Cl₂(10 mL) at -78 °C. A solution of 12

(58.1 mg, 0.14 mmol, 1 eq) in CH₂Cl₂(1 mL) was added. After the

solution had been stirred for 1 h at -78 °C, the mixture was stirred

for 16 h at rt. 2 M NaOH (3 mL) was added and the mixture was

stirred for 30 min. H₂O (30 mL) was added and the layers were

separated. The aqueous layer was extracted with CH₂Cl₂(4 x 20

mL) and the combined organic layers were dried over Na₂SO₄.

The solvent was removed in vacuo and the crude product was

purified by flash column chromatography chromatography (d = 2

cm, h = 15 cm, V = 10 mL, cyclohexane:ethyl acetate = 9:1 + 1 %

N,N-dimethylethanamine) to afford 13. Yellow oil, yield 29.9 mg

(53 %). C₂₇H₃₀FNO (403.5). ¹H NMR (400 MHz, CDCl₃): δ [ppm]

= 1.55 – 1.72 (m, 4H, NCH₂CH₂CH₂CH₂Ph), 2.43 – 3.17 (m, 10H,

NCH₂CH₂CH₂CH₂Ph, 2-CH₂, 4-CH₂and 5-CH₂), 5.06 (s, 2H, O-

CH₂-Ph), 5.60 (d, J = 44.6 Hz, 1H, 1-H), 6.74 – 6.86 (m, 2H, 6-H

and 8-H), 7.16 – 7.45 (m, 11H, 9-H, phenyl and benzyl). ¹⁹F NMR

(376 MHz, CDCl₃): δ [ppm] = -180.8.

A mixture of 10 (143 mg, 0.43 mol, 1 eq), K₂CO₃(475 mg, 3.44

mmol, 8 eq) and (2-fluoroethyl) tosylate (112 mg, 0.52 mmol, 1.2

eq) in CH₃CN (20 mL), was heated to reflux for 72 h. K₂CO₃was

filtered off and the solvent was removed under reduced pressure.

After purification by flash column chromatography (d = 2 cm, h =

15 cm, V = 10 mL, cyclohexane:ethyl acetate = 2:1), 15 was

obtained. Colorless foam, mp 92 °C, yield 127 mg (78 %).

C₁₉H₂₂FNO₄S (379.4). R_f= 0.42 (cyclohexane:ethyl acetate = 1:1).

HPLC: 97.9 %, t_R= 20.46 min. ¹H NMR (400 MHz, CDCl₃): δ [ppm]

= 2.40 (s, 3H, Ph-CH₃), 2.76 – 2.83 (m, 1H, 5-H), 2.87 – 2.96 (m,

1H, 4-H), 3.09 (dd, J = 13.7/2.3 Hz, 1H, 2-H), 3.45 (ddd, J =

15.3/11.8/3.1 Hz, 1H, 5-H), 3.92 (ddd, J = 11.2/4.0/1.9 Hz, 1H, 4-

H), 4.05 (dd, J = 13.8/7.1 Hz, 1H, 2-H), 4.20 (dt, J = 27.8/4.0 Hz,

2H, OCH₂CH₂F), 4.74 (dt, J = 47.4/4.3 Hz, 2H, OCH₂CH₂F), 5.65

(dd, J = 7.2/2.4 Hz, 1H, 1-H), 6.71 (d, J = 7.5 Hz, 1H, 6-H), 6.75

(d, J = 8.3 Hz, 1H, 8-H), 7.12 (t, J = 7.9 Hz, 1H, 7-H), 7.28 (d, J =

8.0 Hz, 2H, 3-H_tosyland 5-H_tosyl), 7.67 (d, J = 8.0 Hz, 2H, 2-H_tosyl

and 6-H_tosyl). A signal for the OH proton is not seen in the spectrum.

¹³C NMR (101 MHz, CDCl₃): δ [ppm] = 21.6 (1C, CH₃), 35.4 (1C,

C-5), 48.2 (1C, C-4), 51.1 (1C, C-2), 64.3 (1C, C-1), 68.4 (d, J =

20.3 Hz, 1C, OCH₂CH₂F), 82.0 (d, J = 170.8 Hz, 1C, OCH₂CH₂F),

111.5 (1C, C-8), 123.9 (1C, C-6), 127.3 (2C, C-2_tosyland C-6_tosyl),

129.2 (1C, C-7), 129.5 (1C, C-9a), 129.8 (2C, C-3_tosyland C-5_tosyl),

135.6 (1C, C-1_tosyl), 140.8 (1C, C-5a), 143.5 (1C, C-4_tosyl), 156.5

(1C, C-9). IR: 푣̃ [cm^-1] = 3503 (O-H), 2924, 2878 (C-H_aliph.), 1585

(C=C_arom.), 1450 (CH₂deform.), 1331 (SO₂), 1258 (C-O), 1153

(SO₂). Exact Mass (ESI): m/z = 402.1138 (calcd. 402.1146 for

C₁₉H₂₂FNO₄SNa [M+Na]⁺).

7-(2-Fluoroethoxy)-2,3,4,5-tetrahydro-3-(4-phenylbutyl)-1H-3-

benzazepine-1-ol (14)

To a solution of 2 (53.5 mg, 0.17 mmol, 1 eq.) in abs. CH₃CN (25

mL), K₂CO₃(23.5 mg, 0.17 mmol, 1 eq) was added and the

mixture was heated to reflux for 1 h. After addition of (2-

fluoroethyl) tosylate (56.2 mg, 0.26 mmol, 1.5 eq) in CH₃CN

(5 mL) the mixture was heated to reflux for 16 h. K₂CO₃was

filtered off and the solvent was removed in vacuo. The residue

was purified by flash column chromatography on silica gel (d = 1

cm, h = 15 cm, V = 5 mL cyclohexane:ethyl acetate = 3:1 + 1 %

N,N dimethylethanamine  cycohexane:ethyl acetate = 1:2 + 1 %

N,N-dimethylethanamine) to afford 14. Colorless solid, mp 82 °C,

yield 18.7 mg (0.05 mmol, 31 %). C₂₂H₂₈FNO₂(357.5). R_f= 0.25

(cyclohexane:ethyl acetate = 1:1 + 1 % N,N-dimethylethanamine).

