Click chemistry based solid phase supported synthesis of dopaminergic phenylacetylenes

Article abstract of DOI:10.1016/j.bmc.2007.08.038

'Click resins' enable solid phase supported reactions to work under nearly perfect conditions fulfilling the requirements of click chemistry. Utilizing the formylpyrrolylmethyltriazole (FPMT) linker 6, which is readily available via copper(I)-catalyzed az

Full text of DOI:10.1016/j.bmc.2007.08.038

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7248–7257
Click chemistry based solid phase supported synthesis
of dopaminergic phenylacetylenes
Pilar Rodriguez Loaiza, Stefan L o¨ ber, Harald H u¨ bner and Peter Gmeiner*
Department of Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander University,
Schuhstrasse 19, D-91052 Erlangen, Germany
Received 16 January 2007; revised 9 August 2007; accepted 21 August 2007
Available online 25 August 2007
Abstract—‘Click resins’ enable solid phase supported reactions to work under nearly perfect conditions fulfilling the requirements of
click chemistry. Utilizing the formylpyrrolylmethyltriazole (FPMT) linker 6, which is readily available via copper(I)-catalyzed azide-
alkyne cycloaddition (CuAAC), a BAL strategy could be successfully applied for a parallel synthesis of dopaminergic phenylacet-
ylens. A focused library of 20 test compounds revealing three points of diversity was generated by a four-step SPOS approach
including microwave assisted Sonogashira coupling. GPCR-ligand binding assays indicated excellent dopamine D3 and D4 receptor
binding affinities which were identified to cause a partial agonist activity for the most potent test compounds 2c,e,i,k.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1
. Introduction
tions fulfilling the requirements of click chemistry very
impressively. Intending to investigate the introduction
of an alkynyl subunit as a pharmacophoric element,
we planned to take advantage of a concept of privileged
The concept of click chemistry, which was established by
K. Barry Sharpless a few years ago, has been exploited
as a highly beneficial tool for the process of lead devel-
opment in drug discovery. Click reactions must be
modular, wide in scope, provide high yields, work in
both small and large scale applications and lack signifi-
cant by-products (or result in inoffensive or easily
8
structures when a careful bioisosteric replacement
should be capable of providing ligands for more than
1
,2
9
–12
one receptor.
carboxamides of type 1 as such a privileged structure
For family A GPCRs, we identified
1
3–16
serving also as potential PET imaging agents.
On
3
removable by-products). The required process charac-
the basis of lead compound 1, we herein present a solid
phase supported construction of a focused library of
type 2. Furthermore, data on their GPCR binding and
functional properties are provided (Chart 1).
teristics include simple reaction conditions, readily avail-
able starting materials and reagents, and should require
no or easily removable solvents. Any purification should
be non-chromatographic, and the products generated
must be stable under physiological conditions. Interest-
ingly, the same requirements hold true for a parallel ap-
proach in drug discovery, since combinatorial chemistry
is highly sensible on the reliability of the reactions used
to construct the new chemical bonds. In conjunction
with a programme to design super-potent dopamine
D3 receptor ligands employing click chemistry, we
herein describe that ‘click resins’ enable solid phase sup-
2. Results and discussion
2.1. Chemistry
4
The backbone amide linker (BAL) strategy for the prep-
aration of C-terminally modified and cyclic peptides, as
well as non-peptidic compounds, has been established
5
,6
7
17,18
ported reactions to work under nearly perfect condi-
by Barany and co-workers.
During the last years,
the versatility of BAL linkage has been largely proved,
especially in solid phase peptide synthesis. On the basis
of this concept, we established a first series of click based
backbone amide linkers, when the electron-donating
properties of the enamine functionality of indole-3-carb-
aldehyde facilitated an extraordinary smooth and high
Keywords: Click resins; Click chemistry; Copper(I)-catalyzed azide-
alkyne cycloaddition (CuAAC); BAL strategy; Microwave assisted
Sonogashira coupling; GPCR; Dopamine D3 receptor binding;
Dopamine D4 receptor binding; Partial agonist activity.
