Synthesis and in silico evaluation of novel compounds for PET-based investigations of the norepinephrine transporter

Article abstract of DOI:10.3390/molecules20011712

Since the norepinephrine transporter (NET) is involved in a variety of diseases, the investigation of underlying dysregulation-mechanisms of the norepinephrine (NE) system is of major interest. Based on the previously described highly potent and selective NET ligand 1-(3-(methylamino)-1-phenylpropyl)-3-phenyl-1,3-dihydro-2H-benzimidaz- ol-2-one (Me@APPI), this paper aims at the development of several fluorinated methylaminebased analogs of this compound. The newly synthesized compounds were computationally evaluated for their interactions with the monoamine transporters and represent reference compounds for PET-based investigation of the NET.

Full text of DOI:10.3390/molecules20011712

Molecules 2015, 20, 1712-1730; doi:10.3390/molecules20011712
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and in Silico Evaluation of Novel Compounds for
PET-Based Investigations of the Norepinephrine Transporter
Catharina Neudorfer 1,2,*, Amir Seddik , Karem Shanab , Andreas Jurik ,
3
1,2
3
1
2
3
1
Christina Rami-Mark , Wolfgang Holzer , Gerhard Ecker , Markus Mitterhauser ,
1
2,
Wolfgang Wadsak and Helmut Spreitzer *
1
Department of Biomedical Imaging and Image-guided Therapy, Division of Nuclear Medicine,
Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria;
E-Mails: karem.shanab@gmail.com (K.S.); christina.rami-mark@meduniwien.ac.at (C.R.-M.);
markus.mitterhauser@meduniwien.ac.at (M.M.); wolfgang.wadsak@meduniwien.ac.at (W.W.)
2
Division of Drug Synthesis, Department of Pharmaceutical Chemistry, Faculty of Life Sciences,
University of Vienna, Althanstraße 14, 1090 Vienna, Austria;
E-Mail: wolfgang.holzer@univie.ac.at
3
Division of Drug Design and Medicinal Chemistry, Department of Pharmaceutical Chemistry,
Faculty of Life Sciences, University of Vienna, Althanstraße 14, 1090 Vienna, Austria;
E-Mails: amir.seddik@univie.ac.at (A.S.); andreas.jurik@univie.ac.at (A.J.);
gerhard.f.ecker@univie.ac.at (G.E.)
*
Authors to whom correspondence should be addressed;
E-Mails: catharina.neudorfer@gmail.com (C.N.); helmut.spreitzer@univie.ac.at (H.S.);
Tel.: +43-4277-55629 (C.N.); +43-4277-55621 (H.S.);
Fax: +43-4277-855629 (C.N.); +43-4277-855621 (H.S.).
Academic Editor: Svend Borup Jensen
Received: 20 November 2014 / Accepted: 14 January 2015 / Published: 20 January 2015
Abstract: Since the norepinephrine transporter (NET) is involved in a variety of diseases,
the investigation of underlying dysregulation-mechanisms of the norepinephrine (NE)
system is of major interest. Based on the previously described highly potent and selective
NET ligand 1-(3-(methylamino)-1-phenylpropyl)-3-phenyl-1,3-dihydro-2H-benzimidaz- ol-
2
-one (Me@APPI), this paper aims at the development of several fluorinated methylamine-
based analogs of this compound. The newly synthesized compounds were computationally

Molecules 2015, 20
evaluated for their interactions with the monoamine transporters and represent reference
1713
compounds for PET-based investigation of the NET.
Keywords: NET; ADHD; cocaine dependence; BAT; PET; FAPPI
1
. Introduction
Abnormal regulation of the norepinephrine transporter (NET) or NET dysfunction, respectively,
cause either increased or decreased levels of norepinephrine (NE) in the synaptic cleft. Since NE is a
fundamental neurochemical messenger, its accurate regulation is of major importance. Thus, the NET,
responsible for NE equilibrium in the synaptic cleft, is representing the reuptake site and considered to
be involved in a variety of neurological/psychiatric disorders [1,2], but also plays a pivotal role in
cardiovascular [1–3] and metabolic diseases [3–5]. Reduced NET levels go along with neurological
disorders like major depression [6,7], Parkinson’s disease (PD), Alzheimer’s disease (AD) [8–18], and
cardiovascular diseases such as hypertension, cardiomyopathy, and heart failure [5,13]. Furthermore, a
dysfunction of the NE system was reported in Attention Deficit Hyperactivity Disorder (ADHD) [9,17,19],
suicide [1,12,20], substance abuse (cocaine dependence) [16], and schizophrenia [10]. A more recent
discovery is the involvement of the NET in diseases like diabetes and obesity, due to its presence in
brown adipose tissue (BAT) and the proposed activation thereof via NE [4,5,21].
Based on the fact that the NET is involved in such a variety of diseases, the investigation of the
underlying dysregulation-mechanism of the NE system is of major interest. For this purpose, information
about the transporter abundance and density in healthy and pathological living human brains is required.
The most suitable and accurate technique to gain this information is positron emission tomography
(
PET). As a non-invasive molecular imaging technique, it represents a suitable approach towards the
collection of missing data in the living organism and direct quantification of receptor/transporter
densities in vivo. To fully gain insight in the molecular changes of the noradrenergic system via PET,
however, prior development of suitable NET PET radioligands is required.
1
1
11
11
So far, radiolabeled NET binding reboxetine analogs [ C]MeNER, [ C]MRB, ((S,S)-2-(α-(2-[ C]-
1
8
18
2
methoxyphenoxy)benzyl)morpholine) and [ F]FMeNER-D
2
((S,S)-2-(α-(2-[ F]fluoro[ H ]methoxy-
2
phenoxy)benzyl)morpholine) have been described, which however display certain limitations such as
metabolic instability, complex radiosyntheses, or late equilibria [22–26].
Recently, Zhang et al. [26] evaluated a series of benzimidazolone-based propanamines with in vitro
inhibitory activity on the human norepinephrine transporter (hNET). The results of these investigations
suggested that compounds containing a phenyl moiety directly attached at the benzimidazolone ring
(
e.g., 1, Figure 1) were the most potent, representing a half maximal inhibitory concentration (IC50)
below 10 nM (IC50 < 10 nM). Furthermore, hNET selectivity over human serotonin transporter (hSERT)
turned out >300-fold superior to those of reboxetine and atomoxetine (16- and 81-fold) [26]. Fluorination
at position 2 of the phenyl moiety attached to the benzimidazolone ring (e.g., 2, Figure 1), indicated
similar hNET potency, comparable to its non-fluorinated analogs (e.g., 1) and additionally exhibited
hNET selectivity over hSERT (80-fold) similar to atomoxetine [26].

