Scholarly article on 2-(4-fluorobutoxy)benzaldehyde 1526935-36-3 from Journal of Medicinal Chemistry p. 391

Article

pubs.acs.org/jmc

Synthesis and Characterization of a Novel Series of Agonist

Compounds as Potential Radiopharmaceuticals for Imaging

Dopamine D_2/3Receptors in Their High-Aﬃnity State

Jan-Peter van Wieringen,^†,#Vladimir Shalgunov,^‡,#Henk M. Janssen,^§P. Michel Fransen,^§

Anton G. M. Janssen,^∥Martin C. Michel,^⊥Jan Booij,*^,†and Philip H. Elsinga^‡

^†Department of Nuclear Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

^‡Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen,

Groningen, The Netherlands

^§SyMO-Chem BV, Eindhoven, The Netherlands

^∥GE Healthcare, Eindhoven, The Netherlands

^⊥Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany

ABSTRACT: Imaging of dopamine D_2/3receptors (D_2/3R) can

shed light on the nature of several neuropsychiatric disorders in

which dysregulation of D_2/3R signaling is involved. Agonist D_2/3

tracers for PET/SPECT imaging are considered to be superior to

antagonists because they are more sensitive to dopamine

concentrations and may selectively label the high-aﬃnity receptor

state. Carbon-11-labeled D_2/3R agonists have been developed, but

these short-lived tracers can be used only in centers with a

cyclotron. Here, we report the development of a series of novel

D₂R agonist compounds based on the 2-aminomethylchromane

(AMC) scaﬀold that provides ample opportunities for the

introduction of longer-lived [¹⁸F] or [¹²³I]. Binding experiments

showed that several AMC compounds have a high aﬃnity and

selectivity for D_2/3R and act as agonists. Two ﬂuorine-containing compounds were [¹⁸F]-labeled, and both displayed speciﬁc

binding to striatal D_2/3R in rat brain slices in vitro. These ﬁndings encourage further in vivo evaluations.

aﬃnity state, receptors are uncoupled from the G-proteins and,

as a result, are inactive. There is substantial evidence that

alterations in the density of D_2/3receptors in the high-aﬃnity

state may be more relevant to the pathophysiology of

neuropsychiatric disorders than alterations in the total receptor

density. This is supported mostly by data from Seeman and co-

workers.¹³In several animal models of neuropsychiatric

disorders, including psychosis, increases in striatal D_2/3high-

aﬃnity receptors of up to 9-fold were registered as compared to

control animals, but total receptor numbers did not increase.¹⁵

Although agonists are selective for the high-aﬃnity state,

antagonists bind with equal aﬃnity to the low- and high-aﬃnity

states of a receptor.¹⁶Therefore, antagonist radioligands cannot

distinguish a shift in the state of receptors from low to high

aﬃnity.¹⁷Numerous compounds have been synthesized and

characterized as agonist tracers for the imaging of D_2/3in the

high-aﬃnity state by means of positron emission tomography

(PET).¹⁸These compounds are of diﬀerent chemical classes,

including ergolines, aporphines, 2-aminotetralins, benzoquino-

lines, and naphthoxazines. Aporphines [¹¹C]NPA (N-propyl-

INTRODUCTION

■

A disturbed dopamine system plays a central role in several

neuropsychiatric disorders, including Parkinson’s disease

(PD),¹dementia with Lewy bodies,²schizophrenia,³and drug

addiction.⁴The prevalence of PD and schizophrenia is about

1% of the total population,^5,6whereas the prevalence of

(alcohol) addiction may be even higher,⁷creating a large

disease burden. For dementia with Lewy body pathology, the

prevalence among people above the age of 65 years is about

10%.⁸

Dopamine receptors belong to the superfamily of G-protein

coupled receptors (GPCRs) and are divided into two

subfamilies based on their action on adenylyl cyclase (AC).

The dopamine D₁-like (D₁and D₅) subfamily stimulates AC to

produce the second messenger cyclic adenosine mono-

phosphate (cAMP), whereas D₂-like receptors (D₂, D₃, and

D₄) inhibit cAMP production by AC.^9,10The D₂and D₃

receptor subtypes are highly involved in the pathophysiology

and treatment of PD and schizophrenia, and, like other GPCRs,

they typically exhibit interconvertible high- and low-aﬃnity

states for agonists in vitro.¹¹⁻¹³In the high-aﬃnity state, the

receptors are coupled to the G-protein, and this state is

considered to be the active form of the receptor.¹⁴In the low-

Received: September 9, 2013

Published: December 10, 2013

391

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Journal of Medicinal Chemistry

Article

a

Chart 1. (R)-2-Aminomethylchroman-7-ols (AMCs) Investigated in This Study

a

Reference compounds 10, iodide series 11, and ﬂuoride series 12. The oxalate salts of the shown amine structures have been used for all assays and

binding studies.

a

Scheme 1. Synthesis of the Unlabelled AMC Compounds

a

Reagents and solvents: (a, e) LiBH₄, THF; (b, f) TsCl, pyridine; (c, i) amine building block, DIPEA, DMSO; (d) MOMCl, DIPEA, CH₂Cl₂; (g)

potassium phthalimide, DMF; (h) H₂NNH₂hydrate, EtOH; (j) (i) aldehyde building block, MeOH and (ii) NaBH₄, MeOH; (k) 4 M HCl, dioxane/

isopropanol. The R group in the series of compounds 10, 11, and 12 denotes a secondary or tertiary amine residue; see Chart 1 for details on the

speciﬁc R groups that were used.

norapomorphine) and [¹¹C]MNPA (2-methoxy-N-propylnor-

apomorphine) and naphthoxazine [¹¹C]PHNO (4-propyl-9-

hydroxynaphthoxazine) turned out to be the most successful

agonists and have also been evaluated as PET tracers in

man.^19,20In humans, agonists [¹¹C]PHNO and [¹¹C]NPA

showed better sensitivity for the detection of endogenous

dopamine release than antagonist [¹¹C]raclopride.²¹⁻²⁴All

three of these tracers also performed better than [¹¹C]-

raclopride in dopamine-release measurement in rodents, pigs,

cats, and nonhuman primates.²⁵This corroborates the notion

that agonist radiotracers are more prone to detect changes in

neurotransmitter concentrations than antagonist tracers.

However, all D_2/3agonist PET ligands successfully evaluated

in humans are [¹¹C]-labeled.^19,20Because of the short half-life

(20.4 min) of carbon-11, these radioligands can be used only

when a cyclotron is present on-site. This expensive device is,

however, available in only a minority of hospitals. To increase

the worldwide availability of such agonist tracers, we are

developing ﬂuorine-18-labeled PET tracers (half-life 109.8 min)

or iodine-123-labeled (half-life 13.2 h) agonist dopamine D_2/3

receptor tracers for single photon emission computed

tomography (SPECT) imaging. Accordingly, we have prepared

and evaluated a new series of agonist compounds based on the

2-aminomethylchroman-7-ol (AMC) scaﬀold. Structure−activ-

ity relationships for this class of compounds²⁶and classes

related to it,²⁷⁻³¹including the preference for the high-aﬃnity

versus low-aﬃnity state of D₂receptor and selectivity for the D₂

receptor over other monoamine receptors such as the serotonin

1A (5-HT_1A) and α₁adrenergic receptor, have already been

examined in detail by Mewshaw and co-workers. Importantly,

the D₂aﬃnity and agonism of AMCs shows considerable

tolerance to structural modiﬁcations in the moiety attached to

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a

Scheme 2. Synthesis of the Phenol Precursors to the [¹⁸F]-Labelled AMCs

a

Reagents and solvents: (a) (i) 4-hydroxybenzaldehyde, AcOH, CH₂Cl₂and (ii) NaB(OAc)₃H; (b) BOC-anhydride, DMF; (c) BnBr, DMF,

K₂CO₃; (d) 4 M HCl, dioxane; (e) tert-butyl N-(2-bromoethyl)carbamate, DIPEA, DMF; (f) TFA, CH₂Cl₂; (g) tosylate 6, DIPEA, DMSO/dioxane;

(h) Pd/C, H₂, EtOH.

the nitrogen atom of the AMC core, which provides ample

opportunities for the introduction of the [¹⁸F] or [¹²³I] label

into the structure without compromising the necessary

pharmacological properties. We have used data from the

Mewshaw studies to design our AMCs that either bear an

iodide or a ﬂuorine atom (Chart 1). We have included the

nonhalogenated reference²⁶compounds 10a and 10b that were

already evaluated in vitro by Mewshaw, as this enables us to put

receptor binding data on the new iodinated (11) and

ﬂuorinated (12) series of compounds into perspective.

Introduction of the piperazine group in some of the AMC

structures was inspired by studies on pramipexole³²and 5-OH-

DPAT,³³piperazines that have shown potent and selective D₃-

receptor binding, as well as by several Mewshaw studies.²⁷⁻³¹

we have prepared molecules 10a, 11b, and 11d via route A.

However, we have found that yields remain low when using

tosylate 3, presumably because 3 can react with itself under

basic conditions as opposed to reacting with the presented

amine reactants. Furthermore, the synthesis of 3 in pyridine

suﬀers from side-product formation, where the ditosylate side

product has been observed in particular. As an alternative, we

used route B that proceeds via tosylate 6, which is MOM-

protected at the 7 OH position of the chroman group. Route B

is longer, as it requires extra (de)protection steps, but the

syntheses have proved to be more reliable. Preparation of 10b,

11a, 11e, 12a, 12c, and 12d was achieved via route B. The

MOM protective group was selected over the methoxy or

benzyloxy group that was applied by Mewshaw because the

methoxy group requires more harsh deprotection conditions

and the benzyloxy group is or may be incompatibile with the

(iodo)benzyl target structures. Route B still gave rise to side-

product formation; for example, in the cases where primary

amines were reacted with tosylate 6, tertiary amine side

products were formed next to the desired secondary amine

main products. We have therefore also employed route C,

which is the longest route and which route proceeds via

primary amine 8. In this case, a reductive amination coupling

step with aldehyde building blocks can be performed so that

side product formation is largely avoided. Compounds 11a,

11c, and 12b were prepared according to route C. Provided

that the aldehyde counterpart for preparing the desired AMC

target molecule is easily accessible, route C is recommended. It

may even be possible to use the unprotected amine (i.e., amine

8 without the MOM group) to prepare AMCs, but we have not

tested this option. The synthesis of the amine or aldehyde

building blocks that are required for coupling to tosylate 3

(route A), tosylate 6 (route B), or amine 8 (route C) are

described in the Experimental Section unless these building

blocks are commercially available. Test compounds 10, 11, and

12 were all converted to their oxalate salts, and these salt end

products were used in all assays and studies described in this

article.

RESULTS

■

Chemical Synthesis. Scheme 1 outlines the synthetic

routes to the prepared cold AMCs (i.e., the unlabeled regular

¹⁹F and ¹²⁷I compounds). Details on speciﬁc transformations

can be found in the Experimental Section. Even though

racemates can be used for initial screening in receptor-binding

studies, we have prepared enantiomerically pure (R)-AMC

compounds, as it is already known that these enantiomers are

the eutomers.²⁶Accordingly, the test compounds 10a, 10b,

11a−e, and 12a−d reported in this work are all (R)

enantiomers, and they have all been prepared from the same

parent compound, 1 (i.e., (R)-7-hydroxychroman-2-carboxylic

acid ethyl ester). The racemate of compound 1 was prepared

from 2,4-dihydroxyacetophenone in two steps, according to

Walenzyk³⁴and Cohen,³⁵and thereafter this racemic mixture

was resolved to 1 (the R enantiomer) using the lipase kinetic

resolution procedure as reported by Kalaritis.³⁶GC analysis on

a chiral capillary column showed two peaks of equal intensity

for the racemate of 1, whereas resolved 1 only showed one peak

(99% ee).

Three routes to AMC target molecule series 10, 11, and 12

were employed. Route A is via unprotected tosylate 3 by

reaction of 3 with the appropriate amine building blocks. This

conveniently short route was used by Mewshaw²⁶as well, and

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Journal of Medicinal Chemistry

Article

a

Table 1. Aﬃnity of Reference and Newly Synthesized Compounds for the Diﬀerent Subtypes of Dopamine Receptors

b

c

b

D₁

dopamine not determined

D₂L_High

D₂L_Low

D₂S_Low

D₃

D₄

D₅

6.97 0.66 (6)

231 nM

5.25 0.62 (6)

11.6 μM

6.06 0.18 (4)

920 nM

7.90 0.59 (5)

24.8 nM

8.15 0.98 (4)

33.8 nM

5.99 0.26 (3)

1.16 μM

PHNO

NPA

10a

not determined

9.47 0.45 (5)

0.45 nM

7.68 0.09 (4)

21.4 nM

7.77 0.24 (3)

18.7 nM

9.00 0.23 (3)

1.08 nM

not determined

10.13 0.08 (2)

0.075 nM

8.79 0.19 (5)

1.73 nM

8.31 0.5 (6)

10.6 nM

8.74 0.15 (3)

1.91 nM

not determined

5.21 0.30 (3)

7.25 μM

8.91 0.62 (4)

2.98 nM

7.40 0.46 (5)

70.2 nM

6.73 0.91 (3)

736 nM

8.50 0.04 (4)

3.18 nM

7.93 0.08 (3)

11.9 nM

5.43 0.11 (3)

3.83 μM

10b

11a

4.98 0.17 (3)

10.9 μM

8.20 0.40 (4)

8.19 nM

6.82 0.22 (5)

168 nM

6.59 0.11 (3)

260 nM

7.72 1.41 (3)

547 nM

8.30 0.26 (3)

5.70 nM

5.56 0.09 (3)

27.7 μM

5.09 0.20 (3)

8.67 μM

8.69 0.64 (4)

3.79 nM

7.45 0.39 (6)

51.8 nM

7.09 0.55 (3)

140 nM

8.25 0.54 (4)

13.9 nM

8.81 0.14 (3)

1.59 nM

5.45 0.18 (3)

3.79 μM

11b

11c

5.41 0.25 (3)

4.29 μM

8.99 0.60 (4)

2.06 nM

7.47 0.56 (6)

72.9 nM

7.54 0.19 (3)

30.8 nM

8.54 0.57 (4)

7.87 nM

9.02 0.07 (3)

0.97 nM

5.34 0.12 (3)

4.64 μM

6.38 0.15 (3)

436 nM

9.79 0.83 (4)

0.68 nM

7.53 0.49 (4)

51.2 nM

7.08 0.67 (3)

166 nM

8.78 0.2 (4)

1.79 nM

not determined

11d

11e

4.52 0.48 (3)

46.5 μM

no meaningful

7.62 0.05 (3)

24.2 nM

7.84 0.02 (3)

14.3 nM

4.76 0.11 (3)

17.8 μM

aﬃnity

5.41 0.11 (3)

3.96 μM

not detected

7.18 0.32 (5)

80.5 nM

7.02 0.41 (3)

131 nM

7.66 0.20 (3)

23.0 nM

8.02 0.12 (3)

9.70 nM

5.46 0.09 (3)

3.48 μM

12a

5.18 0.17 (3)

6.91 μM

8.26 0.13 (2)

5.60 nM

7.21 0.36 (5)

80.5 nM

7.04 0.1 (3)

92.7 nM

8.04 0.21 (3)

12.1 nM

7.50 0.09 (3)

32.1 nM

5.61 0.31 (3)

2.89 μM

12b

12c

5.94 0.04 (3)

1.16 μM

not detected

6,84 0.21 (4)

156 nM

6.45 0.38 (3)

452 nM

7.49 0.1 (3)

33.0 nM

not determined

no meaningful

aﬃnity

no meaningful

aﬃnity

no meaningful

aﬃnity

no meaningful

aﬃnity

7.43 0.17 (4)

39.4 nM

7.32 1.02 (3)

131 nM

4.71 0.05 (3)

19.7 μM

12d

no meaningful

aﬃnity

8.30 0.47 (3)

6.67 nM

6.66 0.18 (5)

235 nM

6.63 0.21 (3)

252 nM

8.06 0.27 (3)

10.0 nM

7.64 0.35 (3)

27.7 nM

5.01 0.01 (3)

9.70 μM

a

Data are shown as the mean SD of −log K_ivalues, with the number of experiments given in parentheses; mean K_ivalues, in nanomolar or

b

micromolar, are given in the bottom row for each compound. Data from experiments using membranes made from HEK293 cells stably transfected

with the concerning dopamine receptor subtypes. Data from experiments using membranes purchased from PerkinElmer.

c

Scheme 2 shows the synthetic route to phenols 13a and 13b,

which are the precursors to radiolabeled compounds [¹⁸F]12a

and [¹⁸F]12d, respectively. MOM-protected amine 8 was

coupled by reductive amination to 4-hydroxy benzaldehyde to

prepare 13a. Commercially available 1-(4-hydroxyphenyl)-

piperazine was converted in ﬁve steps to primary amine

reactant 18, which can be coupled to tosylate 6 to provide 19.

Subsequent debenzylation of 19 aﬀords 13b. Precursors 13a

and 13b both require protection at the 7-OH chroman position

because a selective coupling of the respective [¹⁸F]-

alkyltosylates to the other phenol moiety is desired. Protection

of the NH groups in the precursors is not needed because

coupling under basic conditions with the [¹⁸F]-alkyltosylates is

favored at the phenol position over the NH position. For

details on the preparation of [¹⁸F]12a and [¹⁸F]12d, see the

Radiolabeling section in the Results.

ligands.³⁷See Figure 1 for a typical biphasic binding curve

recorded for dopamine.

Among the AMC compounds tested, 11c had the highest

aﬃnity for the D₂L_highreceptor (pK_i9.79 0.83). Compounds

10a, 10b, 11a, 11b, 12a, and 12d showed high aﬃnities, with

recorded pK_ivalues above 8. The only tertiary amine

Pharmacology. Aﬃnity of the Compounds to the

Dopamine Receptors Determined by Radioligand Binding.

All novel AMC compounds (Chart 1) as well as the reference

ligands dopamine, PHNO, and NPA were tested for their

ability to compete with [³H]spiperone or [³H]SCH23390 for

binding to various receptors of the dopamine receptor family.

In Table 1, the results of these binding studies are compiled.

For almost all compounds, the binding experiments on the D₂L

(D_2long) receptor produced competition curves that were

explained signiﬁcantly better by biphasic models (using

GraphPad Prism). This ﬁnding indicates agonism for these

Figure 1. Representative biphasic competition binding curve for

dopamine binding to the D₂L receptor. The data shown are from one

experiment, where the bound fraction of [³H]spiperone in counts per

minute (cpm) is plotted against the log of the concentration (mol/L)

of dopamine. According to the points of inﬂection (arrows), this

example shows an IC₅₀for the D₂high-aﬃnity state at 10⁻⁶

dopamine and an IC₅₀for the D₂low-aﬃnity state at 10⁻⁴

M

dopamine (these IC₅₀values need to be transformed to K_ivalues using

the Cheng−Prusoﬀ equation).³⁸The fraction of the receptors in the

high-aﬃnity state is 40 7%.

