Fluoro-substituted phenylazocarboxamides: Dopaminergic behavior and N-arylating properties for irreversible binding

Article abstract of DOI:10.1016/j.bmc.2014.12.012

Phenylazocarboxamides can serve as bioisosteres for cinnamides, which are widely occurring substructures in medicinal chemistry. Starting from our lead compound 2, the introduction of additional fluoro substituents and the exchange of the methoxyphenylpip

Full text of DOI:10.1016/j.bmc.2014.12.012

Bioorganic & Medicinal Chemistry 23 (2015) 3938–3947
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluoro-substituted phenylazocarboxamides: Dopaminergic behavior
and N-arylating properties for irreversible binding
⇑
Amelie L. Bartuschat, Tamara Schellhorn, Harald Hübner, Peter Gmeiner, Markus R. Heinrich
Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg, Schuhstraße 19, 91052 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Phenylazocarboxamides can serve as bioisosteres for cinnamides, which are widely occurring substruc-
tures in medicinal chemistry. Starting from our lead compound 2, the introduction of additional fluoro
substituents and the exchange of the methoxyphenylpiperazine head group by an aminoindane moiety
was investigated resulting in dopamine D3 receptor antagonists and agonists with Ki values in the sub-
and low-nanomolar range. As a potentially irreversible ligand, the 3,4,5-trifluoro-substituted phenylazo-
carboxamide 7 was investigated for its N-arylating properties by incubation with the protected lysine
analog 18 and with the L89K mutant of the dopamine D3 receptor. Whereas covalent bond formation with
the lysine unit in TM2 of D3 could not be detected, substantial N-arylation of the side chain of the model
compound 18 has been observed.
Received 24 October 2014
Revised 8 December 2014
Accepted 10 December 2014
Available online 17 December 2014
Keywords:
Covalent ligands
Bioisosteres
Azocarboxamide
Dopamine
Ó 2014 Elsevier Ltd. All rights reserved.
D3 receptor
GPCR
1. Introduction
In parallel to the studies aiming at highly potent and subtype-
selective dopamine D3 receptor ligands, several attempts have
G protein-coupled receptors (GPCRs) of the dopamine D2 family
including the subtypes D2, D3 and D4 are generally known as
important targets for the treatment of CNS disorders including Par-
kinson’s disease, schizophrenia, depression and drug addiction.1 In
recent years, innovative studies have focused on the development
of ligands for the dopamine D3 receptor2 exhibiting an inhibitory
effect on adenylyl cyclase upon activation.3 The particular regional
distribution of the D3 subtype in different brain areas points to a
special role of these receptors in disorders related to drug abuse,4
and D3-subtype selective antagonists are considered to be antipsy-
chotics with a favorable pharmacological profile and less side
effects.5
Among dopamine D3 subtype-selective ligands that have been
synthesized as candidates for therapeutics,6 compounds with phe-
nylpiperazine head groups, such as BP 8973a and FAUC 3467 have
become of particular importance as lead structures (Fig. 1). In
recent studies, the favorable phenylpiperazine unit, mimicking
the dihydroxyphenethylamine structure of dopamine, has been
combined with cinnamic acid amides,8 which led to D3 receptor
ligands of type 1.9
been made to develop 18F-fluorine-labeled derivatives, which
could correctly display the regional distribution of the D3 receptor
subtype in brain.10–12 We found that the cinnamide moiety present
in ligand 1 can be replaced by a para-fluorophenyl substituted azo-
carboxamide unit as it occurs as a substructure of ligand 2.13 Albeit
this replacement led to a decrease in binding affinity to the dopa-
mine D3 subtype by almost one order of magnitude and also to a
lower subtype selectivity over D2, the general suitability of the
azocarboxamide substructure, which was moreover shown to be
reasonably stable regarding metabolism,13 suggests further inves-
tigations on structural modifications.
Moreover, the aromatic core of structurally related phenylazo-
carboxylic esters is highly activated towards nucleophilic aromatic
substitutions.14 Depending on whether a nitro group, a phenoxy
group or a fluorine atom is located in para-position relative to
the azo unit to act as a leaving group, selective substitutions with
phenols, aliphatic or aromatic amines could be achieved under
mild conditions.15 Due to the particularly high activation of the
aromatic core, whereby the azocarbonyl unit exerts an even stron-
ger effect than a nitro group,16 we reasoned that compounds of
type 2 might be valuable prototypes of irreversible ligands, given
that a suitable nucleophile-containing amino acid side chain would
be present in vicinity of the respective binding region of the
receptor.
⇑
Corresponding author. Tel.: +49 9131 85 24115; fax: +49 9131 85 22585.
E-mail address: markus.heinrich@fau.de (M.R. Heinrich).
http://dx.doi.org/10.1016/j.bmc.2014.12.012
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

A.L. Bartuschat et al. / Bioorg. Med. Chem. 23 (2015) 3938–3947
3939
O
N
O
O
N
N
O
O
N
N
O
O
N
H
N
H
F
BP 897
1
Ki [nM]: hD3: 0.46
hD2: 22
N
O
N
N
N
N
H
N
N
H
S
F
2
FAUC 346
Ki [nM]: hD3: 3.5
hD2: 36
Figure 1. Lead compounds BP 897 and FAUC 346, and bioisosteric replacement of a cinnamide by a phenylazocarboxamide substructure.
Covalent binding of ligands to receptors17 has mainly been
accomplished by exploiting the reactivity of carbenes or nitrenes,
generated under irradiation from diazirines18 or azides,19 oxy-
gen-centered radicals photochemically generated from suitable
ketones,20 as well as by employing disulfides,21 alkylating groups22
or Michael acceptors,23 which are able to undergo covalent bond
formation with nucleophilic amino acid side chains. Irreversible
ligand binding based on nucleophilic aromatic substitution, as it
is the well-known reaction principle of Sanger’s reagent,24 has so
far only rarely been exploited.25
In this article, we report the synthesis and biological studies on
novel fluoro-substituted phenylazocarboxamides as well as model
investigations on the 3,4,5-trifluoro-substituted test compound 7
regarding its N-arylating properties towards a protected lysine
equivalent and a mutated dopamine D3 receptor.
difluorinated azoester 11b thereby turned out not to be fully selec-
tive for the ester functionality, and substitution at the aromatic
core was also observed, an alternative route B to the azocarboxa-
mide ligands was investigated. Following this strategy, hydrazines
15a and 15b were first treated with carbonyldiimidazole (CDI) to
give azocarbonyl imidazolides, which were subsequently reacted
with the respective amines 12–14 to provide phenyl semicarbaz-
ides. Final oxidation with manganese dioxide gave the ligands 4,
7 and 10, as well as again 3, 6 and 9. Within the synthesis of the
ligands 2–10 slight variations were made to further optimize the
conditions. A generally applicable, preferred procedure could how-
ever not yet be established, which might partially be due to the dif-
ferent properties of the amines 12–14.
Regarding the results and observations from both routes, it
turned out that the monofluorinated (2, 5, 8) and difluorinated
ligands (3, 6, 9) are accessible via both pathways, whereas the
highly activated trifluorinated compounds (4, 7, 10) can only be
prepared via route B. Attempts to synthesize ligands with a nitro
group on the aromatic core (e.g., R1 = NO2, R2 = H) failed on both
routes A and B, which was due to an overly increased reactivity
of the benzene core towards nucleophiles.
2. Results and discussion
2.1. Chemistry
All azocarboxamide ligands 2–10 included in this study were
prepared following the two routes A and B depicted in Scheme 1.
On route A, the mono- and difluorinated phenylazocarboxylic
tert-butyl esters 11a and 11b, which were readily accessible by
established procedures,14 served as starting materials. Nucleophilic
substitutions with amines 12–14 at the carbonyl unit provided the
desired products 2, 3, 5, 6, 8 and 9. Since the reactions of the
2.2. Receptor binding affinity
The amines 12–14 were chosen to investigate their receptor
binding properties. Amine 12 contains
a
N-(2-methoxy-
phenyl)piperazine substructure, which represents one of the
Route B
Route A
1. CDI
2. H2N
R'
R'
H2N
12 14
-
12 14
3. MnO2
-
O
O
H
N
R1
F
R1
F
R1
F
N
N
R'
K2CO3
N
O
N
N
H
NH2
(EtOH)
r.t.
(DMF)
r.t.
R2
11a
R2
R2
: R , R2 = H
: R1 = H, R2 = F
15b: R1, R2 = F
1
15a
11b: R1 = H, R2 = F
2: R1 = H, R2 = H, R' =
N
N
1
: R = F, R2 = H, R' =
3
4
OMe
1
: R = F, R2 = F, R' =
12
from amine
1
: R = H, R2 = H, R' =
5
N
6: R1 = F, R2 = H, R' =
1
: R = F, R2 = F, R' =
7
13
from amine
N
1
: R = H, R2 = H, R' =
8
9: R1 = F, R2 = H, R' =
10: R1 = F, R2 = F, R' =
from amine 14
Scheme 1. Synthesis of novel phenylazocarboxamide-type dopamine D3 ligands.

3940
A.L. Bartuschat et al. / Bioorg. Med. Chem. 23 (2015) 3938–3947
well-established head groups for dopamine receptor antago-
nists.1a,7 The aminoindane-based amine 13 was included in the
study due to the known effect that related ligands show agonistic
activity at dopamine receptors.26 Inspired by computational stud-
ies, we also considered ligands derived from amine 14 with a
shortened side chain. Competition binding experiments were done
for D3 over D2long, but an increase in selectivity for D3 over D2short.
In general, the trends observed for D3/D2 subtype selectivity follow
known rules for dopamine ligands of this type.4d Whereas a varia-
tion of aliphatic chain length from aminobutyl to aminoethyl leads
to a recognizable effect on subtype selectivity (groups A and B
compared to group C), variations on the lipophilic azophenyl moi-
ety, such as fluorination, as well as on the aminergic head group
(group A vs B) have only minor influence.
27
28
using the human dopamine receptors D2long, D2short
,
D3 and
29
D4.4 stably expressed in CHO cells. Binding affinities to human
D1, the serotonin receptors 5-HT1A and 5-HT2A and the a1 adreno-
receptor were also determined.30 Biological data obtained are sum-
marized in Table 1.