HPLC: 97.1 %, t_R= 17.68 min. ¹H NMR (400 MHz, CDCl₃): δ [ppm]

= 1.53 - 1.62 (m, 2H, NCH₂CH₂CH₂CH₂Ph), 1.63 - 1.71 (m, 2H,

NCH₂CH₂CH₂CH₂Ph), 2.40 (t, J = 12.0 Hz, 1H, 4-H), 2.51 (d, J =

12.1 Hz, 1H, 2-H), 2.58 - 2.67 (m, 5H, NCH₂CH₂CH₂CH₂Ph and

5-H), 3.00 (dd, J = 12.5/6.0 Hz, 1H, 4-H), 3.14 - 3.21 (m, 1H, 2-

H), 3.22 - 3.32 (m, 1H, 5-H), 4.18 (dt, J = 27.9/4.1 Hz, 2H,

OCH₂CH₂F), 4.58 (d, J = 6.7 Hz, 1H, 1-H), 4.73 (dt, J = 47.5/4.0

Hz, 2H, OCH₂CH₂F), 6.64 - 6.70 (m, 2H, 6-H, 8-H), 7.10 (d, J =

8.0 Hz, 1H, 9-H), 7.16 - 7.22 (m, 3H, 2-H_phenyl, 4-H_phenyland 6-

H_phenyl), 7.26 - 7.32 (m, 2H, 3-H_phenyland 5-H_phenyl). A signal for the

OH proton is not seen in the spectrum. ¹³C NMR (101 MHz,

CDCl₃): δ [ppm] = 26.8 (1C, NCH₂CH₂CH₂CH₂Ph), 29.3 (1C,

9-(2-Fluoroethoxy)-2,3,4,5-tetrahydro-1H-3-benzazepin-1-ol

(16)

15 (105 mg, 0.28 mmol, 1 eq) and Mg (150 mg, 6.16 mmol, 22

eq) were suspended in abs. CH₃OH (25 mL) and the mixture was

heated to reflux for 16 h. Conc. H₂SO₄(1.0 mL) was added until a

reaction of Mg was no longer observed. The pH was adjusted with

2 M NaOH to pH 9 and the suspension was filtered. Brine (30 mL)

and CH₂Cl₂(50 mL) were added to the filtrate and the layers were

separated. The aqueous layer was extracted with CH₂Cl₂(3 x 20

mL) and the combined organic layers were dried over Na₂SO₄.

The solvent was removed in vacuo and the crude product was

purified by flash column chromatography (d = 2 cm, h = 15 cm, V

= 10 mL, CH₂Cl₂:CH₃OH = 95:5 + 2 % NH₃ CH₂Cl₂:CH₃OH =

9:1 + 2 % NH₃) to obtain 16. Colorless solid, mp 132 °C, yield 37.2

mg (60 %). C₁₂H₁₆FNO₂(225.3). R_f= 0.23 (CH₂Cl₂:CH₃OH = 9:1

1

+ 2 % NH₃). HPLC: 92.1 %, t_R= 7.77 min. H NMR (400 MHz,

NCH₂CH₂CH₂CH₂Ph),

35.9

56.2

(1C,

C-5),

C-4),

37.0

59.8

(1C,

CDCl₃): δ [ppm] = 2.68 – 2.86 (m, 3H, 2-H, 4-H and 5-H), 3.31 –

3.48 (m, 3H, 2-H, 4-H and 5-H), 4.10 – 4.30 (m, 2H, OCH₂CH₂F),

4.63 – 4.86 (m, 2H, OCH₂CH₂F), 5.59 (d, J = 6.2 Hz, 1H, 1-H),

6.72 – 6.78 (m, 2H, 6-H and 8-H), 7.10 (t, J = 7.9 Hz, 1H, 7-H).

Signals for the OH and NH protons are not seen in the spectrum.

¹³C NMR (101 MHz, CDCl₃): δ [ppm] = 38.2 (1C, C-5), 49.1 (1C,

C-4), 52.5 (1C, C-2), 63.4 (1C, C-1), 68.6 (d, J = 20.5 Hz, 1C,

OCH₂CH₂F), 82.0 (d, J = 170.8 Hz, 1C, OCH₂CH₂F), 111.6 (1C,

C-8), 124.2 (1C, C-6), 128.6 (1C, C-7), 131.9 (1C, C-9a), 142.8

(1C, C-5a), 156.1 (1C, C-9). IR: 푣̃ [cm^-1] = 3298 (N-H), 3063, 3024

(=C-H), 2920 (O-H), 2801 (C-H_aliph.), 1585, 1447 (C=C_arom.), 1254,

1142 (C-O), 1053 (C-OH). Exact Mass (ESI): m/z = 226.1246

(calcd. 226.1238 for C₁₂H₁₇FNO₂[MH]⁺).