394

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compounds, AMCs 11d and 12c, did not show a meaningful

aﬃnity for the D₂L receptor. For 11e and 12b, no biphasic

curves were observed and measured aﬃnities were (well) below

a pK_iof 8 (pK_i7.18 0.32 for 11e and 6.84 0.21 for 12b),

which were comparable to those recorded for D₂L, and all

tested compounds showed aﬃnities for the dopamine D₄

receptor in the same range as that measured for the D₂L

receptor except for tertiary amines 11d and 12c, which had a

high aﬃnity for D₄but no meaningful binding to D₂L. These

two compounds also showed an evident aﬃnity for D₃, with

pK_i’s of 7.62 for 11d and 7.43 for 12c. Finally, all of the tested

compounds lacked reasonable aﬃnity for the dopamine D₁-like

receptors (D₁and D₅) (Table 1).

Table 3. Lipophilicity of AMC Compound Series 10, 11, and

12

a

compound

cLogP

cLogD (cLogP_7.4)

10a

10b

11a

11b

11c

11d

11e

12a

12b

12c

12d

3.42

3.90

4.35

4.74

4.38

4.04

3.86

3.49

1.41

1.46

2.43

2.46

3.30

4.15

2.42

2.12

2.57

3.27

1.54

Ligand-Induced Inhibition of Forskolin-Induced cAMP

Accumulation. In HEK293 cells expressing the dopamine

D₂L receptor, dopamine inhibits the forskolin-induced cAMP

accumulation, where this inhibition becomes apparent at a

certain dopamine concentration. In the cAMP assay (Table 2),

a

Assessed with the BiologP (New cLogP) calculator program from the

University of Massachusetts.

At nanomolar concentration, [¹⁸F]12a showed rather

homogeneous uptake in sagittal rat brain slices, with only

slightly higher binding in the striatum. Striatum-to-cerebellum

and striatum-to-cortex ratios were on average 1.19 and 1.16,

respectively. In the presence of 10 μM of the dopamine D_2/3

antagonist raclopride, the striatum-to-cerebellum ratio was

reduced by 14%, and the striatum-to-cortex ratio, by 11%. At

subnanomolar concentration, however, [¹⁸F]12a demonstrated

moderate but clearly visible speciﬁc uptake in the striatum. At

0.19 nM, striatum-to-cerebellum ratios were, on average, 1.85

and were reduced by 40% in the presence of 10 μM raclopride

and by 35% in the presence of 100 μM guanosinetriphosphate

(GTP). Striatum-to-cortex ratios were, on average, 1.68, and

coincubation with 10 μM raclopride or 100 μM GTP resulted

in 39 and 33% decreases, respectively.

a

Table 2. Potency and Eﬃcacy Maximal Response (E_max

)

ligand

pEC₅₀

E_max(% of dopamine)

N

dopamine

10a

9.2 0.4

10.7 0.3

10.3 0.4

9.2 0.4

10.5 0.4

8.8 0.4

9.4 0.3

9.8 0.2

100

4

3

78

82

86

96

94

5

6

5

6

5

3

10b

11a

11b

11e

12a

12d

a

Expressed as the percent of dopamine response for various ligands at

the human D₂L receptor determined in a cAMP accumulation assay.

All data are expressed as the mean SEM.

At nanomolar concentration, [¹⁸F]12d demonstrated prefer-

ential binding in the striatum and hippocampus. In control

animals, the mean striatum-to-cerebellum ratio was 2.64. In the

presence of 10 μM raclopride or 100 μM GTP, striatum-to-

cerebellum ratios were signiﬁcantly reduced by 37%. Striatum-

to-cortex ratios were, on average, 1.90 in control slices and were

reduced by 37% by raclopride and by 22% by 100 μM GTP.

the AMC test compounds were compared to dopamine with

respect to their inhibition potency, and it was found that 11e

appeared to be less potent than the endogenous agonist

dopamine; 11a and 12a were similarly potent, and all of the

other test compounds were somewhat more potent than

dopamine, with 11b appearing to be the most potent of the

tested halogenated compounds. All compounds had eﬃcacies

comparable to that of dopamine (Table 2).

Lipophilicity. Data regarding lipophilicity are shown in

Table 3. Compounds 10a, 10b, 11a, 11b, 11e, 12a, 12b, and

12d showed a cLogD (cLogP_7.4) in the range between 1 and

3.³⁹

DISCUSSION

■

Our present eﬀort is directed at developing new agonist

radiotracers that can be used in centers without an on-site

cyclotron with the aim of enabling visualization of dopamine

D

_2/3receptors in the agonist high-aﬃnity state. Accordingly, we

Radiolabeling. The two ﬂuorinated compounds, 12a and

12d, were selected for [¹⁸F]-labeling, and this was accomplished

using a three-step two-pot procedure starting with the synthesis

of a [¹⁸F]ﬂuoroalkyl synthon from [¹⁸F]ﬂuoride and the

appropriate ditosylate precursor followed by [¹⁸F]-

ﬂuoroalkylation of phenol precursors 13a or 13b (Scheme 2)

and in situ removal of the MOM-protecting group under acidic

conditions (Scheme 3). The [¹⁸F]-labeled products, [¹⁸F]12a

and [¹⁸F]12d, were puriﬁed by reversed-phase HPLC. A typical

synthesis run lasted for 150 min and started from 5 GBq of

[¹⁸F]ﬂuoride, from which 20−200 MBq of formulated [¹⁸F]12a

or [¹⁸F]12d was obtained.

Decay-corrected yields, radiochemical purities, and speciﬁc

activities of [¹⁸F]12a and [¹⁸F]12d are listed in Table 4.

In Vitro Autoradiography. Results of in vitro auto-

radiography of [¹⁸F]12a and [¹⁸F]12d are presented in Figure

2 and Table 5.

synthesized a novel group of iodinated and ﬂuorinated

compounds based on the AMC scaﬀold, which is known for

its agonistic properties on D_2/3dopamine receptors and for its

good selectivity of binding for these receptor subtypes.²⁶

The primary binding site of the D₂receptor is formed by

transmembrane domains (TMs) 3, 5, and 6 of the receptor.⁴⁰

D₂ligands contain an aromatic core that engages in interactions

with the hydrophobic residues in TM3 and TM6. To this core

are attached an amino group and a hydrogen-bond donating

group to form an ionic bond with the conserved aspartate

residue in TM3 and one or more hydrogen bonds with

conserved serine residues in TM5, respectively (Figure 3).

The optimal distances between these groups and their

optimal mutual orientations have been the subject of numerous

studies that have produced more or less general pharmaco-

phore models ranging from the McDermed criteria developed

in the late 1970s⁴¹to more recent models.^42,43Notably, it was

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a

Scheme 3. Labelling Schemes of [¹⁸F]12a and [¹⁸F]12d

a

Reagents and solvents: (a) K¹⁸F-Kryptoﬁx-K₂CO₃, MeCN; (b) (i) 13a, NaH, DMF and (ii) 0.4 M HCl; (c) (i) 13b, NaH, DMF and (ii) 0.4 M

HCl.

a

Table 4. Decay-Corrected Yields (RCY), Radiochemical Purities (RCP), and Speciﬁc Activities (SA) of [¹⁸F]12a and [¹⁸F]12d

tracer

RCY, %

RCP, % (formulated tracer)

SA (GBq/umol), at end of synthesis

b

[¹⁸F]12a

HPLC: 25 11 (3−47)

94 4 (85−98)

92 96 (1.4−312)

c

n = 10

formulated: 7 8 (1−22)

HPLC: 14 5 (6−20)

formulated: 5 3 (1.4−11)

[¹⁸F]12d

n = 6

92 5 (85−98)

29 12 (8−40)

a

b

Data are expressed as the mean SD, and the range is given in brackets. HPLC fraction containing the tracer vs ¹⁸F-ﬂuoride taken for synthesis

c

Formulated tracer vs ¹⁸F-ﬂuoride taken for synthesis

substituent at the nitrogen, however, cannot be greater than n-

propyl.⁴⁵

Mewshaw et al.²⁶proposed AMCs as a new series of

compounds that meet the criteria for agonists with a high D₂

aﬃnity and an appropriate D₂-over-D₁selectivity. The AMC

scaﬀold oﬀers convenient possibilities to accommodate the

[

¹²³I] or [¹⁸F] label on the N substituent, and the same

substituent can also be used to adjust the lipophilicity of the

compound. On the basis of benzylated secondary amine

compound 10a, which was developed by Mewshaw and was

Figure 2. In vitro autoradiography with [¹⁸F]12a at 0.19 nM (a), [¹⁸F]

12a at 4.5 nM (b), and [¹⁸F]12d at 5.8 nM (c). Note the speciﬁc

binding in the striatum for [¹⁸F]12a (only at lower concentration) and

for [¹⁸F]12d.

high

found to have a high aﬃnity (subnanomolar) for the D₂

receptor, we designed three iodobenzyl and two ﬂuoroalkyloxy-

benzyl candidate ligands. From the conformational and

comparative molecular ﬁeld analysis carried out for the AMC

derivatives by the group of Mewshaw⁴⁶and the modeling data

for the D₂receptor,^47,48we concluded that the benzyl group of

10a is likely to interact with extracellular loop 2 of the receptor

and not with the ancillary pocket of D₂formed by TM2 and

TM7. To probe the spatial requirements of such alignment, we

synthesized para- (11a), meta- (11b), and ortho-iodobenzyl

(11c) as well as para- (12a) and ortho-(4-ﬂuoro)-

found that the nitrogen atom can accommodate a bulky

substituent extending into the ancillary binding pocket of the

receptor formed by TM2 and TM7. The passage between the

primary and ancillary binding pockets is more narrow in D₁-like

receptors than in D₂-like receptors, so the presence of such

bulky substituents imparts D₂-over-D₁selectivity.⁴⁴The second

a

Table 5. In Vitro Autoradiography Results

tracer

Log D

conditions

striatum-to-cerebellum ratio

striatum-to-cortex ratio

[¹⁸F]12a

2.45 0.10

control (n = 3)

1.85 0.23

1.19 0.18

1.10 0.19

1.04 0.22

1.20 0.16

2.64 1.04

1.65 0.37

1.67 0.28

1.68 0.11

1.16 0.16

1.03 0.08

1.05 0.11

1.13 0.09

1.90 0.47

1.20 0.20

1.48 0.17

upper row: 0.19 nM

lower row: 4.5 3.6 nM

raclopride (n = 1)

GTP (n = 1)

[¹⁸F]12d

1.48 0.01

control (n = 4)

raclopride (n = 4)

GTP (n = 1)

5.8 1.6 nM

a

Values are the mean SD from the total number of slides assayed in n independent experiments. The number of slides per condition in each

experiment varied from 4 to 12.

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Several of the prepared compounds indeed showed high

aﬃnity and selectivity for the D_2/3receptors and agonistic

action on the D₂receptor. Dopamine and most of the AMC

compounds consistently showed biphasic competition curves

for the D₂L receptor, which is typical for agonists. The

inﬂection points in these curves reﬂect the agonist high-

(D₂L_high) and low-aﬃnity (D₂L_low) states (Figure 1). In line

with this ﬁnding, the cAMP experiments also showed agonistic

properties for the tested compounds.

Nevertheless, when we compare the aﬃnity of reference

compounds 10a and 10b to the values of Mewshaw et al., our

ﬁndings show aﬃnities that are 1 log scale lower (0.2 vs 3.0 nM

for 10a and 0.7 vs 8.2 nM for 10b). This diﬀerence can be

caused either by interspecies diﬀerence (Mewshaw used

predominantly rat receptors, whereas we used only human

receptors) or by diﬀerences in techniques. The latter is more

likely in our opinion, as discrepancy in the techniques is a well-

known cause of discrepancy in aﬃnity measurements. For

instance, for the D₂L_highreceptor, we found a dopamine

dissociation constant of 231 nM, whereas the literature reports

values of 2 nM using [³H]raclopride,⁵¹4.7 nM using

[³H]spiperone,⁵²and 56 nM using [¹²⁵I]iodosulpiride.⁵³

Reported aﬃnities of dopamine for the D₂S receptor range

between 4.5 and 229 nM using [³H]spiperone as radio-

ligand,⁵⁴⁻⁵⁶whereas 22 nM was found using [¹²⁵I]-

iodosulpiride.⁵⁷Although we did not ﬁnd a high-aﬃnity state

of dopamine for the D₂S receptor in our experiments, a

comparison of the ﬁndings for the D₂S_highand the D₂L_highis still

warranted because these receptors are almost equal structurally.

In our experiments, PHNO showed an aﬃnity for D₂L_highof

0.45 nM, and this is in line with literature ﬁndings of 0.6⁵⁸and

1.5 nM⁵⁹for D₂L_high. NPA exhibited a K_iof 0.08 nM for

D₂L_high, which is reasonably comparable to the literature data

that report K_i’s of 0.38⁶⁰and 0.11 nM.⁶¹

Figure 3. Comparison of the molecular structures of some D₂-agonist

ligands, where their aryl cores are purple, basic nitrogens are red, and

H-bond-donating hydroxyls are blue. The orientation of the functional

groups was chosen to resemble roughly the putative receptor-bound

conformations of the ligands. Note that for conformationally

constrained (+)PHNO the lone electron pair of the nitrogen is

directed roughly normally to the aryl ring and away from view, toward

the conserved aspartate in TM3. In the 3D representation, the TM6−

TM3 axis is directed away from view, that is, TM6 is closer to the

viewer than TM5, whereas TM3 is farther.

butyloxybenzyl (12b) derivatives of 10a. The butyl spacer in

12a and 12b was selected to bring the expected lipophilicity of

the compounds within the Log D range of 2 to 3 (Table 3) to

ensure passage of the blood−brain barrier.⁴⁹

The arylpiperazine motif is known to act as a pharmaco-

phoric core in a number of high-aﬃnity D₂-agonists,³¹and

apparently it can ﬁt into the primary binding pocket of the D₂-

like receptor.⁴⁸Interestingly, Dutta and co-workers synthesized

and evaluated a number of hybrid ligands based on an

aminotetralin or a pramipexol scaﬀold to which an arylpiper-

azine moiety was attached via a spacer.^32,33In these hybrid

ligands, the arylpiperazine moiety is most probably bound in

the ancillary binding pocket of the receptor and not in the

primary binding site.⁵⁰Inspired by these results, we designed

four hybrid AMC derivatives with arylpiperazine side chains

attached to the AMC core either directly (11d and 12c) or

through an ethylene spacer (11e and 12d). Note that

compounds 11d and 12c are the only tertiairy amines in the

series of developed compounds, where all of the other

compounds are secondary amine AMCs.

In the Mewshaw study that evaluated agonist ligands for the

dopamine D_2/3receptor, the aﬃnity was measured in rat striatal

membranes using the agonist [³H]quinpirole, and it was

claimed that this approach speciﬁcally assessed the binding to

the D₂L_highreceptor because [³H]quinpirole should predom-

inantly bind to D₂L_high. Measuring aﬃnities for D₂L_lowwas also

done in rat striatal membranes but in the presence of GTP to

convert D₂L_highto D₂L_lowand with antagonist [³H]spiperone as

the radioligand. In addition, ketanserin was administered to

preclude binding of spiperone to serotonin receptors.²⁶Finally,

a selection of compounds were evaluated in binding assays

using membranes from cells transfected with human D₂S, D₃,

and D₄receptors. We have also used membranes from cloned

human dopamine receptors expressed in cell lines but included

D₁, D₂L, and D₅as well. We also used [³H]spiperone as the

radioligand, resulting in biphasic binding curves from which the

aﬃnities of the compounds for D₂L_highand D₂L_lowwere derived

(Figure 1).

All AMC derivatives designed by us had at least 100-fold

lower aﬃnities toward D₁-like receptors than D₂-like receptors.

This is in agreement with the established notion that bulky

substituents at the nitrogen impart D₂selectivity.

Comparing the aﬃnity toward D₂L_highof the para (11a),

meta (11b) and ortho (11c) iodobenzyl compounds shows that

11a and 11b have about equal aﬃnity (3.8 and 2.1 nM,

respectively), with 11c displaying a 5-fold higher aﬃnity (0.68

nM). This ﬁnding is in accordance with our expectation that

the large iodine atom at the para position of 11a may be

accommodated less easily within the binding pocket (possibly

clashing somewhat with the receptor’s extracellular loop) as

compared to the placement of the meta- and ortho-substituted

iodines in 11b and 11c. Nevertheless, 11a still shows a high

aﬃnity. Compound 12a, with the 4-ﬂuorobutyloxy group at the

para position of the benzyl ring, had a D₂L_highaﬃnity of 5.6 nM,

comparable to that of 11a, whereas its ortho isomer, 12b,

surprisingly did not appear to recognize D₂L_highand had about

3-fold lower aﬃnities for D₂L_lowand D₃than 12a. Apart from

steric interactions, intramolecular hydrogen-bonding interac-

tions between the secondary NH and the O atom of the ﬂuoro-

butoxy chain may also play a role here, where these interactions

can only be conceived for 12b (forming an ideal six-membered

ring) and not for 12a. Similarly, such interactions may exist

with the I atom in 11c but not with those in 11b or 11a. Note

that these intramolecular hydrogen-bonding interactions are

also reﬂected in the increased lipophilicities of compounds 11c

and 12b relative to those of their para- or meta-substituted

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counterparts (Table 3). However, it is diﬃcult to be conclusive

about the binding mode of 12a and particularly 12b.

tracer, but the former never amounted to more than 5% of the

total radioactivity.

In comparison to the benzylamino AMCs, 10a, 11a−c, and

12a−b, the hybrid AMCs, 11d−e and 12c−d, generally showed

lower aﬃnities toward all dopaminergic receptor subtypes, both

D₁-like and D₂-like. This decrease in aﬃnity was, however, not

homogeneous, and the greatest decrease was observed for the

D₂subtype receptors, as tertiary amine hybrids 11d and 12c did

not show a meaningful aﬃnity toward these receptors at all,

whereas ethylene-spaced secondary amine hybrid 11e recog-

nized only the low-aﬃnity state of D₂L and D₂S. This

observation is in agreement with the SAR studies on D₃-over-

D₂selectivity that suggest that the ancillary binding pocket of

D₃is more spacious than that of D₂, so compounds with bulkier

We evaluated labeled compounds [¹⁸F]12a and [¹⁸F]12d by

in vitro autoradiography in rat brain slices. Both ligands

exhibited preferential uptake in the striatum region, where for

[¹⁸F]12a this was visible only at subnanomolar free-ligand

concentrations and for [¹⁸F]12d it was found at low nanomolar

levels (Figure 2). Given that the two compounds had

comparable D₂L_highaﬃnities, the lack of discernible D_2/3

-

speciﬁc binding of [¹⁸F]12a at low nanomolar concentrations

may be due to its higher lipophilicity (Table 3), which may

have led to the D_2/3-speciﬁc signal being buried under the

nonspeciﬁc signal resulting from [¹⁸F]12a binding to

membrane phospholipides.

