Benzyl Phenylsemicarbazides: A Chemistry-Driven Approach Leading to G Protein-Biased Dopamine D4 Receptor Agonists with High Subtype Selectivity

Article abstract of DOI:10.1021/acs.jmedchem.9b01085

Many subtype-selective dopamine receptor ligands developed for the D₂-D₄ family incorporate a 1-arylpiperazine-derived primary recognition motif, which is connected to a lipophilic moiety occupying an extended binding pocket (EBP) of the receptor via an aliphatic linker of variable lengths. The evaluation of a novel group of dopamine receptor ligands now showed that highly subtype-selective ligands [up to K_i(D_4.4) = 0.25 nM, D_2L/D_4.4 = 320, D₃/D_4.4 = 710 for APH199 (17)] can be obtained by choosing a relatively large and conformationally flexible 1-benzyl-1-phenylsemicarbazide substructure to fill the EBP. The novel chemotype APH199 (17) was found to act as a full agonist at the D₄ receptor showing significant bias toward G protein activation over β-arrestin recruitment in comparison to quinpirole.

Full text of DOI:10.1021/acs.jmedchem.9b01085

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Article
Benzyl phenylsemicarbazides: a chemistry-driven approach leading to G
protein-biased dopamine D4 receptor agonists with high subtype selectivity
Anna Pirzer, Roman Lasch, Heike Friedrich, Harald Hübner, Peter Gmeiner, and Markus Rolf Heinrich
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01085 • Publication Date (Web): 15 Oct 2019
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Benzyl phenylsemicarbazides: a chemistry-driven approach leading to G protein-biased
dopamine D receptor agonists with high subtype selectivity
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Anna S. Pirzer, Roman Lasch, Heike Friedrich, Harald Hübner, Peter Gmeiner, Markus R.
Heinrich*
Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander-Universität
Erlangen-Nürnberg, Nikolaus-Fiebiger-Str. 10, 91058 Erlangen, Germany
markus.heinrich@fau.de
Abstract
Many subtype selective dopamine receptor ligands developed for the D -D family incorporate a
2
4
1-arylpiperazine derived primary recognition motif, which is connected to a lipophilic moiety
occupying an extended binding pocket of the receptor via an aliphatic linker of variable length.
The evaluation of a novel group of dopamine receptor ligands now showed that highly subtype
selective ligands (up to K (D ) = 0.25 nM, D /D = 320, D /D = 710 for APH199 (17)) can
i
4.4
2L
4.4
3
4.4
be obtained by choosing a relatively large and conformationally flexible 1-benzyl-1-
phenylsemicarbazide substructure to fill the extended binding pocket. The novel chemotype
APH199 (17) was found to act as a full agonist at the D receptor showing significant bias
4
towards G protein activation over β-arrestin recruitment in comparison to quinpirole.
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Introduction
Dopamine is one of the most prevalent catecholamines in human brain and affects many
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processes in the central nervous system. A variety of disorders can be associated to imbalances
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in the dopamine neurotransmitter system and, thus, dopamine receptors play a crucial role as
drug targets in medicinal chemistry. The family of the human dopamine receptors refers to the
rhodopsin like G protein-coupled receptors and can be divided into the D -like subgroup,
1
consisting of the D and the D receptor, and the D -like subgroup, consisting of the D , D and
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2
-4
the D receptor, respectively.
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When the D receptor was cloned and identified for the first time in 1991, and the preferential
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affinity of the atypical antipsychotic clozapine to this subtype was observed, efforts were
undertaken to design and develop D receptor antagonists as new drugs for the treatment of
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schizophrenia.5-7 A few years later in 1997, the initial enthusiasm to discover improved
antipsychotic drugs with potentially less side effects faded due to a multicenter study revealing
that the D selective receptor antagonist L-745,870 was ineffective in the treatment of
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schizophrenia.8
Nevertheless, subtype-selective D receptor ligands are still developed and investigated with the
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aim to provide better treatment options for addictive disorders associated with cocaine,9,10
1
1
12-16
heroin12-16 and nicotine, eating disorders like anorexia nervosa
17
18-
morphine, amphetamine,
2
0
as well as attention deficit disorder.21-24 Moreover, D selective compounds have been
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discussed as potential drugs with regard to erectile dysfunction25-27 or cognitive deficits. A very
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29
recent publication by Keck et al. further highlights the importance of D subtype selective
4
ligands (K up to 4.3 nM and >100-fold selectivity over D -like receptors).
i
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The continuous interest in highly subtype and also functionally selective ligands for the D4
subtype becomes obvious from a very recent and so far unique approach, in which 138 million
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library molecules were docked against the D receptor. From 589 selected hits, 549 molecules
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were successfully synthesized, and further optimization of the docking hit ZINC621433143 in
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terms of stereochemistry led to the full agonist ZINC621433144 (K = 180 pM). This
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compound did not only show high subtype selectivity towards D , but was also functionally
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selective with a 17-fold preference towards G signaling versus β-arrestin recruitment, compared
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to the classic agonist quinpirole.
Previous studies showed that D selectivity can be achieved by connecting a 1-arylpiperazine
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recognition element, which represents a substitute for the endogenous dopamine, to a lipophilic
31-34
appendage via a short methylene (C ) linker unit.
This structure-subtype selectivity
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relationship is nicely supported by the D receptor antagonists 1a and 1b developed by
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Kulagowski, displaying sub-nanomolar affinity and high subtype selectivity (Figure 1).
O
NC
N
N
N
X
HN
O
N
N
N
N
H
N
Kulagowski et al.35
Glase et al.39
PD168077 (2) (agonist)
R1
30
N
Lyu et al.
H
X = Cl, L-745870 (1a) (antagonist)
X= I (1b)
ZINC621433144 (3) (agonist)
H
N
N
N
n
H
N
N
H
R
N
N
H
X
N
S
N
O
R2
N
N
O
Sampson et al.46
4a: n = 3 4b: n = 4 (antagonists)
Wang et al.47
O
N
X
9-6-24 (5) (agonist)
(this work)
6
Figure 1. Highly subtype selective dopamine D receptor ligands.
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Structurally related antagonists of 1a and 1b, in which the 7-azaindole substructure was replaced
by an indole, a 7a-azaindole (pyrazolopyridine) or an azulene unit showed similar D binding
4
affinity and subtype selectivity, thereby strengthening the suitability of the overall structural
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design.36-38 The D -selective agonist PD168077 (2) developed by Glase and the docking hit
39
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ZINC621433144 (3) are no longer in accordance with the structural requirements mentioned
above. The length of the linker units have been discussed earlier38,40-45 and elongated alkyl chains
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can be found in the D selective ligands 4 and 5, recently developed by Sampson and Wang.
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The new chemotype 5, in which the quinolone moiety acts as a primary recognition motif and the
phenoxy unit represents the lipophilic appendage, was developed using data from the previously
determined D crystal structure (co-crystallized with nemonapride) in combination with virtual
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screening. Taking the D crystal structure as a basis, the detailed docking results from the study
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by Wang47 clearly support that new chemotypes with longer alkyl linker units can be well
tolerated by the D subtype.
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Seeking for a new chemotype that may confer atypical biological effects such as ligand bias, we
found it challenging to investigate if the combination of an elongated linker unit and a
comparably large, but flexible lipophilic appendage – such as in the general structure 6 – might
also be a suitable approach to induce strong D subtype selectivity. Encouraged by the
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remarkable functional selectivity of the atypical chemotype ZINC621433144 (3)29 (17-fold
preference towards G versus β-arrestin), we thought to investigate the effect of our newly
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developed chemotype on the downstream signaling pathways, thereby aiming to get further
insights into the structural determinants for functional selectivity, which are so far poorly
understood.