*
Corresponding author. Tel.: +49 9131 852 9383; fax: +49 9131 852
585; e-mail: gmeiner@pharmazie.uni-erlangen.de
1
9
2
yielding liberation of the final products. A formal
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.038

P. Rodriguez Loaiza et al. / Bioorg. Med. Chem. 15 (2007) 7248–7257
7249
O
N
Y
O
N
Y
N
n
X
N
n
X
(
)
( )
N
H
N
H
(
Het)Ar
R
1
2
Chart 1.
extension of the more aromatic indole moiety (I = 146)
A
by the less aromatic pyrrole (I = 85) should lead to an
A
hydrolysis (2%) resulted in formation of the arylcarbox-
amide 8. Careful analysis by LC/MS indicated high yield
(88%) and, even more important, exceptionally high
purity (>99%).
even greater electron-donating effect of the heterocyclic
2
0
nitrogen. To facilitate a wide application of click based
resins in drug discovery, there is a need for robust and
orthogonally stable linkers that are not only cleaved in
high yields under mild conditions but are also easily
accessible. Thus, our plan of synthesis started from
pyrrole-2-carbaldehyde as a commercially available
building block that could be readily functionalized by
To investigate the reliability and the scope of our solid
phase supported method, we elaborated the synthesis
of a model library. A further objective of this step was
to identify and optimize suitable reaction conditions
for an attachment of alkyne derived subunits which
should play a key role in our structure–activity relationship
studies. As a primary amine, we chose N-benzyl-4-amino-
piperidine which could be reductively immobilized
onto the click resin 6 in presence of NaBH(OAc)3
2
1
N-alkylation. Thus, treatment of the heterocyclic
building block 3 with propargyl bromide afforded the
alkyne 4 which was reacted with azidomethyl polystyrene
resin (5) in presence of copper iodide in THF/DIPEA
2
2–24
(
Scheme 1).
After a preliminary determination of
(Scheme 2). The reaction was considered to be complete
when an absorption band at 1650 cm
À1
the loading by elemental analysis (nitrogen content)
indicated a linker density of 1.0 mmol/g and, thus, com-
plete [3 + 2] azide-alkyne cycloaddition, we tried to
investigate the loading and the reactivity of the new
formylpyrrolylmethyltriazole (FPMT) resin 6 by initiat-
ing a reaction sequence of reductive amination, acyla-
tion and acidic cleavage. As a representative primary
amine, we chose 2-methoxyphenylethylamine which
was added to the functionalized resin 6 in dichorome-
had disap-
peared. Amide bond formation with 4-iodobenzoic acid
and its 3-substituted regioisomer was promoted by using
HOAt/DIC when formation of resin bound carboxam-
ide could be qualitatively ascertained by FTIR display-
ing the appearance of the diagnostic absorption band
À1
at 1620 cm . For the introduction of an alkyne unit,
palladium catalyzed cross-coupling reaction under
Sonogashira conditions had to be elaborated. Employing
phenylacetylene or trimethylsilylacetylene as an
acetylene precursor, cross-coupling reaction could be
observed by FTIR when the catalyst system
thane. After treatment with NaBH(OAc) at room tem-
3
perature and washing, the amine functionalized resin
was acylated with 4-iodobenzoyl chloride to give the
immobilized target compound 7. Finally, TFA induced
2
5
Pd(PPh ) Cl /Cu(I)I was used. To accelerate the reac-
3
2
2
CHO
N
N3
b
+
CHO
N
R
N
N
N
3
4
: R = H
5
6
a
: R = CH C
CH
c, d
2
=
poly-
styrene
I
I
O
O
OMe
N
OMe
N
e
H
N
N
N
N
8
7
Scheme 1. Reagents and conditions: (a) propargyl bromide, K
2
CO
3
, DMF, rt, 16 h; (b) Cu(I)I, THF/DIPEA, 35 ꢁC, 36 h; (c) (1) 2-(2-
N, dichloromethane, rt, 2 h; (d)
methoxyphenyl)ethylamine, dichloromethane, rt, 30 min; (2) Na(OAc)
TFA (2% in dichloromethane), rt, 2 h.
3
BH, rt, 24 h; (d) 4-iodobenzoyl chloride, Et
3

7250
P. Rodriguez Loaiza et al. / Bioorg. Med. Chem. 15 (2007) 7248–7257
I
R
N
O
O
a, b
N
N
c
6
N
N
N
Ph
Ph
N
N
N
N
N
N
e
O
R=
C
C
C
C
Ph or
Ph / H
R=
R=
C
Si(CH3)3
HN
d
e
N
CH
compound
purity yield
Ph
9
9
9
9
a (m-phenylethynyl-) 96
b (p-phenylethynyl-) 94
68
51
57
49
=
poly-
styrene
c (m-ethynyl-)
d (p-ethynyl-)
93
98
Scheme 2. Reagents and conditions: (a) (1) 1-Benzyl-4-aminopiperidine, dichloromethane, rt, 30 min; (2) Na(OAc)
acid, HOAt, DIC, DMF, dichloromethane, rt, 28 h; (c) C1–2, Pd(PPh Cl , CuI, Et
N, DMF, microwave, 120 ꢁC, 15 min; (d) TBAF, THF, rt, 7 h;
e) TFA (2% in dichloromethane), rt, 2 h.