Molecules 2015, 20
1714
Figure 1. Structures of the highly potent and selective NET ligands 1-(3-(methylamino)- 1-
phenylpropyl)-3-phenyl-1,3-dihydro-2H-benzimidazol-2-one (Me@APPI, 1) and 4-fluoro- 1-
2-fluorophenyl)-3-(3-(methylamino)-1-phenylpropyl)-1,3-dihydro-2H-benzimidazol-2-
(
1
1
one (2) as well as radiolabeled analog [ C]Me@APPI (3).
Both benzimidazolone derived propanamines with a phenyl moiety (e.g., 1), as well as a fluorinated
phenyl moiety (as in 2) indicated excellent hNET selectivity over human dopamine transporter (hDAT)
3
with < 50% inhibition of the cocaine analog [ H]WIN-35,428, binding to hDAT at a concentration of
1
0 µM [26]. Given those findings, both described benzimidazolone-based propanamines 1 and 2
represent excellent candidates for selective and potent NET inhibition with high affinity and low
unspecific binding. Thus, on the basis of the results of Zhang et al. [26] the methylamino moiety of the
1
1
core compound 1 has been radiolabeled with C and tested by our research group [27]. All investigated
preclinical parameters, such as affinity, blood brain barrier penetration, lipophilicity, metabolic
1
1
degradation, and selectivity showed excellent results, thus suggesting suitability of C-radiolabeled
1
1
1
-(3-(methylamino)-1-phenylpropyl)-3-phenyl-1,3-dihydro-2H-benzimidazole-2-one ([ C]Me@APPI)
(
3, Figure 1) as a NET radioligand for use in PET.
1
1
Due to successful preclinical testing of [ C]Me@APPI (3) and given the excellent in vitro results of
compound 2, shown by Zhang et al. [26] the aim of this paper is the synthesis and docking studies of
several fluorinated analogs 4–6 of compound 1 (Figure 2) as reference compounds for their later prepared
radioactive analogs. All methylamine-derived benzimidazolone derivatives 4–6 will be subjected to
affinity, selectivity, and lipophilicity studies towards the NET as well as blood brain barrier penetration
experiments at the Medical University of Vienna. The most promising NET ligands will then be selected
1
1
for the development of new, selective PET tracers for the NET and after radiolabeling with both C and
18F, they will be the subject of further experiments.
F
1
1
1
2
2
3
2
N
N
N
N
N
N
H
H
H
N
N
N
O
O
O
F
3
3
4
5
6
F
Figure 2. Chemical structure of envisaged reference compounds 4–6 (FAPPI:1-3).

Molecules 2015, 20
. Results and Discussion
The synthesis of reference compounds 4–6 first required the preparation of side chains 11 and 12, as
1715
2
well as core compounds 16–18. After successful preparation, side chain 11 was merged in a condensation
reaction with core compound 16, whereas side chain 12 was reacted with core compounds 17 and 18,
prior to halogen exchange and substitution with methylamine (Scheme 1).
Reagents and conditions: (i) THF, EtOH, NaBH
00%; (ii) aq. HBr, rt, 3 h, yields for 11: 64%, 12: 86%; (iii) 1,1'-carbonyldiimidazole, anhyd. THF,
rt, overnight, yields for 16: 74%, 17: 61%, 18: 69%; (iv) 1,1'-carbonyldiimidazole, anhyd. DMF,
4
, −10 °C → −5 °C, 10 min, yields for 9: 95%, 10:
1
9
3
7
8
0 °C, 2 h, yields for 17: 80%, 18: 80%; (v) K
0 min, yields for 19: 32%, 20: 63%, 21: 55%; (vi) NaI, acetone, reflux, 24 h, yields for 22: 82%, 23:
6%, 24: 53%; (vii) NH in isopropanol, 80 °C, 3 h, yield for 25: 50%; (viii) methylamine in EtOH,
0 °C, 3 h, yields for 4: 48%, 5: 29%, 6: 30%.
2
CO , DMF, rt, 30 min → addition of 11 and 12 → rt,
3
3
Scheme 1. Synthesis of compounds 4‒6.
For the synthesis of side chains 11 and 12, a protocol of Varney et al. [28] was adopted and the keto
group of commercially available compounds 7 and 8 was reduced with sodium borohydride in order to

Molecules 2015, 20
1716
obtain intermediate alcohols 9 and 10. Subsequent bromination of 9 and 10 with aqueous hydrogen
bromide led to the formation of products 11 and 12, respectively.
Core compound 16 was prepared by the reaction of commercially available N-phenylbenzene-1,2-
diamine (13) with 1,1'-carbonyldiimidazole. For the preparation of 17 and 18 however, 14 and 15 first
had to be made accessible (Scheme 2). Thus, 1-fluoro-2-nitrobenzene (26) reacted with commercially
available fluoroanilines 27 and 28, respectively, to obtain disubstituted amines 29 and 30. Therefore,
two different methods were applied (Scheme 2): The first method (i) was conducted by heating 26 and
2
7 with anhydrous potassium fluoride and potassium carbonate in a microwave oven [29]. Since the
adoption of an alternative method [30]—conventional heating at 180 °C—gave compound 29 in higher
yields, this approach (ii) was chosen for the large scale synthesis of 29 as well as for the preparation of 30.
Reagents and conditions: (i) anhyd. KF, K
2
CO , microwave oven, 900 W, 10 min, yields for 29: 58%;
3
2 3
(ii) anhyd. KF, K CO , 180 °C, 2 d, yields for 29: 68%, 30: 52%; (iii) Zn, AcOH, 0 °C → rt, 2 h, yields
for 14: 93%, 15: n.d.
Scheme 2. Synthesis of compounds 14‒15.
In the next reaction step, the nitro groups of both disubstituted amines 29 and 30 were reduced. For
this purpose 29 or 30 were added to a mixture of zinc/acetic acid. The resulting amines 14 or 15 were
obtained in excellent yields (Scheme 2) [30].
Freshly prepared intermediates 14 and 15 were then subjected to a cyclization reaction with
1
,1'-carbonyldiimidazole in DMF under anhydrous conditions (Scheme 1) by modifying a synthesis
protocol according to Zhang et al. [26]. DMF was preferred over THF to ensure higher yields and shorter
reaction times.
Condensation reactions of benzimidazolones 16, 17, and 18 with side chains 11 and 12, respectively
were performed under basic conditions (Scheme 1) by adapting a procedure of Jona et al. [31]. After
purification, the chloro-substituted derivatives 19–21 were converted into the iodo compounds 22–24 in
a Finkelstein reaction. Target compounds 4–6 (FAPPI:1-3) were then obtained by heating derivatives
2
4
2–24 in a solution of methylamine in ethanol in a sealed tube for 3 h. In addition to reference compounds
–6, free amine 25 was synthesized by dissolving 22 in a solution of ammonia in isopropanol and heating
the resulting mixture in a sealed tube for 3 h. As compound 25 features a free amine moiety, it can be
considered a precursor for radiosynthesis.
Since compounds 4 and 5 comprise a novel fluoro substitution, a computational docking study was
performed to assess if these compounds still would fit in the binding site of the NET. Furthermore, we
aimed at creating a binding mode hypothesis which allows gaining insights into the molecular basis of
binding and selectivity towards the monoamine transporters. As the basic scaffold has been shown to act in