Using a chemistry-based approach, and exploiting a newly developed reductive alkylation
method,48 we now show that the extended binding pocket of the dopamine D receptor can
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indeed tolerate a sterically demanding benzyl phenylsemicarbazide substructure, a crucial
element of our newly developed ligands of type 6. Our study comprises 36 novel ligands, which
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we evaluated for their affinity to the dopamine D4.4 receptor and their subtype selectivity in
comparison to the other D -like receptors, D , D and D . The most promising ligands were
2
2L
2S
3
further studied in functional assays and characterized for their selectivity over a small set of
related GPCRs. Modeling studies finally pointed to new key interactions thereby showing that
the extended binding pocket (EBP) in the dopamine D2-4 receptor family can be a valuable
starting point for future developments with regard to ligands displaying both high subtype and
functional selectivity.
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Results
Synthesis. The synthesis of the target compounds started from the commercially available 1-
arylpiperazines 7a-e which were alkylated with N-phthalyl-protected bromoalkyl amines 8a-c
(Scheme 1). Deprotection with hydrazine provided the free amines 10a-i, which were
subsequently coupled to various phenylazocarboxylic acid tert-butyl esters 11a-f to give the
azocarboxamides 12a-n. The final reductive N-alkylation of 12a-n with benzyl and allyl
bromides 13a-i was carried out according to a protocol recently developed in our group,48
thereby completing the straightforward synthesis of the semicarbazide target compounds 14-47.
Remarkably, the presence of zinc changes the selectivity of the final alkylation, as one would
otherwise expect a selective functionalization at the tertiary aliphatic amino group in the
piperazine subunit of 12. However, and especially for the lower yielding alkylation reactions, we
can currently not exclude that alkylation at the tertiary aliphatic amine also occurs to a
significant extent.
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Scheme 1. Synthesis of target compounds 12a-c and 14-47.a
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PhthN
H2N
HN
N
N
n
n
R1
ii
iii
i
N
X
PhthN
N
X
N
X
+
N
OtBu
+
Br
N
n
R
R
R
X
X
X
O
7a-e
8a-c: n = 1-3
9a-i
10a-i
11a-f
R1
R1
R2
H
N
H
H
N
Br
N
N
N
N
n
iv
N
N
n
+
R2
O
N
X
O
N
X
R
R
X
X
1
2a-n
13a-i
14-47
aReagents and conditions: (i) arylpiperazine hydrochloride 7a-e (1.0 equiv),
bromoalkylphthalimide 8a-c (2.0 equiv.), K CO (4.0 equiv), acetone, reflux, 20 h. 40-100 %; (ii)
2
3
N H (10.0 equiv), ethanol, reflux, 1 h, 65-88 %; (iii) tert-butyl phenylazocarboxylate 11a-f
2
4
(
1.1 equiv.), K CO (4.0 equiv), ethanol, argon atmosphere, 40°C, 24-99 %; (iv) benzyl- or allyl
2 3
bromide 13a-i (2.5-5.0 equiv), zinc powder (2.5-10 equiv), THF, NH Cl, 30 sec., 18-99 %.
4
Notably, compounds that include comparably substituted semicarbazide substructures as the D4
ligands prepared within this work, have, among others, previously appeared in studies focusing
4
9
50
51
on MAP kinase inhibitors, inhibitors of integrin binding and blood coagulation. Compared
to semicarbazides bearing only one substituent on the β-nitrogen atom of the hydrazine subunit,
the β,β-disubstituted derivatives presented herein cannot be directly oxidized to their
corresponding azo compounds and do thus show a high chemical stability, which was also
observed during their preparation. No decomposition occurred even after storing the
semicarbazide ligands in solution and at ambient temperature over several weeks.
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Ligand binding. Radioligand competition binding assays were performed to determine binding
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affinities and the selectivity profiles of 12c and the new dopamine receptor ligands 12a, 12b
and 14-47 in comparison to the previously described compounds L-745870 (1a) and PD168077
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(
2). The binding data were generated by measuring the ligands’ ability to compete with
[3
H]spiperone for the in-house available cloned human dopamine receptor subtypes D , D , D
3
2
L
2S
and D stably expressed in CHO cells (Tables 1-5). As the K values were derived from binding
4.4
i
experiments that have been analyzed as monophasic curves, the selectivity profiles may be
inferred when applying different binding conditions (i.e. using an agonist radioligand or varying
the ratio between receptor and G protein in the membranes.
Already the first series of semicarbazide ligands 14-16 revealed a modified binding profile
compared to the azocarboxamide analogues 12a-c (Table 1). While the azocarboxamides 12a-c
were found to be at least roughly in agreement with the established rule that short linker units
(
12a, n = 1; 12b, n = 2) favor binding to the D subtype, while longer linkers such as C (12c, n =
4
4
3
) shift the affinity towards D3,52 the evaluation of the semicarbazides 14-16 revealed a
remarkable preference for the D subtype independent of the linker length. As the best
4
combination of affinity (K (D ) = 0.55 nM) and selectivity (D /D = 12, D /D = 45) was
i
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2L
4.4
3
4.4
determined for RLH676-1 (15) at a linker length of three carbon atoms (n = 2), the study was
continued with this lead structure.
Table 1. Radioligand binding data of compounds 1a, 2, 12a-c and 14-16 to the subtypes of
the dopamine D receptor family.
2
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F
F
H
H
H
N
N
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OMe
N
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n
OMe
n
O
N
O
N
1
2a: n = 1
12b: n = 2
2c: n = 3
14: n = 1
15: n = 2
16: n = 3
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compound
n =
K
i
(nM±SEM)a
selectivityb
D
2L
D
2S
D
3
D
4.4
D
2L/D4.4
D
2S/D4.4
3
D /D4.4
L-745870
(1a)
-
-
47000±5200 33000±3600 65000±12000 0.70±0.046 67000
47000
93000
PD168077
2300±470
1100±100
4700±1700
2.3±0.56
1000
480
2000
(
2)
1
2a
1
2
3
1
2
3
270±26
110±21
38±4.6
100±35
6.6±1.5
160±21
140±26
51±2.0
34±3.0
71±25
120±30
140±37
3.1±0.38
700±220
25±6.0
36±5.4
20±3.0
7.5
5.5
0.40
11
3.9
2.6
0.36
7.9
7.8
21
3.3
7.0
0.033
78
1
2b
2cc
14
15
1
94±13
9.0±0.41
0.55±0.087
4.4±0.95
4.3±0.76
94±19
12
45
1
6
76±5.2
36
17
a
Values are reported as mean (nM±SEM) of three to five individual experiments each done in triplicate and
b
analyzing the data applying a monophasic curve fitting. Subtype selectivity for D4.4 over D2L, D2S or D
determined by calculating the ratio of K D /K D4.4. The K values determined for 12c are similar to those
previously reported in ref. 52.
3
x
(=D ) was
c
i X i i
Table 2 summarizes the results obtained upon variation of the substituents on the two aromatic
moieties A and B of the benzyl phenylsemicarbazide unit. Compared to RLH676 (15), the
subtype selectivity towards D could be strongly increased by shifting the fluorine atom from the
4
4
-position of ring A to the 4-position of ring B, as in APH212 (18), and even slightly more by
leaving both ring systems A and B unsubstituted, as in compound APH199 (17). Almost all other
substitution patterns, as they are present in ligands 19-36, lead to a reduced D affinity and
4
subtype selectivity. Regarding the trends in D binding affinity for the less potent ligands, most
4
compounds show K values in a quite narrow range from 2 nM to 6 nM, indicating that a variety
i
of substitution patterns is also quite well tolerated. Only unfavorable patterns such as 2,4-
dichloro substitution on ring A (compounds 27 and 28) and/or 4-cyano and 3-methoxy
substitution on ring B (compounds 23, 28, 35 and 36) resulted in K values higher than 10 nM.
i
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2
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4
5
6
7
8
9
Notably, a good subtype selectivity (D /D = 55, D /D = 140) was also observed for ligand
2
L
4.4
3
4.4
3
4, although this was not due to subnanomolar binding to D (K (D ) = 2.9 nM), but due to a
4 i 4.4
much reduced affinity to the D and D subtypes.