3
BH, rt, 24 h; (b) 3-/4-iodobenzoic
3
)
2
2
3
(
tion, microwave assistance turned out to be highly ben-
eficial. Upon exposition to microwave irradiation at
the solid phase supported methodology described above,
five amines, two benzoic acids and two alkyne deriva-
tives were chosen as attractive building blocks. Because
phenylpiperazine moieties are known to effectively bind
to biogenic amine receptors, especially the dopamine D3
subtype, the amines A1–5 were selected for the reductive
amination step. Both para- and meta-iodobenzoic acids
(B1–2) were chosen for the second diversity point, in or-
der to investigate the influence of the orientation of the
acetylene substituent with respect to the amide. For the
Sonogashira coupling, phenylacetylene and trimethylsi-
lylacetylene were selected as commercially available
building blocks when removal of the trimethylsilyl-pro-
tecting group was envisaged after the palladium pro-
moted coupling step. The synthesis was initiated by
reductive amination of the click resin 6 followed by acyl-
ation of the resin bound secondary amines when using
HOAt/DIC as activating agents (Scheme 3). Sonogash-
ira coupling was performed with the corresponding acet-
ylenes C1–2 in presence of Pd(PPh ) Cl , Cu(I)I and
1
1
20 ꢁC, substantial conversion could be observed after
5 min. In order to obtain the free acetylene function-
26
ality, desilylation was promoted by tetrabutylammo-
nium fluoride in THF when a significant absorption at
À1
3
200 cm in the IR spectra indicated a complete reac-
tion. Finally, all the products were cleaved by using
% TFA in dichloromethane. Purities of the crude prod-
2
ucts were assessed by LC/MS. Using the above-men-
tioned synthetic route, all the crude products were
obtained with a high degree of purity (93–98%) as as-
sessed by LC/MS. Any trace impurities present could
be attributed to residual coupling agents. Thus, the
strategy elaborated promised to be a robust methodol-
ogy for the further development of a larger three-dimen-
sional library based on the FPMT resin system.
As an extension of our recent investigations revealing
that conjugated alkynes can substantially improve
molecular recognition between GPCRs and receptor
3
2
2
irradiated via microwave for 15 min at 120 ꢁC. The tri-
methylsilyl group was removed by using a 1 M solution
of tetrabutylammonium fluoride in THF. Finally all the
2
7–29
ligands, a focused library of compounds described
by the general formula 2 should be constructed. Using
Ph / Si(CH3)3
O
d
a
b
c
N
N
6
( )
2a-t
n
N
A1-5
B1-2
C1-2
N
X
N
Y
N
N
Scheme 3. Reagents and conditions: (a) (1) A1–5, dichloromethane, rt, 30 min; (2) Na(OAc)
dichloromethane, rt, 28 h; (c) C1–2, Pd(PPh Cl , CuI, Et
N, DMF, microwave, 120 ꢁC, 15 min; (d) for C1: (1) TBAF, THF, rt, 7 h; (2) TFA (2% in
dichloromethane), rt, 2 h; for C2: TFA (2% in dichloromethane), rt, 2 h.
3
BH, rt, 24 h; (b) B1–2, HOAt, DIC, DMF,
3
)
2
2
3

P. Rodriguez Loaiza et al. / Bioorg. Med. Chem. 15 (2007) 7248–7257
7251
products were released from the solid support by 2%
TFA in dichloromethane. All reaction steps were ran-
domly monitored by IR spectroscopy (see Chart 2).
LC/MS analyses of the crude products are displayed in
Table 1. With exception of the derivative 2c that dis-
plays a purity of 83%, purities >90% were obtained for
N
N
N
N
N
N
NH2
Cl
Cl
OMe
NH2
Cl
Cl
NH2
A1
A2
A3
N
N
N
N
OMe
Cl
Cl
NH2
NH2
A4
A5
O
O
(
CH ) Si
H
H
H
3
3
OH
OH
C1
C2
I
I
H
B1
B2
Chart 2. Building blocks.