For both compounds, binding in the striatum could be

blocked with dopamine D_2/3receptor antagonist raclopride and

could be decreased to a similar extent in the presence of GTP,

which suggests that the studied compounds indeed bind to the

D_2/3receptors in the striatum, showing a strong preference for

the high-aﬃnity state. This promising ﬁnding stimulates

performing future in vivo PET-studies of [¹⁸F]12d in rodents,

especially because the lipophilicity of this compound is in the

requested range for the development of accurate PET tracers.⁶⁴

Most of our novel AMC compounds showed comparable

aﬃnities for both the dopamine D₂and D₃receptor. This is in

line with presently available agonist dopamine receptor PET

tracers such as [¹¹C]NPA and [¹¹C]MNPA. In contrast,

[¹¹C]PHNO, which is D₃-preferring in vivo,²⁴is currently

being used to investigate the regional distribution of D₃

receptors in the brain,^65,66and the latest clinical research data

suggest that D₃-selective tracers might be advantageous over

tracers with mixed D_2/3aﬃnity in some applications.^67,68

Three of our four hybrid compounds (11d−e and 12c) seem

selective for the D₃receptor over the D₂subtype. Although

their D₃aﬃnities are too low to consider them as candidate

tracers, they conﬁrm that high-aﬃnity D₃-selective AMCs can

be made if more studies into structure−activity relationship for

this compound class are undertaken. In addition, several of our

novel compounds display a non-negligible aﬃnity for the D₄R

(Table 1). For instance, 11a and 11b, which are attractive

candidates for [¹²³I] labeling, have low nanomolar K_ivalues on

D₄receptors, on par with their K_i’s at D₂L_high. However, in the

human striatum, the D₄receptor density is 16-fold lower than

that of D_2/3receptors.^69,70In addition, in the rat striatum, the

D₄receptor density is 7- and 5-fold lower than that of the D₂

and D₃receptors, respectively.⁷¹Consequently, we believe that

striatal binding of our tracers will predominantly reﬂect binding

to D_2/3receptors.

groups attached to the nitrogen atom tend to prefer binding to

62,63

D_3.

An interesting exception to this trend is ethylene-

spaced secondary amine hybrid 12d, which has a D₂L_highaﬃnity

comparable to that of ﬂuorobutyloxybenzyl derivative 12a and a

D₂L_high-over-D₂L_lowpreference that is even higher than that of

12a. It is impossible to determine how exactly the

arylpiperazine moiety interacts with the receptor in these

hybrid ligands, but, at least in the case of 12d, the

arylpiperazine must be bound in the ancillary pocket because

arylpiperazines that interact with the primary binding pocket of

the receptors have very low D₂aﬃnities when a bulky group,

such as a ﬂuoroalkoxy group, is placed at the para position of

the aryl ring.^31,55

We successfully labeled two of our compounds (12a and

12d) with ﬂuorine-18, where we made the selection for these

two compounds based on the results of the earlier-described in

vitro experiments: 12a and 12d show a high aﬃnity for

dopamine D_2/3receptors, a low aﬃnity for dopamine D₁-like

receptors, agonism in the cAMP experiment, and have an

appropriate lipophilicity. Scheme 3 shows our approach to

prepare [¹⁸F]12a and [¹⁸F]12d, where we chose to use

[¹⁸F]ﬂuoroalkylation onto a phenol precursor (either 13a or

13b) to arrive at the targeted tracers. The applied [¹⁸F]-labeling

approach is versatile, as we can quickly adapt the procedure for

the preparation of homologous [¹⁸F]ﬂuoroalkyl derivatives of

[¹⁸F]12a or [¹⁸F]12d in case it is necessary to adjust the

hydrophobicity of the ligand. In contrast, an approach in which

one would label compounds by direct [¹⁸F]ﬂuorination of

tosyloxyalkyloxy or bromoalkyloxy precursors would require

the preparation of every individual precursor (but it would

beneﬁt from fewer reaction steps involving [¹⁸F] or [¹⁸F]

intermediates).

As can be seen from Table 4, only 25−30% of the HPLC-

puriﬁed [¹⁸F]12a or [¹⁸F]12d ended up in the formulated

tracer solution. The reason for this was that only 30−50% of

the radioactivity could be eluted from the Oasis HLB SPE

cartridge using the designated portions (1 to 2 mL) of pure

ethanol, with the remainder of the [¹⁸F]12a and [¹⁸F]12d

amines sticking to the cartridge. The elution eﬃciency rose to

80−90% when the ethanol was acidiﬁed with 1% v/v ortho-

phosphoric or acetic acid, but it was decided to use ethanol as

eluent.

Finally, our novel series of compounds may also oﬀer the

opportunity to develop [¹²³I]-labeled SPECT tracers; com-

pound 11a may be an attractive candidate to label with [¹²³I]

because it has appropriate aﬃnity, selectivity, and lipophilicity

and because labeling seems feasible. Future studies will focus on

this possibility.

CONCLUSIONS

■

Tracers [¹⁸F]12a and [¹⁸F]12d were obtained in reasonable

yields and satisfactory speciﬁc activities. Radiochemical purities

were also satisfactory; all radioactive impurities present in

formulated [¹⁸F]12a were much more polar than the tracer

itself (not detectable on QC radio-HPLC, producing a spot

with R_f= 0 on radio-TLC), and formulated [¹⁸F]12d contained

radioactive impurities both more and less hydrophobic than the

We have identiﬁed a new series of agonist radiopharmaceuticals

based on an AMC scaﬀold that bind with high aﬃnity and

selectivity to dopamine D_2/3receptors in vitro. In cAMP assays,

they all show nearly full agonism. In addition, two of these

compounds were labeled successfully with [¹⁸F] and were

evaluated by in vitro autoradiography, where both of them

showed speciﬁc binding in rat striatum: one at subnanomolar

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([¹⁸F]12a) and another at low nanomolar ([¹⁸F]12d)

concentrations. These ﬁndings will stimulate further in vivo

evaluations, for example, animal PET and/or SPECT studies.

Merck. Reversed-phase (RP) chromatography was performed on the

Biotage Isolera One applying a SNAP KP-C18-HS cartridge.

The applied centrifuge was a Thermo Electron corporation Heraeus

Megafuge 1.0.

Synthesis of AMC-Derivative Series 10, 11, and 12. A series of

nine potential agonist compounds for the dopamine D_2/3receptor

incorporating iodine or ﬂuorine atoms in their structures was

synthesized. Iodinated compounds were termed 11a, 11b, 11c, 11d,

and 11e. Fluoridated compounds were numbered 12a, 12b, 12c, and

12d. For internal standardization, two reference compounds without

iodine or ﬂuorine atoms in their structure were also synthesized (10a

and 10b). Structural formulas are shown in Chart 1. All of the test

compound series 10, 11, and 12 reported in this work are (R)

enantiomers, and they all originate from starting compound 1 (i.e.,

(R)-7-hydroxychroman-2-carboxylic acid ethyl ester).

(R)-7-Hydroxychroman-2-carboxylic Acid Ethyl Ester (1). The

racemate of 1^34,35was resolved to 1 (the R enantiomer) using the

lipase kinetic resolution procedure as reported by Kalaritis.³⁶

Accordingly, the racemate of 1 (10 g, 45.5 mmol) was dissolved in

THF (34 mL), and demi-water (270 mL) and a 0.05 M phosphate

buﬀer (68 mL) were added. Finally, amano lipase from Pseudomonas

ﬂuorescens (1.82 g) was added. The reaction mixture was treated with a

1 M NaOH solution (25 mL) by dropwise addition over 10 h. After

NaOH addition, the reaction mixture was stirred for another 8 h. The

mixture was evaporated to remove the THF, and the remaining

aqueous mixture was extracted with ethyl acetate. Washing of the

collected organic layers with saturated NaHCO₃, drying with Na₂SO₄,

and concentrating gave an oil that slowly crystallized. Yield: typically

ca. 45%.

EXPERIMENTAL SECTION

■

Materials (Chemical Synthesis). Reagents, chemicals, materials,

and solvents were obtained from commercial sources, and were used as

received: Biosolve and Merck for solvents, Cambridge Isotope

Laboratories for deuterated solvents, and Aldrich, Acros, ABCR,

Merck, and Fluka for chemicals, materials, and reagents. All solvents

were of AR quality. Moisture or oxygen-sensitive reactions were

performed under an atmosphere of dry N₂or argon.

Methods for Chemical Synthesis and Molecular Character-

1

ization. H and ¹³C NMR spectra were recorded on Varian Mercury

1

(400 MHz for H NMR, 100 MHz for ¹³C NMR) spectrometers at

298 K. Chemical shifts are reported in ppm downﬁeld from

tetramethylsilane (TMS) for ¹H NMR and applying deuterated

chloroform (CDCl₃) or other deuterated solvents as internal reference

for ¹³C NMR. Abbreviations used for splitting patterns are s, singlet; t,

triplet; q, quartet; m, multiplet; dd, double of doublets.

Melting points were measured on a Buchi melting point B-540

apparatus.

̈

LC−PDA/MS analyses were performed on a Shimadzu LC-10 AD

VP series LC coupled to a photo diode array (PDA) detector

(Finnigan Surveyor PDA Plus detector, Thermo Electron corporation)

and an ion-trap detector (LCQ Fleet, Thermo Scientiﬁc). Analyses

were executed at 298 K using an Alltech Alltima HP C18 3μ column

using an injection volume of 1−4 μL, a ﬂow rate of 0.2 mL min⁻¹, and,

typically, a MeCN in H₂O gradient (from 5 to 100% MeCN, where

both MeCN and H₂O contain 0.1% formic acid).

Elemental and UPLC−HRMS analyses were performed on the

aminomethylchroman-7-ol (AMC) test compound series 10, 11, and

12 in their isolated oxalate forms. Elemental analyses were done to

assess the number of oxalate groups that were attached to the AMC

amines and were performed on a PerkinElmer Series II CHNS/O

Analyzer 2400. UPLC−HRMS was used to assess the purity and the

exact molecular weight of the AMCs. Purities were typically ca. 95%,

mostly higher, and are given for every AMC compound. Mass

deviations from the theoretical mass were typically between 0 and 2

ppm. UPLC was performed on a Waters Acquity UPLC equipped with

a sample manager (an autosampler) and a binary solvent manager. An

Acquity UPLC BEH C18 1.7 μm column was used using H₂O and

MeCN both with 0.1% formic acid as the two eluents and applying an

8 min gradient program where the amount of MeCN was increased

from 5 to 60% in 5 min. A sample solution (10% MeCN in H₂O with

0.1% formic acid) with a concentration of ca. 0.1 mg/mL was

prepared, and an injection volume of 0.1 μL was used. For HRMS

detection, a Xevo G2 Qtof detector using Zspray lockspray ionization

was applied.

GC−FID measurements to establish the ee of compound 1 were

performed on a Shimadzu GC-2000 by applying the Varian WCOT

fused silica column (25 m, i.d. 0.25 mm) that is coated with CP

chirasil-dex cb df 0.25 μm particles. An isothermal program was used

(200 °C), applying a carrier gas helium ﬂow of 30 cm/sec.

GC−MS measurements were executed on a Shimadzu GC-17A gas

chromatograph coupled to a Shimadzu GC−MS-QP5000 detector

using a Phenomenex Zebron ZB-35 capillary column (30 m length, i.d.

0.25 mm, 0.25 μm ﬁlm thickness) that was operated using helium as

the carrier gas and typically applying a temperature gradient from 80 to

300 °C.

Note that the reaction conversion (i.e., the hydrolysis of the S

enantiomer to the acid) and the ee of the remaining desired R-

enantiomer ester 1 was monitored using a chiral GC−FID method.

Using this method, the racemate of 1 gives two peaks of equal

intensity, whereas isolated 1 (i.e., the R enantiomer after resolution)

gives one peak with no or very little sign of the S enantiomer (99%

ee). The S enantiomer eluted ﬁrst, and the desired R enantiomer

eluted second. Analytical data of isolated 1 are according to those from

rt

the above-mentioned literature sources, particularly [α]_D= −20.1

(CHCl₃, c 1).

(R)-(7-Hydroxychroman-2-yl)methanol (2). Ethyl ester 1 (3.18

g, 14.4 mmol) was dissolved in THF (200 mL) and cooled to 0 °C. A

2 M LiBH₄in THF solution (18.2 mL, 36.4 mmol, 2.5 equiv) was

added. The reaction mixture was stirred at rt for 3 h, during which

time the mixture became a milky suspension, and it was then poured

into 1:1 water/brine (200 mL) that was brought to pH 2 using a 2N

HCl solution. After stirring for 5 h, the mixture was extracted with

diethyl ether and dried with Na₂SO₄. The fraction of starting

compound (ca. 15%) that was still present was removed by silica

column chromatography (1:1 hexane/ethyl acetate). Yield: 2.1 g

(80%). Analytical data are according to those from literature.²⁶¹H

NMR (CDCl₃): δ 6.9 (m, 1H), 6.35 (m, 2H), 5.35 (bs, 1H), 4.05 (m,

1H), 3.85 (m, 1H), 3.75 (m, 1H), 2.85−2.6 (multiple signals, 2H), 2.2

(bs, 1H), 1.95−1.75 (multiple signals, 2H). LC−MS: m/z 181.1 [M +

H]⁺(calcd 180.20 for C₁₀H₁₂O₃).

Toluene-4-sulfonic Acid, (R)-(7-Hydroxychroman-2-yl)-

methyl Ester (3). A solution of the alcohol 2 (0.5 g, 2.8 mmol) in

pyridine (25 mL) was stirred at 0 °C. 4-Toluenesulfonyl chloride (0.56

g, 2.9 mmol, 1.05 equiv) was added in small portions, and the reaction

mixture was stirred for 6 h at 4 °C. The mixture was then poured in ice

water, and aqueous 2 N HCl was added to acidify to pH < 2.

Extraction with CH₂Cl₂, drying of the collected organic layers with

MgSO₄, and evaporation of the solvents gave a yellowish oil that was

puriﬁed with silica column chromatography (2% MeOH/CHCl₃).

Yield 0.56 g (60%). Analytical data are according to those from

literature.²⁶¹H NMR (CDCl₃): δ 7.8 (m, 2H), 7.35 (m, 2H), 6.82 (m,

1H), 6.35 (m, 1H), 6.20 (m, 1H), 5.55 (bs, 1H), 4.15 (multiple

signals, 3H), 2.75−2.6 (multiple signals, 2H), 2.45 (s, 3H), 1.95 (m,

1H), 1.75 (m, 1H). LC−MS: m/z 335.1 [M + H]⁺(calcd 334.39 for

C₁₇H₁₈O₅S).

Matrix-assisted laser desorption ionization time-of-ﬂight mass

spectrometry (MALDI-TOF MS) was performed on a PerSeptive

Biosystems Voyager-DE PRO spectrometer using an α-cyano-4-

hydroxycinnamic acid matrix.

Analytical thin-layer chromatography (TLC) was performed on

Kieselgel F-254 precoated silica plates. Normal-phase column

chromatography was carried out on ﬂash silica gel (40−63 μm

mesh) or regular silica gel (60−200 μm), both acquired from

Screening Devices B.V., or on standardized aluminum oxide 90 from

399

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Article

(R)-7-(Methoxymethoxy)chroman-2-carboxylic Acid Ethyl

Ester (4). At rt, methoxylmethyl chloride (MOMCl; 0.188 mL, 2.07

mmol, 0.92 equiv) was added to a solution of ester 1 (0.50 g, 2.25

mmol) and DIPEA (1.2 mL, ca. 3 equiv) in CH₂Cl₂(25 mL).

376.4 [M + Na]⁺, 643.3 [2M − H-2CH₃O]⁺(calcd 353.37 for

C₂₀H₁₉NO₅).

(R)-(7-(Methoxymethoxy)chroman-2-yl)-methyl Amine (8).

Phthalimide-protected amine 7 (200 mg, 0.566 mmol) was stirred in

EtOH (3 mL) together with hydrazine monohydrate (0.029 mL, 0.58

mmol). The mixture was heated at an oil bath temperature of 80 °C

for 16 h, during which time the reaction mixture changed from a

suspension to a clear solution and back to a suspension. The mixture

was allowed to cool to rt and was ﬁltered; the ﬁltrate was evaporated

and redissolved in CHCl₃. The organic layer was washed once with a

0.1 M NaOH(aq) solution and once with water. The organic layer was

dried with Na₂SO₄and concentrated to give a colorless oil. Yield: 101

mg (80%). ¹H NMR (CDCl₃): δ 6.90 (m, 1H), 6.51 (multiple signals,

2H), 5.08 (s, 2H), 3.90 (m, 1H), 3.45 (s, 3H), 2.90 (d, 2H), 2.8−2.7

(m, 1H), 2.7−2.6 (m, 1H), 1.85 (m, 1H), 1.75 (m, 1H), 1.4 (bs, 2H).

¹³C NMR (CDCl₃): δ 156.4, 155.3, 129.8, 115.4, 108.8, 104.3, 94.4,

1

Monitoring of the progress of the reaction using H NMR and LC−

MS showed that extra MOMCl was required to complete the reaction,

so another portion of MOMCl (0.188 mL, 0.92 equiv) was added.

Finally, the reaction mixture was washed with water and concentrated,

and the remaining product was puriﬁed by silica column

chromatography (2% MeOH/CHCl₃). Yield: 73%. ¹H NMR

(CDCl₃): δ 6.92 (m, 1H), 6.66 (s, 1H), 6.56 (m, 1H), 5.11 (m,

2H), 4.65 (m, 1H), 4.25 (m, 2H), 3.45 (s, 3H), 2.85−2.6 (multiple

signals, 2H), 2.3−2.2 (m, 1H), 2.2−2.1 (m, 1H), 1.29 (t, 3H). ¹³C

NMR (CDCl₃): δ 170.8, 156.6, 154.0, 129.8, 114.7, 109.4, 104.6, 94.5,

73.7, 61.3, 55.8, 24.7, 22.7, 14.1. LC−MS: m/z 267.3 [M + H]⁺and

235.4 [M − CH₃O]⁺(calcd 266.29 for C₁₄H₁₈O₅).

77.6, 55.8, 46.6, 25.0, 24.0. LC−MS: m/z 224.1 [M + H]⁺(calcd

223.27 for C₁₂H₁₇NO₃).