2
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Binding data of compounds 15 and 17-36 with varying substitution pattern in ring
A and B to the subtypes of the dopamine D receptor family.
2
R1
N
A
B
H
H
N
N
N
OMe
N
O
R2
15,17-36
1
2
(nM±SEM)a
selectivityb
compound
R =
R =
K
i
D
2L
D
2S
D
3
D
4.4
D
2L/ D4.4
12
D
2S/ D4.4
7.8
300
200
13
D
3
/ D4.4
RLH676 (15)
APH199 (17)
APH212 (18)
4-F
H
H
6.6±1.5
77±14
140±31
92±14
73±9.4
77±8.4
4.3±0.76
73±17
25±6.0 0.55±0.087
170±13 0.25±0.054
310±28 0.55±0.088
45
H
320
250
26
710
560
44
23
18
25
43
19
45
36
4.0
23
38
28
71
29
14
140
9.7
14
H
4-F
2-F
3-F
4-F
110±18
47±10
1
2
2
2
9
0
1
2
4-F
160±25
88±16
85±19
150±15
3.6±0.27
3.8±0.52
4.6±0.59
5.9±1.1
15±2.9
4-F
46±12
19
12
4-F
30±3.0
62±7.2
17
6.5
11
4-F
3-OMe 110±12
19
23
24
4-F
4-CN
4-Ph
H
210±36
140±42
32±1.7
41±3.2
35±4.8
150±27
55±15
120±11 640±190
14
8.0
7.1
7.9
10
4-F
42±12
23±3.5
26±1.0
24±3.0
94±13
34±2.4
22±2.2
19±4.4
19±4.6
35±7.2
110±28
130±43
95±18
5.9±2.1
2.9±0.87
2.6±0.21
15±7.1
24
2
2
2
2
5
6
7
8
4-Cl
4-Cl
2,4-Cl
2,4-Cl
4-Br
4-Br
11
2-F
H
16
2
2
61±14
2.3
13
1.6
7.8
10
4-CN
H
280±58
130±27
92±9.1
120±10
76±11
12±2.0
29
30
3.4±0.60
3.3±0.74
1.7±0.50
2.6±0.19
6.2±1.5
2.9±0.77
33±3.8
16
2-F
H
45±6.4
45±2.0
42±12
14
6.7
11
3
3
3
3
1
2
3
4
4-OMe
4-OMe
4-OMe
4-OMe
26
2-F
3-F
4-F
16
7.3
5.6
38
59±4.8
160±11
86±30
10
110±7.3 400±100
110±15 320±120
55
35
36
4-OMe 3-OMe 150±15
4-OMe 4-CN 250±0
4.5
16
3.3
6.9
110±32
220±32
16±3.4
a
Values are reported as mean (nM±SEM) of three to seven individual experiments each done in triplicate and
b
analyzing the data applying a monophasic curve fitting. Subtype selectivity for D4.4 over D2L, D2S or D
3 x
(=D ) was
determined by calculating the ratio of K /K
i
D
X
i 4.4
D .
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Having seen that the combination of an unsubstituted phenyl and an unsubstituted benzyl group
on the semicarbazide, as present in compound APH199 (17), provides high affinity and subtype
selectivity for the D receptor, we replaced the benzyl moiety by smaller allyl or 2-methallyl unit
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to evaluate the effect of decreased size (Table 3). With compound 31 serving as reference for this
modification, it became apparent that allylation as well as 2-methallylation only lead to ligands
3
7 and 38 with significantly reduced D affinity and subtype selectivity. Thus, a certain size of
4
the two units attached to the semicarbazide appears to be necessary to successfully differentiate
between the D -D subtypes.
2
4
Table 3. Binding data for the methoxyphenyl substituted compounds 31, 37 and 38.
MeO
MeO
N
N
H
H
H
H
N
N
N
OMe
N
N
N
OMe
N
N
O
O
31
37: R = H
8: R = Me
R
3
(nM±SEM)a
K
i
selectivityb
compound
R =
D
2L
D
2S
D
3
D
4.4
D
2L/D4.4
26
D
2S/D4.4
9.4
3
D /D4.4
3
3
1
7
---
H
45±2.0
180±43
210±32
19±4.4
340±74
300±28
120±10
660±29
860±73
1.7±0.50
61±2.3
64±8.2
71
11
13
3.0
5.6
38
Me
3.3
4.7
a
Values are reported as mean (nM±SEM) of three to seven individual experiments each done in triplicate and
analyzing the data applying a monophasic curve fitting. Subtype selectivity for D4.4 over D2L, D2S or D (=D ) was
3 x
b
determined by calculating the ratio of K D /K D .
i
X
i
4.4
In the next stage, we turned to the variation of the recognition motif, which was so far
represented by a 1-(2-methoxyphenyl)piperazine. Based on the highly potent and subtype
selective D receptor ligands reported so far (c.f. Figure 1), the 2-methoxyphenyl unit was
4
exchanged for a 4-chlorophenyl, 4-bromophenyl or a pyrimidin-2-yl substructure. The results
obtained in comparison to the so far most APH199 (17) and APH212 (18) are summarized in
Table 4.
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Table 4. Binding data for compounds 17, 18 and 39-44 constituting different structural
variations of the phenylpiperazine moiety.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
X
X
R
R
N
X
N
X
H
H
H
H
N
N
N
N
N
N
N
N
O
O
APH199 (17), 39-41
APH212 (18), 42-44
F
(nM±SEM)a
K
i
selectivityb
compound
R =
X =
D
2L
D
2S
D
3
D
4.4
D
2L/D4.4
320
66
D
2S/D4.4
3
D /D4.4
APH199 (17) 2-OMe CH
77±14
73±17
170±13
0.25±0.054
9.7±5.2
14±7.0
300
42
710
110
110
110
560
50
3
4
9
0
4-Cl
4-Br
H
CH
CH
N
640±120 410±52 1100±320
730±100 520±67 1500±310
580±140 440±96 2000±480
52
37
41
18±5.8
32
24
APH212 (18) 2-OMe CH
140±31
500±46
790±76
110±18
380±35
310±28
0.55±0.088
17±7.1
250
29
200
22
4
4
4
2
3
4
4-Cl
4-Br
H
CH
CH
N
850±100
620±52 1400±120
46±20
17
13
30
430±110 310±47 1900±100
19±4.9
23
16
100
a
Values are reported as mean (nM±SEM) of three to four individual experiments each done in triplicate and
analyzing the data applying a monophasic curve fitting. Subtype selectivity for D4.4 over D2L, D2S or D
b
3 x
(=D ) was
determined by calculating the ratio of K /K
i
D
X
i 4.4
D .
For both series, either starting from the benzyl semicarbazide APH199 (17) or the 4-fluorobenzyl
semicarbazide APH212 (18), these variations resulted in an at least 31-fold loss of D binding
4
affinity for compounds 39-44 combined with a remarkable decrease of selectivity towards the
other subtypes D , D and D . To answer the question whether the increased size of a 4-bromo
2
L
2S
3
or 4-iodo-substituted phenyl ring in the 1-arylpiperazine recognition motif could possibly be
counterbalanced by a shorter linker length (c.f. ligand 1b, Figure 1), we finally prepared the
semicarbazides 45-47 (Table 5). In comparison to 4-bromo derivative 40 (Table 4), the exchange
of the 4-bromo for a 4-iodo substituent as well as the shortening of linker length from three to
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
two methylene units independently led to less binding affinity of the ligands 45-47 to the D4
subtype.