NCH₂CH₂CH₂CH₂Ph), 60.9 (1C, C-2), 67.3 (d, J = 20.6 Hz, 1C,

OCH₂CH₂F), 72.5 (1C, C-1), 82.0 (d, J = 170.7 Hz, 1C,

OCH₂CH₂F), 111.0 (1C, C-8), 117.5 (1C, C-6), 125.9 (1C, C-

4_phenyl), 128.48, 128.53 (4C, C-2_phenyl, C-3_phenyl, C-5_phenyland C-

6_phenyl), 129.9 (1C, C-9), 136.3 (1C, C-9a), 141.5 (1C, C-5a), 142.4

(1C, C-1_phenyl), 157.9 (1C, C-7). IR: 푣̃ [cm^-1] = 3071, 3024 (=C-H),

2932, 2859 (C-H_aliph.), 1609, 1498 (C=C_arom.), 1454 (CH₂deform.),

1246, 1049 (C-O). Exact Mass (APCI): m/z = 358.2175 (calcd.

358.2177 for C₂₂H₂₉FNO₂[MH]⁺).

8

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FULL PAPER

9-(2-Fluoroethoxy)-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-

benzazepin-1-ol (17)

6-H an 8-H), 7.16 - 7.20 (m, 3H, 2-H_phenyl, 4-H_phenyland 6-H_phenyl),

7.25 - 7.30 (m, 3H, 3-H_phenyl, 5-H_phenyland 9-H), 7.31 - 7.45 (m, 5H,

benzyl). ¹³C NMR (151 MHz, CDCl₃): δ [ppm] = 26.3, 29.5 (2C,

NCH₂CH₂CH₂CH₂Ph and NCH₂CH₂CH₂CH₂Ph), 35.8 (1C, C-5),

36.0 (1C, NCH₂CH₂CH₂CH₂Ph), 55.0 (1C, C-4), 59.3 (1C,

NCH₂CH₂CH₂CH₂Ph), 59.8 (1C, C-2), 68.6 (d, J = 19.8 Hz, 1C,

1-Chloro-4-phenylbutane (0.03 mL, 0.21 mmol, 1.5 eq), TBAI

(51.7 mg, 0.14 mol, 1 eq) and K₂CO₃(155 mg, 1.12 mmol, 8 eq)

were added to a solution of 16 (30.5 mg, 0.14 mmol, 1 eq) in

CH₃CN (20 mL). After the suspension was heated to reflux for

72 h, K₂CO₃was filtered off and the solvent was removed in vacuo. OCH₂CH₂F), 70.1 (1C, O-CH₂-Ph), 80.9 (1C, C-1), 83.3 (d, J =

Purification by flash column chromatography (d = 2 cm, h = 15 cm,

V = 10 mL, cyclohexane:ethyl acetate = 3:1 + 1 % N,N-

Dimethylethanamine) led to 17. Colorless solid, mp 68 °C, yield

22.4 mg (46 %). C₂₂H₂₈FNO₂(357.5). R_f= 0.44

(cyclohexane:ethyl acetate = 1:1 + 1 % N,N-dimethylethanamine).

HPLC: 96.7 %, t_R= 18.80 min. ¹H NMR (400 MHz, DMSO-d6): δ

[ppm] = 1.43 – 1.53 (m, 2H, NCH₂CH₂CH₂CH₂Ph), 1.55 – 1.64 (m,

2H, NCH₂CH₂CH₂CH₂Ph), 2.20 (t, J = 11.7 Hz, 1H, 4-H), 2.29 (dd,

169.2 Hz, 1C, OCH₂CH₂F), 111.4 (1C, C-8), 116.7 (1C, C-6),

125.8 (1C, C-4_phenyl), 127.5 (1C, C-9), 127.6 (2C, C-2_benzyland C-

6_benzyl), 128.1 (1C, C-4_benzyl), 128.4 (2C, C-3_phenyland C-5_phenyl),

128.5 (2C, C-2_phenyland C-6_phenyl), 128.7 (2C, C-3_benzyland C-

5_benzyl), 133.4 (1C, C-9a), 137.2 (1C, C-1_benzyl), 141.7 (1C, C-5a),

142.6 (1C, C-1_phenyl), 158.2 (1C, C-7). IR: 푣̃ [cm^-1] = 3024 (=C-H),

2928, 2866, 2800 (C-H_aliph.), 1612, 1582, 1493 (C=C_arom.), 1454

(CH₂deform.), 1254 (C-O), 1115 (C-OH), 1034 (C-O). Exact Mass

(APCI): m/z = 448.2646 (calcd. 448.2646 for C₂₉H₃₅FNO₂[MH]⁺).

J = 12.6, 1.7 Hz, 1H, 2-H), 2.48

–

2.55 (m, 2H,

NCH₂CH₂CH₂CH₂Ph), 2.57 2.64 (m, 3H, 5-H and

–

NCH₂CH₂CH₂CH₂Ph), 2.95 – 3.01 (m, 1H, 4-H), 3.12 (dd, J =

12.6/6.6 Hz, 1H, 2-H), 3.28 – 3.37 (m, 1H, 5-H), 4.14 – 4.25 (m,

2H, OCH₂CH₂F), 4.57 (d, J = 6.4 Hz, 1H, OH), 4.67 – 4.82 (m, 2H,

OCH₂CH₂F), 5.35 (td, J = 6.5/1.6 Hz, 1H, 1-H), 6.72 (d, J = 7.4 Hz,

1H, 6-H), 6.86 (d, J = 8.2 Hz, 1H, 8-H), 7.10 (t, J = 7.8 Hz, 1H, 7-

H), 7.16 – 7.22 (m, 3H, 2-H_phenyl, 4-H_phenyl, 6-H_phenyl), 7.26 – 7.31

(m, 2H, 3-H_phenyland 5-H_phenyl). ¹³C NMR (101 MHz, DMSO-d6): δ

1-(2-Fluoroethoxy)-7-methoxy-3-(4-phenylbutyl)-2,3,4,5-

tetrahydro-1H-3-benzazepine (18b)

19 (113 mg, 0.35 mmol, 1 eq) was dissolved under N₂atmosphere

in DMF (5 mL) and NaH (42.0 mg, 1.05 mmol, 60 %) was added.