(R)-(7-(Methoxymethoxy)chroman-2-yl)methanol (5). Ethyl

ester 4 (0.551 g, 2.07 mmol) was dissolved in THF (30 mL), after

which LiBH₄(2.39 mL of a 2 M solution in THF, 4.8 mmol) was

added, and the solution was cooled using an ice bath. The reaction

mixture was allowed to heat to rt over about 2 h. After veriﬁcation that

the reaction was completed by monitoring with LC−MS and ¹H

NMR, the reaction mixture was poured in water and extracted multiple

times with ethyl acetate. The organic layer was dried using Na₂SO₄

and concentrated to give a colorless oil. This crude product was

puriﬁed using silica column chromatography (2% MeOH/CHCl₃).

Route A: Coupling of Tosylate 3 to the Appropriate Amines.

(R)-2-[(Benzylamino)methyl]chroman-7-ol (10a). Free Amine.

Reference compound 10a was prepared using a similar method to

that described by Mewshaw.²⁶Benzylamine (0.266 g, 2.48 mmol, 3

equiv) was added to a solution of the tosylate 3 (0.285 g, 0.83 mmol)

in DMSO (8 mL). The reaction mixture was stirred for 16 h at 100 °C,

at which point it was poured in water (35 mL). The obtained

suspension was extracted two times with ethylacetate, the collected

organic layers were washed with water to remove DMSO, and the

ethyl acetate solution was dried with Na₂SO₄. The crude free amine

product was puriﬁed with silica column chromatography (5% MeOH/

CHCl₃). Yield: 90 mg (40%). ¹H NMR (CDCl₃): δ 7.45−7.15

(multiple signals, 5H), 6.8 (m, 1H), 6.35 (multiple signals, 2H), 4.05

(m, 1H), 3.95 (d, 1H), 3.85 (d, 1H), 2.85 (m, 1H), 2.75 (m, 1H),

2.7−2.6 (multiple signals, 2H), 1.85 (m, 1H), 1.65 (m, 1H). ¹³C NMR

(CDCl₃): δ 156.2, 154.5, 138.3, 130.0, 128.6, 128.6, 127.4, 112.6,

109.0, 103.3, 74.3, 54.0, 53.6, 26.2, 23.7. LC−MS: m/z 270.3 [M +

H]⁺(calcd 269.34 for C₁₇H₁₉NO₂). MALDI-TOF-MS: m/z 270.18

[M + H]⁺.

Oxalate Salt. To a solution of free amine 10a (90 mg, 0.334 mmol)

in THF (2 mL) was added a solution of oxalic acid dihydrate (84 mg,

0.67 mmol, 2 equiv) in THF (0.5 mL). Immediately after addition, a

solid precipitated. The mixture was stirred for 3 h, a small amount of

ether was added to improve the precipitation, the mixture was ﬁltrated,

and the white solid residue was washed with diethylether. Yield: 82 mg

(68%). ¹H NMR (DMSO-d₆): δ 8.0−6.0 (broad signal acidic protons),

7.55 (m, 2H), 7.4 (m, 3H), 6.8 (m, 1H), 6.28 (m, 1H), 6.23 (s, 1H),

4.27 (m, 1H), 4.23 (s, 2H), 3.2−3.0 (multiple signals, 2H), 2.75−2.5

(multiple signals, 2H), 1.95 (m, 1H), 1.65 (m, 1H). ¹³C NMR

(DMSO-d₆): δ 164.4, 156.5, 153.9, 132.5, 130.0, 129.9, 128.8, 128.6,

111.9, 108.5, 102.9, 71.5, 50.6, 49.9, 24.7, 22.7. mp 212 °C (dec). LC−

MS: m/z 270.2 [M + H]⁺(calcd 269.34 for C₁₇H₁₉NO₂). UPLC−

HRMS [M + H]⁺: calcd 270.1494; found, 270.1491 (1.1 ppm). UPLC:

99% purity. Anal. (C₁₇H₁₉NO₂+ 1 C₂H₂O₄(fw 359.4)) C, H, N:

Found 63.36, 5.27, 3.65; Calcd 63.51, 5.89, 3.90.

1

Yield: 90% (oil). H NMR (CDCl₃): δ 6.92 (m, 1H), 6.54 (multiple

signals, 2H), 5.08 (s, 2H), 4.05 (m, 1H), 3.85−3.75 (m, 1H), 3.75−

3.65 (m, 1H), 3.43 (s, 3H), 2.85−2.6 (multiple signals, 2H), 2.4−2.3

(bs, 1H), 1.95−1.85 (m, 1H), 1.85−1.7 (m, 1H). ¹³C NMR (CDCl₃):

δ 156.4, 155.0, 129.9, 115.4, 109.0, 104.3, 94.4, 76.4, 65.4, 55.8, 23.8,

23.7. LC−MS: m/z 225.0 [M + H]⁺and 193.1 [M − CH₃O]⁺(calcd

224.26 for C₁₂H₁₆O₄). GC−FID analysis gave a single peak when

using the chiral capillary column (see GC−FID analysis of molecule

1).

Toluene-4-sulfonic Acid, (R)-(7-(Methoxymethoxy)chroman-

2-yl)-methyl Ester (6). Alcohol 5 (0.56 g, 2.50 mmol) was dissolved

in pyridine (7.5 mL), and the solution was cooled to 0 °C. p-

Toluenesulfonylchloride (1.05 g, 5.5 mmol, 2.2 equiv) in pyridine was

added slowly, and the reaction mixture was stirred overnight at 4 °C. A

small amount of ice was added to destroy the excess of p-

toluenesulfonylchloride, the pH of the ice-cooled mixture was brought

to 1 using a concentrated hydrochloric acid solution, and the product

was extracted with CH₂Cl₂. The organic layer was washed with

NaHCO₃, with a solution of CuSO₄to remove traces of pyridine, and

ﬁnally with NaCl(aq). Drying with Na₂SO₄and concentration gave a

slightly pinkish colored oil. Yield: 89%. ¹H NMR (CDCl₃): δ 7.83 (m,

2H), 7.38 (m, 2H), 6.95 (m, 1H), 6.55 (multiple signals, 2H), 6.41 (s,

1H), 5.08 (s, 2H), 4.20 (multiple signals, 3H), 3.45 (s, 3H), 2.85−2.6

(multiple signals, 2H), 2.45 (s, 3H), 2.05−1.95 (m, 1H), 1.85−1.7 (m,

1H). ¹³C NMR (CDCl₃): δ 156.5, 154.4, 144.9, 132.7, 129.9, 129.6,

128.0, 114.7, 110.0, 104.4, 94.4, 72.9, 71.1, 55.9, 23.8, 23.3, 21.6. LC−

MS: m/z 401.3 [M + Na]⁺, 347.4 [M − CH₃O]⁺(calcd 378.44 for

C₁₉H₂₂SO₆).

(R)-N-(7-(Methoxymethoxy)chroman-2-yl)-methyl Phthali-

mide (7). Tosylate 6 (300 mg, 0.79 mmol) and potassium

phthalimide (367 mg, 1.98 mmol, 2.5 equiv) were suspended in

DMF (10 mL). The mixture was stirred for 16 h at 80 °C and was then

allowed to cool to rt. Diethyl ether was added, and the mixture was

washed twice with a saturated aqueous NH₄Cl solution, twice with

water, and one more time with a saturated NaCl solution in water. The

organic layer was dried with Na₂SO₄. After removal of the volatiles, the

obtained white solid was puriﬁed with silica column chromatography

(2% MeOH/CHCl₃). Yield: 78%. ¹H NMR (CDCl₃): δ 7.80 (m, 2H),

7.30 (m, 2H), 6.90 (d, 1H), 6.51 (m, 1H), 6.45 (s, 1H), 5.08 (m, 2H),

4.33 (m, 1H), 4.03 (dd, 1H), 3.84 (dd, 1H), 3.45 (s, 3H), 2.74 (m,

2H), 2.03 (m, 1H), 1.78 (m, 1H). ¹³C NMR (CDCl₃): δ 168.2, 156.4,

154.8, 134.0, 132.0, 129.8, 123.4, 115.0, 109.2, 104.5, 94.4, 73.0, 55.9,

41.9, 25.4, 23.5. LC−MS: m/z 322.2 [M − CH₃O]⁺, 354.3 [M + H]⁺,

(R)-2-[(3-Iodobenzylamino)methyl]chroman-7-ol (11b). Free

Amine. Tosylate 3 (0.38 g, 1.1 mmol) was mixed with commercially

available 3-iodo-benzylamine (0.78 g, 3.3 mmol, 2.9 equiv) and DIPEA

(0.6 mL, 3 equiv) in DMSO (10 mL), and this solution was stirred

overnight at an oil bath temperature of 100 °C. According to LC−MS,

no tosylate starting material was present, and the mixture was poured

in water followed by extraction of the water layer with ethyl acetate

(two times). The ethyl acetate layers were collected, dried with

Na₂SO₄, and concentrated. Finally, the crude product was puriﬁed

with silica column chromatography (2% MeOH/CHCl₃), giving 169

1

mg of an oily product (39%). H NMR (CDCl₃): δ 7.7 (s, 1H), 7.6

(m, 1H), 7.35 (m, 1H), 7.05 (m, 1H), 6.8 (m, 1H), 6.35 (multiple

signals, 2H), 4.05 (m, 1H), 3.85 (d, 1H), 3.80 (d, 1H), 2.85 (m, 1H),

2.75 (m, 1H), 2.7−2.55 (multiple signals, 2H), 1.85 (m, 1H), 1.65 (m,

1H).

Oxalate Salt. Free amine 11b (169 mg, 0.43 mmol) was dissolved

in THF (2 mL), and oxalic acid dihydrate (118 mg, 0.94 mmol, 2.2

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Journal of Medicinal Chemistry

Article

equiv) was added. A precipitation formed, and after some stirring, the

suspension was ﬁltrated to give a white solid product. Yield: 152 mg

(73%). ¹H NMR (DMSO-d₆): δ 8.0−6.0 (broad signal acidic protons),

7.9 (s, 1H), 7.75 (m, 1H), 7.55 (m, 1H), 7.25 (m, 1H), 6.85 (m, 1H),

6.28 (m, 1H), 6.25 (s, 1H), 4.25 (m, 1H), 4.15 (s, 2H), 3.2−3.0

(multiple signals, 2H), 2.75−2.5 (multiple signals, 2H), 1.95 (m, 1H),

1.65 (m, 1H). ¹³C NMR (DMSO-d₆): δ 164.0, 156.5, 154.0, 138.4,

137.3, 135.5, 130.7, 129.9, 129.4, 111.9, 108.5, 102.9, 94.9, 71.7, 50.2,

49.9, 24.7, 22.8. mp 214 °C (dec). LC−MS: m/z 396.2 [M + H]⁺

(calcd 395.24 for C₁₇H₁₈NO₂I). UPLC−HRMS [M + H]⁺: calcd

396.0460; found, 396.0455 (1.3 ppm). UPLC: 94% purity. Anal.

(C₁₇H₁₈NO₂I + 1 C₂H₂O₄(fw 485.3)) C, H, N: Found 47.23, 4.33,

2.38; Calcd 47.03, 4.15, 2.89.

poured into water followed by extraction of the aqueous layer ethyl

acetate. Concentration of the organic layer gave a slightly colored oil

(378 mg), which gave 193 mg of pure product after silica column

chromatography (2% MeOH/CHCl₃). Yield 60%. ¹H NMR (CDCl₃):

δ 6.92 (m, 1H), 6.53 (multiple signals, 2H), 5.10 (s, 2H), 4.11 (m,

1H), 3.44 (s, 3H), 2.90−2.55 (multiple signals, 5H), 2.0−1.9 (m, 1H),

1.8−1.6 (multiple signals, 3H), 1.50 (m, 2H), 1.4−1.2 (multiple

signals, 6H), 0.88 (t, 3H). ¹³C NMR (CDCl₃): δ 156.4, 155.2, 129.9,

115.5, 108.8, 104.3, 94.4, 75.2, 55.8, 54.3, 50.1, 31.7, 30.0, 27.0, 25.7,

24.0, 22.6, 14.0. LC−MS: m/z 308.3 [M + H]⁺(calcd 307.43 for

C₁₈H₂₉NO₃).

(R)-N-[7-(Methoxymethoxy)chroman-2-yl]methyl 4-Iodo-

benzyl Amine (9b). Tosylate 6 (327 mg, 0.865 mmol) was added

to a solution of commercially available p-iodobenzylamine (0.4 g; 1.83

mmol) in DMSO (5 mL) and DIPEA (0.8 mL). The mixture was

heated at 85 °C, kept under argon, and stirred overnight. After cooling

to rt, the reaction mixture was poured into water. Extraction with ethyl

acetate and evaporation of the volatiles yielded a slightly colored oil.

This crude material was puriﬁed with silica column chromatography

(2% MeOH/CHCl₃), yielding 283 mg of the product (75%). ¹H NMR

(CDCl₃): δ 7.64 (m, 2H), 7.12 (m, 2H), 6.95 (m, 1H), 6.53 (multiple

signals, 2H), 5.11 (s, 2H), 4.15 (m, 1H), 3.80 (s, 2H), 3.45 (s, 3H),

2.9−2.6 (multiple signals, 4H), 2.0−1.9 (multiple signals, 2H), 1.75

(m, 1H). ¹³C NMR (CDCl₃): δ 156.4, 155.1, 139.9, 137.3, 130.0,

129.8, 115.4, 108.9, 104.3, 94.4, 92.1, 75.2, 55.8, 53.4, 53.1, 25.6, 23.9.

LC−MS: m/z 440.2 [M + H]⁺(calcd 439.29 for C₁₉H₂₂NO₃I).

(R)-1-(4-Iodophenyl)-4-(4-(7-(methoxymethoxy)chroman-2-

yl)-3-azabutyl)-piperazine (9d). Primary amine 24 (0.585 g, 1.77

mmol, 1.5 equiv) was dissolved in DMSO (5 mL) with 4 equiv of

DIPEA (0.8 mL). Tosylate 6 (442 mg, 1.17 mmol) was added, and the

reaction mixture was heated at 85 °C and kept overnight under a

gentle ﬂow of argon. The reaction mixture was then cooled and

poured into water. Extraction with ethyl acetate and evaporation of the

solvent yielded a slightly colored oil that was puriﬁed with alumina

column chromatography (2% MeOH/CHCl₃). Yield: 250 mg (40%).

¹H NMR (CDCl₃): δ 7.50 (m, 2H), 6.95 (m, 1H), 6.67 (m, 2H), 6.53

(R)-1-(4-Iodophenyl)-4-((7-hydroxychroman-2-yl)methyl)-

piperazine (11d). Free Amine. Tosylate 3 (289 mg, 0.865 mmol)

was dissolved in DMSO (10 mL), and to the solution were added

DIPEA (0.334 g, 2.59 mmol, 2.9 equiv) and 1-(4-iodophenyl)-

piperazine (500 mg, 1.74 mmol, 2.0 equiv). 1-(4-Iodophenyl)-

piperazine was synthesized from 1-phenyl-piperazine according to

Hanson et al.⁷²The reaction mixture was stirred overnight at 100 °C

under an argon atmosphere. According to LC−MS, all of the tosylate

reactant was converted. The reaction mixture was cooled to rt and

poured into water. Extraction with ethyl acetate was followed by

drying of the collected organic layers with Na₂SO₄. Evaporation of the

solvent yielded crude product (844 mg) that was puriﬁed with silica

column chromatography (2% MeOH/CHCl₃). A beige solid was

1

obtained. Yield: 120 mg (31%). H NMR (CDCl₃with a drop of

CD₃OD): δ 7.55 (m, 2H), 6.85 (m, 1H), 6.70 (m, 2H), 6.35 (m, 1H),

6.30 (s, 1H), 4.2 (m, 1H), 3.2 (m, 4H), 2.85−2.6 (multiple signals,

7H), 2.55 (m, 1H), 2.0 (m, 1H), 1.75 (m, 1H). LC−MS: m/z 451.3

[M + H]⁺(calcd 450.32 for C₂₀H₂₃N₂O₂I).

Oxalate Salt. To a solution of free amine 11d (120 mg, 0.267

mmol) in THF (4 mL) was added a solution of oxalic acid dihydrate

(74 mg, 0.59 mmol, 2.2 equiv) in THF (1 mL). Immediately, a white

solid precipitated. After 3 h of stirring, the reaction mixture was

centrifuged. The solvent was decanted, and the residue was washed

with a mixture of THF and ether (1:1). After drying, a white solid

residue was isolated (120 mg, 83%). ¹H NMR (DMSO-d₆): δ 7.5 (m,

2H), 6.8 (multiple signals, 3H), 6.25 (m, 1H), 6.20 (s, 1H), 4.3 (m,

1H), 3.25 (m, 4H), 3.1−2.9 (multiple signals, 6H), 2.8−2.6 (multiple

signals, 2H), 2.0 (m, 1H), 1.65 (m, 1H). ¹³C NMR (DMSO-d₆): δ

162.6, 156.5, 154.3, 150.0, 137.4, 129.9, 118.0, 112.1, 108.2, 102.9,

81.3, 71.8, 60.4, 52.3, 46.4, 25.5, 22.9. mp 177 °C (dec). LC−MS: m/z

451.2 [M + H]⁺(calcd 450.32 for C₂₀H₂₃N₂O₂I). UPLC−HRMS [M

+ H]⁺: calcd 451.0882; found,, 451.0882 (0 ppm). UPLC: 96% purity.

Anal. (C₂₀H₂₃N₂O₂I + 1 C₂H₂O₄(fw 540.4)) C, H, N: Found 48.88,

4.12, 4.81; Calcd 48.90, 4.66, 5.18.

Route B: General Procedure for Coupling of MOM-Protected

Tosylate 6 to the Appropriate Amines. Tosylate 6 was added to a

solution of the amine (typically in a molar excess relative to the

tosylate) in DMSO. DIPEA was also present (in molar excess relative

to the tosylate). The reaction mixture was heated to 80−100 °C under

an inert atmosphere of argon, and the progress of the reaction was

followed with LC−MS for 1 or 2 days. In cases where primary amine

starting compounds were used, LC−MS analysis of the reaction

mixture showed the excess of the primary amine starting compound

and the secondary amine desired product as well as tertiary amine

byproduct. After reaction, the mixture was poured into cold water, and

the product was extracted using ethyl acetate. Evaporation yielded an

oily product that requires column chromatographic puriﬁcation. The

obtained secondary amine products can severely stick to silica, so

alumina was sometimes used as the stationary phase. The applied

eluent was a mixture of CHCl₃with a small percentage of MeOH

(typically 1 to 2% v/v) or a gradient with gently increasing MeOH

content starting from pure CHCl₃.

(multiple signals, 2H), 5.08 (s, 2H), 4.15 (m, 1H), 3.44 (s, 3H), 3.17

(m, 4H), 2.95−2.65 (multiple signals, 6H), 2.65−2.55 (multiple

signals, 6H), 2.0−1.9 (multiple signals, 2H), 1.75 (m, 1H). ¹³C NMR

(CDCl₃): δ 156.4, 155.2, 150.9, 137.7, 129.9, 117.9, 115.5, 108.9,

104.3, 94.4, 81.2, 75.0, 57.8, 55.8, 54.2, 53.0, 48.7, 46.4, 25.7, 24.0.