Table 5. Binding data of compounds 40 and 45-47.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
H
H
H
N
N
N
N
N
N
N
N
n
n
O
N
O
N
4
4
0: n = 2
5: n = 1
46: n = 2
47: n = 1
Br
I
(nM±SEM)a
selectivityb
compound
n =
K
i
D
2L
D
2S
D
3
D
4.4
D
2L/D4.4
52
D
2S/D4.4
37
3
D /D4.4
4
4
4
0
5
6
2
1
2
1
730±100
390±92
520±67
430±46
670±17
510±39
1500±310
1800±310
1300±340
1800±290
14±7.0
28±3.8
21±5.2
44±13
110
64
14
15
690±160
490±150
33
32
62
47
11
12
38
a
Values are reported as means (nM±SEM) of three to four individual experiments each done in triplicate and
b
analyzing the data applying a monophasic curve fitting. Subtype selectivity for D4.4 over D2L, D2S or D
3 x
(=D ) was
determined by calculating the ratio of K /K
i
D
X
i 4.4
D .
The biological evaluation of the ligands 17 and 18, possessing the most favorable binding profile
for the D subtype, towards receptors of the D family as well as other non-dopaminergic
4
1
receptors demonstrated that significantly lower binding affinities (higher K values) are found for
i
most other receptors (Table 6).
Table 6. Binding data of compounds 17 and 18 to receptors of the D family as well as other
1
non-dopaminergic receptors.
N
N
H
H
H
H
N
N
N
OMe
N
N
N
OMe
N
N
O
O
APH199 (17)
APH212 (18)
F
compound
K
i
(nM±SD)a
D
1
D
5
α
1A
α
2
β
1
β
2
δ-OR
APH199 (17)
APH212 (18)
200±120
360±99
340±21
1000±91
1.7±0.14
1.6±0.42
400±57
13000±3500
>50000
1800±350
4500±420
10000±7100
19000±5600
860±110
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(nM±SD)a
K
i
compound
κ-OR
μ-OR
5-HT1A
5-HT2A
M
1
M
2
M
3
APH199 (17)
APH212 (18)
3900±1700
4300±1600
2000±850 17±2.1
2300±920 34±12
65±2.1 25000±14000 14000±11000
130±7.1 15000±0 17000±710
Values are reported as mean values in (nM±SD) derived from two individual experiments each done in triplicate.
25000±4900
37000±9200
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
The only exception was observed for the α subtype to which both ligands 17 and 18 showed a
1
A
comparably strong binding with K values of 1.7±0.14 nM and 1.6±0.42 nM, respectively, which
i
corresponds to selectivity ratios of 7.1 and 2.9 over the D subtype. Functional assays at the α
4
1A
receptor led to the observation that 17 and 18 both behave as neutral antagonists (see Supporting
Information for dose-response curves). These assays were performed for G mediated signaling

q
(
IP-One assay) and β-arrestin recruitment (PathHunter assay).
Functional assays subtype to determine agonistic or antagonistic properties at the D subtype
4
were conducted for the most promising ligands RLH676 (15), APH199 (17) and APH212 (18)
using quinpirole as reference compound. The results summarized in Figure 2 and Table 7 show
that all three compounds behave as almost full agonists with bias towards the G protein over the
β-arrestin signaling pathway.
D R stimulated G activation
D R mediated -arrestin 2 recruitment
4
A
4
qi
B
100
75
50
25
0
15
17
100
75
50
25
0
1
5
18
1
7
quinpirole
1
8
15 + 0.3 µM quin
17 + 0.3 µM quin
1
8 + 0.3 µM quin
quinpirole
-12
-11
-10
-9
-8
-7
-6
-5
-11
-10
-9
-8
-7
-6
-5
-4
log [compound], M
log [compound], M
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 2. Dose-response curves for the most potent compounds 15, 17, 18 and the reference
quinpirole measuring G protein or arrestin-mediated D receptor signaling. A) IP-One assay
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
measuring G protein mediated signaling in HEK cells co-expressing the D R and the G protein
4
hybrid G . B) Arrestin recruitment measuring partial agonist properties as well as partial
qi
antagonism of 300 nM quinpirole employing the PathHunter complementation assay in HEK
cells expressing D R-PKA and (EA)-β-arrestin-2. Both graphs show pooled curves from six
4
individual experiments each done in duplicates (agonist effect), antagonist properties are
displayed as mean curves derived from three to four experiments.
Table 7. Key data from functional assays using quinpirole as reference compound.
compound
IP-One-D
4
+Gqia
arrestin-D
4
-PKAb
bias factor
10ΔΔlog(τ/KA)
maximal activation
EC50
maximal activation
EC50
% ± SEM)c
(% ± SEM)c
(nM ± SEM)d
(
(nM ± SEM)
2.2 ± 0.2
1.8 ± 0.39
8.7 ± 4.2
41 ± 15
quinpirole
100
100
83 ± 0.7
340 ± 150
250 ± 150
500 ± 110
1.0
26
RLH676 (15)
APH199 (17)
APH212 (18)
88 ± 4
89 ± 1
86 ± 3
22 ± 2
16 ± 1
17 ± 1
4.9
1.8
a
An IP-One assay with HEK293T cells transiently co-transfected with D
applied for the determination of G protein mediated signaling of the test compounds at D
recruitment was performed using the PathHunter complementation assay with HEK293T cells stably expressing
4
R and the G protein hybrid Gqi was
b
4
R. Measuring of arrestin
c
(
4
EA)-β-arrestin and the transiently transfected D R-PKA construct. Maximum activation in %±SEM relative to the
d
reference quinpirole derived from six individual experiments each done in duplicate. EC50 value ± SEM from six
individual experiments each done in duplicate.
Regarding signaling via the G protein pathway, a strong activation was determined for the three
novel semicarbazides RLH676 (15), APH199 (17) and APH212 (18), which reach 86 to 89% of
the maximum effect attainable by quinpirole. The functional assay on β-arrestin recruitment, in
contrast, revealed that 15, 17 and 18 do by far not reach the same level of activation as quinpriole
and act as weak partial agonists (16 - 22% relative to quinpirole). In presence of an agonist (300
nM of quinpirole) 15, 17 and 18 dose-dependently inhibit this effect with the best potency
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observed for 15 (for 15: IC = 49±19 nM, for 17: IC = 450±140 nM, for 18: IC = 620±220
5
0
50
50
nM) and displayed a partial antagonist effect (Figure 2B).
The fact that the control compound quinpirole shows an approximately 38-fold higher affinity
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
for the G protein assay versus the β-arrestin assay, with EC values of 2.2 nM versus 83 nM, is
5
0
most likely due to an amplification of the G protein response by the spare receptor
phenomenon,53 rather than that the control compound quinpirole exhibits a significant bias.
Comparing the EC (affinity) ratio of 38 [EC (β-arr.)/EC (G )] for quinpirole with the ratios
5
0
50
50
i
calculated in the same way for the semicarbazides 15, 17 and 18, which leads to values of 189,
9 and 12, respectively, shows that only RLH676 (15) displays an affinity- and efficacy-wise
2
bias towards the G protein. For APH199 (17) and APH212 (18), a lower affinity-wise bias (EC50
ratios of 29 and 12 vs. 38 for quinpirole) was determined, so that the overall bias seen in the
signaling of 17 and 18 is primarily driven by a loss of efficacy at stimulating the β-arrestin
pathway - not a loss of affinity. These considerations are supported by the bias factors calculated
54
for 15, 17 and 18 according to the operational model by Black and Leff thereby following
recently published procedures.55,56 From these calculations, a comparably high bias factor of 26
was obtained for RLH676 (15) while lower values of 4.9 and 1.8 were determined for 17 and 18,
respectively. From literature, functional data is available for the recently developed D -selective
4
ligand 621433134 (3) (Figure 1), which was also evaluated using quinpirole as reference, and for
which a G protein over β-arrestin bias factor of 17 was calculated.30
Studies on Metabolic Stability.