Table 1. Building blocks and analytical data for the compound library 2a–t
N
N
H
N
( )n
X
Y
O
R
a
Purity (%)
b
Yield (%)
c
Rt (min)
Compound
Code
Subst. pattern
R =
n =
X =
Y =
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
a
b
c
d
e
f
A1B1C1
A1B1C2
A1B2C1
A1B2C2
A2B1C1
A2B1C2
A2B2C1
A2B2C2
A3B1C1
A3B1C2
A3B2C1
A3B2C2
A4B1C1
A4B1C2
A4B2C1
A4B2C2
A5B1C1
A5B1C2
A5B2C1
A5B2C2
para
para
meta
meta
para
para
meta
meta
para
para
meta
meta
para
para
meta
meta
para
para
meta
meta
H
1
1
1
1
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
93
95
83
97
100
98
95
98
95
97
93
92
96
92
92
93
91
91
92
92
39
42
32
44
50
54
63
61
66
42
63
47
48
57
58
55
32
41
32
32
17.5
19.8
18.9
19.6
15.5
18.6
15.6
18.7
17.2
19.6
17.4
19.7
15.8
18.8
15.8
18.8
17.8
19.9
17.9
19.9
Ph
H
Ph
H
MeO
MeO
MeO
MeO
Cl
Ph
H
H
g
h
i
H
Ph
H
H
Cl
Cl
Cl
Cl
H
j
Ph
H
Cl
Cl
k
l
Ph
H
Cl
m
n
o
p
q
r
MeO
MeO
MeO
MeO
Cl
Ph
H
H
H
Ph
H
H
Cl
Cl
Cl
Cl
Ph
H
Cl
Cl
s
t
Ph
Cl
a
b
c
Purities were determined by LC/MS analysis employing UV detection at 254 nm.
Given values refer to crude yields.
Exact HPLC conditions are given in Section 4.

7252
P. Rodriguez Loaiza et al. / Bioorg. Med. Chem. 15 (2007) 7248–7257
1
4,30
all final products. The yields varied between 32% and
6%. The results demonstrate that the pyrrole resin pro-
in vitro. This was accomplished in a screening sys-
tem by measuring the displacement of the following
6
3
radioligands: [ H]spiperone for D2, D3, D4, [ H]SCH
3
vides an ideal support for preparing libraries of carbox-
amides. The novel BAL type linker 6 proved to be
compatible with the reaction conditions that had to be
applied facilitating a smooth and high yielding cleavage,
as well. Taking also into account the convenient prepa-
ration, of 6, the requirements of click chemistry have
been clearly fulfilled.
3
3
23390 for D1 receptors, [ H]prazosin for a1, [ H]8-hy-
3
droxy-DPAT and [ H]ketanserin for 5-HT1 and 5-
A
HT2, respectively, and using 10 lM, 100 nM and 1 nM
concentrations of the test compounds. The results in
Table 2 reflect the displacement data at a final concen-
tration of 100 nM of the test compound.
2
.2. Biological investigations
Starting from the privileged structure 1, the extension of
the benzamide p-system by introduction of an alkyne
subunit led to substantial and specific GPCR recogni-
tion. The results of the screening indicate that the sub-
family of dichlorophenylpiperazines derived from the
building block A3 exerted the highest relative affinities
for the dopamine D3 receptor. High relative displace-
ments at the D3 receptor were also observed for some
A biogenic amine receptor screening of the phenylacetyl-
enes 2a–t was performed without further purifications
when their ability to bind to the cloned human D2long
D2short, D3 and D4 dopamine receptors, the porcine
D1 dopamine and a1 adrenergic receptors, and the sero-
tonin receptor subtypes 5-HT1 , 5-HT2 was evaluated
,
A
Table 2. Screening of receptor binding: relative displacement of the radioligand by the test compounds 2a–t utilizing human D2long, D2short, D3 and
a
D4.4 receptors as well as porcine D1, 5-HT1
A
, 5-HT2 and a1 receptors
displacement of radioligand [%]
Cmpd.