After 30 min, (2-fluoroethyl) tosylate (68, 113 mg, 0.52 mmol, 1.5

eq) in DMF (2 mL) was added and the solution stirred for 24 h at

room temperature. Water (10 mL), CH₂Cl₂(30 mL) and brine (20

mL) were added and the layers were separated. The aqueous

layer was extracted with CH₂Cl₂(4 x 10 mL) and the combined

organic layers were washed with brine (3 x 10 mL) and over

Na₂SO₄. The solvent was removed under reduced pressure and

the residue was purified by flash column chromatography (d = 2

cm, h = 15 cm, V = 10 mL, cyclohexane:ethyl acetate = 2:1 + 1 %

N,N-dimethylethanamine) to obtain 18b. Yellow oil, yield 43.8 mg

(34 %). C₂₃H₃₀FNO₂(371.5). R_f= 0.51 (cyclohexane:ethyl acetate

= 1:1 + 1 % N,N-dimethylethanamine). HPLC: 97.0 %, t_R= 20.24

[ppm]

=

26.1 (1C, NCH₂CH₂CH₂CH₂Ph), 28.8 (1C,

NCH₂CH₂CH₂CH₂Ph), 35.0 (1C, NCH₂CH₂CH₂CH₂Ph), 35.2 (1C,

C-5), 55.0 (1C, C-4), 58.4 (1C, NCH₂CH₂CH₂CH₂Ph), 59.4 (1C,

C-2), 62.4 (1C, C-1), 68.3 (d, J = 19.0 Hz, 1C, OCH₂CH₂F), 82.3

(d, J = 166.6 Hz, 1C, OCH₂CH₂F), 111.3 (1C, C-8), 122.8 (1C, C-

6), 125.6 (1C, C-4_phenyl), 128.0 (1C, C-7), 128.19 (2C, C-3_phenyland

C-5_phenyl), 128.24 (2C, C-2_phenyl, and C-6_phenyl) , 131.7 (1C, C-9a),

142.2 (1C, C-1_phenyl), 142.6 (1C, C-5a), 155.4 (1C, C-9). IR: 푣̃ [cm^-

¹] = 3433 (O-H), 3086, 3024 (=C-H), 2928, 2855, 2808 (C-H_aliph.),

1585, 1467, 1447 (C=C_arom.), 1258 (C-O), 1096 (C-OH), 1045 (C-

O). Exact Mass (ESI): m/z = 358.2207 (calcd. 358.2177 for

C₂₂H₂₉FNO₂[MH]⁺).

1

min. H NMR (400 MHz, CDCl₃): δ [ppm] = 1.53 - 1.68 (m, 4H,

NCH₂CH₂CH₂CH₂Ph), 2.48 - 2.93 (m, 9H, NCH₂CH₂CH₂CH₂Ph,

2-H, 4-H and 5-H), 3.02 (dd, J = 14.8/8.2 Hz, 1H, 5-H), 3.67 - 3.79

(m, 2H, OCH₂CH₂F), 3.79 (s, 3H, OCH₃), 4.49 - 4.52 (m, 1H, 1-H),

(dt, J = 47.7/4.3 Hz, 2H, OCH₂CH₂F), 6.66 - 6.75 (m, 2H, 6-H, 8-

H), 7.15 - 7.20 (m, 3H, 2-H_phenyl, 4-H_phenyland 6-H_phenyl), 7.25 - 7.30

(m, 3H, 3-H_phenyl, 5-H_phenyland 9-H). ¹³C NMR (101 MHz, CDCl₃) δ

7-(Benzyloxy)-1-(2-fluoroethoxy)-3-(4-phenylbutyl)-2,3,4,5-

tetrahydro-1H-3-benzazepine (18a)

12 (233 mg, 0.58 mmol, 1 eq) was dissolved under N₂atmosphere

in DMF (20 mL) and NaH (34.8 mg, 0.87 mmol, 60 %) was added.

After 45 min, (2-fluoroethyl) tosylate (139 mg, 0.64 mmol, 1.1 eq)

[ppm]

NCH₂CH₂CH₂CH₂Ph),

=

26.2, 29.5 (2C, NCH₂CH₂CH₂CH₂Ph and

35.7 (1C, C-5), 36.0 (1C,

in DMF (5 mL) was added and the solution stirred for 24 h at 70 °C. NCH₂CH₂CH₂CH₂Ph), 55.0 (1C, C-4), 55.4 (1C, OCH₃), 59.3 (1C,

An additional amount of (2-fluoroethyl) tosylate (63.0 mg,

0.29 mmol, 0.5 eq) in DMF (5 mL) and NaH (34.8 mg, 0.87 mmol,

60 %) was added and the solution was stirred for 16 h at 70 °C.

At room temperature, water (10 mL), CH₂Cl₂(30 mL) and brine

(20 mL) were added and the layers were separated. The aqueous

layer was extracted with CH₂Cl₂(4 x 10 mL) and the combined

organic layers were washed with brine (3 x 10 mL). After drying

over Na₂SO₄the solvent was removed in vacuo. The crude

product was purified by flash column chromatography (d = 2 cm,

h = 15 cm, V = 10 mL, cyclohexane:ethyl acetate = 2:1 + 1 % N,N

dimethylethanamine) to yield 18a. Colorless solid, mp 73 °C, yield

32.9 mg (13 %). C₂₉H₃₄FNO₂(447.6). R_f= 0.55

(cyclohexane:ethyl acetate = 1:1 + 1 % N,N-dimethylethanamine).