LC−MS: m/z 538.3 [M + H]⁺(calcd 537.44 for C₂₄H₃₂N₃O₃I).

(R)-N-[7-(Methoxymethoxy)chroman-2-yl]methyl 4-(4-

Fluorobutoxy)benzyl Amine (9e). Amine 21 (0.198 g, 1.00

mmol, 1.2 equiv) was dissolved in DMSO (5 mL) with 5 equiv of

DIPEA (0.75 mL). Tosylate 6 (318 mg, 0.84 mmol) was added, and

the mixture was heated at 95 °C and left to react for 2 days under an

argon atmosphere. The reaction was cooled and poured in water.

Extraction with ethyl acetate yielded a slightly colored oil that was

puriﬁed using silica column chromatography (2% MeOH/CHCl₃) and

subsequent RP C18 chromatography using an aqueous eluent gradient.

1

Yield: 124 mg (36%). H NMR (CDCl₃): δ 7.25 (m, 2H), 6.90 (m,

1H), 6.85 (m, 2H), 6.53 (multiple signals, 2H), 5.05 (s, 2H), 4.6 (m,

1H), 4.45 (m, 1H), 4.15 (m, 1H), 3.95 (m, 2H), 3.8 (s, 2H), 3.4 (s,

3H), 2.9−2.65 (multiple signals, 4H), 2.0−1.7 (multiple signals, 7H).

LC−MS: m/z 404.3 [M + H]⁺(calcd 403.49 for C₂₃H₃₀NO₄F).

(R)-1-(4-(2-Fluoroethoxy)phenyl)-4-((7-(methoxymethoxy)-

chroman-2-yl)-methyl)-piperazine (9g). 1-(4-(2-Fluoroethoxy)-

phenyl)-piperazine was prepared from commercially available 1-(4-

hydroxyphenyl)-piperazine according to Tietze et al.⁷³using the 1-

ﬂuoro-2-tosyloxy-ethane building block that was prepared⁷⁴from

commercially available 2-ﬂuoroethanol. 1-(4-(2-Fluoroethoxy)-phe-

nyl)-piperazine (0.305 g, 1.36 mmol, 1.2 equiv) was dissolved in

DMSO (5 mL) with 3 equiv of DIPEA (0.6 mL). Tosylate 6 (428 mg,

1.13 mmol) was added, and the reaction mixture was heated at 95 °C.

Overnight stirring under an inert argon atmosphere was followed by

cooling the reaction mixture and pouring it in water. Extraction with

ethyl acetate and concentration yielded a slightly colored crude oil that

was puriﬁed using silica column chromatography (2% MeOH/

CHCl₃). Yield: 320 mg (66%). ¹H NMR (CDCl₃): δ 7.0−6.8

(R)-N-[7-(Methoxymethoxy)chroman-2-yl]methyl 1-Hexyl

Amine (9a). n-Hexylamine (0.321 g, 3.18 mmol) was dissolved in

DMSO (5 mL) with 5 equiv of DIPEA (0.9 mL). Tosylate 6 (397 mg,

1.05 mmol) was added, and the reaction mixture was heated overnight

at 95 °C under an argon atmosphere. The mixture was cooled and

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(multiple signals, 5H), 6.56 (multiple signals, 2H), 5.12 (s, 2H), 4.79

(m, 1H), 4.67 (m, 1H), 4.20 (m, 2H), 4.15 (m, 1H), 3.46 (s, 3H), 3.13

(m, 4H), 2.9−2.55 (multiple signals, 8H), 2.1−2.0 (m, 1H), 1.85−1.7

(m, 1H). LC−MS: m/z 431.4 [M + H]⁺(calcd 430.52 for

C₂₄H₃₁N₂O₄F).

221 mg of product (60%). H NMR (CDCl₃): δ 7.26 (m, 1H), 7.23

(m, 1H), 6.85 (multiple signals, 2H), 6.80 (m, 1H), 6.53 (multiple

signals, 2H), 5.10 (s, 2H), 4.56 (bs, 1H), 4.45 (t, 1H), 4.15 (m, 1H),

4.06 (t, 2H), 3.85 (dd, 2H), 3.44 (s, 3H), 2.90−2.6 (multiple signals,

4H), 2.05 (bs, 1H), 2.0−1.85 (multiple signals, 5H), 1.8−1.7 (m, 1H).

¹³C NMR (CDCl₃): δ 156.9, 156.4, 155.3, 129.9, 129.8, 128.3, 128.1,

( R ) - 1 - ( 4 - ( 2 - F l u o r o e t h o x y ) p h e n y l ) - 4 - ( 4 - ( 7 -

(methoxymethoxy)chroman-2-yl)-3-azabutyl)-piperazine (9h).

Primary amine 26 (0.364 g, 1.36 mmol, 1.2 equiv) was dissolved in

DMSO (5 mL) with 3 equiv of DIPEA (0.6 mL). Tosylate 6 (428 mg,

1.13 mmol) was added, and the reaction mixture was heated overnight

at 100 °C under a gentle ﬂow of argon. The reaction mixture was

cooled and poured into water. Extraction with ethyl acetate and

concentration gave a slightly colored oil (0.36 g). Finally, puriﬁcation

with alumina column chromatography (2% MeOH/CHCl₃) gave a

yield of 47% of pure product (250 mg). ¹H NMR (CDCl₃): δ 7.0−6.8

(multiple signals, 5H), 6.54 (multiple signals, 2H), 5.10 (s, 2H), 4.79

(m, 1H), 4.67 (m, 1H), 4.20 (m, 1H), 4.13 (m, 2H), 3.44 (s, 3H), 3.12

(m, 4H), 3.0−2.5 (multiple signals, 12H), 2.05−1.9 (multiple signals,

2H), 1.75 (m, 1H). LC−MS: m/z 474.4 [M + H]⁺(calcd 473.59 for

C₂₆H₃₆N₃O₄F).

120.4, 115.5, 111.0, 108.8, 104.3, 94.5, 84.5, 82.8, 75.6, 67.1, 55.8, 53.6,

49.4, 27.4, 27.2, 25.7, 25.4, 25.3, 24.0. LC−MS: m/z 404.3 [M + H]⁺

(calcd 403.49 for C₂₃H₃₀NO₄F).

Routes B and C: General Procedure for Deprotection of the

MOM Group. The appropriate MOM-protected compound 9 (100 to

300 mg) was dissolved in an 4 M HCl solution in dioxane (2−5 mL;

HCl in high molar excess) and isopropanol (2−5 mL). The reaction

mixture was stirred for about 3 h at rt, and a N₂ﬂow was maintained

over the reaction mixture. From time to time, N₂was bubbled through

the solution to remove volatile reaction products from the mixture.

The reaction mixture may turn hazy because of the precipitation of the

ammonium salt. After completion of the reaction (the reaction was

monitored by LC−MS), the mixture was added to water, and the pH

was brought to slightly basic by addition of a NaOH solution. The

product was isolated from the water layer by several extractions using

CHCl₃or CH₂Cl₂with some added THF followed by drying of the

collected organic layers with Na₂SO₄and evaporation of the solvent.

General Procedure for Preparation of the Oxalate Salt. The

isolated free amine compound was dissolved in THF, and a solution of

oxalic acid dihydrate in THF was added. The reaction mixture turned

hazy because of precipitation of the oxalate ammonium salt. After

about 3−5 h of stirring, the reaction mixture was centrifuged, and the

solvent was pipetted or decanted oﬀ. The residue was stirred in a fresh

1:1 mixture of diethyl ether and THF and was centrifuged again. The

solvent was removed, and the precipitate was dried to yield a white,

oﬀ-white, or slightly brownish powder product.

Route C: General Procedure for Coupling MOM-Protected

Amine 8 to the Appropriate Aldehydes. MOM-protected primary

amine 8 and the appropriate aldehyde (1 equiv) were mixed in MeOH

and stirred overnight at rt. During this step, the imine was formed. The

1

conversion was followed with H NMR by inspecting the aldehyde

signal (because of the reversibility of the reaction in the presence of

water, monitoring by LC−MS was not possible). After completion of

imine formation, 1.1 equiv of NaBH₄was added, and the reaction

1

mixture was stirred at rt for 1 to 3 h. H NMR was used to monitor

imine conversion. The reaction mixture was poured in water, and the

aqueous solution was stirred for 1 h and then extracted with CH₂Cl₂

or, alternatively, with CHCl₃. Because of the presence of boron salts, it

may be necessary to ﬁrst adjust the pH of the water layer to become

acidic (pH 1) and then back to slightly basic (pH 8) and then perform

the extraction at this higher pH. Drying of combined organic layers

with Na₂SO₄was followed by removal of the solvents to yield the

product.

(R)-N-[7-(Methoxymethoxy)chroman-2-yl]methyl 4-Iodo-

benzyl Amine (9b). 4-Iodobenzaldehyde was prepared from

commercially available p-iodo benzoic acid using a two-step

procedure⁷⁵via the p-iodo benzyl alcohol. Amine 8 (418 mg, 1.87

mmol) and freshly prepared 4-iodobenzaldehyde (433 mg, 1.87

mmol) were stirred in MeOH (10 mL). NaBH₄(77.7 mg, 2.06 mmol)

was added followed by extraction with CHCl₃and then CH₂Cl₂. An

oily product was obtained (766 mg, 93%). The analytical data are

identical to those reported for the same 9b product prepared and

isolated via route B (vide supra).

(R)-N-[7-(Methoxymethoxy)chroman-2-yl]methyl 2-Iodo-

benzyl Amine (9c). 2-Iodobenzaldehyde was prepared from its

commercially available benzyl alcohol precursor.⁷⁶Amine 8 (192 mg,

0.86 mmol) was stirred with 2-iodobenzaldehyde (200 mg, 0.86

mmol) in MeOH (5 mL). According to the ¹H NMR, ca. 4% of

aldehyde was still present; nevertheless, NaBH₄(42 mg, 1.11 mmol,

1.3 equiv) was added. After extraction, the crude yellow oil (331 mg)

was puriﬁed by silica column chromatography (ethyl acetate/hexane

1:1) to yield 209 mg (56%) of product. ¹H NMR (CDCl₃): δ 7.80 (m,

1H), 7.40 (m, 1H), 7.28 (m, 1H), 6.95 (multiple signals, 2H), 6.53

(multiple signals, 2H), 5.08 (s, 2H), 4.15 (m, 1H), 3.87 (s, 2H), 3.45

(s, 3H), 2.9−2.6 (multiple signals, 4H), 2.05−1.85 (multiple signals,

2H), 1.85−1.75 (m, 1H). ¹³C NMR (CDCl₃): δ 156.4, 155.2, 142.0,

139.3, 129.8, 129.4, 128.7, 128.2, 115.5, 108.9, 104.4, 99.5, 94.4, 75.2,

58.0, 55.8, 53.2, 25.6, 23.9. LC−MS: m/z 440.2 [M + H]⁺(calcd

439.29 for C₁₉H₂₂NO₃I).

(R)-N-[7-(Methoxymethoxy)chroman-2-yl]methyl 2-(4-

Fluorobutoxy)benzyl Amine (9f). Amine 8 (205 mg, 0.92 mmol)

was stirred with 2-(4-ﬂuorobutoxy) benzaldehyde 22 (179 mg, 0.92

mmol) in MeOH (5 mL). According to ¹H NMR, 10% of the

aldehyde was still present; nevertheless, NaBH₄(45 mg, 1.19 mmol,

1.3 equiv) was added. The crude yellowish oil (340 mg) was puriﬁed

with silica column chromatography (ethyl acetate/hexane 1:1) to yield

(R)-2-[(Hexylamino)methyl]chroman-7-ol (10b). Free Amine.

Reference compounds 10b was already reported by Mewshaw,²⁶

although the racemic mixture was prepared and not the (R)

enantiomer. Furthermore, we prepared 10b via its MOM-protected

precursor, whereas Mewshaw prepared the racemate via its benzyl-

protected precursor. Compound 9a (121 mg, 0.39 mmol) was

dissolved in a solution of 4 M HCl in dioxane (2 mL) and isopropanol

1

(3 mL). Yield after work up: 100 mg (96%). H NMR (CDCl₃): δ

6.78 (m, 1H), 6.45 (m, 1H), 6.32 (m, 1H), 4.07 (m, 1H), 2.90 (t, 1H),

2.8−2.5 (multiple signals, 5H), 1.85 (m, 1H), 1.75−1.5 (multiple

signals, 3H), 1.4−1.2 (multiple signals, 6H), 0.88 (t, 3H). ¹³C NMR

(CDCl₃): δ 156.7, 154.5, 130.0, 112.2, 109.4, 103.0, 74.2, 54.2, 50.0,

31.6, 29.2, 26.9, 26.3, 23.7, 22.5, 14.0. LC−MS: m/z 264.3 [M + H]⁺

(calcd 263.38 for C₁₆H₂₅NO₂).

Oxalate Salt. Free amine 10b (120 mg, 0.456 mmol) was dissolved

in THF (2 mL), and oxalic acid dihydrate (86 mg, 0.68 mmol, 1.5

equiv) in THF (1 mL) was added. A white solid precipitate was

isolated (115 mg, 71%). ¹H NMR (DMSO-d₆): δ 9.0−6.0 (broad

signal, acidic protons), 6.85 (m, 1H), 6.28 (m, 1H), 6.23 (s, 1H), 4.25

(m, 1H), 3.25−3.05 (multiple signals, 2H), 2.9 (m, 2H), 2.75−2.5

(multiple signals, 2H), 1.95 (m, 1H), 1.65 (multiple signals, 3H), 1.25

(m, 6H), 0.9 (t, 3H). ¹³C NMR (DMSO-d₆): δ 164.6, 156.6, 153.9,

129.9, 111.9, 108.5, 102.9, 71.3, 50.1, 47.3, 30.7, 25.7, 25.2, 24.7, 22.7,

21.9, 13.9. mp 152 °C (dec). LC−MS: m/z 264.3 [M + H]⁺(calcd

263.38 for C₁₆H₂₅NO₂). UPLC−HRMS [M + H]⁺: calcd 264.1964;

found, 264.1966 (0.8 ppm). UPLC: 92% purity. Anal. (C₁₆H₂₅NO₂+ 1

C₂H₂O₄(fw 353.4)) C, H, N: Found 60.59, 7.68, 3.55; Calcd 61.18,

7.70, 3.96.

(R)-2-[(4-Iodobenzylamino)methyl]chroman-7-ol (11a). Free

Amine. Compound 9b (171 mg, 0.389 mmol) was deprotected in 4 M

HCl in dioxane (3.5 mL) and isopropanol (3.5 mL). After workup, an

1

oil was obtained in 80% yield (123 mg). H NMR (CD₃OD): δ 7.65

(m, 2H), 7.15 (m, 2H), 6.80 (m, 1H), 6.27 (m, 1H), 6.23 (m, 1H),

4.05 (m, 1H), 3.80 (bs, 2H), 2.9−2.5 (multiple signals, 4H), 1.93 (m,

1H), 1.65 (m, 1H). ¹³C NMR (CDCl₃): δ 156.0, 154.4, 137.8, 137.7,

130.6, 130.1, 112.7, 109.1, 103.2, 93.1, 74.2, 53.6, 53.5, 26.2, 23.6. LC−

MS: m/z 396.2 [M + H]⁺(calcd 395.24 for C₁₇H₁₈NO₂I). MALDI-

TOF-MS: m/z 396.0 [M + H]⁺.

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Oxalate Salt. Free amine 11a (118 mg, 0.299 mmol) was dissolved

in THF (4 mL), and oxalic acid dihydrate (49 mg, 0.389 mmol) in

THF (1 mL) was added. A white solid was isolated (90 mg, 62%). ¹H

NMR (DMSO-d₆): δ 8.0−6.0 (broad signal acidic protons), 7.75 (m,

2H), 7.30 (m, 2H), 6.80 (m, 1H), 6.28 (m, 1H), 6.25 (s, 1H), 4.25 (m,

1H), 4.15 (s, 2H), 3.2−3.0 (multiple signals, 2H), 2.75−2.5 (multiple

signals, 2H), 1.95 (m, 1H), 1.65 (m, 1H). ¹³C NMR (DMSO-d₆): δ

164.2, 156.5, 154.0, 137.4, 132.6, 132.2, 129.9, 111.9, 108.5, 102.9,

95.3, 71.7, 50.1, 50.0, 24.7, 22.8. mp 211 °C (dec). LC−MS: m/z

396.1 [M + H]⁺(calcd 395.24 for C₁₇H₁₈NO₂I). UPLC−HRMS [M +

H]⁺: calcd 396.0460; found, 396.0466 (1.5 ppm). UPLC: 95% purity.

Anal. (C₁₇H₁₈NO₂I + 1 C₂H₂O₄(fw 485.3)) C, H, N: Found 48.39,

4.53, 2.32; Calcd 47.03, 4.15, 2.89.

(2 mL). No starting compound was present according to LC−MS

analysis, but there were some impurities present. After aqueous

workup, the crude product was puriﬁed by silica column

chromatography (eluent: heptane/THF 1:1). Yield: 45 mg (41%) of

1

a solid. H NMR (CDCl₃): δ 7.35 (d, 2H), 6.90 (d, 2H), 6.78 (m,

1H), 6.38 (multiple signals, 2H), 4.60 (m, 1H), 4.45 (m, 1H), 4.08 (m,

1H), 4.00 (t, 2H), 3.90 (dd, 2H), 2.9−2.5 (multiple signals, 4H), 2.0−

1.75 (multiple signals, 5H), 1.7 (m, 1H). ¹³C NMR (CDCl₃): δ 158.3,

156.3, 154.5, 130.2, 130.1, 130.0, 114.6, 112.4, 109.2, 103.3, 84.6, 82.9,

74.2, 67.2, 53.6, 53.5, 27.3, 27.1, 26.3, 25.2, 23.7. LC−MS: m/z 360.0

[M + H]⁺(calcd 359.44 for C₂₁H₂₆NO₃F). MALDI-TOF-MS: m/z

360.1 [M + H]⁺.