Molecules 2015, 20
1717
an enantioselective manner, the respective (R) enantiomers were used throughout the docking studies [26].
The ligands were docked in the substrate binding site (S1) of the outward-open conformation of the
transporter models (see Experimental Section for details), since related inhibitors, such as nortriptyline,
sertraline, mazindol, etc. were also shown to fit in the S1 of the Drosophila DAT (dDAT) and the
“
SERT”-ized leucine transporter (“LeuBAT”) in the same protein conformation [32,33]. Interestingly,
the co-crystallized ligand in dDAT, nortriptyline (31), has the same ranking of human NET, SERT and
DAT activity as reference compound 1, i.e., 4.4, 18 and 1149 nM K vs. 9, 2995 and >10,000 nM IC50
D
,
respectively [26,34]. Additionally, nortriptyline shares important structural features with the
benzimidazolones, i.e., two aromatic moieties and an N-methyl-ethylenamine side chain. Therefore their
binding mode can be expected to be similar.
Common scaffold clustering [35] revealed two binding hypotheses (see Experimental Section) which
indicated that compounds 4–6 fit in the S1 of all three transporters. Hence, additional fluorination does
not seem to cause steric clashes. In both hypotheses, the most prominent protein-ligand interaction was
the cationic nitrogen atom placed in the A sub pocket [36], located between the central Asp75/98/79 side
chain as a salt-bridge and the Phe72/95/76 side chain as a cation-pi interaction in NET/SERT/DAT,
respectively. This is well in accordance with the X-ray structures of the templates. Additional pi-pi
stacking interactions with Phe152/176/156 and Phe323/355/341 further promote the binding in both
hypotheses obtained:
Hypothesis 1: the benzimidazolone heterocycle (ring 2) is placed in the B sub pocket and ring 3 in
the C pocket, whereas ring 1 is solvent exposed (see figure in Experimental Section).
Hypothesis 2: Ligand ring 1 is placed in the B pocket whereas ring 2 is placed at the same height
(measured from the membrane-water interface) and overlap with the rings of nortriptyline (31, Figure 3).
The solvent exposed Tyr151/Tyr175/Phe155 in NET/SERT/DAT, resp. T-stacks with ring 2 whereas
ring 3 points extracellularly (Table 1, Figure 4).
Figure 3. Chemical structure of nortriptyline (co-crystallized ligand from the template, PDB
code 4M48) [28].
Table 1. Sequence alignment of all distinct monoamine transporter residues located in sub
sites (reference [32]) in the vicinity of the docked compounds. Red: hydrophilic side chain,
green: lipophilic side chain, bold: bulkier side chain.
B Site
S420
T439
A423
C Site
A145
A177
S149
Outer Site
Y151
hNET
hSERT
hDAT
M424
L443
M427
G149
A173
G153
V148
I172
F72
Y95
F76
D473
E493
D476
A477
T497
A480
Y175
V152
F155

Molecules 2015, 20
1718
Figure 4. Overlay of compounds 1, 2, and 4–6 (maroon) in binding hypothesis 2 in hNET
showing agreement with the co-crystal pose of nortriptyline (31) (green). Val148 and
Asp473 allow more space than Ile172 and Glu439 in hSERT, resp., whereas Tyr151 might
induce a more potent stacking interaction as compared to Phe155 in hDAT. The angle between
ligand ring 2 and 3 is almost 90° in all poses. The extracellular space is located above.
As binding hypothesis 2 is in close agreement with the co-crystallized ligands in dDAT and LeuBAT,
we focus further analysis on this proposed binding mode. Binding hypothesis 2 indicates why the
investigated compounds (4–6) show weaker affinity to SERT and DAT than to NET: lower SERT
affinity may be due to Ile172 and Glu439, allowing less space for the ligand to be accommodated as
compared to in NET, that comprises a valine and an aspartate at the homologous positions, respectively.
Lower DAT affinity could be ascribed to weaker T stacking interactions of Phe155 as compared to
Tyr151 in NET, based on previous findings that a Tyr-Phe pair has a stronger binding energy than a
Phe-Phe pair [37].
Since the docking studies indicate that fluorinated methyl amines 4–6 (FAPPI:1-3) bind in an
analogous way to the NET as reference compound 1 [26], compounds 4–6 will be employed in future
studies and evaluated for affinity and selectivity towards the NET. Additionally, lipophilicity studies and
blood brain barrier penetration experiments are planned for compounds 4–6 at the Medical University
of Vienna. The most promising derivatives regarding their suitability as NET ligands will then be
selected for the further development of new and selective PET tracers for the NET, which will comprise
1
1
18
18
either a [ C]methylamine, [ F]fluoroalkyl amine or [ F]fluorobenzene radiolabel, respectively. The
results of ongoing studies on affinity, selectivity and lipophilicity of the discussed compounds, will be
published in a subsequent paper.
3. Experimental Section
3.1. General
The NMR spectra were recorded from CDCl
3
or DMSO-d solutions on a Bruker DPX200
6
1
13
spectrometer (200 MHz for H, 50 MHz for C) or on a Bruker Avance III 400 spectrometer (400 MHz

Molecules 2015, 20
1719
1
13
15
19
for H, 100 MHz for C, 40 MHz for N, 376 MHz for F) at 25 °C. The center of the solvent (residual)
1
signal was used as an internal standard which was related to TMS with δ 7.26 ppm ( H in CDCl
3
),
1
13
13
15
δ 2.49 ppm ( H in DMSO-d
6
), δ 77.0 ppm ( C in CDCl
3
) and δ 39.5 ppm ( C in DMSO-d
6
). N NMR
1
9
spectra (gs-HMBC, gs-HSQC) were referenced against neat, external nitromethane, F NMR spectra by
1
absolute referencing via Ξ ratio. Digital resolutions were 0.25 Hz/data point in the H and 0.3 Hz/data
1
3
point in the C-NMR spectra. Coupling constants (J) are quoted in Hz. The following abbreviations
were used to show the multiplicities: s: singlet, d: doublet, t: triplet, q: quadruplet, dd: doublet of doublet,
m: multiplet. Mass spectra were obtained on a Shimadzu QP 1000 instrument (EI, 70 eV),
high-resolution mass spectrometry (HRMS) was carried out on a Finnigan MAT 8230 (EI, 10 eV) or
Finnigan MAT 900 S (ESI, 4 kV, 3 μA, CH
a micro-TOF analyzer. Microwave experiments were carried out in a Synthos 3000 microwave oven
SXQ80 rotor, Anton Paar, Graz, Austria) with an internal temperature probe. Compound purity: all
compounds synthesized featured a purity of at least 95%.
3
CN/MeOH) electrospray ionization mass spectrometer with
(
3
.2. Syntheses
3
.2.1. General Procedure for the Synthesis of 9 and 10
Starting materials 7 or 8, respectively (1 mmol) was dissolved in THF (1 mL) and EtOH (1 mL) was
added. The mixture was cooled to −10 °C and NaBH (1.05 mmol) was slowly added at this temperature.
4
The solution was stirred at −5 °C for 10 min and thereafter, poured into a mixture of saturated aqueous
ammonium chloride (3 mL) in ice (1.5 g). The product was extracted with diethyl ether, dried over
Na
2
SO and evaporated to dryness. The crude product was employed directly in the subsequent reaction
4
step without further purification.
3
-Chloro-1-(4-fluorophenyl)propan-1-ol (9). Yield: 4.78 g (95%), pale yellow oil, analytical data are in
complete accordance with literature values [38].
3
-Chloro-1-phenylpropan-1-ol (10). Yield: 4.61 g (99%), light yellow oil, analytical data are in complete
accordance with literature values [28].
3
.2.2. General Procedure for the Synthesis of 11 and 12
To starting material 9 or 10, respectively (1 mmol) was added 48% aqueous HBr (3 mL) and the
mixture was stirred for 3h at room temperature. Thereafter, the solution was poured into a mixture of
CO (1 g) in ice (5.5 g) and additional solid K CO was added for neutralization (pH 7). The crude
reaction product was extracted with diethyl ether, the combined organic layers were dried with MgSO
K
2
3
2
3
4
and evaporated to dryness. The crude product was employed directly in the subsequent reaction step
without further purification.
1
-(1-Bromo-3-chloropropyl)-4-fluorobenzene (11). Yield: 64%, pale yellow oil, analytical data are in
complete accordance with literature values [39].