Code
D1
D2long
D2short
D3
D4
α1
5-HT1
A
5-HT2
2
2
2
2
2
a
b
c
d
e
A1B1C1
A1B1C2
A1B2C1
A1B2C2
A2B1C1
A2B1C2
A2B2C1
A2B2C2
A3B1C1
A3B1C2
A3B2C1
A3B2C2
A4B1C1
A4B1C2
A4B2C1
A4B2C2
A5B1C1
A5B1C2
A5B2C1
A5B2C2
4
4
21
1
31
7
64
61
63
28
90
66
78
83
97
100
97
91
70
45
35
46
80
47
80
22
81
9
64
30
57
9
14
15
76
32
52
3
0
0
10
3
21
5
41
7
94
8
13
0
6
3
7
35
15
69
46
34
29
56
18
53
35
35
45
30
11
26
5
63
14
66
55
43
5
8
2
f
1
4
14
15
31
51
27
53
6
9
2
2
g
h
6
12
6
86
29
33
30
49
41
40
0
1
2
0
2
i
j
11
16
9
39
19
40
8
33
0
2
2
k
45
9
15
5
2
l
0
2
m
10
7
27
3
43
10
23
34
57
11
31
0
71
36
69
88
40
8
14
14
19
4
2
n
o
p
q
2
10
7
18
29
35
2
17
1
2
2
3
12
6
32
12
19
8
2
r
0
2
s
4
12
2
33
7
2
2
t
0
28
displacement %
color code
<30%
31-70%
71-79%
80-90%
91-100%
a
Displacement of the radioligand at a final concentration of the test compound of 100 nM determined as triplicate in (%).

P. Rodriguez Loaiza et al. / Bioorg. Med. Chem. 15 (2007) 7248–7257
7253
members of the methoxyphenylpiperazine A2 (A2B1C1,
A2B2C2) and the five-carbon spacer containing amine
A5 (A5B1C1, A5B2C1) derived subfamilies. In addition,
high levels of displacements for other library members
were observed for other biogenic amine receptor sub-
types. For example, the test compounds 2a (A1B1C1)
and 2c (A1B2C1) with a two-carbon spacer showed
strong binding at the D4 receptor, while the meta-substi-
tuted alkynylcarboxamides 2p (A4B2C2) and 2g
well by the D3 receptor. Thus, the test compound 2j
(A3B1C2) displayed a K of 3.2 nM for D3 and excep-
i
tional subtype selectivity (>200). The piperazinylethyl
substituted carboxamide 2c (A1B2C1) turned out to ex-
hibit high D4 selectivity (50-fold over D3, and ꢀ100-
fold over D2). To exclude that the superior D4 binding
of 2c was due to a chemical impurity, determination of
the binding affinity was repeated after chromatographic
purification revealing a similar K value of 0.45 nM.
i
(
HT1 receptors, respectively.
A2B2C1) showed significant affinities for a1 and 5-
As a measure of functional activity, ligand efficacy of the
test compounds 2c, 2e, 2i and 2k displaying exceptional
A
The equilibrium binding constants (K ) were then deter-
i
high binding (K values 61 nM) was determined by a
i
3
mitogenesis assay measuring the rate of [ H]thymidine
mined for members for the subfamilies A1, A2, A3, and
for those compounds showing a percentage displace-
À
incorporation into growing CHO dhfr cells and a
CHO10001 cell line stably expressing the human D3
3
1
ment greater than 80%. The affinity constant (K ) val-
i
1
5
ues are shown in Table 3. Test compounds 2e
and D4.2 receptor, respectively. The data listed in
Table 4 clearly show substantial D3 ligand efficacy of
the test compounds 2e, 2i and 2k with EC50 values in
(A2B1C1), 2i (A3B1C1), 2j (A3B1C2), 2k (A3B2C1)
and 2q (A5B1C1) display high affinity for the dopa-
mine D3 receptor, especially the 4-ethynylcarboxamide
2
(
i
displaying
sub-nanomolar
binding
affinity
K = 0.31 nM). Thus, as has been noted in previous
i
Table 4. Intrinsic activities of selected test compounds and the
reference quinpirole derived from the stimulating effect on mitogenesis
1
1
studies, a 2,3-dichlorophenylpiperazine moiety with
a four-carbon spacer proved to be the scaffold leading
to the highest affinity for the D3 receptor. It is impor-
tant to note that D3 affinity is superior for the terminal
acetylenes when compared to the phenylacetylene
derivatives. Furthermore, it was interesting to see that
para-substituted phenylacetylene derivatives display
higher binding affinities than the respective meta-regioi-
somers. Two obvious explanations can apply for this
fact; the electronic density distribution due to the reso-
nance effect between the carbonyl function and the
triple bond in para position is different to the meta-iso-
mers. Additionally, the geometry of the two p-systems
is different, where probably the meta-derivatives could
induce repulsive steric interactions with the GPCR
binding sites. On the other hand in para position, even
a sterically demanding phenylacetylene unit is tolerated
a
of D3 and D4.2 receptor expressing cells
b
c
Compound Code
Receptor EC50 value
subtype
Intrinsic act.