HPLC: 96.9 %, t_R= 22.61 min. ¹H NMR (600 MHz, CDCl₃): δ [ppm]

= 1.54 - 1.67 (m, 4H, NCH₂CH₂CH₂CH₂Ph), 2.49 - 2.94 (m, 9H,

NCH₂CH₂CH₂CH₂Ph), 59.8 (1C, C-2), 68.0 (d, J = 19.9 Hz, 1C,

OCH₂CH₂F), 80.9 (1C, C-1), 83.3 (d, J = 169.2 Hz, 1C,

OCH₂CH₂F), 110.5 (1C, C-8), 115.8 (1C, C-6), 125.8 (1C, C-

4_phenyl), 127.6 (1C, C-9), 128.4 (2C, C-3_phenyland C-5_phenyl), 128.5

(2C, C-2_phenyland C-6_phenyl), 133.1 (1C, C-9a), 141.7 (1C, C-5a),

142.6 (1C, C-1_phenyl), 158.9 (1C, C-7). IR: 푣̃ [cm^-1] = 2936, 2859,

2808 (C-H_aliph.), 1607, 1582, 1497 (C=C_arom.), 1454 (CH₂deform.),

1254, 1115, 1034 (C-O). Exact Mass (APCI): m/z = 372.2350

(calcd. 372.2333 for C₂₃H₃₁FNO₂[MH]⁺).

1-(2-Fluoroethoxy)-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-

benzazepin-7-ol (18c)

18a (52.4 mg, 0.12 mmol, 1 eq) was dissolved in abs. CH₃OH (5

mL) and added to a suspension of Pd/C (10.5 mg, 10 %) in

CH₃OH (15 mL). After the mixture had been stirred for 30 min

NCH₂CH₂CH₂CH₂Ph, 2-H, 4-H and 5-H), 3.01 (dd, J = 14.6/8.1 Hz, under H₂atmosphere (1 bar) at room temperature, the catalyst

1H, 5-H), 3.67 - 3.80 (m, 2H, OCH₂CH₂F), 4.48 - 4.65 (m, 3H,

OCH₂CH₂F and 1-H), 5.05 (s, 2H, O-CH₂-Ph), 6.75 - 6.82 (m, 2H,

was removed by filtration over Celite^®. The solvent was removed

in vacuo and the residue was purified by flash column

9

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FULL PAPER

chromatography (d = 2 cm, h = 15 cm, V = 10 mL,

cyclohexane:ethyl acetate = 1:2 + 1 % N,N-dimethylethanamine)

to afford 18c. Yellow oil, yield 33.0 mg (79 %). C₂₂H₂₈FNO₂

(357.5). R_f= 0.23 (cyclohexane:ethyl acetate = 1:1 + 1 % N,N-

For the binding assay, the cell pellet was resuspended in PBS

solution and the number of cells was determined using a Scepter^®

cell counter (MERCK Millipore, Darmstadt, Germany).

Subsequently, the cells were lysed by sonication (4 °C, 6 x 10 s

cycles with breaks of 10 s). The resulting cell fragments were

centrifuged with a high performance cool centrifuge (23,500 x g,

4 °C). The supernatant was discarded and the pellet was

resuspended in a defined volume of PBS yielding cell fragments

of approximately 500,000 cells/mL. The suspension of membrane

homogenates was sonicated again (4 °C, 2 x 10 s cycles with a

break of 10 s) and stored at -80 °C.

1

dimethylethanamine). HPLC: 90.6 %, t_R= 18.48 min. H NMR

(600 MHz, CDCl₃):

δ

[ppm]

=

1.55

– 1.65 (m, 4H,

NCH₂CH₂CH₂CH₂Ph), 2.52 – 2.64 (m, 5H, NCH₂CH₂CH₂CH₂Ph

and 4-H), 2.70 – 2.84 (m, 3H, 2-H, 4-H and 5-H), 2.87 – 2.94 (m,

1H, 2-H), 2.96 – 3.02 (m, 1H, 5-H), 3.63 – 3.76 (m, 2H,

OCH₂CH₂F), 4.49 – 4.62 (m, 3H, OCH₂CH₂F and 1-H), 6.59 –

6.68 (m, 2H, 6-H an 8-H), 7.14 – 7.29 (m, 6H, 9-H and phenyl). A

signal for the OH proton is not seen in the spectrum. ¹³C NMR

(151 MHz, CDCl₃) δ [ppm] = 26.0, 29.5 (2C, NCH₂CH₂CH₂CH₂Ph),

35.2 (1C, C-5), 35.9 (1C, NCH₂CH₂CH₂CH₂Ph), 54.9 (1C, C-4),

59.3 (1C, NCH₂CH₂CH₂CH₂Ph), 59.6 (1C, C-2), 68.6 (d, J = 19.9

Hz, 1C, OCH₂CH₂F), 80.6 (1C, C-1), 83.3 (d, J = 169.1 Hz, 1C,

OCH₂CH₂F), 112.7 (1C, C-8), 116.9 (1C, C-6), 125.9 (1C, C-

4_phenyl), 127.9 (1C, C-9), 128.4 (2C, C-3_phenyland C-5_phenyl), 128.5

(2C, C-2_phenyland C-6_phenyl), 132.5 (1C, C-9a), 141.7 (1C, C-5a),

142.5 (1C, C-1_phenyl), 155.4 (1C, C-7). IR: 푣̃ [cm^-1] = 3024 (=C-H),

2940, 2913, 2828 (C-H_aliph.), 1616, 1582, 1497 (C=C_arom.), 1466,

1454 (CH₂deform.), 1281 (C-OH), 1157, 1103, 1038 (C-O). Exact

Mass (ESI): m/z = 358.2182 (calcd. 358.2177 for C₂₂H₂₉FNO₂

[MH]⁺).