Molecules 2015, 20
1720
(
1-Bromo-3-chloropropyl)benzene (12). Yield: 5.64 g (86%), yellow oil, analytical data are in complete
accordance with literature values [28].
3
.2.3. General Procedure for the Synthesis of 29 and 30
4
-Fluoroaniline or 2-fluoroaniline, respectively (1 mmol), anhydrous KF (1 mmol), and K
1 mmol) were well powdered with a mortar and a pestle, then 1-fluoro-2-nitrobenzene (1 mmol) was
added and the mixture was stirred for 2 days at 180 °C. Thereafter, water (5 mL) and CH Cl (5 mL) were
added and the organic layer was washed with 10% HCl (5 mL) and brine (5 mL). The combined organic
layers were dried over Na SO and evaporated to dryness prior to purification by column chromatography.
2
CO
3
(
2
2
2
4
N-(4-Fluorophenyl)-2-nitroaniline (29). Yield: 68%, dark orange crystals, mp. 82–83 °C, purification:
silica gel 60, petroleum ether/ethyl acetate 9:1 and RP-18 silica gel, methanol/water 7:3, analytical data
are in complete accordance with literature values [29].
2
-Fluoro-N-(2-nitrophenyl)aniline (30). Yield: 52%, orange crystals, mp. 79–80 °C, purification: silica
gel 60, petroleum ether/ethyl acetate 9:1, analytical data are in complete accordance with literature
values [40].
3
.2.4. Alternative Procedure for the Synthesis of 29
-Fluoroaniline (7.89 g, 6.73 mL, 70.87 mmol), anhydrous KF (4.13 g, 70.87 mmol), and K
4
2
CO
3
(
9.81 g, 70.87 mmol) were well powdered with a mortar and a pestle, then 1-fluoro-2-nitrobenzene
10.00 g, 7.47 mL, 70.87 mmol) was added and the mixture was irradiated in the microwave (900 W,
(
1
0 min). Thereafter, water (8 mL) and CH
2
Cl
2
(10 mL) were added and the organic layer was washed
SO and
with 10% HCl (5 mL) and brine (5 mL). The combined organic layers were dried over Na
2
4
evaporated to dryness prior to purification by column chromatography (silica gel 60, petroleum
ether/ethyl acetate 9.5:0.5). Yield: 9.51 g (58%), dark orange crystals, mp. 82 °C–83 °C.
3
.2.5. General Procedure for the Synthesis of 14 and 15
0
To a solution of Zn (13.8 mmol) in glacial acetic acid (1 mL) was added starting material 28 or 29
(
1 mmol) at 0 °C under argon atmosphere. After the addition, the mixture was allowed to warm to room
0
temperature and was stirred for 2 h. Zn was filtered off and the pH of the solution was adjusted to pH 9
with 2N NaOH. Thereafter, the aqueous layer was extracted three times with CH
2
Cl , the combined
2
organic layers were dried over MgSO and evaporated to dryness.
4
N-(4-Fluorophenyl)benzene-1,2-diamine (14). Yield: 93%, dark orange-reddish oil, purification: silica
gel 60, petrol ether/ethyl acetate 9:1, analytical data are in complete accordance with literature values [30].
N-(2-Fluorophenyl)benzene-1,2-diamine (15). The crude reaction product was subjected to the next
reaction step without further purification.

Molecules 2015, 20
.2.6. General Procedure for the Synthesis of 16–18
1721
3
To a solution of starting materials 13, 14, or 15 (1 mmol) in THF was added 1,1'-carbonyldiimidazole
1.4 mmol) under argon atmosphere and the mixture was stirred at room temperature overnight.
Thereafter, the crude reaction product was purified by column chromatography.
(
1-Phenyl-1,3-dihydro-2H-benzimidazol-2-one (16). Yield: 0.85 g (74%), pink crystals, mp.
2
01 °C–202 °C, THF: 10 mL, purification: silica gel 60, petroleum ether/ethyl acetate 1:1, analytical
data are in complete accordance with literature values [41].
1
-(4-Fluorophenyl)-1,3-dihydro-2H-benzimidazol-2-one (17). Yield: 61%, brown resin, THF: 25 mL,
1
purification: silica gel 60, petroleum ether/ethyl acetate 9:1, H-NMR (200 MHz, CDCl
3
): δ (ppm)
6
.74–6.81 (m, 2H), 6.89–7.11 (m, 2H), 7.14–7.23 (m, 2H), 7.48–7.59 (m, 1H), 7.73–7.78 (m, 1H), 9.03
1
3
(
br s, 1H), C-NMR (50 MHz, CDCl
3
): δ (ppm) 108.5, 110.0, 116.3, 116.8, 121.4, 121.7, 122.3, 128.0,
+
1
7
28.2, 130.4, 135.1, 155.1, 159.3, 164.2, MS: m/z (%) 228 (M , 100), 199 (31), 172 (8), 114 (9), 95 (10),
+
5 (17), 51 (10), HRMS: m/z calculated for C13
H
10FN O [M + H] : 229.0772. Found: 229.0769.
2
1
-(2-Fluorophenyl)-1,3-dihydro-2H-benzimidazol-2-one (18). Yield: 69%, brown resin, THF: 20 mL,
1
purification: silica gel 60, petroleum ether/ethyl acetate 9:1, H-NMR (200 MHz, CDCl
3
): δ (ppm)
6
.82–6.85 (m, 1H), 7.00–7.19 (m, 3H), 7.26–7.88 (m, 2H), 7.43–7.60 (m, 2H), 10.54 (br s, 1H),
13C-NMR (50 MHz, CDCl
): δ (ppm) 108.9, 110.1, 117.0, 117.4, 121.5, 121.9, 122.4, 124.9, 125.0,
3
+
1
28.2, 129.6, 130.4, 154.8, 155.4, 160.5, MS: m/z (%) 228 (M , 33), 199 (9), 181 (15), 149 (17), 111
(22), 97 (20), 71 (41), 69 (100), 55 (53), 43 (56), HRMS: m/z calculated for C13
H
9
FN
NaO [M + Na]+:
2
2
51.0597. Found: 251.0592.
3
.2.7. Alternative Procedure for the Synthesis of 17 and 18
A solution of 1,1'-carbonyldiimidazole (1.4 mmol) in DMF (4 mL) was slowly added to a mixture of
1
4 or 15 (1 mmol) in DMF (4 mL) under argon atmosphere. The resulting solution was stirred at 90 °C
for 2 h. After completion of the reaction, the solvent was evaporated in vacuo, the slurry was taken up
in water, filtered and dried.
1
1
3
-(4-Fluorophenyl)-1,3-dihydro-2H-benzimidazol-2-one (17). Yield: 80%, brown resin.
-(2-Fluorophenyl)-1,3-dihydro-2H-benzimidazol-2-one (18). Yield: 80%, brown resin.
.2.8. General Procedure for the Synthesis of 19–21
Starting materials 16–18 (1 mmol) and K
2
CO (2 mmol) were suspended in DMF (1.8 mL) and stirred
3
at 25 °C for 30 min. 11 and 12, respectively (1.5 mmol) were added after 30 min and the solution was
stirred at room temperature overnight. To the mixture was added ethyl acetate (5 mL) and water (5 mL).
The aqueous layer was extracted several times with ethyl acetate (10 mL) and the combined organic
layers were washed with brine, dried over MgSO and evaporated to dryness.
4