The metabolic stability of semicarbazide APH199 (17) was studied using male rat liver
microsomes and imipramine as a reference57 under previously established conditions.58 The
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reliability of the assay could be confirmed, as product patterns similar to those reported for
imipramine in literature were obtained after comparable incubation times with N-demethylation
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and hydroxylation occuring as main metabolic pathways. The results obtained for APH199 (17)
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in comparison with imipramine are summarized in Figure 3.
Figure 3. Oxidative metabolism of APH199 (17) and imipramine upon exposure to male rat liver
microsomes. Results from LC-MS analysis are depicted in percentage of non-metabolized
APH199 (17) and imipramine.
As shown in Figure 3, APH199 (17) was found to be more stable than imipramine under the
assay conditions, whereat imipramine is typically classified as a moderately stable positive
control for phase I metabolism.57,58 After 120 minutes incubation time, analysis of the reaction
mixture by LC-ESI-MS showed approximately 50% hydroxylation of 17 (detected as
+
+
[
M+O+H] : m/z = 490), approximately 20% of O-demethylated 17 (detected as [M−CH +H] :
2
+
m/z = 460), approximately 20% of N-debenzylated 17 (detected as [M−C H +H] : m/z = 384),
7
6
+
and approximately 10% of unchanged APH199 (17) (detected as [M+H] : m/z = 474). These
results suggest that 17 should possess sufficient metabolic stability to be considered for in vivo
testing in the future. Furthermore, no warnings in the PAINS screening were obtained for the
metabolites of APH199 (17) resulting from O-demethylation and N-debenzylation. The
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hydroxylated metabolite could not be screened since the actual position of the hydroxy group is
so far unknown.
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Docking. Docking studies59-61 were performed to get insights into the binding poses of the novel
semicarbazide ligands, especially the possible interactions in the extended binding pocket (EBP).
Moreover, explanations for subtype selectivity can be derived. Advantageously, crystal
structures are available for the dopamine receptor subtypes D , D and D , which were recently
2
3
4
6
2
63
47
co-crystalized with the antagonists risperidone, eticlopride and nemonapride, respectively.
The extended binding pocket (Figure 4, left) in the D subtype is mainly surrounded by amino
4
acids bearing non-polar side chains with the exception of Arg186, whereat the hydrophilic
guanidine moiety is however not orientated towards the lipophilic benzyl unit of the ligand 17.
From the comparably long distance between the benzyl unit of 17 and the guanidine side chain of
Arg186, which is around 5.2Å, only a very weak cation-aryl interaction could possibly occur.64
Lipophilic amino acids that are in vicinity to the phenyl moiety of APH199 (17) are Val87,
Leu90, Phe91 and Leu111 (highlighted in pink). A lipophilic residue, which is closely located to
the benzyl moiety on the semicarbazide is the side chain of Val184, and Trp101 might act as a
further boundary element for the EBP.
EBP
N
H
H
N
N
H
N
MeO
O
N
APH-199 (16)
green)
(
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Figure 4. Calculated binding pose of ligand APH199 (17) (green) to the dopamine D receptor
4
4
7
(
PDB-D : 5WIU). View from the extracellular side on the phenyl and benzyl units occupying
4
the extended binding pocket (EBP) (left), view on the semicarbazide unit (center).
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Key interactions of the polar residues of ligand 17 are shown in the center of Figure 4. The
protonated tertiary aliphatic amine forms a salt bridge with Asp115 from TM3 and the carbonyl
unit of the semicarbazide is engaged in a hydrogen bond with Thr434 from TM7. Due to these
two anchor groups, we assume that the C alkyl spacer present in 17 provides the necessary
3
flexibility so that both interactions to Asp115 and Thr434 can effectively occur, while the large
lipophilic benzyl phenylsemicarbazide of 17 is at the same time correctly directed into the EBP.
The fixation of the 3-methoxyphenyl unit of the 1-arylpiperazine recognition element by Val116
from TM3 and Phe411 from TM6 has earlier been observed for the corresponding recognition
4
7
element of nemonapride in its co-crystal with the D receptor. Insights into the effects that are
4
responsible for the subtype selectivity of the D ligand 17 over the subtypes D and D are given
4
2
3
in Figure 5.
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Figure 5. Subtype selectivity of ligand 17 (green). Docking into the D subtype (left) compared
4
with an overlay of the D (purple) and D (turquoise) subtype (right) (PDB-D : 5WIU; PDB-D :
2
3
4
2
4
7,62,63
6CM4; PDB-D :3PBL).
3
1
1
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From the computational results depicted in Figure 5, one can conclude that the EBP is likely to
play a key role in the context of subtype selectivity of the novel semicarbazide ligands. As nicely
6
2
shown by Wang, an extended binding pocket (EBP) is available in all three subtypes D , D
2
3
and D , but its orientation within the overall seven transmembrane structure changes
4
significantly. An important amino acid, which leads to this reorientation, is Leu111 from TM3 in
D , which is exchanged for a much bulkier Phe110 in the D subtype and for a Phe106 in the D
4
2
3
subtype (Figure 4, right). Although the EBP of the D and D subtypes is on the other hand larger
2
3
at the original position of Phe91 in D , as this amino acid is replaced by either Val91 in D or
4
2
Val86 in D , the semicarbazide ligand 17 appears to be unable to follow this reorientation as the
3
polar key interactions shown in Figure 4 (center) only allow a favorable placement of the bulky
benzyl phenylsemicarbazide unit in the EBP of the D subtype. So if no simple reorientation is
4
possible for ligand 17 in the other subtypes, the phenyl moiety on the semicarbazide is likely to
lead to a significant steric clash with Phe110 in D and Phe106 in D thereby explaining the
2
3
observed subtype selectivity. Moreover, the importance of the key interactions (Figure 4, center)
also provide an explanation for the reduced influence of the chain length connecting the
recognition element and the lipophilic appendage (c.f. Figure 1) in the semicarbazide ligands. As
shown in Table 1, all three semicarbazides 14-16 displayed a remarkable selectivity towards D4
although the length of the alkyl linker was varied from two to three, and finally four methylene
units. In contrast, the key interactions of the novel semicarbazide ligands to Asp115 and Thr434
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(
Figure 4, center) are significantly disturbed when a more bulky nitrile group is placed on the
benzyl moiety of the semicarbazide, such as in ligand 23, since either the salt bridge to Asp115
can then no longer be formed or the size of the EBP in the D subtype will become insufficient to
4
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host the enlarged benzyl phenylsemicarbazide.
Finally, the docking study was extended to the known D selective ligands 1b and 9-6-24 (5)
4
(Figure 6). Since these computational experiments led to a similar docking pose for 9-6-24 (5) as
4
7
previously determined by Wang et al., the obtained results can serve as a control for the
reliability of our docking studies.
O
I
N
N
H
N
O
N
H
N
N
H
1b
9
-6-24 (5)
Kulagowski et al.35
Wang et al.47
Figure 6. Docking of the known D selective ligands 1b and 9-6-24 (5) into the D subtype
4
4
47
(PDB-D : 5WIU).
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As shown in Figure 6, both ligands 1b and 5 only partially occupy the EBP available in the D4
subtype, whereat the lipophilic 4-iodophenyl group of ligand 1b points to TM6 and TM7 (Figure
6, left) and the phenoxy unit in ligand 5 is oriented towards TM2 and TM3 (Figure 6, right).