(%)
(nM)
2
2
2
2
c
e
i
A1B2C1 D4
0.73
2.1
2.0
3.5
5.2
10
49
57
A2B1C1 D3
A3B1C1 D3
A3B2C1 D3
D3
48
57
k
Quinpirole
100
100
D4
a
b
c
À
Determined with CHO dhfr mutant and CHO10001 cells stably
expressing the human D3 and D4.2 receptor, respectively.
EC50 values derived from a mean curve of four independent
experiments.
3
Rate of incorporation of [ H]thymidine as evidence for mitogenesis
activity relative to the maximal effect of the full agonist quinpirole
(
=100%) used as a reference.
A
Table 3. Receptor binding data of selected compounds at human D2long, D2short, D3 and D4.4 receptors as well as porcine D1, 5-HT1 and a1
receptors
a
Compound
Code
K
i
values in (nM)
3
3
[ H]spiperone
3
[ H]prazo-sin
3
[ H]WAY 100635
[
H]SCH 23390
pD1
hD2long
hD2short
hD3
hD4
pa1
p5-HT1A
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
a
A1B1C1
A1B1C2
A1B2C1
A1B2C2
A2B1C1
A2B1C2
A2B2C1
A2B2C2
A3B1C1
A3B1C2
A3B2C1
A3B2C2
A4B2C2
A5B1C1
A5B2C1
3500
5500
1500
3100
3000
16,000
1500
5900
910
69
48
31
29
8.0
22
8.4
48
n.d.
n.d.
11
b
b
4700
120
1200
250
1400
140
1200
15
9900
83
c
d
e
f
50
68
0.97 (0.45)
12
12
74
1400
160
190
78
n.d.
17
1.0
10
110
220
16
6.5
98
n.d.
5.4
g
h
i
6.2
22
4.9
35
280
27
170
120
330
40
n.d.
99
0.31
3.2
0.83
7.5
37
16
j
4700
730
780
15
840
24
200
15
n.d.
35
k
l
1800
590
210
31
240
27
190
35
88
n.d.
n.d.
n.d.
n.d.
p
q
s
1.5
14
770
860
23
51
11
23
1.5
5.6
69
100
30
a
K
i
values in nM are based on the means of 2–4 experiments each done in triplicate. n.d. not determined.
b
Binding affinity of compound 2c was also determined after flash-chromatographic purification. Resulting K
i
values are given in parentheses.

7254
P. Rodriguez Loaiza et al. / Bioorg. Med. Chem. 15 (2007) 7248–7257
the low nanomolar range (48–57%, EC50 = 2.0 À 3.5
nM). The alkynylbenzamide 2c proved to behave as a
D4 partial agonist (49%, EC50 = 0.73 nM). Due to
recent observations indicating that D3 and D4 partial
agonists are of potential therapeutical interest for the
treatment of cocain addiction and erectile dysfunction,
respectively, our findings could be a starting point for
further biomedical investigations.
as a yellow solid. Analytical data are identical to those
reported in Ref. 21.
4
.3. FPMT resin (6)
Merrifield resin (3.0 g, 1.1 mmol/g) was reacted with
NaN (0.60 g, 9.2 mmol), in DMSO (35 mL) at 70 ꢁC
3
for 48 h. After being cooled to room temperature the re-
sin was sequentially washed with DMSO (3· 50 mL),
H O (3· 50 mL), MeOH (5· 50 mL), CH Cl (3·
2
2
2
3. Conclusion
5
0 mL) and Et O (3· 50 mL), dried under vacuum
2
and analyzed by IR spectroscopy showing a strong
absorption at 2090 cm . The resulting azidomethyl
In conclusion, utilizing the FPTM resin 6, which is read-
ily available via [3 + 2]azide-alkyne cycloaddition, a
BAL strategy could be successfully applied for a parallel
synthesis of dopaminergic phenylacetylens. A focused
library of 20 test compounds revealing three points of
diversity was generated by a four-step SPOS approach
including microwave assisted Sonogashira coupling.