Oxalate Salt. Free amine 12a (87 mg, 0.242 mmol) was dissolved

in THF (2 mL), and oxalic acid dihydrate (46 mg, 0.365 mmol, 1.5

equiv) in THF (1 mL) was added. The residue was a white solid (68

mg, 68%). ¹H NMR (DMSO-d₆): δ 7.5−5.0 (broad signal acidic

protons), 7.45 (d, 2H), 6.95 (d, 2H), 6.8 (m, 1H), 6.3 (m, 1H), 6.25

(s, 1H), 4.55 (m, 1H), 4.45 (m, 1H), 4.25 (m, 1H), 4.1 (s, 2H), 4.0

(m, 2H), 3.2−3.0 (multiple signals, 2H), 2.75−2.6 (multiple signals,

2H), 1.95 (m, 1H), 1.85−1.7 (multiple signals, 4H), 1.65 (m, 1H). ¹³C

NMR (DMSO-d₆): δ 164.4, 158.9, 156.5, 153.9, 131.6, 129.9, 124.0,

114.5, 111.9, 108.5, 102.9, 84.4, 82.8, 71.5, 67.1, 50.0, 49.5, 26.7, 26.5,

24.7, 22.7. mp 177 °C. LC−MS: m/z 360.1 [M + H]⁺(calcd 359.44

for C₂₁H₂₆NO₃F). UPLC−HRMS [M + H]⁺: calcd 360.1975; found,

360.1970 (1.4 ppm). UPLC: 95% purity. Anal. (C₂₁H₂₆NO₃F +

C₂H₂O₄fw 449.5)) C, H, N: Found 60.40, 6.00, 2.76; Calcd 61.47,

6.28, 3.12.

(R)-2-[(2-Iodobenzylamino)methyl]chroman-7-ol (11c). Free

Amine. Compound 9c (209 mg, 0.48 mmol) was dissolved in a

mixture of 4 M HCl dioxane (2.4 mL) and isopropanol (2.4 mL).

After 1 h, extra amounts of 4 M HCl in dioxane (1 mL) and

isopropanol (l mL) were added. After workup, a glassy product was

1

obtained in quantitative yield (188 mg). H NMR (CDCl₃): δ 7.82

(m, 1H), 7.40 (m, 1H), 7.35 (m, 1H), 6.95 (m, 1H), 6.76 (m, 1H),

6.35 (m, 1H), 6.30 (m, 1H), 4.10 (m, 1H), 4.05 (d, 1H), 3.90 (d, 1H),

2.9−2.75 (multiple signals, 2H), 2.75−2.5 (multiple signals, 2H), 1.8

(m, 1H), 1.65 (m, 1H). ¹³C NMR (CDCl₃): δ 155.9, 154.7, 140.6,

139.7, 130.2, 130.0, 129.3, 128.5, 112.8, 108.8, 103.4, 99.7, 74.4, 57.8,

53.0, 26.1, 23.7. LC−MS: m/z 396.2 [M + H]⁺(calcd 395.24 for

C₁₇H₁₈NO₂I).

Oxalate Salt. Free amine 11c (188 mg, 0.48 mmol) was dissolved

in THF (2 mL), and oxalic acid dihydrate (132 mg, 1.05 mmol, 2.2

equiv) in THF (1 mL) was added. A white solid residue was isolated

(219 mg, 94%). ¹H NMR (DMSO-d₆): δ 8.0−6.0 (broad signal acidic

protons), 7.95 (m, 1H), 7.60 (m, 1H), 7.45 (m, 1H), 7.13 (m, 1H),

6.80 (m, 1H), 6.30−6.25 (multiple signals, 2H), 4.3−4.1 (multiple

signals, 3H), 3.1 (m, 2H), 2.7−2.6 (multiple signals, 2H), 2.05 (m,

1H), 1.65 (m, 1H). ¹³C NMR (DMSO-d₆): δ 163.6, 156.5, 154.1,

139.4, 136.8, 130.3, 130.2, 129.9, 128.6, 112.0, 108.4, 103.0, 100.9,

72.3, 55.5, 50.9, 24.9, 22.8. mp 209 °C (dec). LC−MS: m/z 396.2 [M

+ H]⁺(calcd 395.24 for C₁₇H₁₈NO₂I). UPLC−HRMS [M + H]⁺:

calcd 396.0460; found, 396.0466 (1.5 ppm). UPLC: 97% purity. Anal.

(C₁₇H₁₈NO₂I + 1 C₂H₂O₄(fw 485.3)) C, H, N: Found 46.81, 4.03,

2.56; Calcd 47.03, 4.15, 2.89.

(R)-2-[(2-(4-Fluorobutoxy)benzylamino)methyl]chroman-7-

ol (12b). Free Amine. Compound 9f (227 mg, 0.563 mmol) was

dissolved in a mixture of 4 M HCl dioxane (2.4 mL) and isopropanol

(2.4 mL). After 1 h, an extra amount of 4 M HCl in dioxane (1.5 mL)

was added. After workup, an oily product was obtained (202 mg,

1

100%). H NMR (CDCl₃): δ 7.25 (multiple signals, 2H), 6.95 (m,

1H), 6.90 (m, 1H), 6.75 (m, 1H), 6.40 (m, 1H), 6.27 (m, 1H), 4.45

(m, 1H), 4.36 (m, 1H), 4.1−4.0 (multiple signals, 4H), 3.80 (d, 1H),

2.85−2.5 (multiple signals, 4H), 2.0−1.75 (multiple signals, 5H), 1.65

(m, 1H). ¹³C NMR (CDCl₃): δ 157.0, 156.5, 154.6, 130.3, 129.9,

128.8, 126.5, 120.6, 112.4, 111.1, 109.1, 103.3, 84.5, 82.8, 74.4, 67.2,

53.2, 49.1, 27.4, 27.2, 26.3, 25.4, 25.3, 23.7. LC−MS: m/z 360.3 [M +

H]⁺(calcd 359.44 for C₂₁H₂₆NO₃F).

(R) 1-(4-Iodophenyl)-4-(4-(7-hydroxychroman-2-yl)-3-azabu-

tyl)-piperazine (11e). Free Amine. Compound 9d (115 mg, 0.214

mmol) was dissolved in 4 M HCl in dioxane (3.5 mL) and isopropanol

(4 mL). The reaction was not complete after 3 h, so the reaction

mixture was evaporated to yield an oily product. A fresh solution of 4

M HCl in dioxane was added, and after a further 90 min of stirring

under N₂, the reaction was complete. A quantitative yield of an oil was

Oxalate Salt. Free amine 12b (202 mg, 0.563 mmol) was dissolved

in THF (2 mL), and oxalic acid dihydrate (156 mg, 1.24 mmol, 2.2

equiv) in THF (1 mL) was added. In this case, no precipitate was

formed after addition or after 3 h of stirring at rt, so diethylether was

added to induce precipitation. Centrifugation, washing, and drying

1

gave a white solid residue (159 mg, 63%). H NMR (DMSO-d₆): δ

1

8.0−6.0 (broad signal acidic protons), 7.45 (m, 1H), 7.4 (m, 1H), 7.05

(m, 1H), 7.0 (m, 1H), 6.85 (m, 1H), 6.28 (m, 1H), 6.23 (s, 1H), 4.5

(m, 1H), 4.4 (m, 1H), 4.3 (m, 1H), 4.2 (q, 2H), 4.1 (m, 2H), 3.2−3.1

(multiple signals, 2H), 2.75−2.6 (multiple signals, 2H), 1.95 (m, 1H),

1.85−1.7 (multiple signals, 4H), 1.65 (m, 1H). ¹³C NMR (DMSO-d₆):

δ 164.2, 156.9, 156.5, 153.9, 131.3, 130.6, 129.9, 125.1, 120.4, 111.9,

111.8, 108.5, 102.9, 84.4, 82.8, 71.5, 67.4, 50.2, 45.6, 26.7, 26.5, 24.7,

22.7. mp 54 °C. LC−MS: m/z 360.3 [M + H]⁺(calcd 359.44 for

C₂₁H₂₆NO₃F). UPLC−HRMS [M + H]⁺: calcd 360.1975; found,

360.1978 (0.8 ppm). UPLC: 99% purity. Anal. (C₂₁H₂₆NO₃F +

C₂H₂O₄(fw 449.5)) C, H, N: Found 61.27, 6.10, 2.76; Calcd 61.47,

6.28, 3.12.

(R)-1-(4-(2-Fluoroethoxy)phenyl)-4-((7-hydroxychroman-2-

yl)-methyl)-piperazine (12c). Free Amine. Compound 9g (120 mg,

0.279 mmol) was dissolved in a solution of 4 M HCl in dioxane (2.5

mL) and isopropanol (4 mL). A slightly colored solid was obtained.

¹H NMR (THF-d₈): δ 7.75 (bs, 1H), 6.8−6.6 (multiple signals, 5H),

obtained after workup. H NMR (CDCl₃): δ 7.50 (m, 2H), 6.77 (m,

1H), 6.64 (m, 2H), 6.49 (s, 1H), 6.28 (m, 1H), 4.08 (m, 1H), 3.10 (m,

2H), 3.05 (m, 2H), 2.9−2.3 (multiple signals, 14H), 1.83 (m, 1H),

1.65 (m, 1H). ¹³C NMR (CDCl₃): δ 156.4, 154.5, 150.9, 137.7, 129.9,

117.7, 112.2, 109.2, 103.4, 81.0, 74.5, 56.4, 54.0, 52.6, 48.7, 46.0, 26.3,

23.7. LC−MS: m/z 494.2 [M + H]⁺(calcd 493.39 for C₂₂H₂₈N₃O₂I).

MALDI-TOF-MS: m/z 494.0 [M + H]⁺.

Oxalate Salt. Free amine 11e (105 mg, 0.214 mmol) was dissolved

in THF (4 mL), and oxalic acid dihydrate (63 mg, 0.50 mmol, 2.3

equiv) in THF (1 mL) was added. A white solid was isolated (93 mg,

57%). ¹H NMR (DMSO-d₆): δ 8.0−6.0 (broad signal acidic protons),

7.5 (m, 2H), 6.85 (m, 1H), 6.8 (m, 2H), 6.28 (m, 1H), 6.23 (s, 1H),

4.3 (m, 1H), 3.35−3.1 (multiple signals, 8H), 2.8−2.6 (multiple

signals, 8H), 2.0 (m, 1H), 1.65 (m, 1H). ¹³C NMR (DMSO-d₆): δ

162.9, 156.5, 153.8, 150.4, 137.3, 130.0, 117.8, 111.9, 108.6, 102.9,

80.7, 71.2, 52.9, 52.2, 50.2, 47.4, 43.7, 24.7, 22.7. mp 201 °C. LC−MS:

m/z 494.2 [M + H]⁺(calcd 493.39 for C₂₂H₂₈N₃O₂I). UPLC−HRMS

[M + H]⁺: calcd 494.1304; found, 494.1294 (2.0 ppm). UPLC: 99%

purity. Anal. (C₂₂H₂₈N₃O₂I + 3 C₂H₂O₄(fw 763.5)) C, H, N: Found

43.37, 4.77, 5.44; Calcd 44.05, 4.49, 5.50.

6.08 (m, 1H), 6.03 (m, 1H), 4.58 (m, 1H), 4.45 (m, 1H), 4.1−3.9

(multiple signals, 3H), 2.95 (bs, 4H), 2.7−2.4 (multiple signals, 8H),

1.98 (m, 1H), 1.6 (m, 1H). ¹³C NMR (THF-d₈): δ 157.7, 156.5, 153.4,

147.2, 130.2, 118.3, 115.7, 113.2, 108.5, 103.8, 83.5, 81.9, 74.6, 68.5,

68.3, 63.1, 55.0, 51.1, 27.1, 24.5. LC−MS: m/z 387.3 [M + H]⁺(calcd

386.47 for C₂₂H₂₇N₂O₃F).

(R)-2-[(4-(4-Fluorobutoxy)benzylamino)methyl]chroman-7-

ol (12a). Free Amine. Compound 9e (124 mg, 0.308 mmol) was

dissolved in a solution of 4 M HCl in dioxane (2 mL) and isopropanol

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Oxalate Salt. Free amine 12c (108 mg, 0.279 mmol) was dissolved

in THF (4 mL), and oxalic acid dihydrate (155 mg, 1.23 mmol, ca. 4.5

equiv) in THF (1 mL) was added. A white solid residue was obtained

(119 mg, 90%). ¹H NMR (DMSO-d₆): δ 8.0−6.0 (broad signal acidic

protons), 6.95−6.8 (multiple signals, 5H), 6.28 (m, 1H), 6.2 (s, 1H),

4.75 (m, 1H), 4.65 (m, 1H), 4.35 (m, 1H), 4.15 (m, 1H), 4.1 (m, 1H),

3.25−3.0 (multiple signals, 10H), 2.75−2.6 (multiple signals, 2H),

1.98 (m, 1H), 1.6 (m, 1H). ¹³C NMR (DMSO-d₆): δ 163.0, 156.5,

154.2, 152.1, 144.8, 129.9, 117.6, 115.1, 112.1, 108.3, 102.9, 83.1, 81.4,

71.5, 67.4, 67.2, 60.2, 52.5, 47.8, 25.4, 22.8. mp 105 °C. LC−MS: m/z

387.3 [M + H]⁺(calcd 386.47 for C₂₂H₂₇N₂O₃F). UPLC−HRMS [M

+ H]⁺: calcd 387.2084; found, 387.2078 (1.5 ppm). UPLC: 95%

purity. Anal. (C₂₂H₂₇N₂O₃F + C₂H₂O₄(fw 476.5)) C, H, N: Found

60.33, 5.73, 5.54; Calcd 60.50, 6.14, 5.88.

(R)-1-(4-(2-Fluoroethoxy)phenyl)-4-(4-(7-hydroxychroman-

2-yl)-3-azabutyl)-piperazine (12d). Free Amine. Compound 9h

(200 mg, 0.422 mmol) was dissolved in a 4 M HCl solution in dioxane

(2 mL) and isopropanol (2 mL). The reaction was not complete after

3 h, so an extra portion of 4 M HCl solution in dioxane was added (2

mL), and the mixture was stirred overnight at rt under N₂. A slightly

colored oil was obtained after workup. Yield: 160 mg (88%). ¹H NMR

(CDCl₃): δ 6.85 (m, 4H), 6.8 (m, 1H), 6.5 (m, 1H), 6.3 (m, 1H), 4.8

(m, 1H), 4.7 (m, 1H), 4.2 (m, 1H), 4.15 (multiple signals, 2H), 3.1−

2.4 (multiple signals, 16H), 1.8 (m, 1H), 1.65 (m, 1H). LC−MS: m/z

430.3 [M + H]⁺(calcd 429.54 for C₂₄H₃₂N₃O₃F).

in water. The suspension was ﬁltrated, and the residue was puriﬁed

with silica column chromatography (2% MeOH/CHCl₃). Yield: 1.94 g

(73%). Analytical data were in accordance with those reported in a

patent.⁷⁷¹H NMR (CDCl₃): δ 7.5−7.3 (multiple signals, 5H), 6.9

(multiple signals, 4H), 5.02 (s, 2H), 3.56 (t, 4H), 3.0 (t, 4H), 1.48 (s,

9H). LC−MS: m/z 369.2 [M + H]⁺(calcd 368.48 for C₂₂H₂₈N₂O₃).

1-(4-Benzyloxyphenyl)piperazine (16). Compound 15 (1.75 g,

4.75 mmol) was mixed with a 4 N HCl dioxane solution (30 mL) and

was stirred for 1 h at rt. The reaction mixture was ﬁltrated and washed

with ethyl acetate to aﬀord the HCl salt, a white solid residue.

Extraction using chloroform and a basic aqueous solution gave the

amine product. Yield: 1.3 g (100%). Analytical data were in accordance

with those reported in a patent.⁷⁷¹H NMR (CDCl₃): δ 7.5−7.3

(multiple signals, 5H), 6.9 (multiple signals, 4H), 5.0 (s, 2H), 3.0 (bs,

8H), 1.5 (bs, 1H). LC−MS: m/z 269.3 [M + H]⁺(calcd 268.36 for

C₁₇H₂₀N₂O).

1-(4-Benzyloxyphenyl)-4-(2-N-(tert-butyl-carbamate)-ethyl)-

piperazine (17). tert-Butyl N-(2-bromoethyl)carbamate was prepared

according to a literature procedure.⁷⁸Secondary amine 16 (0.4 g; 1.49

mmol) was mixed with DMF (5 mL), DIPEA (2.6 mL, 14.9 mmol, 10

equiv), and tert-butyl N-(2-bromoethyl)carbamate (0.33 g, 1.49

mmol), yielding a suspension. The mixture was stirred at 40 °C for

72 h and turned into a clear brown solution. Pouring into water gave a

suspension that was extracted with diisopropyl ether. The organic layer

was dried with Na₂SO₄and evaporated to dryness; the crude product

residue was puriﬁed using silica column chromatography (2:1

Oxalate Salt. Free amine 12d (160 mg, 0.373 mmol) was dissolved

in THF (4 mL), and oxalic acid dihydrate (300 mg, 2.38 mmol, ca. 6

equiv) in THF (1 mL) was added. The isolated residue was a white

1

pentane/THF). Yield: 369 mg (60%). H NMR (CDCl₃): δ 7.5−7.3

(multiple signals, 5H), 6.9 (multiple signals, 4H), 5.0 (s, 2H), 4.95 (bs,

1H), 3.25 (m, 2H), 3.05 (m, 4H), 2.6 (m, 4H), 2.5 (t, 2H), 1.45 (s,

9H). ¹³C NMR (CDCl₃): δ 152.9, 145.9, 137.3, 128.5, 127.8, 127.4,

125.5, 118.0, 115.5, 79.2, 70.5, 57.2, 53.0, 50.5, 37.1, 28.4. LC−MS: m/

z 412.3 [M + H]⁺(calcd 411.55 for C₂₄H₃₃N₃O₃).

1

solid (225 mg, 86%). H NMR (DMSO-d₆): δ 8.0−6.0 (broad signal

acidic protons), 6.9−6.8 (multiple signals, 5H), 6.28 (m, 1H), 6.24 (s,

1H), 4.75 (m, 1H), 4.64 (m, 1H), 4.30 (m, 1H), 4.20 (m, 1H), 4.12

(m, 1H), 3.3−3.1 (multiple signals, 4H), 3.1 (m, 4H), 2.8−2.5

(multiple signals, 8H), 1.95 (m, 1H), 1.65 (m, 1H). ¹³C NMR

(DMSO-d₆): δ 162.4, 156.5, 153.8, 151.8, 145.4, 130.0, 117.3, 115.1,

111.9, 108.6, 102.9, 83.1, 81.5, 71.2, 67.5, 67.3, 52.9, 52.4, 50.3, 49.0,

43.6, 24.7, 22.7. mp 198 °C (dec). LC−MS: m/z 430.3 [M + H]⁺

(calcd 429.54 for C₂₄H₃₂N₃O₃F). UPLC−HRMS [M + H]⁺: calcd

430.2506; found, 430.2499 (1.6 ppm). UPLC: 99% purity. Anal.

(C₂₄H₃₂N₃O₃F + 3 C₂H₂O₄(fw 699.6)) C, H, N: Found 52.61, 5.40,

5.46; Calcd 51.51, 5.48, 6.01.