Notably, the alternative orientation of ligand 1b, in which the 4-iodophenylpiperazine acts as
recognition element and the 7-azaindole moiety occupies the EBP, would lead to a much larger
distance between the protonated piperazine nitrogen and Asp115 (5.7 Å) and can thus be
considered as much less likely (see Supporting Information). All three ligands 1b, 9-6-24 (5) and
APH199 (17) show a similar key interaction of their protonated amine to Asp115 from TM3
1
1
1
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(Figure 6 and Figure 4, center), but only 5 and 17 display a second polar interaction of their
lipophilic appendages directed to the EBP. Whereas this second polar interaction of the
semicarbazide APH199 (17) is an intermolecular one (semicarbazide carbonyl to Thr434 in the
D subtype, Figure 4, center) the favorable placement of the phenoxy unit of 9-6-24 (5) in the
4
EBP is secured by an intramolecular interaction of the ether oxygen atom to a hydrogen atom on
the protonated secondary amine. Besides these favorable polar key interactions, the novel
semicarbazide ligands nevertheless demonstrate that high subtype selectivities for the D subtype
4
over D and D can also be achieved by compounds fully occupying the EBP of D (Figure 4,
2
3
4
left).
Discussion and Conclusions
In summary, it has been shown that 1-arylpiperazines that are linked to a novel 1-benzyl-1-
phenylsemicarbazide motif can display high affinity and subtype selectivity towards the
dopamine D receptor in combination with a significant bias towards G protein activation.
4
Taking advantage of a recently developed Barbier-type reaction, which is applicable for the
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reductive alkylation of phenylazocarboxamides, a series of 34 new semicarbazide ligands could
be prepared via a straightforward synthetic sequence. The resulting compounds were
subsequently evaluated in competition binding assays towards the three subtypes of the D2
family and structure-activity relationships as summarized in Figure 7 were observed.
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D4 binding affinity
R1 = H, R2 = H
prefered over
R1 = H, R2 = 4-F and R1 = 4-F, R2 = H
prefered over all
D4 binding affinity
R1
R3 = 2-OMe
prefered over
4-Br, 4-I
double substitutions on R1 and R2
H
R3
N
H
X
N
N
R2
(for X = CH)
N
O
N
X
X = CH (R3 = 2-OMe)
prefered over
n
G protein over -arrestin bias
D4 binding affinity
3
X = N (R = H)
R1 = 4-F, R2 = H
prefered over
n = 1
prefered over
n = 0 or 2
1
= H, R2 = H
R
prefered over
1
2
R
= H, R = 4-F
for n = 1, X = CH, R3 = 2-OMe
Figure 7. Summary of structure activity relationships (SARs) and structure-functional selectivity
relationships (SFSRs) for the novel semicarbazide ligands.
According to docking studies, the favorable binding profile of the most promising ligand
APH199 (17) (K (D ) = 0.25 nM, D /D = 320, D /D = 710) results from two main factors.
i
4.4
2L
4.4
3
4.4
Firstly, a comparably strong fixation of the ligand in the D receptor is likely to be achieved via a
4
salt bridge linking the protonated piperazine to Asp115 combined with a hydrogen bond between
Thr434 and the carbonyl oxygen of the novel semicarbazide substructure. Secondly, the
particular combination of a phenyl and benzyl substituent on the semicarbazide unit leads to a
full occupation of the extended binding pocket (EBP) and, due to an insufficient flexibility of the
1
-benzyl-1-phenylsemicarbazide in the subtypes D and D , also to a clash with the bulkier side
2 3
chains of Phe110 (in D ) and Phe106 (in D ), which can serve as a plausible explanation for
2
3
subtype selectivity. These results demonstrate that the full occupation of the EBP in the
dopamine receptor subtype D by ligands bearing bulky lipophilic appendages can offer a
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valuable starting point for future developments towards G protein-biased D -selective agonists,
4
which has so far not been exploited. To date, little is known about G protein-biased D -selective
4
agonists besides the recently published ligand 621433134 (3) (Figure 1) and the semicarbazides
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
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2
2
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8
9
0
developed within this work. Within the D receptor family, most examples for biased ligands
2
have so far been reported for the D subtype.65 For research groups interested in the
2
pharmacology of the newly developed semicarbazide ligands, we would be happy to provide
samples within a future collaboration.
Experimental Section
Synthesis.
1
Solvents and reagents are obtained from commercial sources and used as received. H-NMR and
1
3
1
13
C-NMR spectra are recorded on Bruker Avance 600 ( H: 600 MHz; C: 151 MHz) and Bruker
1
13
1
Avance 400 ( H: 400 MHz; C: 101 MHz) spectrometers. For H-NMR spectra CDCl is used as
3
solvent referenced to TMS (0 ppm): CHCl (7.26 ppm), (CD ) SO (2.5 ppm), CD OD
3
3 2
3
(3.31 ppm). Chemical shifts are reported in parts per million (ppm). Coupling constants are in
Hertz (Hz). The following abbreviations are used for the description of signals: s (singlet), d
1
3
(
doublet), t (triplet), q (quartet), m (multiplet), bs (broad singlet). C-NMR (DEPTQ) spectra are
recorded in CDCl using CDCl (77.16 ppm) and in (CD ) SO using (CD ) SO (39.52 ppm) as
3
3
3 2
3 2
standard, respectively. Chemical shifts are given in parts per million (ppm). Mass spectra are
recorded using electron spray ionization (ESI) and Atomospheric Pressure Photoionization
(APPI) and a sector field mass analyzer for HRMS measurements. Analytical TLC is carried out
on Merck silica gel plates using short wave (254 nm) UV light to visualize components. Silica
gel (Kieselgel 60, 40-63mm, Merck) is used for flash column chromatography. Infrared spectra
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5
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6
were recorded on a JASCO FT/IR-4100 spectrometer using a NaCl crystal (25 mm×4 mm)
purchased at Sigma Aldrich. The bands were classified due to their intensity in strong (s), middle
(m) and weak (w). The purity of the substances was determined using an analytical HPLC
0
1
2
3
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5
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9
0
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9
0
(10:90→100:0 methanol/water + 0.1% formic acid over 18 min, Eclipse XDB-C8; 5µm; 4,6×150
mm). The purity of all target compounds 12a-c and 14-47 was determined as > 95%.
Synthesis and characterization of compounds
General Procedure 1: Alkylation of phenylpiperazine (GP 1)
A
suspension of the phenylpiperazine hydrochloride
7
(4.00 mmol, 915 mg),
bromoalkylphthalimide 8 (8.00 mmol) and potassium carbonate (16.0 mmol, 2.21 g) was
refluxed in dry acetone (25 mL) for 20 hours. After completion of the reaction, the mixture was
cooled to room temperature, filtered and the solid was washed with acetone (10 mL). The
organic solvent was removed under reduced pressure and the crude product 9 was purified by
flash chromatography.
2
-(2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (9a). Compound 9a was
prepared from 1-(2-methoxyphenyl)piperazine-hydrochloride (7a) (4.00 mmol, 915 mg), N-(2-
bromethyl)phthalimide (8a) (8.00 mmol, 2.03 g) and potassium carbonate (16.0 mmol, 2.21 g) in
acetone (25 mL) according to GP 1. Purification by column chromatography (hexane/ethyl
acetate = 3:1 → 2:1) gave 9a (3.11 mmol, 1.13 g, 78%) as a slightly yellow solid. R : 0.2
f
1
(hexane / ethyl acetate = 3:1) [UV]; H NMR (400 MHz, CDCl ): δ (ppm) = 2.69 - 2.81 (m,
3
6
7
H), 3.04 (m, 4 H), 3.85 (s, 3 H), 3.87 - 3.93 (m, 2 H), 6.82 - 7.01 (m, 4 H), 7.67 - 7.74 (m, 2 H),
1
3
.82 - 7.87 (m, 2 H); C NMR (151 MHz, DEPTQ, CDCl ): δ (ppm) = 35.2 (CH ), 50.6
3
2
(2 × CH ), 53.3 (2 × CH ), 55.3 (CH ), 55.8 (CH ), 111.1 (CH), 118.2 (CH), 121.0 (CH), 122.8
2
2
3
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(
CH), 123.1 (2 × CH), 132.2 (2 × C ), 133.8 (2 × CH), 141.3 (C ), 152.2 (C ), 168.3 (2 × C ).