Molecules 2015, 20
1722
1
-(3-Chloro-1-(4-fluorophenyl)propyl)-3-phenyl-1,3-dihydro-2H-benzimidazol-2-one (19). Yield: 32%,
1
white oil, purification: silica gel 60, petroleum ether/ethyl acetate 8:2, H-NMR (200 MHz, CDCl
3
): δ
(
ppm) 2.70–2.87 (m, 1H), 3.12–3.30 (m, 1H), 3.60–3.66 (m, 2H), 5.73 (m, 1H), 7.00–7.11 (m, 6H),
1
3
7
1
1
.38–7.57 (m, 7H), C-NMR (50 MHz, CDCl
3
): δ (ppm) 34.3, 42.0, 53.8, 108.7, 109.0, 115.5, 115.9,
+
+
21.7, 122.0, 126.0, 127.8, 129.2, 129.4, 129.5, MS: m/z (%) 380 (M , 21), 210 (100) (M -C
9
9
H ClF),
81 (8), 167 (12), 135 (9), 115 (5), 109 (58), 77 (12), HRMS: m/z calculated for C22
H
18ClFN
2
ONa
+
[M + Na] : 403.0989. Found: 403.0989.
1
-(3-Chloro-1-phenylpropyl)-3-(4-fluorophenyl)-1,3-dihydro-2H-benzimidazol-2-one (20). Yield: 63%,
dark orange resin, purification: silica gel 60, petroleum ether/ethyl acetate 9:1 and RP-18 silica gel,
1
methanol, H-NMR (400 MHz, CDCl
3
): δ (ppm) 2.80–2.88 (m, 1H, 2'-CH
), 5.79 (dd, J = 10.0 Hz and 5.6 Hz, 1H, 1'-CH), 7.04–7.09 (m, 4H, benzim 4-CH,
benzim 5-CH, benzim 6-CH, benzim 7-CH), 7.21–7.26 (m, 2H, f-phen 3-CH, f-phen 5-CH), 7.31–7.33
m, 1H, phen 4-CH), 7.36–7.40 (m, 2H, phen 3-CH, phen 5-CH), 7.52–7.56 (m, 4H, f-phen 2-CH,
2
), 3.17–3.26 (m, 1H, 2'-CH
2
),
3
.62–3.70 (m, 2H, 3'-CH
2
(
1
3
f-phen 6-CH, phen 2-CH, phen 6-CH), C-NMR (100 MHz, CDCl
3
): δ (ppm) 34.1 (2'-CH
2
), 42.0
2
(3'-CH ), 54.4 (1'-CH), 108.6 (benzim 4-CH), 109.0 (benzim 7-CH), 116.4 (d, J = 22.9 Hz, f-phen 3-CH,
1
1
4
16.4 (d, J = 22.9 Hz, f-phen 5-CH), 121.6 (benzim 5-CH), 122.1 (benzim 6-CH), 127.4 (phen 2-CH),
27.4 (phen 6-CH), 127.9 (d, J = 8.6 Hz, f-phen 2-CH), 127.9 (d, J = 8.6 Hz, f-phen 6-CH), 128.1 (phen
-CH), 128.7 (benzim 7a-C), 128.8 (phen 3-CH), 128.8 (phen 5-CH), 129.4 (benzim 3a-C), 130.3 (d,
J = 3.1 Hz, f-phen 1-C), 138.4 (phen 1-C), 153.2 (benzim 2-CO), 161.6 (d, J = 247.7 Hz, f-phen 4-CF),
19F-NMR (471 MHz, CDCl
): δ (ppm) -113.31 (m, 5-CF), MS: m/z (%) 380 (M , 2), 228 (100)
+
3
+
(
M -C
9
H
10Cl), 199 (11), 185 (16), 153 (6), 117 (14), 91 (73), 75 (8), HRMS: m/z calculated for
+
C
22
H
19ClFN
2
O [M + H] : 381.1170. Found: 381.1176.
1
-(3-Chloro-1-phenylpropyl)-3-(2-fluorophenyl-1,3-dihydro-2H-benzimidazol-2-one (21). Yield: 55%,
1
orange resin, purification: silica gel 60, petroleum ether/ethyl acetate 9:1, H-NMR (400 MHz, CDCl
3
):
δ (ppm) 2.78–2.86 (m, 1H, 2'-CH
2
), 3.23 (br s, 1H, 2'-CH
2
), 3.64–3.68 (m, 2H, 3'-CH
2
), 5.79 (br s, 1H,
1
'-CH), 6.85–6.87 (m, 1H, benzim 4-CH) 7.05–7.06 (m, 3H, benzim 5-CH, benzim 6-CH, benzim 7-CH),
.28–7.40 (m, 5H, f-phen 3-CH, f-phen 6-CH, phen 3-CH, phen 4-CH, phen 5-CH), 7.43–7.48 (m, 1H,
7
1
3
f-phen 4-CH), 7.52–7.56 (m, 3H, f-phen 5-CH, phen 2-CH, phen 6-CH), C-NMR (100 MHz, CDCl
3
):
δ (ppm) 34.2 (2'-CH ), 41.9 (3'-CH ), 54.5 (1'-CH), 108.8 (d, J = 1.7 Hz, benzim 4-CH), 109.0 (benzim
2
2
7-CH), 117.1 (d, J = 19.5 Hz, f-phen 3-CH), 121.6 (benzim 5-CH), 121.9 (f-phen 1-C), 122.0 (benzim
6-CH), 124.8 (d, J = 3.9 Hz, f-phen 6-CH), 127.3 (phen 2-CH), 127.3 (phen 6-CH), 128.1 (phen 4-CH),
128.8 (phen 3-CH), 128.8 (phen 5-CH), 129.4 (benzim 3a-C), 129.5 (f-phen 5-CH), 130.2 (d, J = 7.8 Hz,
f-phen 4-CH), 138.4 (phen 1-C), 153.0 (benzim 2-CO), 157.9 (d, J = 253.2 Hz, f-phen 2-CF), due to
limited resolution of the measuring apparatus, quaternary carbon benzim 7a-C could not be detected,
19F-NMR (471 MHz, CDCl
): δ (ppm) -118.39 (m, f-phen 2-CF), MS: m/z (%) 380 (M , 12), 228 (100)
+
3
+
(
[
M -C
9
H
10Cl), 199 (5), 153 (3), 117 (7), 91 (49), 75 (5), HRMS: m/z calculated for C22
H
19ClFN O
2
+
M + H] : 381.1170. Found: 381.1164.