2.3. Studies on irreversible binding and functional assays
D3 binding affinity of the phenylpiperazine derivative 2 was sig-
nificantly reduced by the introduction of the N@N-unit (as com-
pared to cinnamide 1), but was largely regained when the degree
of fluorination was increased, with a maximum affinity for the
difluorinated azocarboxamide ligand 3 (Ki = 0.72 nM). Within the
aminobutyl substituted ligands 16 and 5–7, no significant loss of
D3 binding affinity occured through the replacement of the cinna-
mide 16 by the azocarboxamide 5. However, the introduction of
further fluorine atoms on the aromatic core, as in compounds 6
and 7, led to a remarkable loss in affinity towards D3. Comparison
of the D3 binding affinity of the aminoethyl substituted carboxam-
ides (17, 8–10)7 displayed an increase of affinity through the intro-
duction of the N@N-unit. Whereas the related difluoro derivative 9
showed almost unchanged properties towards all dopamine recep-
tor subtypes compared to 8, the trifluorinated compound 10
displayed much lower binding affinities. Replacement of the
C@C- for the N@N-unit led in most cases to a decrease of selectivity
For initial experiments on the reactivity of the ligands towards
nucleophilic aromatic substitution under assay conditions, ligand 7
was chosen as a representative structure (Scheme 2).32 The reac-
tion of 7 with N-acetyl lysine methyl ester 18 in DMF provided
19 as reference compound for further investigations in high yield.
Under the conditions of a biological assay in PBS buffer at nearly
neutral pH values, the same reaction proceeded much slower,
but the desired conjugate 19 could still be identified unambigu-
ously by HPLC-MS analysis (see Supporting information).33
Increased reaction rates were determined upon addition of DMSO
(1% or 1:1 v/v mixture) to the buffered solution, whereby the latter
conditions gave 19 in 10% yield.
To incorporate a nucleophilic position into the binding pocket,
the amino acid leucine at position 89 in the extracellular region
of transmembrane helix 2 of the dopamine D3 receptor was
exchanged for lysine. The amino acid side chain in this position
points to the binding pocket and may allow a covalent bond
Table 1
Receptor binding data at the dopamine D1–D5, the serotonin 5-HT1A and 5-HT2A and the adrenergic
a
1 receptors for the phenylazocarboxamides 2–10 and the cinnamides 1, 16
and 1731
O
N
O
R1
F
X
N
OMe
R1
F
X
N
n
X
N
H
X
N
H
R2
R2
: X = CH, R1 = R2 = H
16: X = CH, n = 3, R1 = R2 = H
5: X = N, n = 3, R1 = R2 = H
6: X = N, n = 3, R1 = H, R2 = F
7: X = N, n = 3, R1 = R2 = F
group A:
group B:
1
2
: X = N,
R
1 = R2 = H
3: X = N, R1 = H, R2 = F
4: X = N, R1 = R2 = F
: X = CH, n = 1, R1 = R2 = H
: X = N, n = 1, R1 = R2 = H
group C:
17
8
9: X = N, n = 1, R1 = H, R2 = F
10: X = N, n = 1, R1 = R2 = F
Ki valuesa (nM SEM)
hD3 hD4.4
[3H]spiperone
Ratio D2/D3
b
Compd
hD1
hD5
hD2long
hD2short
p5-HT1A
h5-HT2A
pa1
[3H]prazo-
sin
[3H]SCH23390
[3H]WAY 600135 [3H]ketan-
serin
19
213
3
4
16
5
6
7
17
8
9
760 120c
1300 320 2800 550
nd
31 4.1
38 4.6
22 1.1
34 3.0
0.46 0.077 75 23
28 1.4c
3.3 1.1
nd
330 160c
540 75
nd
5.2 1.4c
5.2 0.38
nd
67 (48)
13 (11)
10 (16)
3.0 0.41
0.72 0.18c
1.4 0.074c
0.31 0.12c
0.85 0.22
2.0 0.28c
4.9 1.6c
94 13
nd
nd
7.5 0.92c 12 4.4c
36 5.7c
47 24c
16 5.0c
34 5.4
31 5.7c
40 2.8c
1.4 0.10
nd
nd
6.6 3.1c
19 5.0c
23 3.4
25 7.1c
40 14c
9.5 3.5c
2.9 0.21c
36 10
nd
nd
nd
4.7 (6.8)
61 (9.4)
27 (42)
13 (6.5)
8.2 (2.4)
3.1 (0.93)
3.6 (2.9)
1.4 (3.0)
1.3 (0.84)
1300 70c
2900 71c
5.0 2.1c
5.8 0.60
nd
89 7.8c
550 75
nd
120 29c
170 13
nd
3300 750 7000 2200
nd
nd
nd
nd
13 4.2c
12 3.0c
13 4.0
nd
4.3 1.0
nd
210 23
nd
nd
510 54
nd
58 4.1
nd
nd
900 61
2200 370 11000 35000 43 2.8
14 4.6
nd
nd
nd
nd
5.0 2.0c
3.9 1.2c
110 31
4.0 0.071c 1.4 0.21c
0.99 0.041c nd
8.5 5.0c
73 9.6
2.8 1.3c
87 14
2.0 0.21c
10 3.0
nd
3.3 1.5
10
2600 540 3800 88
nd = not determined.
a
Ki-values in nM SEM are mean values of 3–7 independent experiments each done in triplicate.
Selectivity of binding for D3 over D2 displayed as ration of Ki(D2long)/Ki(D3) and Ki(D2short)/Ki(D3) in parentheses.
Ki-values in nM SD are mean values of two independent experiments each done in triplicate.
b
c

A.L. Bartuschat et al. / Bioorg. Med. Chem. 23 (2015) 3938–3947
3941
NH2
O
F
N
N
O
N
N
H
N
H
O
O
O
HN
F
F
N
N
18
N
N
H
F
K2CO3 (DMF), r.t., 20 h
(85%)
7
19
F
or
O
PBS buffer
N
O
(H2O), r.t., 24 h
(detected by HPLC-MS)
H
O
Scheme 2. Nucleophilic aromatic substitution of 7 with protected lysine 18 in dimethylformamide and under conditions of a biological assay.
formation with an appropriately positioned ligand. Covalent link-
age at the analogous position His89 in the b2AR has been
reported.34
antagonist haloperidol (10 lM) was added and incubation was
continued for further 180 min. Accumulated radioactivity was
determined and the resulting IP-response was expressed as fold
over basal.
HEK293T cells transiently transfected with the Dw3 t receptor and
the DL389K receptor mutant, respectively, were stimulated with the
reference agonist quinpirole in an IP accumulation assay21a,35 to
determine the ability of the mutant to be stimulated similarly to
the wild type. The mutant thereby showed no loss in receptor func-
tion compared to the wild-type, even a slightly improved activa-
tion profile could be observed (see Supporting information).
The azocarboxamides 5, 6 and 7 were then investigated for their
efficacy and potency to activate the D3 wild-type receptor and the
DL389K receptor mutant. In general, the activation pattern of all three
compounds did not notably differ. At the wild-type receptor,
potencies in a low nanomolar range (2.6–11 nM) and nearly full
agonist properties were observed, which is similar to the potency
of the reference agent quinpirole with an EC50 of 2.4 nM (Fig. 2,
left). A slightly different pattern of receptor activation could be
observed at the DL389K mutant, whereby the mutation caused a
rightward shift to the dose–response curves. The activity of quinpi-
role was not significantly influenced by that mutation. The poten-
cies of the aminoindanes 5, 6 and 7 at the wild-type receptor
decreased 3–6-fold compared to the reference but indicated still
nearly full agonist properties (Fig. 2, right). At the DL389K mutant,
the test compounds 5 and 6 showed strong partial agonism
whereas the trifluoro-substituted azocarboxamide 7 was able to
fully activate the receptor. (Fig. 2, see also Supporting information)
The study on the irreversibility of binding was performed by an
IP accumulation assay which allows the detection of continued
[3H]IP production in the presence of an antagonist in cells express-
ing the receptor of interest.21a,35 HEK cells transiently expressing
In these experiments the reference agent quinpirole caused an
activation level of 3.2-fold over basal after 60 min further increas-
ing to 6.8-fold over basal after 4 h of incubation time. Adding hal-
operidol after 60 min, the IP-response of the reference remained at
3.7-fold over basal representing approximately the level after
60 min incubation without antagonist. For the test compounds 5,
6 and 7, a similar behavior could be observed (see Supporting
information). Any irreversible blocking of the receptor by an ago-
nist should result in an increased formation of IP also after addition
of an antagonist. As we could not observe any further increase of IP
accumulation for 5–7, this result lead us to the conclusion that
these compounds were not able to conjugate within the receptor
under the investigated conditions.
3. Summary
Based on the lead structure of phenylazocarboxamide 2, the
introduction of additional fluoro substituents on the aromatic core
combined with a replacement of the methoxyphenylpiperazine
head group by an aminoindane moiety led to new dopamine D3
receptor antagonists and agonists, of which compound 3 and 5
even showed Ki values in the subnanomolar range. The present
study therefore strongly supports the assumption that phenylazo-
carboxamides represent a so far unknown group of bioisosteres for
cinnamides. The remarkable potential of phenylazocarboxamides
for future studies was further underlined by functional assays with
the aminoindane derivatives 5–7. Attempts to exploit the
particularly high reactivity at the aromatic core of the 3,4,5-tri-
fluoro-substituted phenylazocarboxamide 7 were successful with
the protected lysine analog 18, since irreversible conjugation
the DL389K mutant were stimulated with 3
lM of each of the fluori-
nated phenylazocarboxamides 5, 6 and 7 for 60 min or 240 min,
respectively. In parallel after preincubation for 60 min, the D3
IP formation - HEK with Dw3 t + GαqG66Di5
IP formation - HEK with D3L89K + GαqG66Di5
125
125
quinpirole
quinpirole
5
6
7
5
6
7
100
100
75
50
25
0
75
50
25
0
-12 -11 -10 -9
-8
-7
-6
-5
-4
-12 -11 -10 -9
-8
-7
-6
-5
-4
compound [logM]
compound [logM]
Figure 2. Dose–response curves of the fluorinated phenylazocarboxamides 5–7 and the reference quinpirole at Dw3 t or D3L89K expressing cells. In an IP accumulation assay the
functional properties of the test compounds were measured at cells transiently expressing Dw3 t and the promiscuous G-protein GaqG66Di5 (left) or the mutant D3L89K and
G
aqG66Di5 (right). Curves represent pooled and normalized means SEM of one to three independent experiments each done in triplicate.