q
q
q
q
2
-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)propyl)isoindoline-1,3-dione (9b). Compound 9b
was prepared from 1-(2-methoxyphenyl)piperazine-hydrochloride (7a) (4.00 mmol, 915 mg), N-
2-brompropyl)phthalimide (8b) (8.00 mmol, 2.14 g) and potassium carbonate (16.0 mmol,
2.21 g) in acetone (25 mL) according to GP 1. Purification by column chromatography
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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(
(hexane/ethyl acetate = 3:1) gave 9b (3.40 mmol, 1.29 g, 85%) as a slightly yellow solid. R : 0.2
f
1
(hexane / ethyl acetate = 3:1) [UV]; H NMR (400 MHz, CDCl ): δ (ppm) = 1.91 (p, J = 6.9 Hz,
3
2 H), 2.49 (t, J = 6.9 Hz, 2 H), 2.50 - 2.68 (m, 4 H), 2.18 - 2.79 (m, 4 H), 3.79 (t, J = 6.9 Hz,
H), 3.82 (s, 3 H), 6.82 (ddd, J = 1.5 Hz, J = 8.0 Hz, J = 9.5 Hz, 2 H), 6.88 (td, J = 1.5 Hz,
J = 7.6 Hz, 1 H), 6.96 (td, J = 1.5 Hz, J = 7.6 Hz, 1 H), 7.67 - 7.71 (m, 2 H), 7.82 - 7.85 (m,
2
1
3
2
H); C NMR (151 MHz, DEPTQ, CDCl ): δ (ppm) = 25.2 (CH ), 36.7 (CH ), 50.4 (CH ), 53.3
3 2 2 2
(2 × CH ), 55.3 (CH ), 56.1 (2 × CH ), 111.1 (CH), 118.1 (CH), 120.9 (CH), 122.8 (CH), 123.1
2 3 2
(2 × CH), 132.3 (2 × C ), 133.8 (2 × CH), 141.3 (C ), 152.2 (C ), 168.5 (2 × C ).
q
q
q
q
2
-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butyl)isoindoline-1,3-dione (9c). Compound 9c was
prepared from 1-(2-methoxyphenyl)piperazine-hydrochloride (7a) (4.00 mmol, 915 mg), N-(2-
bromobutyl)phthalimide (8c) (8.00 mmol, 2.26 g) and potassium carbonate (16.0 mmol, 2.21 g)
in acetone (25 mL) according to GP 1. Purification by column chromatography (hexane/ethyl
acetate = 3:1) gave 9c (2.57 mmol, 1.01 g, 64%) as a slightly yellow solid. R : 0.2 (hexane / ethyl
f
1
acetate = 1:1) [UV]; H NMR (400 MHz, CDCl ): δ (ppm) = 1.60 (bs, 2 H), 1.74 (td, J = 7.2 Hz,
3
J = 14.9 Hz, 2 H), 2.31 - 3.36 (m, 10 H), 3.73 (t, J = 7.2 Hz, 2 H), 3.86 (s, 3 H), 6.85 (dd,
J = 1.2 Hz, J = 8.3 Hz, 1 H), 6.90 - 6.95 (m, 2 H), 6.98 - 7.01 (m, 1 H), 7.70 - 7.73 (m, 2 H),
1
3
7
.83 - 7.86 (m, 2 H); C NMR (91 MHz, CDCl ): δ (ppm) = 24.2 (CH ), 26.7 (CH ), 37.9 (CH ),
3 2 2 2
50.6 (2 × CH ), 53.5 (2 × CH ), 55.4 (CH ), 58.1 (CH ), 111.2 (CH), 118.2 (CH), 121.0 (CH),
2
2
3
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22.9 (CH), 123.2 (2 × CH), 132.2 (2 × C ), 133.9 (2 × CH), 141.4 (C ), 152.3 (C ), 168.4
q
q
q
(2 × Cq).
2
-(3-(4-(4-Chlorophenyl)piperazin-1-yl)propyl)isoindoline-1,3-dione (9d). Compound 9d was
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prepared from 1-(4-chlorophenyl)piperazine-hydrochloride (7a) (5.00 mmol, 1.35 g), N-(2-
brompropyl)phthalimide (8b) (10.0 mmol, 2.68 g) and potassium carbonate (20.0 mmol, 2.76 g)
in acetone (60 mL) according to GP 1. Purification by column chromatography (methylene
chloride/methanol = 50:1) gave 9d (2.85 mmol, 1.09 g, 57%) as a white solid. R : 0.4 (methylene
f
1
chloride / methanol = 50:1) [UV]; H NMR (400 MHz, CDCl ): δ (ppm) = 1.84 (dp, J = 7.1 Hz,
3
J = 13.8 Hz, 2 H), 2.32 - 2.51 (m, 6 H), 2.90 (dd, J = 7.5 Hz, J = 12.6 Hz, 4 H), 3.70 (t,
J = 6.9 Hz, 2 H), 6.62 - 6.70 (m, 2 H), 7.03 - 7.12 (m, 2 H), 7.55 - 7.62 (m, 2 H), 7.69 - 7.77 (m,
1
3
2
5
H); C NMR (101 MHz, DEPTQ, CDCl ): δ (ppm) = 25.3 (CH ), 36.7 (CH ), 49.1 (2 × CH ),
3 2 2 2
3.1 (2 × CH ), 56.1 (CH ), 117.3 (2 × CH), 123.3 (2 × CH), 124.6 (C ), 129.0 (2 × CH), 132.4
2
2
q
(2 × C ), 134.0 (2 × CH), 149.9 (C ), 168.6 (2 × C ). The analytical data obtained is in agreement
q q q
with those reported in literature.66
-(3-(4-(4-Bromophenyl)piperazin-1-yl)propyl)isoindoline-1,3-dione (9e). Compound 9e was
2
prepared from 1-(4-bromophenyl)piperazine-hydrochloride (7c) (1.25 mmol, 302 mg), N-(2-
bromopropyl)phthalimide (8b) (2.50 mmol, 670 mg) and potassium carbonate (5.00 mmol,
6
91 mg) in acetone (3.1 mL) according to GP 1. Purification by column chromatography
(methylene chloride/methanol = 50:1) gave 9e (1.01 mmol, 434 mg, 81%) as a slightly yellow
1
solid. R : 0.3 (chloroform / methanol = 50:1) [UV]; H NMR (400 MHz, CDCl ): δ (ppm) = 1.91
f
3
(dp, J = 6.9 Hz, J = 13.7 Hz, 2 H), 2.40 - 2.61 (m, 6 H), 2.99 (dd, J = 7.5 Hz, J = 12.4 Hz, 4 H),
3
.75 - 3.84 (m, 2 H), 6.64 - 6.74 (m, 2 H), 7.24 - 7.33 (m, 2 H), 7.62 - 7.74 (m, 2 H), 7.78 - 7.88
1
3
(m, 2 H); C NMR (101 MHz, DEPTQ, CDCl ): δ (ppm) = 25.2 (CH ), 36.6 (CH ), 48.8
3
2
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(
2 × CH ), 52.9 (2 × CH ), 56.0 (CH ), 111.6 (C ), 117.5 (2 × CH), 123.1 (2 × CH), 131.8
2
2
2
q
(2 × CH), 132.3 (2 × C ), 133.9 (2 × CH), 150.3 (C ), 168.5 (2 × C ).
q
q
q
2
-(3-(4-(Pyrimidin-2-yl)piperazin-1-yl)propyl)isoindoline-1,3-dione (9f). Compound 9f was
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prepared from 2-(piperazine-1-yl)pyrimidine-hydrochloride (7d) (1.42 mmol, 432 µL), N-(2-
bromopropyl)phthalimide (8b) (2.84 mmol, 761 mg) and potassium carbonate (5.68 mmol,
7
85 mg) in acetone (9 mL) according to GP 1. Purification by column chromatography (ethyl
acetate+3% NH ) gave 9f (1.42 mmol, 499 mg, 100%) as a slightly yellow solid. R : 0.54 (ethyl
3
f
1
acetate+3%NH ) [UV]; H NMR (400 MHz, CDCl ): δ (ppm) = 1.85 - 1.95 (m, 2 H), 2.32 - 2.53
3
3
(m, 6 H), 3.66 (bs, 4 H), 3.79 (t, J = 6.9 Hz, 2 H), 6.45 (t, J = 4.7 Hz, 1 H), 7.67 - 7.72 (m, 2 H),
7
.81 - 7.86 (m, 2 H), 8.26 (d, J = 4.7 Hz, 2 H); 13C NMR (101 MHz, DEPTQ,
CDCl ): δ (ppm) = 25.3 (CH ), 36.8 (CH ), 43.5 (CH ), 53.1 (2 × CH ), 56.3 (2 × CH ), 109.9
3
2
2
2
2
2
(CH), 123.3 (2 × CH), 132.4 (2 × C ), 134.0 (2 × CH), 157.8 (2 × CH), 161.6 (C ), 168.6
q q
(
2 × Cq).