Preparation of membrane homogenates from pig brain

cortex

Fresh pig brain cortex was homogenized with the potter (500-

800 rpm, 10 up and down strokes) in 6 volumes of cold 0.32 M

sucrose. The suspension was centrifuged at 1,200 x g for 10 min

at 4 °C. The supernatant was separated and centrifuged at

31,000 x g for 20 min at 4 °C. The pellet was resuspended in 5-6

volumes of TRIS/EDTA buffer (5 mM TRIS/1 mM EDTA, pH 7.5)

and centrifuged again at 31,000 x g (20 min, 4 °C). The final pellet

was resuspended in 5-6 volumes of buffer and frozen (-80 °C) in

1.5 mL portions containing about 0.8 mg protein/mL.

Receptor binding studies

Materials

Preparation of membrane homogenates from guinea pig

brain

Guinea pig brains and rat livers were commercially available

(Harlan-Winkelmann, Borchen, Germany). Pig brains were a

donation of the local slaughterhouse (Coesfeld, Germany). The

recombinant L(tk-) cells stably expressing the GluN2B receptor

were obtained from Prof. Dr. Dieter Steinhilber (Frankfurt,

Germany). Homogenizers: Elvehjem Potter (B. Braun Biotech

International, Melsungen, Germany) and Soniprep^®150, MSE,

London, UK). Centrifuges: Cooling centrifuge model Rotina^®35R

(Hettich, Tuttlingen, Germany) and High-speed cooling centrifuge

model Sorvall^®RC-5C plus (Thermo Fisher Scientific,

Langenselbold, Germany). Multiplates: standard 96 well

multiplates (Diagonal, Muenster, Germany). Shaker: self-made

device with adjustable temperature and tumbling speed (scientific

workshop of the institute). Harvester: MicroBeta^®FilterMate 96

Harvester. Filter: Printed Filtermat Typ A and B. Scintillator:

Meltilex^®(Typ A or B) solid state scintillator. Scintillation analyzer:

MicroBeta^®Trilux (all Perkin Elmer LAS, Rodgau-Jügesheim,

Germany).

5 guinea pig brains were homogenized with the potter (500-

800 rpm, 10 up and down strokes) in 6 volumes of cold 0.32 M

sucrose. The suspension was centrifuged at 1,200 x g for 10 min

at 4 °C. The supernatant was separated and centrifuged at

23,500 x g for 20 min at 4 °C. The pellet was resuspended in 5-6

volumes of buffer (50 mM TRIS, pH 7.4) and centrifuged again at

23,500 x g (20 min, 4 °C). This procedure was repeated twice.

The final pellet was resuspended in 5-6 volumes of buffer and

frozen (-80 °C) in 1.5 mL portions containing about 1.5 mg

protein/mL.

Preparation of membrane homogenates from rat liver

Two rat livers were cut into small pieces and homogenized with

the potter (500-800 rpm, 10 up and down strokes) in 6 volumes of

cold 0.32 M sucrose. The suspension was centrifuged at

1,200 x g for 10 min at 4 °C. The supernatant was separated and

centrifuged at 31,000 x g for 20 min at 4 °C. The pellet was

resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 8.0) and

incubated at rt for 30 min. After the incubation, the suspension

was centrifuged again at 31,000 x g for 20 min at 4 °C. The final

pellet was resuspended in 5-6 volumes of buffer and stored

at -80 °C in 1.5 mL portions containing about 2 mg protein/mL.

Cell culture and preparation of membrane homogenates from

GluN2B cells

Mouse L(tk-) cells stably transfected with the dexamethasone-

inducible eukaryotic expression vectors pMSG GluN1a, pMSG

GluN2B (1:5 ratio) were grown in Modified Earl’s Medium (MEM)

containing 10 % of standardized FCS (Biochrom AG, Berlin,

Germany). The expression of the NMDA receptor at the cell

surface was induced after the cell density of the adherent growing

cells had reached approximately 90 % of confluency. For the

induction, the original growth medium was replaced by growth

medium containing 4 µM dexamethasone and 4 µM ketamine

(final concentration). After 24 h, the cells were rinsed with

phosphate buffered saline solution (PBS, Biochrom AG, Berlin,

Germany), harvested by mechanical detachment and pelleted

(10 min, 5,000 x g).

Protein determination

The protein concentration was determined by the method of

Bradford.^[39]modified by Stoscheck.^[40]The Bradford solution was

prepared by dissolving 5 mg of Coomassie Brilliant Blue G 250 in

2.5 mL of EtOH (95 %, v/v). 10 mL deionized H₂O and 5 mL

phosphoric acid (85 %, m/v) were added to this solution, the

mixture was stirred and filled to a total volume of 50 mL with

deionized water. The calibration was carried out using bovine

serum albumin as a standard in 9 concentrations (0.1, 0.2, 0.4,

0.6, 0.8, 1.0, 1.5, 2.0 and 4.0 mg /mL). In a 96 well standard

multiplate, 10 µL of the calibration solution or 10 µL of the

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membrane receptor preparation were mixed with 190 µL of the

Bradford solution, respectively. After 5 min, the UV absorption of

the protein-dye complex at  = 595 nm was measured with a plate

reader (Tecan Genios^®, Tecan, Crailsheim, Germany).

membrane preparation of guinea pig brain cortex (about 100 μg

of the protein) was incubated with various concentrations of test

compounds, 2 nM [³H]-(+)-pentazocine, and TRIS buffer (50 mM,

pH 7.4) at 37 °C. The non-specific binding was determined with

10 μM

unlabeled

(+)-pentazocine.