3942
A.L. Bartuschat et al. / Bioorg. Med. Chem. 23 (2015) 3938–3947
through N-arylation could be observed under the conditions of a
biological cell assay. Initial experiments with ligand 7 and the
L89K mutant of the dopamine D3 receptor did not lead to covalent
binding, which might however be due to the protonation of the
newly incorporated lysine unit by unfavorably placed glutamic
acid residues at positions 90 (TM2) and 363 (TM7). Having shown
that the reactivity of the aromatic core of phenylazocarboxamides
is generally sufficient to achieve N-arylation, our current efforts
focus on the design and the synthesis of ligands for receptor
subtypes naturally presenting lysine at their binding sites.
Moreover, experiments are underway to explore an extension of
the principle towards a selective labeling of tyrosine residues with
4-nitrophenylazocarboxamides.14,15
was stirred for 1 h at 0 °C. The chilled solution was filtered and
then a solution of tin(II)–chloride dihydrate (20.0–22.0 mmol) in
concentrated hydrochloric acid (10 mL) was added dropwise at
0 °C. The precipitate was filtered off and washed with a saturated
aqueous solution of sodium chloride (30 mL). Due to stability rea-
sons hydrazines which were not used immediately, were dried and
stored as hydrochlorides. They were later extracted under basic
conditions directly before using them in a reaction. Therefore the
solid hydrochloride (1.00 mmol) was dissolved in a saturated
aqueous solution of sodium carbonate (40.0 mL) and then
extracted with diethyl ether (4 ꢁ 50 mL). The combined organic
phases were washed with a saturated aqueous solution of sodium
chloride (20 mL) and dried over sodium sulfate. After filtration the
solvent was removed under reduced pressure. The phenylhydr-
azine could be used without further purification.
4. Experimental
4.1. Chemistry
4.1.1.1. 3,4-Difluorophenylhydrazine hydrochloride (15a). Com-
pound 15a was prepared from 3,4-difluoroaniline (1.03 g,
8.01 mmol), sodium nitrite (0.83 g 12.0 mmol) and tin(II)–chloride
dihydrate (4.51 g, 20.0 mmol) in hydrochloric acid (20 mL). Hydro-
chloride 15a (1.15 g, 6.36 mmol, 79%) was obtained as a white
solid. 1H NMR (600 MHz, CDCl3 as free base) d 3.57 (br s, 2H),
5.16 (br s, 1H), 6.48 (dtd, J = 1.6 Hz, J = 3.2 Hz, J = 7.9 Hz, 1H), 6.70
(ddd, J = 2.8 Hz, J = 6.7 Hz, J = 12.0 Hz, 1H), 7.00 (td, J = 8.6 Hz,
J = 10.0 Hz, 1H); 19F NMR (339 MHz, HCl salt, D2O) d ꢀ138.0 (m,
1F), ꢀ146.8 (m, 1F), (the analytical data obtained is in agreement
with those reported in literature).37
Solvents and reagents were obtained from commercial sources
and used as received. NMR spectra were recorded on Bruker
Avance 600 (1H: 600 MHz, 13C: 151 MHz) and Bruker Avance 360
(1H: 360 MHz, 13C: 91 MHz). For 1H NMR CDCl3, CD3OD, D2O and
CD3CN were used as solvents referenced to TMS (0 ppm), CHCl3
(7.26 ppm) or CHD2CN (1.94 ppm). For 13C NMR CDCl3, CD3OD
and CD3CN were used as solvents with CDCl3 (77.0 ppm) and
CD3CN (1.24 ppm) as standard. 19F NMR spectra were recorded at
339 MHz using C6F6 (ꢀ164.9 ppm) as standard. Chemical shifts
were reported in parts per million (ppm). Coupling constants are
reported in Hertz (J Hz). The following abbreviations are used for
the description of signals: s (singlet), d (doublet), t (triplet), q (qua-
druplet), quin (quintet), m (multiplet), br s (broad signal). Mass
spectra were recorded on Jeol GC mate II GC–MS-system using
electron impact (EI) or electron spray ionization (ESI) and a sector
field mass analyzer for MS and HRMS measurements. High-perfor-
mance liquid chromatography (HPLC) is performed on a Varian
4.1.1.2. 3,4,5-Trifluorophenylhydrazinehydrochloride(15b). Com-
pound 15b was prepared from 3,4,5-trifluoroaniline (1.47 g,
10.0 mmol), sodium nitrite (0.76 g, 11.0 mmol) and tin(II)–chloride
dihydrate (5.00 g, 22.0 mmol) in hydrochloric acid (50 mL). Hydro-
chloride 15b (576 mg, 2.91 mmol, 29%) was obtained as a white
solid. 1H NMR (360 MHz, CDCl3 + 1% CD3OD) d 6.67–6.76 (m, 2H);
13C NMR (151 MHz, CDCl3 + 1% CD3OD) d 99.4 (dd, JCF = 6.1 Hz,
JCF = 19.9 Hz, 2ꢁ CH), 135.3 (td, JCF = 15.3 Hz, JCF = 246.0 Hz, Cq),
140.5 (dt, JCF = 3.4 Hz, JCF = 10.5 Hz, Cq), 151.3 (ddd, JCF = 5.2 Hz,
JCF = 10.5 Hz, JCF = 248.9 Hz, 2ꢁ Cq).
940-LC with a XRs C8 column (21.2 ꢁ 150 mm ꢁ 5
lm) and PDA
detection. (Solvent A: 0.1% aqueous TFA solution, solvent B: aceto-
nitrile, flow rate 20 mL/min, Method A: Gradient A/B: min 0–7, 90/
10% ? 80/20%, min 7–15, 80/20% ? 0/100%, min 15–22, 0/100%;
Method B: Gradient A/B: min 0–10, 100/0%? 70/30%, min 10–18,
70/30% ? 20/80%, min 18–25, 20/80%, min 25–26, 20/80% ? 0/
100%, min 26–30 0/100%). HPLC–MS is performed on an Agilent
4.1.2. tert-Butyl (E)-2-(3,4-difluorophenyl)diazene-1-carboxylate
(11b)
To a solution of hydrazine 15a (440 mg, 3.05 mmol) in dry ace-
tonitrile (30 mL) was added di-tert-butyldicarbonate (800 mg,
3.66 mmol) under argon atmosphere at room temperature. The
mixture was stirred at room temperature and after complete con-
sumption of the reactants, as monitored by TLC, the solvent was
removed under reduced pressure. The crude product was purified
by column chromatography (silica gel, hexane/ethyl acetate = 6:1)
to give the difluorophenylhydrazine carboxylate (542 mg,
2.22 mmol, 73%) as a light orange solid. Rf = 0.2 (hexane/ethyl ace-
tate = 4:1) [UV, KMnO4]; 1H NMR (600 MHz, CDCl3) d 1.47 (s, 9H),
6.37 (br s, 1H), 6.49–6.53 (m, 1H), 6.66 (ddd, J = 2.7 Hz, J = 6.7 Hz,
J = 11.9 Hz, 1H), 7.01 (td, J = 2.7 Hz, J = 8.7 Hz, 1H); 13C NMR
(151 MHz, CDCl3) d 28.2 (3ꢁ CH3), 81.7 (Cq), 102.4 (d, JCF = 21.4 Hz,
CH), 108.2–108.3 (m, Cq), 117.5 (dd, JCF = 1.3 Hz, JCF = 18.3 Hz, CH),
145.0 (dd, JCF = 12.9 Hz, JCF = 239.6 Hz, Cq), 145.4 (d, JCF = 7.5 Hz,
CH), 150.8 (dd, JCF = 13.6 Hz, JCF = 246.4 Hz, Cq), 156.1 (Cq); 19F
1100 series with
a
Zorbax Eclipse XDB-C8 column
(4.6 ꢁ 150 mm ꢁ 5 m) and VWL detection coupled with a Bru-
l
ker-Esquire 2000 mass spectrometer with EI-ionisation. (Solvent
A: 0.1% aqueous HCOOH solution, solvent B: methanol, flow rate
0.5 mL/min. Gradient A/B: 0–3, 90/10%, min 3–18, 90/10% ? 100/
0%, min 18–24, 100/0%, min 24–26, 100/0% ? 10/90%, 26–30 10/
90%.) Analytical TLC was carried out on Merck silica gel plates
using short wave (254 nm) UV light, KMnO4 [3.0 g KMnO4, 20 g
potassium carbonate, 5.0 mL aqueous sodium hydroxide (5% w/
w) in 300 mL H2O] and ninhydrin [200 mg ninhydrin in 100 mL
ethanol] to visualise components. Silica gel (Kieselgel 60, 40–
63 lm, Merck) was used for flash column chromatography. The
phosphate buffered saline (PBS) was adjusted to a pH value of
7.3 [137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 ꢁ 7 H2O,
1.3 mM KH2PO4 in 200 mL H2O]. Compounds 1,9 2,13 11a,14 129
and 1326 were prepared according to literature procedures.
NMR (339 MHz, D2O)
d
ꢀ139.8 (ddd, J = 9.0 Hz, J = 11.8 Hz,
J = 21.0 Hz, 1F), ꢀ151.9 (dddd, J = 3.4 Hz, J = 6.8 Hz, J = 10.0 Hz,
J = 13.1 Hz, 1F). In the second step manganese dioxide (535 mg,
6.15 mmol) was added to a solution of the difluorophenylhydr-
azine carboxylate (300 mg, 1.23 mmol) in dry dichloromethane
(10 mL) under argon atmosphere at room temperature. After com-
plete consumption of the reactants, as monitored by TLC, the mix-
ture was filtered over CeliteÒ and washed with dichloromethane.