Molecules 2015, 20
.2.9. General Procedure for the Synthesis of 22–24
1723
3
A solution of starting materials 19, 20, or 21 (1 mmol) and NaI (1.03 g, 6.89 mmol) in acetone (7 mL)
was refluxed for 24 h. The precipitate formed was filtered and the solvent was removed in vacuo.
1
-(1-(4-Fluorophenyl)-3-iodopropyl)-3-phenyl-1,3-dihydro-2H-benzimidazol-2-one (22). Yield: 82%,
1
yellow resin, H-NMR (200 MHz, CDCl
3
): δ (ppm) 2.77–2.92 (m, 1H), 3.14–3.31 (m, 3H), 5.63–5.70
1
3
(
m, 1H), 7.01–7.10 (m, 6H), 7.36–7.56 (m, 7H), C-NMR (50 MHz, CDCl
3
): δ (ppm) 2.3, 35.3, 57.0,
1
1
08.8, 108.9, 115.5, 115.9, 121.6, 122.0, 126.0, 127.7, 128.5, 129.1, 129.3, 129.4, 134.0, 134.1, 134.3,
+
+
53.0, 159.9, 164.8, MS: m/z (%) 472 (M , 32), 317 (8), 210 (100) (M -C
9
H
9
F), 181 (11), 167 (23), 140
ONa [M + Na]+:
(
3), 135 (43), 115 (8), 109 (34), 77 (15), 51 (7), HRMS: m/z calculated for C22
H18FIN
2
4
95.0346. Found: 495.0353.
1
-(4-Fluorophenyl)-3-(3-iodo-1-phenylpropyl)-1,3-dihydro-2H-benzimidazol-2-one (23). Yield: 76%,
1
yellow crystals, mp. 39 °C–41 °C, purification: silica gel 60, petrol ether/ethyl acetate 9:1, H-NMR
(
200 MHz, CDCl
J = 6 Hz and 2 Hz, 1H, 1'-CH), 7.04–7.09 (m, 4H, benzim 4-CH, benzim 5-CH, benzim 6-CH, benzim
-CH), 7.17–7.42 (m, 5H, f-phen 3-CH, f-phen 5-CH, phen 4-CH, phen 2-CH, phen 5-CH), 7.51–7.57
3
): δ (ppm) 2.81–2.99 (m, 1H, 2'-CH
2
), 3.11–3.35 (m, 3H, 2'-CH
2
, 3'-CH
2
), 5.70 (dd,
7
1
3
(
m, 4H, f-phen 2-CH, f-phen 6-CH, phen 2-CH, phen 6-CH), C-NMR (50 MHz, CDCl
3
): δ (ppm) 2.4
(
2'-CH ), 35.2 (3'-CH ), 57.6 (1'-CH), 108.6 (benzim 4-CH), 109.2 (benzim 7-CH), 116.4 (d, J = 23 Hz,
2
2
f-phen 3-CH), 116.4 (d, J = 23 Hz, f-phen 5-CH), 121.6 (benzim 5-CH), 122.1 (benzim 6-CH), 127.3
phen 2-CH), 127.3 (phen 6-CH), 127.8 (phen 4-CH), 128.1 (d, J = 5 Hz, f-phen 2-CH), 128.1 (d, J = 5 Hz,
f-phen 6-CH), 128.5 (benzim 7a-C), 128.8 (phen 3-CH), 128.8 (phen 5-CH), 129.4 (benzim 3a-C), 130.3
d, J = 3Hz, f-phen 1-C), 138.1 (phen 1-C), 153.2 (benzim 2-CO), 161.6 (d, J = 247 Hz, f-phen 4-CF),
(
(
+
+
MS: m/z (%) 472 (M , 13), 317 (5), 228 (100) (M -C
9
H
10I), 199 (8), 185 (15), 117 (47), 103 (2), 91 (40),
+
7
5 (8), 55 (5), HRMS: m/z calculated for C22
H19FIN
2
O [M + H] : 473.0526. Found: 473.0506.
1
-(2-Fluorophenyl)-3-(3-iodo-1-phenylpropyl)-1,3-dihydro-2H-benzimidazol-2-one (24). Yield: 53%,
1
yellow crystals, mp. 38 °C–39 °C, purification: silica gel 60, petrol ether/ethyl acetate 9:1, H-NMR
(200 MHz, CDCl
3
): δ (ppm) 2.80–2.98 (m, 1H, 2'-CH
2
), 3.13–3.36 (m, 3H, 2'-CH
2
, 3'-CH
2
), 5.67–5.74
(
m, 1H, 1'-CH), 6.85–6.88 (m, 1H, benzim 4-CH) 6.99–7.07 (m, 3H, benzim 5-CH, benzim 6-CH,
benzim 7-CH), 7.26–7.45 (m, 5H, f-phen 3-CH, f-phen 6-CH, phen 3-CH, phen 4-CH, phen 5-CH),
1
3
7
.47–7.58 (m, 4 H, f-phen 4-CH, f-phen 5-CH, phen 2-CH, phen 6-CH), C-NMR (50 MHz, CDCl
3
): δ
(
ppm) 2.3 (2'-CH ), 35.4 (3'-CH ), 57.6 (1'-CH), 108.9 (d, J = 1.5 Hz, benzim 4-CH), 109.1 (benzim
2
2
7
6
1
-CH), 117.1 (d, J = 19 Hz, f-phen 3-CH), 121.6 (benzim 5-CH), 121.9 (f-phen 1-C), 122.0 (benzim
-CH), 124.8 (d, J = 3.5 Hz, f-phen 6-CH), 127.3 (phen 2-CH), 127.3 (phen 6-CH), 128.1 (phen 4-CH),
28.8 (phen 3-CH), 128.8 (phen 5-CH), 129.5 (f-phen 5-CH), 130.2 (d, J = 8 Hz, f-phen 4-CH), 138.2
+
(
(
phen 1-C), 153.0 (benzim 2-CO), 157.8 (d, J = 251.5 Hz, f-phen 2-CF), MS: m/z (%) 472 (M , 9), 317
+
5), 241 (4), 228 (100) (M -C
9
H
10I), 199 (5), 185 (10), 117 (37), 91 (29), 75 (7), HRMS: m/z calculated
+
for C22
H19FIN
2
O [M + H] : 473.0526. Found: 473.0532.