GPCR-ligand binding assays indicated excellent dopa-
mine D3 and D4 receptor binding affinities which were
identified to cause a partial agonist activity for the most
potent test compounds 2c,e,i,k.
À1
polystyrene 5 was shaken together with 1-propargylpyr-
role-2-carbaldehyde 4 (1.0 g, 7.5 mmol) and CuI (20 mg,
0
3
.11 mmol) in a mixture of THF/DIPEA 2:1 (35 mL) at
5 ꢁC for 36 h whereas a disappearance of the IR-
À1
absorption at 2090 cm could be observed. After being
cooled to room temperature, the solvents were removed
by filtration and a washing procedure including pyridine
(
3· 50 mL), THF (3· 50 mL), MeOH (3· 50 mL),
CH Cl (5· 50 mL) and Et O (3· 50 mL) followed dry-
ing of the resin under vacuum afforded the resin 6 (IR:
1
2
2
2
À1
650 cm for the C@O group).
4. Experimental
4
.4. Determination of resin capacity
4
.1. General
Resin 6 (100 mg) was distributed in a Teflon vessel and
reacted with a solution of 2-methoxyphenylethylamine
Polystyrene resins were purchased from NovaBiochem.
SPOS were performed manually in a Heidolph Synthe-
sis I equipped with PFA vessels. Microwave assisted
syntheses were conducted with a CEM Discover fo-
cussed microwave oven. Absolute solvents were pur-
chased from Acros. Commercially available starting
material was used without further purification. IR
spectra were registered on JASCO model FTIR 410
(83 mg, 0.55 mmol) in CH Cl (10 mL) for 30 min. After
addition of NaBH(OAc) (117 mg, 0.55 mmol) the resin
2 2
3
was agitated at room temperature for 24 h. The resin
was thoroughly washed with MeOH (3· 25 mL), CH Cl
2
2
(
3· 25 mL), Et O (2· 25 mL) and dried by suction. IR
2
analysis revealed the disappearance of the band at
1
was accomplished by the addition of 4-iodobenzoyl chlo-
À1
650 cm
indicating a complete reaction. Acylation
1
instrument via KBr pellet. H NMR (360 MHz) spectra
ride (147 mg, 0.55 mmol) and Et N (57 mg, 0.55 mmol)
3
were determined on a BRUCKER AM 360 or a
BRUCKER AVANCE spectrometer in solution. LC/
MS analyses were conducted in an Agilent Binary
Gradient System (MeOH/0.1 N aq HCOOH, 10:90–
in CH Cl (5 mL) and agitation for 18 h at room temper-
2
2
ature. After a sequentially filtering and washing proce-
dure with MeOH (3· 25 mL), CH Cl (3· 25 mL) and
2
2
Et O (3· 25 mL) the IR spectra showed an absorption
9
0:10) in combination with ChemStation Software
and UV detection at 254 nm using a Zorbax SB-C8
4.6 mm · 150 mm, 5 lm) with a flow rate of 0.5 mL/
2
À1
band at 1617 cm for the C@O of the carboxamide.
Subsequently, the resin was treated with 2% TFA in
CH Cl (10 mL) for 2 h. After collection of the cleavage
solution the resin was rinsed with CH Cl and the com-
(
min. Mass detection was pointed out with a Brucker
Esquire 2000 ion-trap mass spectrometer using an
APC ionization source. EI-MS spectra were recorded
on FINNIGAN MAT TSQ 70 spectrometer. Flash
chromatography was done using Silica gel (40–63 lm)
as stationary phase. TLC analyses were done on Merck
2
2
2
2
bined filtrates were washed with an aq solution of NaH-
CO , dried with Na SO and evaporated under reduced
3
2
4
pressure. The residue was dried under high vacuum over-
night to afford 33.5 mg (88%) of crude product 8 which
was analyzed by LC/MS system with UV detection
60 F254 glass plates and analyzed by UV light (254 nm)
or by iodine vapour.
(254 nm) indicating a purity of >99%.