The solvent was removed under reduced pressure and the crude
4.1.1. General procedure for the synthesis of
phenylhydrazines36
A solution of the respective aniline (8.01–10.0 mmol) in glacial
acetic acid (5.0 mL) was treated with concentrated hydrochloric
acid (20–50 mL, 36%) at room temperature and cooled to 0 °C. Sub-
sequently
a pre-cooled solution of sodium nitrite (11.0–
12.0 mmol) in water (3.0 mL) was added slowly and the mixture

A.L. Bartuschat et al. / Bioorg. Med. Chem. 23 (2015) 3938–3947
3943
product was purified by column chromatography (silica gel, hex-
ane/ethyl acetate = 19:1) to give 11b (296 mg, 1.22 mmol, 99%)
as a light orange solid. Rf = 0.5 (hexane/ethyl acetate = 19:1) [UV,
KMnO4]; 1H NMR (600 MHz, CDCl3) d 1.66 (s, 9H), 7.33 (ddd,
J = 7.9 Hz, J = 8.8 Hz, J = 9.5 Hz, 1H), 7.71 (ddd, J = 2.4 Hz,
J = 7.4 Hz, J = 10.5 Hz, 1H), 7.78–7.81 (m, 1H); 13C NMR (151 MHz,
CDCl3) d 27.8 (3ꢁ CH3), 85.5 (Cq), 110.3 (dd, JCF = 1.7 Hz, JCF = 18.6 -
Hz, CH), 117.7 (d, JCF = 18.7 Hz, CH), 123.0 (dd, JCF = 3.4 Hz,
JCF = 7.2 Hz, CH), 148.1 (dd, JCF = 3.5 Hz, JCF = 4.6 Hz, Cq), 150.9
(dd, JCF = 14.1 Hz, JCF = 252.6 Hz, Cq), 153.6 (dd, JCF = 13.5 Hz,
NEt3, hexane/ethyl acetate = 1:3 ? 100% ethyl acetate). Azocarbox-
amide 3 (52.2 mg, 121 mol, 41%) was obtained as red-brown vis-
l
cous oil. For biological testing it was additionally purified using
HPLC method A. Rf = 0.4 (100% ethyl acetate desact. NEt3) [UV];
1H NMR (360 MHz, TFA salt, CDCl3) d 1.70–1.85 (m, 4H), 2.54 (t,
J = 6.4 Hz, 2H), 2.72 (br s, 4H), 3.10 (br s, 4H), 3.54 (q, J = 6.0 Hz,
2H), 3.85 (s, 3H), 6.79 (dd, J = 2.0 Hz, J = 7.6 Hz, 1H), 6.83–6.90
(m, 2H), 6.96–7.03 (m, 1H), 7.27 (ddd, J = 7.9 Hz, J = 8.8 Hz,
J = 9.5 Hz, 1H), 7.71 (ddd, J = 2.4 Hz, J = 7.4 Hz, J = 10.5 Hz, 1H),
7.80 (dddd, J = 1.5 Hz, J = 2.4 Hz, J = 4.1 Hz, J = 8.7 Hz, 1H), 8.06 (br
s, 1H); 13C NMR (151 MHz, TFA salt, CDCl3) d 24.2 (CH2), 27.4
(CH2), 40.7 (CH2), 50.1 (2ꢁ CH2), 53.2 (2ꢁ CH2), 55.3 (CH2), 57.8
(CH3), 110.5 (dd, JCF = 1.6 Hz, JCF = 18.6 Hz, CH), 111.2 (CH), 117.7
(d, JCF = 18.7 Hz, CH), 118.0 (CH), 120.9 (CH), 123.1 (CH), 123.2
(dd, JCF = 3.3 Hz, JCF = 7.1 Hz, CH), 140.8 (Cq), 147.8 (dd, JCF = 3.3 Hz,
JCF = 4.8 Hz, Cq), 150.8 (dd, JCF = 13.9 Hz, JCF = 252.5 Hz, Cq), 152.1
(Cq), 153.6 (dd, JCF = 13.3 Hz, JCF = 257.9 Hz, Cq), 160.6 (Cq); 19F
NMR (339 MHz, TFA salt, CDCl3) d ꢀ78.8 (TFA), ꢀ131.8 (dd,
J = 9.4 Hz, J = 21.7 Hz, 1F), ꢀ137.7 (m, 1F); MS (EI) m/z (%): 431
(22) [M+], 290 (100), 205 (34), 193 (24), 150 (27), 120 (21), 70
(67); HRMS (ESI) m/z calcd for C22H28F2N5O2 [MH+] 432.2206,
found 432.2199.
JCF = 257.9 Hz, Cq), 160.7 (Cq); 19F NMR (339 MHz, CDCl3)
ꢀ132.2 (m, 1F), ꢀ137.8 (m, 1F).
d
4.1.3. General procedures for the synthesis of phenylazocarbox-
amides 3–10
From phenylazocarboxylic acid tert-butyl esters (Route A,
Scheme 1): To a stirred solution of the phenylazocarboxylic acid
tert-butyl ester (11a or 11b, 0.3–0.4 mmol) and K2CO3 (4 equiv)
or triethyl amine (2.5 equiv) in dry ethyl acetate (5.0 mL) or dry
ethanol (2.0 mL) was added the amine (12, 13 or 14) (0.5 equiv)
and the resulting mixture was stirred at room temperature. The
reaction course was monitored by TLC. The reaction mixture was
either concentrated under reduced pressure or diluted with water
(30 mL) and extracted with ethyl acetate (3ꢁ 30 mL). The com-
bined organic phases were then washed with a saturated aqueous
solution of sodium chloride (20 mL) and dried over sodium sulfate.
After filtration the solvent was removed under reduced pressure.
The crude product was purified by column chromatography, and
for the biological testing additionally via preparative HPLC
(Method A or B) to give the required product.
From phenylhydrazines (Route B, Scheme 1): A solution of phen-
ylhydrazine hydrochloride (15a or 15b, 0.3–0.7 mmol) in dry
N,N-dimethylformamide (2.0–3.0 mL) was added dropwise to a
solution of 1,10-carbonyldiimidazole (1 equiv) in dry N,N-dimethyl-
formamide (3.0–9.0 mL) over a period of 15–30 min at room tem-
perature under nitrogen atmosphere and stirring was continued
for 2 h. To the resulting mixture a solution of the respective amine
(0.6–1.3 equiv) in dry N,N-dimethylformamide (2.0–3.0 mL) was
added over a period of 30 min and stirring was continued for
2.5 h to overnight. Subsequently manganese dioxide (4.0–
8.0 equiv) was added and the mixture was stirred for 2 h. The solid
was filtered off and washed with dichloromethane or chloroform.
The combined organic phases were concentrated under reduced
pressure and the crude product was purified via column chroma-
tography as described for every product. For the biological testing
an additional purification via preparative HPLC (Method A or B), to
give the required product, was made. Variations from this proce-
dure are described with every product.
4.1.3.2. (E)-N-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butyl)-2-
(3,4,5-trifluoro-phenyl)diazene-1-carboxamide (4).
According
to the general procedure for Route B, a solution of 3,4,5-trif-
luorophenylhydrazine hydrochloride (15b) (85.2 mg, 0.53 mmol)
in dry N,N-dimethylformamide (2.5 mL) was added dropwise to a
solution of 1,10-carbonyldiimidazole (84.9 mg, 0.52 mmol) in dry
N,N-dimethylformamide (4.0 mL) over a period of 15 min at room
temperature under nitrogen atmosphere and stirring was continued
for 2 h. To the resulting mixture a solution of 4-(4-(2-methoxy-
phenyl)piperazin-1-yl)butan-1-amine (12) (140 mg, 0.53 mmol) in
dry N,N-dimethylformamide (2.0 mL) was added over a period of
15 min and stirring was continued overnight. In this particular case
no MnO2 was added. The organic solution was concentrated under
reduced pressure and the crude product was purified via column
chromatography (silica gel, desact. with NEt3, hexane/ethyl ace-
tate = 1:2 ? 100% ethyl acetate). Azocarboxamide
4 (24.0 mg,
5.3 mol, 10%) was obtained as brown viscous oil. For biological
l
testing it was additionally purified using HPLC method A. Rf = 0.4
(100% ethyl acetate desact. NEt3) [UV, KMnO4], 1H NMR (360 MHz,
CDCl3) d 1.68–1.86 (m, 4H), 2.53 (t, J = 6.4 Hz, 2H), 2.71 (br s, 4H),
3.08 (br s, 4H), 3.54 (dd, J = 5.6 Hz, J = 11.3 Hz, 2H), 3.85 (s, 3H),
6.77 (dd, J = 1.7 Hz, J = 8.2 Hz, 1H), 6.82–6.88 (m, 2H), 6.96–7.02
(m, 1H), 7.60 (dd, J = 6.5 Hz, J = 7.8 Hz, 2H), 8.26 (br s, 1H); 13C
NMR (91 MHz, CDCl3) d 24.4 (CH2), 27.4 (CH2), 40.1 (CH2), 50.1
(2ꢁ CH2), 53.2 (2ꢁ CH2), 55.3 (CH2), 57.8 (CH3), 108.4 (d, JCF = 22.7 -
Hz, 2ꢁ CH), 108.4 (JCF = 9.7 Hz, Cq), 111.3 (CH), 117.9 (CH), 120.9
(CH), 123.1 (CH), 140.8 (Cq), 152.1 (Cq), 160.3 (Cq), (three Cq signals
missing); 19F NMR (339 MHz, CDCl3) d ꢀ134.7 (dd, J = 7.7 Hz,
J = 20.1 Hz, 2F), ꢀ154.9 (tt, J = 6.3 Hz, J = 20.1 Hz, 1F).