The

K_dvalue

of

General procedures for the binding assays

(+)-pentazocine is 2.9 nM.^[42]

The test compound solutions were prepared by dissolving

approximately 10 µmol (usually 2-4 mg) of test compound in

DMSO so that a 10 mM stock solution was obtained. To obtain

the required test solutions for the assay, the DMSO stock solution

was diluted with the respective assay buffer. The filtermats were

presoaked in 0.5 % aqueous polyethylenimine solution for 2 h at

rt before use. All binding experiments were carried out in

duplicates in the 96 well multiplates. The concentrations given are

the final concentration in the assay. Generally, the assays were

performed by addition of 50 µL of the respective assay buffer,

50 µL of test compound solution in various concentrations (10^-5,

10^-6, 10^-7, 10^-8, 10^-9and 10^-10mol/L), 50 µL of the corresponding

radioligand solution and 50 µL of the respective receptor

preparation into each well of the multiplate (total volume 200 µL).

The receptor preparation was always added last. During the

σ₂receptor

The assays were performed with the radioligand [³H]di-o-

tolylguanidine (specific activity 50 Ci/mmol; ARC, St. Louis, MO,

USA). The thawed rat liver membrane preparation (about 100 µg

protein) was incubated with various concentrations of the test

compound, 3 nM [³H]di-o-tolylguanidine and buffer containing (+)-

pentazocine (500 nM (+)-pentazocine in TRIS buffer (50 mM

TRIS, pH 8.0)) at rt. The non-specific binding was determined with

10 μM non-labeled di-o-tolylguanidine. The K_dvalue of di-o-

tolylguanidine is 17.9 nM.^[43]

Radiosynthesis

Cyclotron produced ¹⁸F-fluoride was trapped on a Waters Sep-

Pak Accell Plus QMA Carbonate Plus Light cartridge and eluted

with TBA-OH solution (1 mL, 46.7 mg/mL in MeOH). The eluate

was collected in a sealed Wheaton reactor (size: 5 mL) and was

azeotropically dried at 90 °C by addition of CH₃CN (3 × 1 mL)

under reduced pressure and a stream of nitrogen, followed by full

vacuum without nitrogen stream for 5 min at 90 °C. Ethylene di-

tosylate (5 mg, 13.5 µmol) was dissolved in anhydrous CH₃CN

(500 µL), added to the dried fluoride and reacted for 4 min at 90 °C.

After complete reaction, CH₃CN (600 µL) was added to the

reaction vial and the whole volume was transferred through a

Waters Sep-Pak Silica Plus Light cartridge (preconditioned with 5

mL Et₂O). The reaction vial was rinsed with CH₃CN (500 µL) and

transferred through the cartridge. The collected CH₃CN phase

was diluted with H₂O (1.5 mL) and injected into a semi-preparative

incubation, the multiplates were shaken at

a speed of

500-600 rpm at the specified temperature. Unless otherwise

noted, the assays were terminated after 120 min by rapid filtration

using the harvester. During the filtration, each well was washed

five times with 300 µL of water. Subsequently, the filtermats were

dried at 95 °C. The solid scintillator was melted on the dried

filtermats at a temperature of 95 °C for 5 min. After solidifying of

the scintillator at rt, the trapped radioactivity in the filtermats was

measured with the scintillation analyzer. Each position on the

filtermat corresponding to one well of the multiplate was

measured for 5 min with the [³H]-counting protocol. The overall

counting efficiency was 20 %. The IC₅₀values were calculated

with the program GraphPad Prism^®3.0 (GraphPad Software, San

Diego, CA, USA) by non-linear regression analysis. Subsequently, HPLC system, equipped with a Smartline Pump 1000, Smartline

the IC₅₀values were transformed into K_ivalues using the equation

of Cheng and Prusoff.^[41]The K_ivalues are given as mean value

± SEM from three independent experiments.

Manager 5000, Smartline UV detector 2500 (Knauer), 5 mL-loop

and a GabiStar radio detector (Raytest). A semi-preparative

reversed phase column was used (Waters Sunfire C18 10×150

mm, 5 µm) under isocratic conditions (50% 0.01 M H₃PO₄in

CH₃CN at 5 mL/min). The desired radio peak eluted between 5.78

and 6.15 min.

GluN2B binding site of the NMDA receptor

The competitive binding assay was performed with the

radioligand [³H]ifenprodil (60 Ci/mmol; BIOTREND, Cologne,

Germany). The thawed cell membrane preparation from the

transfected L(tk-) cells (about 20 μg protein) was incubated with

various concentrations of test compounds, 5 nM [³H]-ifenprodil,

The collected radio peak was diluted with H₂O (25 mL) and loaded

on a Water Sep-Pak C18 Plus Light cartridge (preconditioned with

5 mL EtOH, followed by 10 mL H₂O). The cartridge was washed

and TRIS/EDTA-buffer (5 mM TRIS/1 mM EDTA, pH 7.5) at 37 °C. with H₂O (2.5 mL) before it was dried with air (5 mL). 2-

The non-specific binding was determined with 10 μM unlabeled

ifenprodil. The K_dvalue of ifenprodil is 7.6 nM.^[29]