Molecules 2015, 20
1724
3
.2.10. General procedure for the synthesis of 1-(3-amino-1-(4-fluorophenyl)propyl)-3-phenyl-1,3-
dihydro-2H-benzimidazol-2-one (25)
1-(1-(4-fluorophenyl)-3-iodopropyl)-3-phenyl-1,3-dihydro-2H-benzimidazol-2-one (0.26 g, 0.55 mmol)
and a solution of NH in isopropanol (2 M, 22 mL) were heated in a sealed tube for 3 h at 80 °C. After
3
evaporation of the solvent, the crude product was purified by column chromatography (silica gel 60,
1
CH
2
Cl
2
/MeOH 9:1). Yield: 0.10 g (50%), light brown crystals, mp. 87–88 °C. H-NMR (400 MHz,
CDCl
3
): δ (ppm) 2.74–2.84 (m, 3H, 2'-CH
'-CH), 6.79–6.81 (m, 1H, benzim 7-CH), 6.96–7.01 (m, 5H, benzim 4-CH, benzim 5-CH, benzim
-CH, f-phen 3-CH, f-phen 5-CH), 7.30–7.33 (m, 1H, phen 4-CH), 7.40–7.48 (m, 4H, f-phen 2-CH,
2
, 3'-CH
2
), 2.98–3.04 (m, 1H, 3'-CH
2
), 5.74–5.78 (m, 1H,
1
6
f-phen 6-CH, phen 3-CH, phen 5-CH), 7.52–7.54 (m, 2H, phen 2-CH, phen 6-CH), due to limited
1
3
resolution of the instrumentation, the NH
δ (ppm) 30.3 (2'-CH ), 37.8 (3'-CH ), 52.7 (1'-CH), 109.2 (benzim 4-CH), 110.0 (benzim 7-CH), 115.7
d, J = 21.5 Hz, f-phen 3-CH), 115.7 (d, J = 8.2 Hz, f-phen 5-CH), 122.0 (benzim 5-CH), 122.3 (benzim
2
protons could not be detected, C-NMR (100 MHz, CDCl
3
):
2
2
(
6
-CH), 126.4 (phen 2-CH), 126.4 (phen 6-CH), 127.5 (benzim 7a-C), 128.1 (phen 4-CH), 129.2 (d,
J = 8.2 Hz, f-phen 2-CH), 129.2 (d, J = 8.2 Hz, f-phen 6-CH), 129.5 (benzim 3a-C), 129.7 (phen 3-CH),
29.7 (phen 5-CH), 133.3 (d, J = 3.2 Hz, f-phen 1-C), 134.0 (phen 1-CH), 153.8 (benzim 2-CO), 162.3
1
1
9
(
(
d, J = 247.4 Hz, f-phen 4-CF), F-NMR (471 MHz, CDCl ): δ (ppm) -113.68 (m, f-phen CF), MS: m/z
3
+
+
%) 361 (M , 17), 331 (10), 210 (100) (M -C
9
H
11FN), 181 (15), 167 (16), 149 (29), 128 (17), 77 (19),
+
5
7 (20), 43 (12), HRMS: m/z calculated for C22
.2.11. General Procedure for the Synthesis of 4–6
Starting materials 18, 19 or 20 (1 mmol) and a solution of methylamine in EtOH (12.5 mL, 8 M) were
H
21FN
3
O [M + H] : 362.1669. Found: 362.1674.
3
heated in a sealed tube for 3 h at 80 °C. After evaporation of the solvent, the crude reaction product was
purified by column chromatography.
1
-(1-(4-Fluorophenyl)-3-(methylamino)propyl)-3-phenyl-1,3-dihydro-2H-benzimidazol-2-one
(4).
Yield: 48%, light orange resin, purification: silica gel 60, dichloromethane/methanol 9:1 and
1
dichloromethane/ethyl acetate/methanol 7:2:1, H-NMR (400 MHz, CDCl
3
): δ (ppm) 2.42 (s, 3H,
), 5.76–5.79 (m, 1H, 1'-CH), 6.88–6.90
m, 1H, benzim 7-CH), 6.97–7.05 (m, 4H, benzim 5-CH, benzim 6-CH, f-phen 3-CH, f-phen 5-CH),
NHCH
3
), 2.57–2.74 (m, 4H, 2'-CH
2
, 3'-CH
2
), 3.15 (br s, 1H, NHCH
3
(
7
.07–7.10 (m, 1H, benzim 4-CH), 7.39–7.43 (m, 1H, phen 4-CH), 7.46–7.57 (m, 6H, f-phen 2-CH, f-phen
1
3
6
-CH, phen 2-CH, phen 3-CH, phen 5-CH, phen 6-CH), C-NMR (100 MHz, CDCl
), 35.9 (NHCH ), 48.3 (3'-CH ), 53.1 (1'-CH), 108.9 (benzim 4-CH), 109.5 (benzim 7-CH), 115.5
d, J = 21.5 Hz, f-phen 3-CH), 115.5 (d, J = 21.5 Hz, f-phen 5-CH) 121.4 (benzim 5-CH), 121.8 (benzim
-CH), 126.0 (phen 2-CH), 126.0 (phen 6-CH), 127.7 (phen 4-CH), 128.0 (benzim 7a-C), 129.0 (d,
J = 8.1 Hz, f-phen 2-CH), 129.0 (d, J = 8.1 Hz, f-phen 6-CH), 129.4 (benzim 3a-C), 129.5 (phen 3-CH),
29.5 (phen 5-CH), 134.4 (phen 1-CH), 134.6 (d, J = 3.4 Hz, f-phen 1-CH), 153.5 (benzim 2-CO), 162.1
3
): δ (ppm) 30.6
(2'-CH
2
3
2
(
6
1
1
9
(
d, J = 246.7 Hz, f-phen 4-CF), F-NMR (471 MHz, CDCl ): δ (ppm) -114.36 (m, f-phen 4-CF), MS:
3
+
+
m/z (%) 375 (M , 16), 210 (57) (M -C10
H
13FN), 181 (10), 167 (12), 150 (10), 109 (22), 97 (16), 71 (27),
+
5
7 (78), 44 (100), HRMS: m/z calculated for C23
H23FN
3
O [M + H] : 376.1825. Found: 376.1821.