4.1.3.1. (E)-2-(3,4-Difluorophenyl)-N-(4-(4-(2-methoxyphenyl)
piperazin-1-yl)-butyl)diazene-1-carboxamide (3).
ing to the general procedure for Route B, a solution of 3,4-dif-
luorophenylhydrazine hydrochloride (15a) (42.3 mg, 293 mol)
in dry N,N-dimethylformamide (3.0 mL) was added dropwise to a
solution of 1,10-carbonyldiimidazole (47.5 mg, 293
mol) in dry
Accord-
l
4.1.3.3. (E)-N-(4-((2,3-Dihydro-1H-inden-2-yl)(prop-1-yl)amino)-
butyl)-2-(4-fluoro-phenyl)diazene-1-carboxamide (5). Accord-
ing to the general procedure for Route A, azocarboxamide 5 was
prepared from tert-butyl 2-(4-fluorophenyl)azocarboxylate (11a)
(91.9 mg, 0.41 mmol), K2CO3 (150 mg, 1.02 mmol) and N1-(2,3-
l
N,N-dimethylformamide (3.0 mL) over a period of 15 min at room
temperature under nitrogen atmosphere and stirred for further
2 h. To this mixture
piperazin-1-yl)butan-1-amine (12) (47.5 mg, 293
a
solution of 4-(4-(2-methoxyphenyl)
mol) in dry
l
dihydro-1H-inden-2-yl)-N1-(prop-1-yl)butane-1,4-diamine
(13)
N,N-dimethylformamide (2.0 mL) was added over a period of
15 min and the resulting mixture was stirred overnight. Subse-
quently manganese dioxide (209 mg, 2.40 mmol) was added and
stirring was continued for 1 h. The solid was filtered off and was
washed with dichloromethane. The combined organic solutions
were concentrated under reduced pressure and the crude product
was purified via column chromatography (silica gel, desact. with
(50.0 mg, 0.20 mmol) in dry ethyl acetate (5.0 mL). The reaction
mixture was diluted with water (30 mL) and extracted with ethyl
acetate (3 ꢁ 30 mL). The combined organic phases were washed
with a saturated aqueous solution of sodium chloride (20 mL) and
dried over sodium sulfate. After filtration the solvent was removed
under reduced pressure. The crude product was purified via
column chromatography (silica gel desact. with NEt3, hexane/ethyl

3944
A.L. Bartuschat et al. / Bioorg. Med. Chem. 23 (2015) 3938–3947
acetate = 2:1 ? 100% ethyl acetate) to give azocarboxamide 5
(41.4 mg, 107 mol, 53%) as brown viscous oil. For biological testing
1H-inden-2-yl)-N1-(prop-1-yl)butane-1,4-diamine (13) (78.8 mg,
0.32 mmol) in dry N,N-dimethylformamide (3.0 mL) was added
over a period of 30 min and stirring was continued for 2.5 h. Sub-
sequently manganese dioxide (116 mg, 1.33 mmol) was added
and the mixture was stirred for 2 h. The solid was filtered off and
washed with chloroform. The combined organic solutions were
concentrated under reduced pressure and the crude product was
purified via column chromatography (silica gel, desact. with NEt3,
hexane/ethyl acetate = 1:1). Purification using HPLC method B gave
l
it was additionally purified using HPLC method B. Rf = 0.5 (ethyl ace-
tate/methanol = 9:1) [UV, KMnO4]; 1H NMR (360 MHz, CDCl3) d 0.87
(t, J = 7.4 Hz, 3H), 1.45–1.58 (m, 2H), 1.69 (dd, J = 6.1 Hz, J = 12.6 Hz,
2H), 1.72–1.79 (m, 2H), 2.51–2.58 (m, 2H), 2.61 (t, J = 6.5 Hz, 2H),
2.91 (dd, J = 8.8 Hz, J = 15.3 Hz, 2H), 3.02 (dd, J = 7.7 Hz, J = 15.2 Hz,
2H), 3.52 (dd, J = 6.2 Hz, J = 12.2 Hz, 2H), 3.62–6.72 (m, 1H), 7.08–
7.15 (m, 6H), 7.87 (dd, J = 5.2 Hz, J = 9.2 Hz, 2H), 8.03 (br s, 1H);
13C NMR (91 MHz, CDCl3): d 11.9 (CH3), 18.9 (CH2), 25.2 (CH2),
28.0 (CH2), 36.3 (2ꢁ CH2), 40.9 (CH2), 51.1 (CH2), 52.9 (CH2), 63.0
(CH), 116.4 (d, JCF = 23.1 Hz, 2ꢁ CH), 124.4 (2ꢁ CH), 126.1 (d,
JCF = 9.5 Hz, 2ꢁ CH), 126.4 (2ꢁ CH), 141.5 (2ꢁ Cq), 147.79 (d,
JCF = 3.0 Hz, Cq), 160.8 (Cq), 165.7 (d, JCF = 255.7 Hz, Cq); 19F NMR
(339 MHz, CDCl3) d ꢀ108.4 (m, 1F); HRMS (ESI) m/z calcd for C23H30-
FN4O [MH+] 397.2398, found 397.2398.
the TFA salt of 7 (26.8 mg, 49.1 lmol, 15%) as light brown solid.
Rf = 0.5 (ethyl acetate/methanol = 9:1) [UV, KMnO4]; HPLC-MS
tR = 17.6 min, m/z 433.8; 1H NMR (600 MHz, CDCl3) d 0.88 (t,
J = 7.4 Hz, 3H), 1.52 (dt, J = 7.4 Hz, J = 14.8 Hz, 2H), 1.67–1.74 (m,
2H), 1.78 (td, J = 7.4 Hz, J = 14.8 Hz, 2H), 2.51–2.60 (m, 2H), 2.63
(t, J = 6.3 Hz, 2H), 2.88 (dd, J = 8.7 Hz, J = 15.5 Hz, 2H), 3.02 (dd,
J = 7.8 Hz, J = 15.4 Hz, 2H), 3.52 (dd, J = 5.8 Hz, J = 12.1 Hz, 2H),
3.67 (td, J = 8.3 Hz, J = 16.9 Hz, 1H), 7.04–7.21 (m, 4H), 7.53 (dd,
J = 6.5 Hz, J = 7.9 Hz, 2H), 8.35 (br s, 1H); 13C NMR (151 MHz, TFA
salt, CD3CN) d 11.3 (CH3), 17.5 (CH2), 21.5 (CH2), 27.0 (CH2), 35.4
(CH2), 35.5 (CH2), 40.3 (CH2), 51.3 (CH2), 53.1 (CH2), 64.3 (CH),
109.1 (dd, JCF = 4.7 Hz, JCF = 18.3 Hz, 2ꢁ CH), 125.5 (2ꢁ CH), 128.3
(2ꢁ CH), 140.2 (2ꢁ Cq), 162.6 (Cq), (four Cq-signals missing); 19F
NMR (339 MHz, CDCl3) d ꢀ76.3 (TFA), ꢀ134.7 (dd, J = 8.2 Hz,
J = 19.5 Hz, 2F), ꢀ155.6 (m, 1F); HRMS (ESI) m/z calcd for C23H28F3-
N4O [MH+] 433.2210, found 433.2214.
4.1.3.4. (E)-2-(3,4-Difluorophenyl)-N-(4-((2,3-dihydro-1H-inden-
2-yl)(prop-1-yl)-amino)butyl)diazene-1-carboxamide
(6).
According to the general procedure for Route B, a solution of 3,4-
difluorophenylhydrazine hydrochloride (15a) (130 mg, 0.72 mmol)
in dry N,N-dimethylformamide (2.0 mL) was added dropwise to a
solution of 1,10-carbonyldiimidazole (113 mg, 0.72 mmol) in dry
N,N-dimethylformamide (3.0 mL) over a period of 20 min at room
temperature under nitrogen atmosphere and stirring was contin-
ued for 2 h. To the resulting mixture a solution of N1-(2,3-dihy-
dro-1H-inden-2-yl)-N1-(prop-1-yl)butane-1,4-diamine
(13)
4.1.3.6. (E)-N-(2-((2,3-Dihydro-1H-inden-2-yl)(prop-1-yl)amino)
(230 mg, 0.93 mmol) in dry N,N-dimethylformamide (2.0 mL) was
added over a period of 20 min and stirring was continued for 2 h.
Subsequently manganese dioxide (250 mg, 2.88 mmol) was added
and the mixture was stirred overnight. The solid was filtered off
and was washed with chloroform. The combined organic solutions
were concentrated under reduced pressure and the crude product
was purified via column chromatography (silica gel, desact. with
ethyl)-2-(4-fluoro-phenyl)diazene-1-carboxamide
(8).
According to the general procedure for Route A, azocarb-
oxamide 8 was synthesized from tert-butyl 4-fluorophenylazo-
carboxylate (11a) (86.7 mg, 0.36 mmol), amine 14 (39.0 mg,
0.18 mmol) and triethyl amine (1.12 mL, 0.90 mg, 0.89 mmol) in
dry ethanol (2.0 mL). After concentration of the reaction mixture
under reduced pressure the crude product was purified by column
chromatography (silica gel desact. with NEt3, hexane/ethyl ace-
NEt3, hexane/ethyl acetate = 1:1) to give azocarboxamide
6
(55.0 mg, 0.13 mmol, 18%) as a brown viscous oil. For biological
testing it was additionally purified using HPLC method B. Rf = 0.4
(ethyl acetate/methanol = 9:1) [UV, KMnO4]; 1H NMR (600 MHz,
TFA salt, CD3CN) d 0.86 (t, J = 7.4 Hz, 3H), 1.48 (qd, J = 7.4 Hz,
J = 14.8 Hz, 2H), 1.56–1.61 (m, 2H), 1.63–1.68 (m, 2H), 2.49 (t,
J = 7.7 Hz, 2H), 2.56 (t, J = 6.9 Hz, 2H), 2.81 (dd, J = 8.6 Hz,
J = 15.4 Hz, 2H), 2.99 (dd, J = 7.7 Hz, J = 15.2 Hz, 2H), 3.37 (dd,
J = 6.6 Hz, J = 12.4 Hz, 2H), 3.60–3.66 (m, 1H), 7.08–7.11 (m, 2H),
7.12–7.15 (m, 2H), 7.43–7.48 (m, 1H), 7.71 (ddd, J = 2.4 Hz,
J = 7.5 Hz, J = 11.0 Hz, 1H), 7.75–7.78 (m, 1H); 13C NMR (151 MHz,
TFA salt, CD3CN) d 12.2 (CH3), 20.6 (CH2), 25.7 (CH2), 28.4 (CH2),
37.2 (2ꢁ CH2), 41.4 (CH2), 51.7 (CH2), 53.7 (CH2), 63.9 (CH), 111.2
(dd, JCF = 1.4 Hz, JCF = 18.8 Hz, CH), 119.1 (d, JCF = 18.9 Hz, CH),
123.7 (dd, JCF = 3.4 Hz, JCF = 7.7 Hz, CH), 125.4 (2ꢁ CH), 127.2 (2ꢁ
CH), 143.1 (2ꢁ Cq), 149.2 (dd, JCF = 3.4 Hz, JCF = 5.0 Hz, Cq), 151.8
(dd, JCF = 14.3 Hz, JCF = 249.8 Hz, Cq), 154.2 (dd, JCF = 13.3 Hz,
tate = 10:1 ? 100% ethyl acetate) to give azocarboxamide
8
(20.4 mg, 53.3 mol, 30%) as a brown oil. For the biological testing
l
it was additionally purified using preparative HPLC method B.