1-(4-Benzyloxyphenyl)-4-(2-aminoethyl)-piperazine (18).

Boc-protected amine 17 (0.74 g; 1.79 mmol) was dissolved in

CH₂Cl₂(8 mL) and TFA (2 mL), and the solution was stirred for 1 h

at rt. Evaporation of the solvents and redissolution into (8 mL) and

1

TFA (2 mL) was followed by further stirring at rt until H NMR and

TLC analyses showed that the Boc group was deprotected. Dilution

with CHCl₃and washing of the organic layer with a 0.1 M NaOH (pH

10) to which NaCl was added to enhance the phase separation was

followed by drying of the organic layer with Na₂SO₄and evaporation

of the volatiles to yield a beige solid (528 mg, 95%). ¹H NMR

(CDCl₃): δ 7.5−7.3 (multiple signals, 5H), 6.9 (multiple signals, 4H),

5.0 (s, 2H), 3.15 (m, 4H), 2.8 (m, 2H), 2.6 (m, 4H), 2.5 (t, 2H), 1.65

(bs, 2H). LC−MS: m/z 312.3 [M + H]⁺(calcd 311.43 for

C₁₉H₂₈N₃O).

Synthesis of the Precursors to [¹⁸F]12a and [¹⁸F]12d:

Phenols 13a and 13b. Precursor to [¹⁸F]12a: (R)-N-[7-

(Methoxymethoxy)chroman-2-yl]methyl 4-Hydroxybenzyl Amine

(13a). Primary amine 8 (146 mg, 0.65 mmol), commercially available

4-hydroxybenzaldehyde (80 mg, 0.65 mmol), and acetic acid (43 mg,

0.72 mmol, 1.1 equiv) were stirred in CH₂Cl₂(4 mL) for 2.5 h at rt.

According to ¹H NMR, the reaction was complete, so sodium

triacetoxyborohydride (305 mg, 1.44 mmol, 2.2 equiv) was added, and

the reaction mixture was left to stir for 18 h at rt. Monitoring with

LC−MS showed that conversion to the desired product was ca. 80%.

The mixture was brought to a pH of 7 with a 0.1 M NaOH aqueous

solution and was then extracted with CH₂Cl₂. Drying of the organic

layer with Na₂SO₄, evaporation of the solvent, and puriﬁcation with

silica column chromatography (hexane/ethyl acetate 1:1) gave the

(R)-1-(4-Benzyloxyphenyl)-4-(4-(7-(methoxymethoxy)-

chroman-2-yl)-3-azabutyl)-piperazine (19). A solution of tosylate

6 (300 mg, 0.793 mmol) in 1:1 dioxane/DMSO (1 mL) was added to

a mixture of DIPEA (1.4 mL, 7.93 mmol, 10 equiv) and amine 18 (306

mg, 0.952 mmol, 1.2 equiv) in 1:1 dioxane/DMF (1 mL). This

mixture was stirred overnight at 90 °C. According to LC−MS, there

was still some tosylate present, so DIPEA (0.5 mL) was added, and the

reaction mixture was stirred at 90 °C for another 24 h. The mixture

was then poured into water (pH became ca. 9 to 10), and the aqueous

layer was extracted with CHCl₃. The organic layer was washed with

NaCl(aq), dried with Na₂SO₄, and concentrated to give the crude

product that was puriﬁed with alumina column chromatography (1:1

1

product in a yield of 76% (164 mg). H NMR (CDCl₃): δ 7.12 (m,

2H), 6.9 (m, 1H), 6.65 (m, 2H), 6.55 (multiple signals, 2H), 5.1 (s,

2H), 5.05 (bs, 2H), 4.2 (m, 1H), 3.75 (m, 2H), 3.4 (s, 3H), 2.9−2.6

(multiple signals, 4H), 1.9 (m, 1H), 1.7 (m, 1H). ¹³C NMR (CDCl₃):

δ 156.4, 156.0, 155.0, 130.0, 129.9, 129.8, 115.8, 115.5, 109.0, 104.3,

94.5, 74.5, 55.9, 53.3, 53.2, 25.7, 23.9. LC−MS: m/z 330.1 [M + H]⁺

(calcd 329.40 for C₁₉H₂₃NO₄).

1

ethyl acetate/pentane). Yield: 156 mg (32%). H NMR (CDCl₃): δ

7.45−7.3 (multiple signals, 5H), 6.9 (multiple signals, 5H), 6.55

(multiple signals, 2H), 5.1 (s, 2H), 5.0 (s, 2H), 4.15 (m, 1H), 3.4 (s,

3H), 3.1 (m, 4H), 2.9−2.7 (multiple signals, 6H), 2.6 (multiple

signals, 6H), 1.95 (multiple signals, 2H), 1.75 (m, 1H). ¹³C NMR

(CDCl₃): δ 156.5, 155.2, 152.9, 146.0, 137.4, 129.9, 128.5, 127.8,

127.4, 118.0, 115.5, 114.9, 108.9, 104.4, 94.5, 75.1, 70.5, 57.8, 55.9,

54.3, 53.4, 50.6, 46.5, 25.8, 24.0. LC−MS: m/z 540.4 [M + Na]⁺, 518.5

[M + H]⁺and 259.8 [M+2H]²⁺(calcd 517.67 for C₃₁H₃₉N₃O₄).

(R)-1-(4-Hydroxyphenyl)-4-(4-(7-(methoxymethoxy)-

chroman-2-yl)-3-azabutyl)-piperazine (13b). Benzylated mole-

cule 19 (156 mg, 0.30 mmol) was dissolved in EtOH, and Pd/C

Precursor to [¹⁸F]12d: Multistep Synthesis to Phenol 13b.

Compounds 15 and 16 were prepared according to a patent

procedure.⁷⁷

4-(4-Benzyloxy-phenyl)-piperazine-1-carboxylic Acid tert-

Butyl Ester (15). 4-(4-Hydroxyphenyl)-piperazin-1-carboxylic acid

tert butyl ester (14) was prepared according to Tietze.⁷³Compound

14 (2 g, 7.19 mmol), benzyl bromide (1.23 g, 7.19 mmol), and K₂CO₃

(0.99 g, 7.19 mmol) were mixed in DMF (2 mL). The reaction

mixture was stirred at 50 °C under argon for 16 h and was then poured

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catalyst (Degussa type) was added. The mixture was shaken under

hydrogen at 70 psi, and the conversion was monitored by H NMR.

4H), 2.6 (m, 4H), 2.5 (t, 2H), 1.45 (s, 9H). LC−MS: m/z 432.1 [M +

H]⁺(calcd 431.32 for C₁₇H₂₆N₃O₂I).

1

1-(2-Aminoethyl)-4-(4-iodophenyl)-piperazine (24). Boc-pro-

tected amine 23 (1.58 g, 3.65 mmol) was added slowly to a

concentrated 10 M HCl solution in water (2 mL). The reaction was

stirred for 15 min at rt, diluted with water (50 mL), and adjusted to a

pH of 9 to 10 using a NaOH solution in water. The product was

isolated by extraction of the water layer with ethyl acetate (twice),

drying of the organic layer over Na₂SO₄, and evaporation of the

volatiles. Yield: 0.78 g (65%). ¹H NMR (CDCl₃): δ 7.45 (m, 2H), 6.75

(m, 2H), 3.1 (m, 4H), 3.0 (bs, 2H), 2.65 (t, 2H), 2.5 (m, 4H), 2.35

(m, 2H). ¹³C NMR (CDCl₃): δ 150.9, 137.7, 118.0, 81.2, 61.1, 53.0,

48.7, 38.8. LC−MS: m/z 332.1 [M + H]⁺(calcd 331.20 for

C₁₂H₁₈N₃I).

Filtration over two paper ﬁlters and one using a syringe ﬁlter (45 μm

regenerated cellulose) gave a yield of 121 mg product (94%). ¹H NMR

(CDCl₃): δ 6.9 (m, 1H), 6.8 (m, 2H), 6.75 (m, 2H), 6.5 (multiple

signals, 2H),5.1 (s, 2H), 4.2 (m, 1H), 3.4 (s, 3H), 3.1 (m, 4H), 2.9−

2.7 (multiple signals, 6H), 2.6 (multiple signals, 6H), 1.95 (m, 1H),

1.75 (m, 1H). ¹³C NMR (CDCl₃): δ 156.5, 155.1, 150.3, 145.4, 130.0,

118.5, 116.0, 115.5, 109.0, 104.4, 94.5, 74.7, 57.4, 55.9, 54.1, 53.3, 50.8,

46.1, 25.8, 24.0. LC−MS: m/z 428.4 [M + H]⁺and 214.7 [M + 2H]²⁺

(calcd 427.55 for C₂₄H₃₃N₃O₄).

Preparation of Amine and Aldehyde Building Blocks for

Coupling to Tosylate 3 (Route A), Tosylate 6 (Route B), or

Amine 8 (Route C). tert-Butyl (4-(4-Fluorobutoxy)-benzyl)-

carbamate (20). tert-Butyl (4-hydroxybenzyl)-carbamate, as pre-

pared⁷⁹from the HBr salt of 4-hydroxy-benzylamine (3.17 g, 14.2

mmol), was mixed with commercially available 1-bromo-4-ﬂuoro-

butane (2.62 g, 17 mmol, 1.2 equiv) and K₂CO₃(2.36 g, 17 mmol, 1.2

equiv) in MeCN (75 mL). The reaction mixture was stirred overnight

at 80 °C under an inert argon atmosphere, cooled to rt, and ﬁltered to

remove salts. The ﬁltrate was concentrated, and the crude product was

puriﬁed with silica column chromatography (2% MeOH/CHCl₃).

Drying under vacuum removed traces of 1-bromo-4-ﬂuorobutane.

1-(2-N-(tert-Butylcarbamate)ethyl)-4-(4-(4-ﬂuoroethoxy)-

phenyl)-piperazine (25). 1-(4-(2-Fluoroethoxy)-phenyl)-pipera-

zine⁷³(1.88 g, 8.4 mmol), tert-butyl N-(2-bromoethyl)carbamate⁷⁸

(2.07 g, 9.24 mmol, 1.1 equiv), and DIPEA (mL) were mixed in

MeCN (20 mL). The mixture was stirred overnight under argon at an

oil bath temperature of 50 °C. The reaction mixture was concentrated

and redissolved in CH₂Cl₂to enable washing with water (twice) and

with a saturated NaCl solution (once). The organic layer was dried

with Na₂SO₄and concentrated to aﬀord crude product that was

puriﬁed using silica column chromatography (elution gradient starting

with CHCl₃and ﬁnally using 3% MeOH/CHCl₃). After evaporation of

1

Yield: 2.9 g (68%). H NMR (CDCl₃): δ 7.2 (m, 2H), 6.85 (m, 2H),

4.8 (bs, 1H), 4.55 (m, 1H), 4.45 (t, 1H), 4.2 (m, 2H), 3.95 (t, 2H),

1.95−1.8 (multiple signals, 4H), 1.45 (s, 9H). ¹³C NMR (CDCl₃): δ

158.2, 155.8, 131.0, 128.8, 114.5, 84.5, 82.9, 79.1, 67.2, 44.1, 28.3, 27.2,

27.0, 25.2, 25.1. LC−MS: m/z 242.0 [M − C₄H₈]⁺(calcd 297.37 for

C₁₆H₂₄NO₃F).

1

the solvent, a yellowish solid was obtained. Yield: 2.42 g (78%). H

NMR (CDCl₃): δ 6.85 (multiple signals, 4H), 4.95 (bs, 1H), 4.75 (m,

1H), 4.65 (m, 1H), 4.2 (m, 1H), 4.15 (m, 1H), 3.25 (m, 2H), 3.05 (m,

4H), 2.6 (m, 4H), 2.5 (t, 2H), 1.45 (s, 9H). ¹³C NMR (CDCl₃): δ

155.9, 152.5, 146.2, 118.0, 115.5, 82.9, 81.2, 79.0, 67.8, 67.6, 57.2, 53.0,

50.4, 37.1, 28.4.

4-(4-Fluorobutoxy)-benzyl Amine (21). Boc-protected amine

20 (0.56 g, 1.87 mmol) was stirred at rt for 10 min in 4 M HCl in

dioxane (0.6 mL). The reaction mixture developed into a hazy

suspension. The mixture was evaporated to dryness, and the residue

was dissolved in water that was adjusted to a basic pH using a NaOH

solution. The product was extracted using CH₂Cl₂. The organic layer

was dried using Na₂SO₄and evaporated to yield the desired amine

product. Yield: 261 mg (71%). ¹H NMR (CDCl₃): δ 7.2 (m, 2H), 6.85

(m, 2H), 4.55 (m, 1H), 4.45 (t, 1H), 4.0 (t, 2H), 3.8 (s, 2H), 1.9

(multiple signals, 4H), 1.4 (bs, 2H). ¹³C NMR (CDCl₃): δ 157.8,

135.6, 128.2, 114.5, 84.5, 82.9, 67.2, 45.9, 27.3, 27.1, 25.3, 25.2. LC−

MS: m/z 198.1 [M + H]⁺, 181.3 [M − NH₂]⁺(calcd 197.25 for

C₁₁H₁₆NOF).

1-(2-Aminoethyl)-4-(4-(4-ﬂuoroethoxy)phenyl)-piperazine

(26). Boc-protected amine 25 (2.42 g, 6.58 mmol) was stirred in

concentrated 10 M HCl solution in water (20 mL). A thick white foam

formed, and after 20 min, water (75 mL) was added as well as solid

NaOH to adjust the pH to about 10. The product was extracted from

the water layer using CH₂Cl₂(twice). Drying of the collected organic

layers (Na₂SO₄) and evaporation of the solvent gave a white solid.

Yield: 1.47 g (84%). ¹H NMR (CDCl₃): δ 6.85 (multiple signals, 4H),

4.75 (m, 1H), 4.65 (m, 1H), 4.2 (m, 1H), 4.15 (m, 1H), 3.05 (m, 4H),

2.8 (t, 2H), 2.6 (m, 4H), 2.45 (t, 2H), 1.4 (bs, 2H). ¹³C NMR

(CDCl₃): δ 152.4, 146.2, 117.9, 115.4, 82.8, 81.1, 67.7, 67.5, 61.1, 53.3,

50.4, 38.7. LC−MS: m/z 268.3 [M + H]⁺(calcd 267.35 for

C₁₄H₂₂N₃OF).

2-(4-Fluorobutoxy)-benzaldehyde (22). 2-Hydroxybenzalhyde

(1 g, 8.18 mmol) and 1-bromo-4-ﬂuorobutane (1.52 g, 9.8 mmol, 1.2

equiv), both commercially available, were mixed with K₂CO₃(2.26 g,

16.3 mmol, 2 equiv) in DMF (40 mL). The suspension was stirred

overnight under argon at an oil bath temperature of 50 °C. The next

day, the reaction mixture was ﬁltrated and diluted with water so that

the product could be extracted from the water layer using diethyl ether

(twice). The ether layer was washed twice with water and once with a

saturated NaCl solution and was then dried with Na₂SO₄. Starting

bromide was still present in the product, but it could be removed by

Preparation of Radiochemistry [¹⁸F]Fluoroalkylation Pre-

cursors and References Compounds: Tosylates 27−30. 1,4-

Butanediol Ditosylate (27). p-Toluenesulfonyl chloride (3.17 g,

16.6 mmol) was dissolved in 10 mL of dry CH₂Cl₂. To this solution

was added 3.0 mL (2.29 g, 22.7 mmol) of dry triethylamine. The

mixture was cooled to 0 °C in an ice bath, and 610 μL (616 mg, 7.6

mmol) of dry 1,4-butanediol was added. After 30 min of stirring,

another 60 μL of 1,4-butanediol was added. The mixture was left to

stir overnight, with the reaction mixture temperature coming to rt. On

the next day, the mixture was diluted with an extra portion of CH₂Cl₂

(15 mL), cooled to 0 °C, and acidiﬁed by slowly adding 50 mL of 0.8

M HCl. The organic layer was separated and washed with 0.3 M HCl

(3 × 50 mL). The CH₂Cl₂solution of the crude product was then

concentrated in vacuo, and the product was puriﬁed on a silica column

eluted with hexane/CH₂Cl₂/ethyl acetate (6:6:1) to yield 1.93 g of

ﬁnal product as white crystals. Yield: 58%. ¹H NMR (300 MHz,

CDCl₃) δ: 7.76 (d, J = 8.4 Hz, 4H), 7.35 (d, J = 8.4 Hz, 4H), 3.95−

4.03 (m, 4H), 2.45 (s, 6H), 1.65−1.74 (m, 4H).

1,2-Ethanediol Ditosylate (28). This compound was prepared

and isolated in as similar fashion as its butane-spaced counterpart, 27.

Analytical data were according to expectations.

4-Fluorobutyl Tosylate (29). Tetrabutylammonium ﬂuoride

trihydrate (66 mg, 0.25 mmol) was dried by coevaporation in vacuo

with three 3 mL portions of acetonitrile (the temperature in the ﬂask

was not allowed to rise above 30 °C to prevent decomposition of the

tetrabutyl ammonium ﬂuoride). The dried tetrabutyl ammonium

1

coevaporation with diisopropylether. Yield: 1.43 g (89%). H NMR

(CDCl₃): δ 10.5 (s, 1H), 7.8, (m, 1H), 7.55 (m, 1H), 7.0 (m, 2H),

4.60 (m, 1H), 4.46 (m, 1H), 4.13 (t, 2H), 2.05−1.8 (multiple signals,

4H). ¹³C NMR (CDCl₃): δ 189.6, 161.2, 135.9, 128.3, 124.8, 120.6,

112.4, 84.3, 82.7, 67.8, 27.2, 27.0, 25.2, 25.1. GC-MS: m/z 121 [M −

C₄H₈F]⁺(calcd 196.22 for C₁₁H₁₃O₂F).