[¹⁸F]fluoroethyl tosylate was eluted with anhydrous DMSO (1 mL)

into a new Wheaton reactor (size: 5 mL), containing compound 2

(2 mg, 6.4 µmol) and 5 M NaOH (2.44 µL, 12.2 µmol). The mixture

was reacted at 120 °C for 12 min and allowed to cool down for 5

min. The crude was diluted with H₂O (2 mL) and injected into the

same HPLC system, using the following gradient: 0-20 min from

5 % B to 80 % B, 25 min: 80 % B; with A=0.01 M H₃PO₄and B =

CH₃CN at 4 mL/min. comment

PCP binding site of the NMDA receptor

The assay was performed with the radioligand [³H]-(+)-MK-801

(22.0 Ci/mmol; Perkin Elmer). The thawed membrane preparation

of pig brain cortex (about 100 μg of the protein) was incubated

with various concentrations of test compounds, 2 nM

[³H]-(+)-MK-801, and TRIS/EDTA buffer (5 mM TRIS/1 mM EDTA,

pH 7.5) at rt. The non-specific binding was determined with 10 μM

unlabeled (+)-MK-801. The K_dvalue of (+)-MK-801 is 2.26 nM.

Quality control of the radiolabeled compound was performed on

an analytical Agilent 1100 series HPLC system with a 100 µL-loop,

equipped with a GabiStar radiodetector (Raytest). An analytical

HPLC column (Atlantis T3, C18, 3 µm, 150×4.6 mm, Waters) was

used at a flow rate of 1 mL/min with a gradient method as

follows:0-10 min from 40 % B to 60 % B, 10-11 min to 40 % B, 11-

σ₁receptor

The assay was performed with the radioligand [³H]-(+)-

pentazocine (22.0 Ci/mmol; Perkin Elmer). The thawed

11

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FULL PAPER

15 min 40 % B; with 0.1% aqueous TFA (solvent A) and CH₃CN

(solvent B).

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Autoradiography

Slices (20 µm) of a Wistar rat brain (535 g, male rat) were pre-

incubated for 10 min in HEPES buffer, containing 0.1% BSA at

0 °C. After pre-incubation time, radio tracer solutions of [¹⁸F]14

(10 nM) with and without blocking compounds were added to the

brain slices (700 µL). For selectivity/ sensitivity, the following

blockers were used: 1 mM (S)-glutamate, 100 µM eliprodil,

100 µM Ro 25-6981 and 100 µM [¹⁹F]14. Incubation time was 45

min at room temperature. Slices were washed at 0 °C with HEPES

buffer, 0.1 % BSA for 5 min, followed by 2 × 3 min HEPES buffer

and 2 × 5 sec. H₂O. Brain slices were dried, placed in an imaging

cassette, exposed to the film for 15 min and analyzed on a

phosphor imager.

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Animal experiments were in accordance with the Swiss legislation

on animal welfare and approved by the Veterinary Office of the

Canton Zurich, Switzerland (license ZH 02/2012).

B. Tewes, B. Frehland, D. Schepmann, K. Schmidtke, T. Winckler, B.

Wünsch, Conformationally constrained NR2B selective NMDA

receptor antagonists derived from ifenprodil: Synthesis and biological

evaluation of tetrahydro-3-benzazepine-1,7-diols. Bioorg. Med. Chem.

2010, 18, 8005-8015.

[18]

[19]

[20]

F. Sobrio, G. G. C. Perrio, L. Barre, D. Debruyne, D. PET and SPECT

imaging of the NMDA receptor system: An overview of radiotracer

development. Mini Rev. Med. Chem. 2010, 110, 870-886.

S. W. Gruber, S. M. Ametamey. Imaging the glutamate receptor

syubtypes – Much achieved, and still much to do. Drug. Discov. Today

Tech. 2017, 25, 27-36.

Acknowledgements:

This

work

was

supported

by

the

Deutsche

Forschungsgemeinschaft (DFG) and the Schweizerischer

Nationalfonds zur Förderung der Wissenschaftlichen Forschung

(SNF), which is gratefully acknowledged.

A.-S. Salabert, C. Fonta, C. Fontan, D. Adel, M. Alonso, C. Pestourie,

H. Belhadj-Tahar, M. Tafani, P. Payoux, Radiolabeling of [¹⁸F]-

fluoroethylnormemantine and initial in vivo evaluation of this innovative

PET tracer for imaging the PCP sites of NMDA receptors. Nucl Med

Biol 2015, 42, 643–653.

Keywords:

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Bottlaender, M. Kassiou, Radiosynthesis andin vivo evaluation of

NMDA receptor • ifenprodil binding site • GluN2B subunit • 3-

benzazepines • F-containing • radioligand receptor binding

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¹¹C]Ro-647312: a novel NR1/2B subtype selective NMDA receptor

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Boninsegni, C. Keller, M. Szermerski, R. Schibli, B. Wünsch, S. M.

studies

• GluN2B affinity • selectivity • radiosynthesis •

autoradiography • displacement experiments

Ametamey,

J.

Nucl.

Med.

2017,

accepted.

DOI:

10.2967/jnumed.117.200451.

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wuensch@uni-muenster.de

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Graphical Abstract

Tetrahydro-3-bnzazepin-1,7-diols with high affinity and selectivity

served as lead compounds for the development of fluorinated

PET tracers for labeling of GluN2B subunit containing NMDA

receptors. The 7-(2-fluoroethoxy) derivative showed moderate

GluN2B affinity (K_i= 162 nM) but high selectivity over related

receptors. The [¹⁸F]labeled analog (radiochemical purity >98.9 %)

was used for autoradiography of brain slices. Removal of the

radioligand from its specific binding sites by different competitors

(e.g. Ro 25-6981) confirmed the selective labeling of GluN2B

receptors.

14

Article Doi

Fluorinated GluN2B Receptor Antagonists with a 3-Benzazepine Scaffold Designed for PET Studies

DOI: 10.1002/cmdc.201700819

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