Rf = 0.5 (hexane/ethyl acetate = 1:1) [UV, KMnO4]; 1H NMR
(600 MHz, TFA salt, CDCl3) d 1.01 (t, J = 7.3 Hz, 3H), 1.75–1.92 (m,
2H), 3.00–3.20 (m, 2H), 3.27–3.39 (m, 5H), 3.48 (br s, 1H), 3.86
(br s, 1H), 3.94 (br s, 1H), 4.23 (quin, J = 7.6 Hz, 1H), 7.18–7.24
(m, 6H), 7.99 (dd, J = 5.2 Hz, J = 9.0 Hz, 2H), 8.88 (br s, 1H), 12.51
(br s, 1H); 13C NMR (91 MHz, TFA salt, CDCl3) d 11.2 (CH3), 16.9
(CH2), 34.2 (CH2), 34.6 (CH2), 36.7 (CH2), 51.2 (CH2), 54.4 (CH2),
64.2 (CH), 116.4 (d, JCF = 23.2 Hz, 2ꢁ CH), 124.6 (2ꢁ CH), 126.6
(d, JCF = 9.7 Hz, 2ꢁ CH), 127.9 (2ꢁ CH), 138.2 (2ꢁ Cq), 147.8 (Cq),
162.0 (Cq), 166.2 (d, JCF = 256.4 Hz, Cq); 19F NMR (339 MHz, TFA
salt, CDCl3) d ꢀ78.8 (TFA), ꢀ107.9 (s, 1F); HRMS (ESI) m/z calcd
for C21H26FN4O [MH+] 369.2085, found 369.2080.
JCF = 254.3 Hz, Cq), 162.9 (Cq); 19F NMR (339 MHz, CDCl3)
d
4.1.3.7. (E)-2-(3,4-difluorophenyl)-N-(2-((2,3-dihydro-1H-inden-2-
yl)(prop-1-yl)-amino)ethyl)diazene-1-carboxamide (9). According
to the general procedure for Route A, azocarboxamide 9 was syn-
thesized from tert-butyl 3,4-difluorophenylazocarboxylate (11b)
(78.3 mg, 0.32 mmol), amine 14 (35.3 mg, 0.16 mmol) and triethyl
amine (1.11 mL, 0.82 mg, 0.81 mmol) in dry ethanol (2.0 mL). After
concentration of the reaction mixture under reduced pressure the
crude product was purified by column chromatography (silica gel
desact. with NEt3, hexane/ethyl acetate = 10:1 ? 100% ethyl ace-
ꢀ137.4 (m, 1F), ꢀ133.0 (dddd, J = 4.5 Hz, J = 7.3 Hz, J = 9.9 Hz,
J = 20.3 Hz, 1F); HRMS (ESI) m/z calcd for C23H29F2N4O [MH+]
415.2304, found 415.2307.
4.1.3.5. (E)-N-(4-((2,3-Dihydro-1H-inden-2-yl)(prop-1-yl)amino)bu-
tyl)-2-(3,4,5-tri-fluorophenyl)diazene-1-carboxamide (7). Accord-
ing to the general procedure for Route B, a solution of 3,4,
5-trifluorophenylhydrazine hydrochloride (65.9 mg, 0.33 mmol)
in dry N,N-dimethylformamide (3.0 mL) was added dropwise to a
solution of 1,10-carbonyldiimidazole (53.8 mg, 0.32 mmol) and
tate) to give azocarboxamide 9 (30.5 mg, 78.9 lmol, 48%) as a
brown oil. For the biological testing it was additionally purified
using preparative HPLC method B. Rf = 0.5 (hexane/ethyl ace-
tate = 1:1) [UV, KMnO4], 1H NMR (360 MHz, TFA salt, CDCl3) d
1.01 (t, J = 7.3 Hz, 3H), 1.78–1.91 (m, 2H), 3.05 (br s, 1H), 3.14 (br
s, 1H), 3.35 (br s, 5H), 3.48 (br s, 1H), 3.86 (br s, 1H), 3.93 (br s,
diisopropylethylamine (58 lL, 64.4 mg, 0.50 mmol) in dry N,N-
dimethylformamide (9.0 mL) over a period of 20 min at room tem-
perature under nitrogen atmosphere and stirring was continued
for 1.5 h. To the resulting mixture a solution of N1-(2,3-dihydro-

A.L. Bartuschat et al. / Bioorg. Med. Chem. 23 (2015) 3938–3947
3945
1H), 4.23 (quin, J = 7.6 Hz, 1H), 7.20–7.24 (m, 4H), 7.33 (dd,
J = 8.8 Hz, J = 17.3 Hz, 1H), 7.77 (t, J = 8.1 Hz, 1H), 7.83–7.86 (m,
1H), 9.08 (br s, 1H); 13C NMR (151 MHz, CDCl3) d 11.2 (CH3), 16.9
(CH2), 34.2 (CH2), 34.6 (CH2), 36.7 (CH2), 51.2 (CH2), 54.4 (CH2),
64.2 (CH), 110.9 (dd, JCF = 1.7 Hz, JCF = 18.6 Hz, CH), 117.7 (d,
JCF = 18.7 Hz, CH), 123.6 (dd, JCF = 3.2 Hz, JCF = 7.4 Hz, CH), 124.6
(2ꢁ CH), 127.9 (2ꢁ CH), 138.1 (2ꢁ Cq), 147.8 (Cq), 150.9 (dd,
JCF = 14.1 Hz, JCF = 252.9 Hz, Cq), 153.9 (dd, JCF = 13.6 Hz,
JCF = 258.4 Hz, Cq), 161.8 (Cq); 19F NMR (339 MHz, CDCl3) d ꢀ78.8
(TFA), ꢀ131.6 (m, 1F), ꢀ137.7 (m, 1F); MS (EI) m/z (%): 386 [M+]
(5), 245 (19), 189 (13), 188 (100), 146 (11), 117 (60), 116 (17),
115 (19), 113 (12), 85 (10), 83 (12), 72 (12), 32 (14), 28 (25); HRMS
(ESI) m/z calcd for C21H25F2N4O [MH+] 387.1991, found 387.1990.
7.12–7.18 (m, 4H); 13C NMR (151 MHz, CDCl3) d 11.8 (CH3), 20.3
(CH2), 28.5 (3ꢁ CH3), 36.2 (2ꢁ CH2), 38.6 (CH2), 50.5 (CH2), 53.7
(CH2), 62.8 (CH), 79.0 (Cq), 124.4 (2ꢁ CH), 126.4 (2ꢁ CH), 141.6
(2ꢁ Cq), 156.1 (Cq); HRMS (ESI) m/z calcd for C19H31N2O2 [MH+]
319.2380, found 319.2378. In the second step, a solution of the
above prepared N-Boc-protected amine (238 mg, 0.75 mmol) in
dichloromethane (10 mL) was treated with trifluoroacetic acid
(0.58 mL, 854 mg, 7.49 mmol) and the resulting mixture was stir-
red at room temperature. The reaction course was monitored by
TLC after micro-workup with an aqueous solution of sodium
carbonate and ethyl acetate. Upon completion of the reaction a sat-
urated aqueous solution of sodium carbonate (30 mL) was added.
After extraction with chloroform (5 ꢁ 30 mL) the combined
organic phases were washed with a saturated solution of sodium
chloride (30 mL) and dried over sodium sulfate. After filtration
the solvent was removed under reduced pressure and amine 14
(164 mg, quant.) was obtained as a clear, light brown oil and was
used without further purification. 1H NMR (360 MHz, CDCl3) d
0.89 (t, J = 7.4 Hz, 3H), 1.45–1.55 (m, 2H), 2.48–2.52 (m, 2H), 2.61
(t, J = 6.1 Hz, 2H), 2.80 (t, J = 6.0 Hz, 2H), 2.89 (dd, J = 8.0 Hz,
J = 15.9 Hz, 2H), 3.03 (dd, J = 7.9 Hz, J = 15.5 Hz, 2H), 3.69–3.77
(m, 1H), 7.12–7.18 (m, 4H).
4.1.3.8.
(E)-N-(2-((2,3-Dihydro-1H-inden-2-yl)(prop-1-yl)amino)
ethyl)-2-(3,4,5-tri-fluorophenyl)diazene-1-carboxamide (10). Accord-
ing to the general procedure for Route A, a solution of 3,4,
5-trifluorophenylhydrazine (15b) (50.0 mg, 0.26 mmol) in dry
N,N-dimethylformamide (3.0 mL) was added dropwise to a solu-
tion of 1,10-carbonyldiimidazole (40.2 mg, 0.25 mmol) in dry
N,N-dimethylformamide (5.0 mL) over a period of 30 min at room
temperature under nitrogen atmosphere and stirring was contin-
ued for 3 h. To the resulting mixture a solution of N1-(2,3-
dihydro-1H-inden-2-yl)-N1-(prop-1-yl)ethane-1,4-diamine
(36.0 mg, 0.17 mol) in dry N,N-dimethylformamide (3.0 mL) was
(14)
4.1.5. General procedure for the synthesis of cinnamides 16 and
17
l
added over a period of 30 min and stirring was continued over-
night. Subsequently manganese dioxide (120 mg, 1.40 mmol) was
added and the mixture was stirred for 4 h. The solid was filtered
off and washed with chloroform. The combined organic solutions
were concentrated under reduced pressure and the crude product
was purified via column chromatography (silica gel, desact. with
NEt3, hexane/ethyl acetate = 1:1). After purification using HPLC
1-Chloro-N,N,2-trimethylpropenylamine (79.0 lL, 0.60 mmol)
was added to a solution of (E)-3-(4-fluorophenyl)acrylic acid
(100 mg, 0.60 mmol) in dry dichloromethane (1.5 mL) and the
resulting mixture was stirred for 2 h at room temperature under
nitrogen atmosphere. Subsequently a solution of the amine 13 or
14 (0.60 mmol) in dry dichloromethane (1.0 mL) was added to
the in situ generated acid chloride and the mixture was stirred
for 24 h at room temperature. A saturated aqueous solution of
ammonium chloride (20 mL) was added and the aqueous layer
was extracted with dichloromethane (8 ꢁ 20 mL). The combined
organic phases were washed with a saturated aqueous solution
of sodium chloride (20 mL) and dried over sodium sulfate. After fil-
tration the solvent was removed under reduced pressure and the
crude product was purified via column chromatography as
described for each product.