1-(2-N-(tert-Butylcarbamate)ethyl)-4-(4-iodophenyl)-pipera-

zine (23). 1-(4-Iodophenyl)-piperazine⁷²(4.6 g, 15.1 mmol), tert-

butyl N-(2-bromoethyl)carbamate⁷⁸3.7 g, 16.6 mmol, 1.1 equiv), and

DIPEA (5.3 mL, 30.4 mmol, 2 equiv) were dissolved in MeCN (30

mL). The mixture was stirred overnight at 50 °C and kept under

argon. The reaction mixture was evaporated to dryness and was

redissolved in CHCl₃. The solution was washed two times with water

and once with a NaCl solution and was then dried over Na₂SO₄. After

concentration, the crude product was puriﬁed with silica column

chromatography (1% MeOH/CHCl₃). Yield 80%. ¹H NMR (CDCl₃):

δ 7.50 (m, 2H), 6.65 (m, 2H), 4.95 (bs, 1H), 3.25 (m, 2H), 3.15 (m,

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ﬂuoride was redissolved in a small quantity of dry acetonitrile, and this

solution was added to a solution of 1,4-butanediol ditosylate (100 mg,

0.25 mmol). The reaction was conducted in reﬂuxing acetonitrile for

24 h, and the conversion was monitored by TLC on silica plates

(hexane/ethyl acetate 2:1, R_f= 0.75). After the reaction ended,

acetonitrile was evaporated in vacuo, the residue was redissolved in

hexane/ethyl acetate (2:1 v/v), and the product was puriﬁed on a silica

column eluted with hexane/ethyl acetate (2:1 v/v) to yield 30 mg of

concentrations of the dopamine agonists. After the stimulation

solutions were added to the well plate, the cell suspensions (diluted

to 6000 cells/well) were added in a 1:1 ratio so that the ﬁnal

concentrations of IBMX and forskolin in the assay were 0.5 mM and

31.6 μM, respectively. The plate was incubated for 30 min at room

temperature for the stimulation, and subsequently diﬀerent detection

reagents and antibody solutions were added to a total volume of 20

μL/well. Measurements were carried out on a Victor 2 plate reader

(Wallac, PerkinElmer, Zaventum, Belgium) 3 h after adding the

detection mixtures.

Lipophilicity. Lipophilicity (cLogP and cLogD) of the unlabeled

compounds was calculated using the BiologP (New cLogP) calculator

program from the University of Massachusetts. LogD of the [¹⁸F]-

labeled compounds was determined using radioactivity remaining on

the HLB cartridge after its elution with ethanol during the formulation

procedure (vide infra). The cartridge was dried for 5 min in strong N₂

ﬂow and eluted with at least 1.5 mL of n-octanol. Then, 500 μL of

octanol eluate was mixed with 500 μL of 1 M phosphate buﬀer, pH

7.4, and vigorously vortexed for 5 min at rt. After that, the mixture was

centrifuged at 17 000g for 10 min and three 100 μL aliquots of organic

and aqueous phase were loaded into a gamma-counter for radioactivity

measurement. To correct for the possible presence of radioactive

impurities in the tracer, aliquots of organic and aqueous phases were

later analyzed by radio-TLC, and the counts obtained from the

gamma-counter were corrected according to the percentage of

impurities found in aqueous and organic phases. LogD was calculated

with the formula

1

ﬁnal product as brownish crystals. Yield: 48%. H NMR (300 MHz,

CDCl₃) δ: 7.79 (d, J = 8.1 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 4.44 (dt,

J₁= 47.0 Hz, J₂= 5.6 Hz, 2H), 4.08 (t, J = 4.08 Hz, 2H), 2.46 (s, 3H),

1.67−1.84 (m, 4H).

2-Fluoroethyl Tosylate (30). 2-Fluoroethyl tosylate was synthe-

sized according to the same protocol as 1,4-butanediol ditosylate.

Instead of 1,4-butanediol, 2-ﬂuoroethanol (240 μL, 268 mg, 4.2 mmol)

was reacted with p-toluenesulfonyl chloride (755 mg, 4.0 mmol).

Yield: 760 mg of ﬁnal product was obtained (86%) as a colorless

slightly yellowish oil. ¹H NMR (200 MHz, CDCl₃) δ: 7.82 (d, J = 7.6

Hz, 2H), 7.37 (d, J = 6.8 Hz, 2H), 4.58 (dt, J₁= 47.0 Hz, J₂= 4.2 Hz,

2H), 4.27 (dt, J₁= 27.6 Hz, J₂= 4.0 Hz, 2H), 2.48 (s, 3H).

Cell Culture and Preparation of Membranes. Human

embryonic kidney 293 cells (HEK293) stably expressing the human

dopamine receptor subtypes were cultured in RPMI 1640 medium

with 2 mM ^L-glutamine in the presence of 100 U/mL of penicillin and

100 U/mL of streptomycin. To maintain selection pressure, 50 μg/mL

of neomycin and 5 μg/mL of puromycin were also added to the

medium of cells expressing dopamine D₂L, D₂S, D₃, and D₄receptors

and D₁and D₅receptors, respectively. Cells were harvested by

scraping the culture dishes, washed by centrifugation at 200g, and lysed

in ice-cold buﬀer containing 5 mM Tris and 5 mM MgCl₂at pH 7.4.

The suspension was homogenized with 8 strokes in a glass/Teﬂon

homogenizer and centrifuged twice for 1 h at 30 000g at 4 °C. Pellets

were resuspended in buﬀer (50 mM Tris, pH 7.4) and rehomogenized

with 4 strokes in the glass/Teﬂon homogenizer, and aliquots were

frozen in liquid nitrogen and stored at −80 °C. Protein content was

measured using the method of Bradford.⁸⁰For experiments on

dopamine D₂S and D₃receptors, membrane preparations were

obtained from PerkinElmer (Waltham, MA, USA).

tracer

⎛

⎝

⎞

⎠

CPM_org

X

org

⎜

⎟

LogD = lg

CPM X^tracer

aq aq

tracer

org

where CPM_orgis the mean cpm in the organic aliquots, X

is the

mean percent activity accounting for intact tracer in organic aliquots

tracer

org

(as determined from radio-TLC), and CPM_organd X

are the

corresponding values for the aqueous phase.

For each tracer batch used for LogD determination, the experiment

was conducted in duplicate. LogD values are the mean calculated from

at least two independent experiments.

Receptor Binding Assays. For the radioligand experiments, the

samples were thawed and rehomogenized in Mg-containing buﬀer (50

mM Tris, 5 mM KCl, 2 mM CaCl₂, and 2 mM MgCl₂pH 7.4) to allow

formation of the agonist high-aﬃnity state. Rehomogenization was

done with a short burst (10 s) of an ultra turrax at full speed. Binding

experiments were performed with 40−60 μg of protein per assay, an

assay volume of 1000 μL, and a radioligand concentration of 0.15 nM

for the [³H]spiperone (D₂L, D₂S, D₃, and D₄receptors) assays and 1.5

nM for [³H]SCH23390 (D₁and D₅receptor) assays, with an

incubation time of 2 h at 25 °C and a preincubation of 15 min at 25

°C. Nonspeciﬁc binding was deﬁned using 1 μM (+)-butaclamol

(antagonist for dopamine receptors). To avoid oxidation of the

compounds, 0.05% ascorbic acid was added to all solutions. All

experiments were performed in duplicate in a 96-well platform, and

reactions were terminated by rapid vacuum ﬁltration over Whatman

GF/C ﬁlter plate with ice-cold buﬀer (50 mM Tris). For the D₃

receptor binding experiments, plates were precoated with 0.1% PEI.

Filter radioactivity was measured in a PerkinElmer Topcount with

Microscint-O cocktail as scintillant. The competition experiments were

analyzed using the Prism program (version 5.01, GraphPad Software,

San Diego, CA, USA) by ﬁtting sigmoidal curves to the data. Resulting

IC₅₀values were converted to K_ivalues on the basis of the Cheng−

Prusoﬀ equation.³⁸

Materials (in Vitro Experiments). The transfected dopamine

receptor cell lines were a generous gift of Prof. Gross (Abbott).

Human D₂S and D₃membranes were obtained from PerkinElmer

(Waltham, MA, USA). [³H]Spiperone (speciﬁc activity 16.2 Ci/

mmol) and [³H]SCH23390 (speciﬁc activity 73.1 Ci/mmol) were

obtained from PerkinElmer. (+)-Butaclamol, (R)-(−)-propylnorapo-

morphine HCl, and dopamine HCl were obtained from Sigma-Aldrich

(Zwijndrecht, The Netherlands). (+)-PHNO HCl was obtained from

Axon Medchem (Groningen, The Netherlands).

Radiochemistry. Preparation of [¹⁸F]12a. ¹⁸F-ﬂuoride in enriched

H₂¹⁸O water was adsorbed on an anion-exchange column (QMA) and

later eluted into a reaction vial with 1−1.5 mL of 1.5 mg/mL of

K₂CO₃and 5 mg/mL of Kryptoﬁx solution in 9:1 v/v mixture of

acetonitrile and water. After the elution of QMA, acetonitrile and

water were evaporated at 130 °C under nitrogen ﬂow. For further

water removal, 1 mL of anhydrous acetonitrile was added into the

reaction vial and evaporated. Then, 5 mg of 1,4-butanediol ditosylate

(27) in 1 mL of anhydrous acetonitrile was added into the vial, the vial

was sealed, and the reaction was carried out for 4 min at 130 °C

followed by cooling for 5 min . One milliliter of water was added to the

cooled reaction mixture, and the resulting mixture was loaded into the

HPLC loop. 4-[¹⁸F]ﬂuorobutyltosylate was puriﬁed by reverse-phase

HPLC using Alltima C18 5u 250 × 10 mm column eluted with

acetonitrile/10 mM H₃PO₄(65:35 v/v) at 5 mL/min (t_R= 7.0 min, k′

= 3.9, as conﬁrmed using the reference compound 4-ﬂuorobutyltosy-

late 29). The collected radioactive fraction was diluted 2- to 3-fold

with water, and 4-[¹⁸F]ﬂuorobutyltosylate was adsorbed on a tC18

light Seppak cartridge. The cartridge was rinsed with 5 mL of pure

water and dried for 5 min in strong nitrogen ﬂow. Afterward, 4-

[¹⁸F]ﬂuorobutyltosylate was eluted from the cartridge with 0.7 mL of

dry DMF into a v-vial containing 0.6 mg of precursor 13a, 30 μL of

dry DMF, and 2 to 3 mg of NaH. The reaction vial was sealed, and the

cAMP Accumulation. To determine the concentrations of cAMP

formed in the assay, the LANCE cAMP 384 kit from PerkinElmer was

used according to the manufacturer’s protocol. Transfected HEK293

cells stably expressing the dopamine D₂L were cultured as mentioned

earlier. They were detached from the surface of the culture dish with 2

mL of dissociation solution, washed with HBSS, and resuspended in a

standard buﬀer (SB) containing HBSS, 0.05% BSA, and 5 mM

HEPES. The stimulation solutions contained SB with 1.0 mM 3-

isobutyl-1-methylxanthine (IBMX), 63.2 μM forskolin, and diﬀerent

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alkylation reaction was conducted for 10 min at 120 °C; then, 1 mL of

0.4 M HCl was added into the vial with a syringe. The MOM group

was then deprotected, which lasted 10 min at 120 °C. [¹⁸F]12a was

puriﬁed by reverse-phase HPLC using Alltima C18 5u 250 × 10 mm

column eluted with acetonitrile/10 mM H₃PO₄(40:60 v/v) at 5 mL/

min (t_R= 9.8 min, k′ = 5.4; Scheme 3).

On the day of the experiment, the slides with mounted slices were

taken out of storage and allowed to come to room temperature for 5−

10 min. After that, incubation buﬀer (50 mM Tris-HCl, 5 mM KCl, 2

mM CaCl₂, 2 mM MgCl₂, and 120 mM NaCl, pH 7.4, 25 °C) was

applied to the slides using an automatic pipet (1−1.2 mL/slide), and

the slides were preincubated for 15 min at room temperature. The

preincubation buﬀer was then removed, and the slides were placed

into staining jars containing incubation buﬀer and radioligand ([¹⁸F]

12a or [¹⁸F]12d) at a concentration of about 5 nM (4.5 3.6 nM for

[¹⁸F]12a and 5.8 1.6 nM for [¹⁸F]12d, n = 4). Four conditions were

tested: radioligand only, radioligand in NaCl-free buﬀer, radioligand

and 10 μM raclopride (nonspeciﬁc binding only), and radioligand and

100 μM GTP (low-aﬃnity state only). The slides were incubated for

35 min at 37 °C, washed once with ice-cold incubated buﬀer (3.5

min), and dipped for 30 s into ice-cold sterile water to remove buﬀer

salts.

After drying the slides in a stream of room-temperature air, they

were exposed on phosphor storage screens for 6−10 h. Afterward, the

storage screens were read by a Cyclone Storage Phosphor System

(Packard Instruments Co). Quantiﬁcation of plate readings was done

with Optiquant software (version 3.00, Packard Instruments Co) by

drawing regions of interest on the striatum and cerebellum manually.

The animal experiments were performed by licensed investigators in

compliance with the Law on Animal Experiments of The Netherlands.

The protocol was approved by the Committee on Animal Ethics of the

University of Groningen. The rats were maintained on a 12 h light/12

h dark regime and were fed standard laboratory chow ad libitum.

The collected HPLC fraction was diluted with 2 to 3 volumes of

water, and [¹⁸F]12a was adsorbed on a Oasis HLB SPE cartridge. The

cartridge was rinsed with 5 mL of pure water, and [¹⁸F]12a was then

eluted with 1 to 2 mL of absolute ethanol (typically 20−40%

recovery). After the partial or complete evaporation of ethanol in

nitrogen ﬂow (with or without application of vacuum), the tracer was

formulated in physiological saline (ethanol content in the formulated

tracer was kept below 10%). The identity of [¹⁸F]12a was conﬁrmed

by coelution with the unlabeled 12a on reverse-phase HPLC and

radio-TLC. From the same data, radiochemical purity was assessed.

Reverse-phase HPLC was carried out on Platinum EPS 5u 250 × 4.6

column eluted with acetonitrile/physiological saline/formic acid

(70:30:0.1 v/v) at 2 mL/min (t_R= 6.4 min, k′ = 4.5). For quality

control, radio-TLC was carried out on silica plates eluted with

dichloromethane/methanol/ammonia (100:5:1, R_f= 0.25) and ethyl

acetate/methanol/triethylamine (100:5:1, R_f= 0.5).

Preparation of [¹⁸F]12d. The procedure described above for [¹⁸F]

12a was also used for the preparation of [¹⁸F]12d, with the following

modiﬁcations: ethylene glycol ditosylate (28) and precursor 13b were

used instead of 1,4-butanediol ditosylate (27) and precursor 13a,

respectively. 2-[¹⁸F]ﬂuoroethyl tosylate puriﬁcation conditions were as

follows: Alltima C18 5u 250 × 10 mm column eluted with

acetonitrile/10 mM sodium acetate buﬀer pH 4.0 (60:40 v/v) at 5

mL/min (t_R= 6.8 min, k′= 3.8, as conﬁrmed using the reference

tosylate 30). At the end of the deprotection step, the reaction mixture

was diluted with 10 mL of water, titrated with 1 M NaOH to pH 4−7,

and then put through the Oasis HLB SPE cartridge. Adsorbed activity

was then eluted from the cartridge with 1 mL of an acetonitrile/DMF

mixture (1:1 v/v). The eluate was further diluted with water to a total

volume of 1.5−2 mL and injected onto the Alltima C18 5u 250 × 10

mm HPLC column for puriﬁcation of [¹⁸F]12d (Scheme 3).

[¹⁸F]12d puriﬁcation conditions were as follows: Alltima C18 5u

250 × 10 mm column eluted with a gradient of acetonitrile in 10 mM

sodium acetate buﬀer: isocratic 45% acetonitrile during the ﬁrst 2 min,

then a linear decrease from 45% at 2 min to 30% acetonitrile at 15 min

(t_R[¹⁸F]12d = 10.6 min, k′ = 5.9).

HPLC conditions for the identity conﬁrmation and RCP assessment

were as follows: platinum EPS 5u 250 × 4.6 column eluted with

acetonitrile/10 mM phosphate buﬀer pH 7 (65:35 v/v) at 2 mL/min

(t_R= 11.0 min, k′ = 7.3).

Quality control TLC conditions were as follows: dichloromethane/

methanol/ammonia 100:5:1 (R_f= 0.25) and ethyl acetate/methanol/

ammonia/triethylamine 100:5:5:1 (R_f= 0.3). Before putting radio-

active samples onto the TLC plates, drops of 12d oxalate standard

solution containing 2 μg of compound were put onto the start

positions of each lane to exclude on-plate decomposition of no-carrier-

added [¹⁸F] compound.

Autoradiography. Frozen brains of young (10−12 weeks of age;

300−350 g body weight) male Sprague−Dawley rats (Harlan, The

Netherlands) were cut into halves along the sagittal symmetry plane.

Each half was mounted, lateral side up, on a paper slide pretreated by

Tissue-Tek ﬁxing cocktail (Sakura, The Netherlands) and ﬁxed by

placing it onto a petri dish ﬂoating on liquid nitrogen. After ﬁxing,

brains were cut at −12 °C into sagittal slices 20 μm thick using a Leica

microtome, and the slices were thaw-mounted on Superfrost (70 × 22

mm, Fischer) adhesive slides. Only slices containing both striatal and

cerebellar regions (representing nonspeciﬁc binding) were used. Slides

were organized in pairs, and in each pair, new slices were mounted

alternately on the ﬁrst or the second slide of the pair so that slices in

corresponding positions on the two slides forming a pair would

represent adjacent tissue layers. The slices were allowed to dry and

were then put into storage boxes with desiccator (silicagel) bags and

kept at −80 °C until they were used (no longer than 1 week).

AUTHOR INFORMATION

■

Corresponding Author

*E-mail: j.booij@amc.uva.nl. Tel: +31 20 5662397.

Author Contributions

^#These authors contributed equally to this work.

Notes

The authors declare the following competing ﬁnancial

interest(s): A.G.M.J. is an employee of GE Healthcare, a

company marketing the DA D_2/3receptor antagonist radio-

pharmaceutical [¹²³I]IBZM. M.C.M. is an employee of

Boehringer Ingelheim, a company marketing the DA D₂

agonist pramipexole. J.B. is consultant at GE Healthcare. The

other authors declare that they have no competing interest.

ACKNOWLEDGMENTS

■

J.A.J.M. (Jef) Vekemans, Eindhoven University of Technology,

(TU/e), is thanked for his advice on synthetic design; J.L.J.

(Joost) van Dongen and R.A.A. (Ralf) Bovee (both TU/e) are

thanked for their expertise on and execution of the HRMS

measurements and elemental analyses, respectively. Prof. E.W.

(Bert) Meijer (TU/e and ICMS Eindhoven) is thanked for

enabling the execution of the synthetic part of the presented

research. Jan Keijser (Center for Behavior and Neurosciences,

University of Groningen) is thanked for his advice on the

design and execution of in vitro autoradiography experiments.

Jan Koerts (Laboratory of Pathology and Medical Biology,

UMCG) is thanked for providing access to the equipment

needed for the preparation of brain slices. This work was

supported by a grant from the Dutch Technology Foundation

STW (grant 10127).

ABBREVIATIONS USED

■

AMC, 2-aminomethylchromane; DIPEA, diisopropylethyl-

amine; UPLC, ultra-performance liquid chromatography

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D2/D3 receptors in vivo with positron emission tomography. Curr.

Top. Med. Chem. 2010, 10, 1477−1498.

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Synthesis and characterization of a novel series of agonist compounds as potential radiopharmaceuticals for imaging dopamine D2/3 receptors in their high-affinity state

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