Molecules 2015, 20
-(4-Fluorophenyl)-3-(3-(methylamino)-1-phenylpropyl)-1,3-dihydro-2H-benzimidazol-2-one
1725
1
(5).
Yield: 29%, light yellow crystals, mp. 100 °C–102 °C, purification: silica gel 60,
1
dichloromethane/methanol 9:1 and RP-18 silica gel methanol/water 9:1 and 7:3, H-NMR (200 MHz,
CDCl
3
): δ (ppm) 2.53 (s, 3H, NHCH
.96–7.03 (m, 4H, benzim 4-CH, benzim 5-CH, benzim 6-CH, benzim 7-CH), 7.16–7.34 (5H, f-phen
-CH, f-phen 5-CH, phen 4-CH, phen 2-CH, phen 5-CH), 7.49–7.56 (m, 4H, f-phen 2-CH, f-phen 6-CH,
3
), 3.13 (br s, 4H, 2'-CH
2
, 3'-CH
2
), 5.77–5.81 (m, 1H, 1'-CH),
6
3
phen 2-CH, phen 6-CH), due to limited resolution of the instrumentation, the NH proton could not be
1
3
detected, C-NMR (50 MHz, CDCl
1'-CH), 108.8 (benzim 4-CH), 109.9 (benzim 7-CH), 116.6 (d, J = 23 Hz, f-phen 3-CH), 116.6 (d,
J = 23 Hz, f-phen 5-CH), 122.1 (benzim 5-CH), 122.6 (benzim 6-CH), 127.2 (phen 2-CH), 127.2 (phen
-CH), 127.7 (phen 4-CH), 128.3 (d, J = 6 Hz, f-phen 2-CH), 128.3 (d, J = 6 Hz, f-phen 6-CH), 128.4
benzim 7a-C), 128.9 (phen 3-CH), 128.9 (phen 5-CH), 129.2 (benzim 3a-C), 129.7 (d, J = 3 Hz, f-phen
-C), 136.9 (phen 1-C), 153.4 (benzim 2-CO), 161.7 (d, J = 248 Hz, f-phen 4-CF), MS: m/z (%) 375
3
): δ (ppm) 27.6 (2'-CH
2
), 33.1 (NHCH
3
), 47.0 (3'-CH
2
), 54.2
(
6
(
1
+
+
(
M , 11), 330 (7), 228 (50) (M -C10
H
14N), 199 (7), 185 (9), 147 (17), 128 (26), 117 (8), 91 (13), 58 (34),
+
4
4 (100), HRMS: m/z calculated for C23
H23FN
3
O [M + H] : 376.1825. Found: 376.1828.
1
-(2-Fluorophenyl)-3-(3-(methylamino)-1-phenylpropyl)-1,3-dihydro-2H-benzimidazol-2-one
(6).
Yield: 30%, yellow crystals, mp. 92 °C–93 °C, purification: silica gel 60, dichloromethane/methanol 9:1
1
and RP-18 silica gel methanol/water 9:1 and 7:3, H-NMR (200 MHz, CDCl
3
): δ (ppm) 2.57 (s, 3H,
NHCH
3
), 3.01–3.15 (m, 4H, 2'-CH
.98–7.05 (m, 3H, benzim 5-CH, benzim 6-CH, benzim 7-CH), 7.25–7.42 (m, 5H, f-phen 3-CH, f-phen
-CH, phen 3-CH, phen 4-CH, phen 5-CH), 7.48–7.61 (m, 4 H, f-phen 4-CH, f-phen 5-CH, phen 2-CH,
proton could not be detected,
), 33.2 (NHCH3), 46.9 (3'-CH ), 53.5 (1'-CH), 109.0
benzim 4-CH), 110.1 (benzim 7-CH), 117.1 (d, J = 19.5 Hz, f-phen 3-CH), 122.2 (benzim 5-CH), 122.7
benzim 6-CH), 125.1 (d, J = 3.0 Hz, f-phen 6-CH), 127.3 (phen 2-CH), 127.1 (phen 6-CH), 128.3 (phen
2
, 3'-CH ), 5.75–5.86 (m, 1H, 1'-CH), 6.83–6.87 (m, 1H, benzim 4-CH)
2
6
6
phen 6-CH), due to limited resolution of the instrumentation, the NHCH
13C-NMR (50 MHz, CDCl
): δ (ppm) 27.7 (2'-CH
3
3
2
2
(
(
4
-CH), 128.9 (phen 3-CH), 128.9 (phen 5-CH), 129.5 (f-phen 5-CH), 130.7 (d, J = 4.5 Hz, f-phen 4-CH),
36.5 (phen 1-C), 153.6 (benzim 2-CO), 157.6 (d, J = 250.5 Hz, f-phen 2-CF), due to limited resolution
1
of the measuring apparatus, quaternary carbon f-phen 1-C could not be detected, MS: m/z (%) 375 (M+,
+
1
7), 318 (10), 228 (82) (M -C10
H
14N), 199 (9), 185 (9), 147 (16), 128 (35), 117 (9), 91 (20), 58 (43), 44
+
(
100), HRMS: m/z calculated for C23
H23FN
3
O [M + H] : 376.1825. Found: 376.1822.
3
.3. Computational Methods
The ligand structures were built in the protonated form using Molecular Operating Environment
MOE) 2013 [42]. Homology models of human NET, SERT and DAT were obtained from the
(
Drosophila dopamine transporter template (dDATcryst, PDB id 4M48 [32]), by selecting the model with
the most favorable Discrete Optimized Protein Energy (DOPE) of 250 generated by Modeller 9.11 [43].
The co-crystallized inhibitor nortriptyline was retained during model generation and the compounds
were docked in the same site using Genetic Optimization for Ligand Docking (GOLD) 5.2 [44]. One
hundred poses per ligand (i.e., five hundred poses per protein target) were generated based on the
GoldScore scoring function, while keeping the ligand flexible and the protein rigid.

Molecules 2015, 20
1726
The common chemical scaffold, i.e., the reference compound, was extracted from the resulting poses,
analogous to the methods of our previous study [35]. Cluster analysis was performed based on Euclidian
distance and complete linkage of the root-mean square deviation of the ligand’s heavy atoms matrix
using XLStat [45]. The dendrogram was cut at eight clusters and the ones containing all five ligands
were selected (Figure 5).
Figure 5. Left column: Overlay of compounds 1, 2, and 4–6 (maroon) in binding hypothesis
1
and comparison with the co-crystal pose of nortriptyline (31) (green). In NET, V148 allows
more space than I172 in SERT. The angle between ligand ring 2 and 3 is ca. 60° in all poses.
Right column: Overlay of compounds 1, 2, and 4–6 (maroon) in binding hypothesis 2. In
NET, V148 still allows more space than in hSERT, whereas E439 might disrupt ligand ring
3
. DAT lacks a more potent stacking interaction due to F155 as compared to NET[Y151]
and SERT[Y175]. Binding mode 2 poses are more in agreement with the co-crystal pose.
The angle between ligand ring 2 and 3 is almost 90° in all poses. The extracellular space is
located above in all figures.

Molecules 2015, 20
. Conclusions
In conclusion, ten new compounds have been synthesized within the scope of this work, which aimed
1727
4
at the development of new, selective, high affinity references for the imaging of the NET system via PET.
Four of these new compounds (4–6 and 25) will be employed in future studies. Whilst methylamines 4–6
(
FAPPI:1-3) represent reference compounds for their later prepared radioactive analogs, additionally
prepared free amine 25 (APPI:1) will serve as precursor for radiolabeling. Since docking studies indicate
that fluorinated methyl amines 4–6 (FAPPI:1-3) bind in an analogous way to the NET as reference
compound 1, these compounds 4–6 are promising candidates for biological evaluation.
Acknowledgments
AS, AJ, and GFE acknowledge financial support provided by the Austrian Science Fund, grants
AW/0123221 and F3502. This article was supported by the Open Access Publishing Fund of the
University of Vienna.
Author Contributions
Catharina Neudorfer: Responsible for the performance of the syntheses and writing; Amir Seddik:
Structure activity relationships calculations; Karem Shanab: Contributions to syntheses and experimental
procedures; Andreas Jurik: Structure activity relationships calculations; Christina Rami-Mark:
Participated in design and performance of the experiments; Wolfgang Holzer: Performance of the NMR
analyses; Gerhard Ecker: Conceived and supervised the SAR experiments; Markus Mitterhauser:
Designed parts of the research and proofread the manuscript; Wolfgang Wadsak: Designed parts of the
research and proofread the manuscript; Helmut Spreitzer: Conceived and supervised the syntheses.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 4–6, 9–25, and 29 and 30 are available from the authors.
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