method B the TFA salt of azocarboxamide 10 (3.8 mg, 9.40 lmol,
6%) was obtained as light brown solid. Rf = 0.5 (hexane/ethyl ace-
tate = 1:1) [UV, KMnO4]; 1H NMR (600 MHz, TFA salt, CDCl3) d
1.01 (t, J = 7.3 Hz, 3H), 1.75–1.92 (m, 2H), 3.00–3.20 (m, 2H),
3.27–3.39 (m, 5H), 3.45–3.52 (m, 1H), 3.80 (br s, 2H), 4.23 (quin,
J = 7.6 Hz, 1H), 7.18–7.24 (m, 6H), 7.99 (dd, JHF = 5.2 Hz, J = 9.0 Hz,
2H), 8.88 (br s, 1H), 12.51 (br s, 1H); 13C NMR (151 MHz, TFA salt,
CD3CN) d 11.2 (CH3), 14.5 (CH2), 32.3 (2ꢁ CH2), 37.1 (CH2), 52.1
(CH2), 54.3 (CH2), 64.9 (CH), 125.5 (2ꢁ CH), 128.4 (2ꢁ CH), (nine
signals missing); 19F NMR (339 MHz, TFA salt, CDCl3) d ꢀ76.4
(TFA), ꢀ134.7 (dd, J = 8.0 Hz, J = 19.5 Hz, 2F), ꢀ155.2 (m, 1F); HRMS
(ESI) m/z calcd for C21H24F3N4O [MH+] 405.1897, found 405.1881.
4.1.5.1. (E)-N-(4-((2,3-Dihydro-1H-inden-2-yl)(prop-1-yl)ami-
no)butyl)-3-(4-fluoro-phenyl)acrylamide (16).
Cinnamide
16 was synthesized from N1-(2,3-dihydro-1H-inden-2-yl)-N1-
(prop-1-yl)butane-1,4-diamine (13) (177 mg, 0.72 mmol) accord-
ing to the general procedure described above. The crude product
was purified via column chromatography (silica gel, desact. with
4.1.4. Synthesis of N1-(2,3-dihydro-1H-inden-2-yl)-N1-prop-1-
ylethane-1,2-diamine (14)
N-(2,3-Dihydro-1H-inden-2-yl)-N-(prop-1-yl)amine (416 mg,
2.37 mmol) was added to a solution of N-Boc-2-aminoacetalde-
hyde (754 mg, 4.74 mmol) in dry dichloromethane (20 mL) under
argon atmosphere at room temperature and the resulting mixture
was stirred for 20 min. Subsequently sodium triacetoxyborohy-
dride (1.00 g, 4.74 mmol) was added and the mixture was stirred
overnight. Water (30 mL) and a saturated aqueous solution of
sodium carbonate (30 mL) were added. After extraction with
dichloromethane (3 ꢁ 80 mL) the combined organic phases were
washed with a saturated solution of sodium chloride (30 mL) and
dried over sodium sulfate. After filtration the solvent was removed
under reduced pressure and the crude product was purified via col-
umn chromatography (silica gel, desact. with NEt3, hexane/ethyl
acetate = 4:1) to give tert-butyl (2-((2,3-dihydro-1H-inden-2-
yl)(prop-1-yl)amino)ethyl)carbamate (733 mg, 2.30 mmol, 97%)
as a colorless oil. Rf = 0.1 (ethyl acetate/hexane = 4:1) [UV, KMnO4];
1H NMR (600 MHz, CDCl3) d 0.89 (t, J = 7.3 Hz, 3H), 1.45 (s, 9H),
1.51 (br s, 2H), 2.49 (br s, 2H), 2.61 (br s, 2H), 2.87 (br s, 2H),
3.02 (br s, 2H), 3.19 (br s, 2H), 3.71 (br s, 1H), 4.96 (br s, 1H),
NEt3, ethyl acetate/methanol = 9:1) to give 16 (23.6 mg, 59.8 lmol,
10%) as a beige solid. For biological testing it was additionally puri-
fied using HPLC method B. Rf = 0.2 (ethyl acetate/methanol = 9:1)
[UV, ninhydrin]; 1H NMR (600 MHz, TFA salt, CDCl3) d 0.99 (t,
J = 7.3 Hz, 3H), 1.60–1.67 (m, 1H), 1.73–1.92 (m, 4H), 1.95–2.03
(m, 1H), 2.97–3.04 (m, 2H), 3.08 (dt, J = 4.8 Hz, J = 12.5 Hz, 1H),
3.23–3.41 (m, 6H), 3.53 (td, J = 6.4 Hz, J = 12.0 Hz, 1H), 3.59 (br s,
1H), 4.09–4.15 (m, 1H), 6.51 (d, J = 15.7 Hz, 1H), 7.04 (t,
J = 8.7 Hz, 2H), 7.19–7.25 (m, 4H), 7.38 (br s, 1H), 7.49 (dd,
J = 5.4 Hz, J = 8.8 Hz, 2H), 7.58 (d, J = 15.7 Hz, 1H); 13C NMR
(151 MHz, TFA salt, CDCl3) d 11.1 (CH3), 16.4 (CH2), 21.2 (CH2),
25.9 (CH2), 34.3 (CH2), 34.5 (CH2), 37.6 (CH2), 50.6 (CH2), 52.0
(CH2), 63.3 (CH), 115.8 (d, JCF = 21.9 Hz, 2ꢁ CH), 120.4 (d, JCF = 2.3
Hz, CH), 124.5 (2ꢁ CH), 127.7 (2ꢁ CH), 129.6 (d, JCF = 8.4 Hz, 2ꢁ
CH), 131.1 (d, JCF = 3.4 Hz, Cq), 138.2 (2ꢁ Cq), 139.6 (CH), 163.4
(d, JCF = 250.0 Hz, Cq), 166.7 (Cq); 19F NMR (339 MHz, TFA salt,
CDCl3) d ꢀ78.7 (TFA), ꢀ114.2 (m, 1F); MS (EI) m/z (%): 394 [M+]
(3), 366 (14), 365 (55), 278 (13), 189 (18), 188 (100), 149 (63),
121 (18), 117 (70), 116 (17), 115 (22), 101 (13), 72 (22), 70 (11),

3946
A.L. Bartuschat et al. / Bioorg. Med. Chem. 23 (2015) 3938–3947
43 (12); HRMS (EI) m/z calcd for C25H31FN2O [M+] 394.2420, found
assays were run at protein concentrations of 4–6
lg/well, 1–4 lg/
394.2422.
well, 1–6 g/well and 5–6 g/well with KD values of 0.058–
l
l
0.10 nM, 0.062–0.095 nM, 0.098–0.32 nM, and 0.15–0.35 nM and
4.1.5.2. (E)-N-(2-((2,3-Dihydro-1H-inden-2-yl)(prop-1-yl)ami-
Bmax values of 610–140 fmol/mg, 1500–4400 fmol/mg, 1400–
no)ethyl)-3-(4-fluoro-phenyl)acrylamide (17).
Cinnamide
8000 fmol/mg and 650–2000 fmol/mg protein for the D2long, D2short,
17 was prepared from N1-(2,3-dihydro-1H-inden-2-yl)-N1-(prop-
1-yl)ethane-1,4-diamine (14) (70 mg, 0.32 mmol) according to
the general procedure described above. The crude product was
purified via column chromatography (silica gel, desact. with
NEt3, hexane/ethyl acetate = 1:1 ? ethyl acetate) to give 17
D3 and D4.4 receptors, respectively. D1, D5 and 5-HT2A binding was
achieved using homogenates of membranes from HEK 293 cells,
which were transiently transfected with the pcDNA3.1 vector con-
taining the appropriate human gene (all purchased from Missouri
S&T cDNA Resource Center (UMR), Rolla, MO) by the calcium phos-
phate method.38 Membranes were incubated at final concentra-
(23.6 mg, 51.8 lmol, 16%) as a beige solid. For biological testing
it was additionally purified using HPLC method B. Rf = 0.4 (ethyl
acetate = 9:1) [UV, ninhydrin]; 1H NMR (600 MHz, TFA salt, CDCl3)
d 1.01 (t, J = 6.4 Hz, 3H), 1.75–1.89 (m, 2H), 3.00–3.07 (m, 1H),
3.08–3.15 (m, 1H), 3.25–3.40 (m, 5H), 3.42–3.52 (m, 1H), 3.72–
3.83 (br s, 1H), 3.84–3.94 (m, 1H), 4.17–4.25 (m, 1H), 5.04 (br s,
2H), 6.52 (d, J = 15.0 Hz, 1H), 7.04 (t, J = 8.4 Hz, 2H), 7.20–7.23
(m, 4H), 7.51 (dd, JHF = 5.4 Hz, J = 8.2 Hz, 2H), 7.60 (d, J = 15.0 Hz,
1H); 13C NMR (151 MHz, TFA salt, CDCl3) d 11.1 (CH3), 17.0
(CH2), 34.4 (2ꢁ CH2), 36.1 (CH2), 52.4 (CH2), 54.3 (CH2), 64.2
(CH), 115.8 (d, JCF = 22.6 Hz, 2ꢁ CH), 119.3 (CH), 124.6 (2ꢁ CH),
127.9 (2ꢁ CH), 129.9 (d, JCF = 8.1 Hz, 2ꢁ CH), 130.9 (d, JCF = 3.6 Hz,
Cq), 138.0 (2ꢁ Cq), 140.9 (CH), 165.7 (d, JCF = 250.8 Hz, Cq), 168.1
(Cq); 19F NMR (339 MHz, CDCl3) d ꢀ78.7 (TFA), ꢀ113.4 (s, 1 F);
HRMS (ESI) m/z calcd for C23H28FN2O [MH+] 367.2180, found
367.2182.