-(2-(4-(4-Bromophenyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (9g). Compound 9g was
2
prepared from 1-(4-bromophenyl)piperazine-hydrochloride (7c) (1.24 mmol, 300 mg), N-(2-
bromoethyl)phthalimide (8a) (2.49 mmol, 670 mg) and potassium carbonate (4.98 mmol,
6
88 mg) in acetone (3.1 mL) according to GP 1. Purification by column chromatography
(methylene chloride/methanol = 50:1) gave 9g (0.501 mmol, 208 mg, 40%) as a white solid. Rf:
1
0
.2 (methylene chloride / methanol = 50:1) [UV]; H NMR (400 MHz, CDCl ): δ (ppm) = 2.64 -
3
2.72 (m, 6 H), 3.06 - 3.12 (m, 4 H), 3.86 (t, J = 6.5 Hz, 2 H), 6.72 - 6.78 (m, 2 H), 7.28 - 7.33
(
m, 2 H), 7.69 - 7.74 (m, 2 H), 7.82 - 7.86 (m, 2 H); 13C NMR (101 MHz, DEPTQ, CDCl3):
δ (ppm) = 35.3 (CH ), 48.9 (2 × CH), 52.9 (2 × CH ), 55.7 (CH ), 111.7 (C ), 117.6 (2 × CH),
2
2
2
q
123.3 (2 × CH), 131.9 (2 × CH), 132.3 (2 × C ), 134.0 (2 × CH), 150.4 (C ), 168.5 (2 × C ).
q
q
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-(3-(4-(4-Iodophenyl)piperazin-1-yl)propyl)isoindoline-1,3-dione (9h). A suspension of 1-
(4-iodophenyl)piperazine-hydrochloride (7e) (0.826 mmol, 268 mg) and potassium carbonate
(1.65 mmol, 228 mg) in DMF (5 mL) was heated at 80°C for 10 min. N-(2-
bromopropyl)phthalimide (8b) (0.909 mmol, 244 mg) was added to the mixture and the reaction
was stirred at the same temperature for 6 hours. After cooling to room temperature the solvent
was evaporated and the residue was partioned between water (50 mL) and ethyl acetate (50 mL).
The aqueous phase was further extracted with ethyl acetate (3 × 30 mL). The combined organic
layers were dried over sodium sulfate and evaporated to dryness. The product 9h (0.735 mmol,
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39 mg, 48%) was obtained as a white solid by crystallization from ethyl acetate.67 Rf: 0.5
1
(methylene chloride / methanol = 50:1) [UV]; H NMR (400 MHz, CDCl ): δ (ppm) = 1.90 (p,
3
J = 6.8 Hz, 2 H), 2.48 (t, J = 6.9 Hz, 2 H), 2.50 – 2.55 (m, 4 H), 2.97 - 3.04 (m, 4 H), 3.79 (t,
J = 6.9 Hz, 2 H), 6.58 - 6.64 (m, 2 H), 7.44 - 7.50 (m, 2 H), 7.69 (dd, J = 3.0 Hz, J = 5.5 Hz,
2 H), 7.83 (dd, J = 3.0 Hz, J = 5.5 Hz, 2 H); 13C NMR (151 MHz, DEPTQ, CDCl ): δ (ppm)
3
=
25.2 (CH ), 36.7 (CH ), 48.6 (2 × CH ), 52.9 (2 × CH2), 56.1 (CH ), 81.4 (C ), 118.1
2 2 2 2 q
(2 × CH), 123.2 (2 × CH), 132.4 (2 × C ), 134.0 (2 × CH), 137.8 (2 × CH), 150.9 (C ), 168.6
q q
(
2 × Cq).
2
-(2-(4-(4-Iodophenyl)piperazin-1-yl)ethyl)isoindoline-1,3-dione (9i). A suspension of 1-(4-
iodophenyl)piperazine-hydrochloride (7e) (1.54 mmol, 500 mg) and potassium carbonate
3.08 mmol, 426 mg) in DMF (10 mL) was heated at 80°C for 10 min. N-(2-
(
bromoethyl)phthalimide (8a) (1.69 mmol, 429 mg) was added to the mixture and the reaction
was stirred at the same temperature for 6 hours. After cooling to room temperature the solvent
was evaporated and the residue was partioned between water (50 mL) and ethyl acetate (50 mL).
The aqueous phase was further extracted with ethyl acetate (3 × 30 mL). The combined organic
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layers were dried over sodium sulfate and evaporated to dryness. The product 9i (0.517 mmol,
2
46 mg, 63%) was obtained as a white solid by crystallization from ethyl acetate. R : 0.5
f
1
(methylene chloride / methanol = 50:1) [UV]; H NMR (400 MHz, CDCl ): δ (ppm) = 2.61 -
3
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.76 (m, 6 H), 3.04 - 3.16 (m, 4 H), 3.87 (t, J = 6.4 Hz, 2 H), 6.62 - 6.68 (m, 2 H), 7.45 - 7.52
1
3
(
m, 2 H), 7.71 (dd, J = 3.0 Hz, J = 5.5 Hz, 2 H), 7.84 (dd, J = 3.0 Hz, J = 5.5 Hz, 2 H); C NMR
(
151 MHz, DEPTQ, CDCl ): δ (ppm) = 35.2 (CH ), 48.7 (CH ), 52.9 (2 × CH ), 55.7 (2 × CH ),
3
2
2
2
2
8
1
1.4 (C ), 118.1 (2 × CH), 123.4 (2 × CH), 132.3 (2 × C ), 134.0 (2 × CH), 137.9 (2 × CH),
q
q
50.9 (C ), 168.5 (2 × C ).
q
q
General Procedure 2: Deprotection of the alkylamines (GP 2)
A solution of the phthalimide 9 (3.00 mmol, from GP 1) in ethanol (25 mL) was refluxed with
hydrazine hydrate (30.0 mmol) for one hour. Upon completion of the reaction, hydrochloric acid
(5 mL, 2 M) was added and the solution was let to come to room temperature while stirring. After
removing the solvents under reduced pressure, the obtained solid was dissolved with a sodium
hydroxide solution (2 M) and was extracted with dichloromethane (6 × 15 mL). The combined
organic phases were washed with brine (20 mL) and dried over sodium sulfate. The drying agent
was separated by filtration and the solvents were removed under vacuum. The amine 10 could be
used without further purification.
2-(4-(2-Methoxyphenyl)piperazin-1-yl)ethan-1-amine (10a). Compound 10a was prepared
from 9a (3.11 mmol, 1.13 g, from GP 1) and hydrazine hydrate (31.1 mmol, 1.60 mL) according
to GP 2. 10a (2.68 mmol, 631 mg, 86%) is obtained as a pure colorless oil. R : 0.2
f
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