tions of 3–4 lg/well, 5–10 lg/well or 4–10 lg/well with receptor
densities (Bmax values) of 2200–5000 fmol/mg, 350–450 fmol/mg
and 880–3200 fmol/mg protein and specific affinities (KD values)
of 0.22–0.75 nM, 0.35–0.37 nM and 0.19–0.45 nM for D1, D5 and
5-HT2A,
respectively. The radioligands [3H]SCH23390 (specific
activity 80 Ci/mmol; Biotrend, Cologne, Germany) and [3H]ketan-
serin (specific activity 53 Ci/mmol; PerkinElmer, Rodgau, Germany)
were used at concentrations of 0.3–0.7 nM for D1, 0.4–0.5 nM for D5
and 0.3–0.5 nM for 5-HT2A, respectively. Receptor binding experi-
ments at 5-HT1A and a1 were performed with homogenates pre-
pared from porcine cerebral cortex.30 Assays were run with
membranes at a protein concentration of 60–80
lg/well or 20–
40 g/well, Bmax values of 50–130 fmol/mg and 160–210 fmol/mg,
l
KD values of 0.060–0.30 nM and 0.060–0.17 nM and radioligand
concentrations of 0.2–0.33 nM for [3H]WAY600135 (specific activ-
ity 80 Ci/mmol; Biotrend, Cologne, Germany) and 0.2 nM for
[3H]prazosin (specific activity 85 Ci/mmol; PerkinElmer, Rodgau,
4.1.6. Methyl (E)-N2-acetyl-N6-(4-(((4-((2,3-dihydro-1H-inden-2-
yl)(prop-1-yl)amino) butyl)carbamoyl)diazenyl)-2,6-difluoro-
Germany) for 5-HT1A and
ing was determined in the presence of haloperidol (10
WAY600135 (10 M), ketanserin (10 M) or prazosin (10 M) for
D1–D5, 5-HT1A, 5-HT2A or 1, respectively. Protein concentration
a
1 receptor, respectively. Unspecific bind-
phenyl)-
D
-lysinate (19)
solution of azocarboxamide
mol) and N -acetyl- -lysine methyl ester hydrochloride 18
(2.91 mg, 12.2 mol) in dry N,N-dimethylformamide (0.5 mL) was
treated with potassium carbonate (5.00 mg, 36.2 mol) under
lM),
Conditions A:
A
7
(3.26 mg,
l
l
l
a
5.97
l
L
a
l
was established by the method of Lowry using bovine serum albu-
l
min as standard.39
argon atmosphere at room temperature. The mixture was stirred
for 12 h at room temperature, stored for two days at ꢀ18 °C and
then stirred for additional 7 h at room temperature. Subsequently
the solvent was removed under reduced pressure and the residue
Data analysis of the competition curves from the radioligand
binding experiments was accomplished by non-linear regression
analysis using the algorithms in PRISM5.0 (GraphPad Software,
San Diego, CA). EC50 values derived from the resulting dose
response curves were transformed into the corresponding Ki values
utilizing the equation of Cheng and Prusoff.40
was purified using HPLC method B to give 19 (3.70 mg, 5.08
85%) as a deep red solid. Conditions B: A mixture of 7 (4.04 mg,
7.39 mol) was dissolved in dimethylsulfoxide (0.25 mL), and
PBS buffer (pH = 7.4, 0.25 mL) and subsequently N -acetyl- -lysine
methyl ester hydrochloride 18 (22.3 mg, 93.4 mol) were added.
lmol,
l
a
L
4.3. Preparation of receptor mutants by site directed
mutagenesis
l
The mixture was stirred at room temperature. The reaction course
and product formation were monitored via LCMS. Compound 19
could be detected by mass analysis and retention time. Rf = 0.1
(ethyl acetate/methanol = 9:1) [UV, KMnO4]; HPLC-MS tR = 16.8 -
min, m/z 616.2; 1H NMR (600 MHz, TFA salt, CDCl3) d 0.96 (br s,
3H), 1.40–1.45 (m, 2H), 1.62–1.68 (m, 3H), 1.71–1.83 (m, 5H),
1.87 (s, 3H), 1.95–2.03 (m, 2H), 3.26–3.40 (br s, 6H), 3.45 (t,
J = 6.0 Hz, 2H), 3.65 (s, 3H), 4.20 (br s, 1H), 4.34 (dd, J = 7.9 Hz,
J = 13.1 Hz, 1H), 5.34 (br s, 1H), 6.71 (br s, 1H), 7.18–7.27 (m,
6H), 8.02 (br s, 1H), 10.70 (br s, 1H), 19F NMR (339 MHz, TFA salt,
CDCl3) d ꢀ133.7 (2 F); HRMS (ESI) m/z calcd for C32H45F2N6O4
[MH+] 615.3465, found 615.3474.
The hDRDw3 t cDNA subcloned into a pcDNA3.1(+) expression
vector was used as described previously.41 Oligonucleotidic prim-
ers were purchased from Biomers.net. Point mutations were intro-
duced by polymerase chain reaction with oligonucleotidic primers
bearing the desired mutation and the hDRDw3 t receptor cDNA as a
template. All mutations were verified by DNA sequence analysis
using the service of LGC Genomics, Berlin.
4.4. Determination of D3 activation via inositol phosphate
assays
Agonist-induced activation of the human Dopamine D3 wild-
type receptor and receptor mutants was studied in inositol phos-
phate (IP) accumulation assays as described previously.35 In brief,
HEK293T cells were transiently co-transfected with cDNAs encod-
ing the human Dopamine D3 wild-type receptor and receptor
4.2. Binding assays
Receptor binding studies were carried out as described.30 In
brief, radioligand displacement experiments with human D2long
,
D2short
,
27 D35,28 and D4.429 receptors were carried out with prepara-
mutants, respectively and the G protein GaqG66Di5. (The GaqG66Di5-
tions of membranes from CHO cells stably expressing the corre-
sponding receptor and [3H]spiperone (specific activity 73 Ci/
mmol; PerkinElmer, Rodgau, Germany) at final concentrations of
0.1–0.2 nM, 0.2 nM, 0.2–0.4 nM and 0.15–0.35 nM for D2long, D2short
D3 and D4.4, respectively according to the particular KD value. The
protein was a kind gift from Prof. Dr. Evi Kostenis42 of the Univer-
sity of Bonn.) Twenty-four h after transfection, cells were seeded
into 24-well plates and incubated with myo-[3H]inositol. On the
next day, test compounds (diluted in binding buffer containing
50 mM Tris, 1 mM EDTA, 5 mM MgCl2, 0.1 mM dithiothreitol,
,

A.L. Bartuschat et al. / Bioorg. Med. Chem. 23 (2015) 3938–3947
3947
Brain Mapp. 2008, 29, 400; (b) Wilson, A. A.; McCormick, P.; Kapur, S.; Willeit,
M.; Garcia, A.; Hussey, D.; Houle, S.; Seeman, P.; Ginovart, N. J. Med. Chem.
2005, 48, 4153.
100 lg/ml Bacitracin, 5 lg/ml Soybean Trypsin Inhibitor, pH 7.4,
supplemented with10 mM LiCl) were added at 37 °C for 120 min.
After lysis, cell extract was separated by anion-exchange chroma-
tography and total IP was eluted. Radioactivity was measured by
scintillation counting. Data were analyzed by normalizing disinte-
grations per minute (dpm) values with 0% for the unstimulated
receptor and 100% for the full effect of the reference quinpirole.
Concentration–response curves were fitted by nonlinear regression
using the GraphPad Prism 5 software.
11. Mach, R. H.; Tu, Z.; Xu, J.; Li, S.; Jones, L. A.; Taylor, M.; Luedtke, R. R.; Derdeyn,
C. P.; Perlmutter, J. S.; Mintun, M. A. Synapse 2011, 65, 724.
12. (a) Hocke, C.; Maschauer, S.; Hübner, H.; Löber, S.; Utz, W.; Kuwert, T.;
Gmeiner, P.; Prante, O. ChemMedChem 2010, 5, 941; (b) Hocke, C.; Prante, O.;
Löber, S.; Hübner, H.; Gmeiner, P.; Kuwert, T. Bioorg. Med. Chem. Lett. 2005, 15,
4819; (c) Hocke, C.; Prante, O.; Salama, I.; Hübner, H.; Löber, S.; Kuwert, T.;
Gmeiner, P. ChemMedChem 2008, 3, 788; (d) Hocke, C.; Cumming, P.;
Maschauer, S.; Kuwert, T.; Gmeiner, P.; Prante, O. Nucl. Med. Biol. 2014, 41,
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Kuwert, T.; Gmeiner, P. J. Med. Chem. 2007, 50, 489.
Irreversible activation of the D3 receptor mutants was tested at
13. Fehler, S. K.; Maschauer, S.; Höfling, S. B.; Bartuschat, A. L.; Tschammer, N.;
Hübner, H.; Gmeiner, P.; Prante, O.; Heinrich, M. R. Chem. Eur. J. 2014, 20, 370.
14. Höfling, S. B.; Bartuschat, A. L.; Heinrich, M. R. Angew. Chem., Int. Ed. 2010, 49,
9769.
15. Jasch, H.; Höfling, S.; Heinrich, M. R. J. Org. Chem. 2012, 77, 1520.
16. Unpublished results.
17. For reviews covalent ligand binding, see: (a) Dormán, G.; Prestwich, G. D.
Trends Biotechnol. 2000, 18, 64; (b) Vodovozova, E. L. Biochemistry (Mosc.) 2007,
72, 1; (c) Sinz, A. Angew. Chem., Int. Ed. 2007, 46, 660; (d) Weber, P. J.; Beck-
Sickinger, A. G. J. Pept. Res. 1997, 49, 375.
18. (a) Chee, G.-L.; Yalowich, J. C.; Bodner, A.; Wu, X.; Hasinoff, B. B. Bioorg. Med.
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3 lM test compounds in comparison to the reversible reference
ligand quinpirole (3
lM). After incubation for 60 min, the antago-
nist haloperidol (10
lM) was added to one-half of the sample (buf-
fer was added to the other half) and incubations were continued
for an additional 180 min. Total IP accumulation was determined
as described above. Data were analyzed by defining dpm values
for the unstimulated control as 1. IP-response of the test com-
pounds was expressed as x-fold over basal.
Acknowledgements
The authors would like to thank the Deutsche Forschungsgeme-
inschaft DFG for financial support within the projects HE5413/3-1
and GRK1910/B3. The experimental assistance by Karina Wicht,
Markus Lang, Sarah Höfling and Stefanie Fehler is gratefully
acknowledged. The authors are further grateful for computational
support by Ralf Kling.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.12.012.
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