4
.2. 1-Propargylpyrrole-2-carbaldehyde (4)
4.5. Model library
Pyrrole-2-carbaldehyde (8.0 g, 84 mmol), propargyl bro-
mide (12.0 g, 0.10 mol) and K CO (5.0 g, 36 mmol) in
4.5.1. Reductive amination. Resin 6 (4· 100 mg) was dis-
tributed into six vessels and a solution of 1-benzyl-4-
2
3
DMF (50 mL) were stirred at room temperature for
6 h. The mixture was treated with a saturated aqueous
solution of NaHCO and extracted with EtOAc. The or-
ganic layer was washed with brine, dried with Na SO4
2
and concentrated under vacuum to give 4 (6.0 g, 71%)
aminopiperidine (4· 105 mg, 4· 0.55 mmol) in CH
(6 mL) was added. Agitation for 30 min at room temper-
ature was followed by addition of NaBH(OAc)
233 mg, 4· 1.1 mmol). The mixture was agitated at
room temperature for 24 h. After filtration of the
2 2
Cl
1
(4·
3
3

P. Rodriguez Loaiza et al. / Bioorg. Med. Chem. 15 (2007) 7248–7257
7255
solvent, the resins were thoroughly washed with MeOH,
CH Cl and Et O and dried by suction.
corresponding primary amines 4 · A1, 4 · A2, 4 · A3,
4 · A4, 4 · A5 (each 0.5 mmol) in CH Cl (5 mL).
After agitation for 30 min at room temperature NaB-
2
2
2
2
2
4.5.2. Acylation. The resulting six batches of immobi-
lized amines were acylated using 3-iodobenzoic acid
H(OAc) (0.233 g, 1.0 mmol) was added and shaking
3
was continued for 24 h. The resins were extensively
washed with MeOH, CH Cl , Et O and the corre-
(
1
0
2· 135 mg, 2· 0.55 mmol) or 4-iodobenzoic acid (2·
2
2
2
35 mg, 2· 0.55 mmol), HOAt (4· 75 mg, 4·
sponding acids 10 · B1 and 10 · B2 (each 0.5 mmol),
HOAt (20· 74 mg, 20· 0.5 mmol) and DIC (20·
63 mg, 0.5 mmol) in a mixture of DMF/CH Cl (2:3,
.55 mmol), DIC (4· 69 mg, 4· 0.55 mmol) dissolved
in a mixture of DMF/CH Cl (2:3, 5 mL). Reaction mix-
2
2
2
2
tures were agitated for 28 h at room temperature,
washed with DMF, MeOH, CH Cl , Et O and dried
by suction. FTIR spectra showed signal at 1650 cm
5 mL) were added. The resins were agitated at room
temperature for 28 h, washed with DMF, MeOH,
CH Cl and Et O. The resins were transferred into
2
2
2
À1
2
2
2
for the C@O of the carboxamide group.
10 mL microwave glass tubes (pyrex) and the corre-
sponding acetylenes 10 · C1 or 10 · C2 (each
0.2 mol) were added together with Pd(PPh ) Cl (20·
4
.5.3. Pd-coupling. The resins were transferred to four
3
2
2
microwave tubes and Pd(PPh ) Cl (4· 3.9 mg, 4·
3.5 mg, 20· 0.005 mmol), CuI (20· 1.9 mg, 20·
3
2
2
5
2
.5 lmol), CuI (4· 2.1 mg, 4· 11 lmol), Et N (4·
0.011 mmol), DMF (0.5 mL) and Et N (2 mL). The
3
3
mL), DMF (4· 0.5 mL) and the corresponding alkyne
tubes were sealed and heated under microwave irradi-
ation at 120 ꢁC for 15 min. After being cooled to
ambient temperature, the reaction mixtures were fil-
tered and washed with DMF, THF, MeOH, CH Cl
C1–C2 (4· 0.22 mmol) were added. The tubes were
capped, heated in the microwave cavity for 15 min at
1
and washed with DMF, THF, CH Cl and Et O.
20 ꢁC, transferred to six vessels and sequentially filtered
2
2
and Et O. In case of the 10 (trimethylsilyl)acetylenes,
2
2
2
2
deprotection was accomplished by addition of tetrabu-
tylammonium fluoride (1.0 M in THF, 10· 0.2 mmol)
in THF (2 mL) and agitation at room temperature for
7 h. The resins were washed with THF, MeOH,
CH Cl and Et O. Finally, all products were cleaved
4
.5.4. Deprotection. The three C1-derived silylalkynes
were deprotected by addition of tetrabutylammonium
fluoride (1.0 M in THF, 2· 2.2 mL) followed by agita-
tion at room temperature for 7 h. The resins were
washed with THF, MeOH, CH Cl and Et O and dried
2
2
2
from the solid support by reaction with 2% TFA in
CH Cl (10 mL) at room temperature for 2 h. Each
resin was filtered and rinsed with CH Cl , the com-
2
2
2
under reduced pressure.
2
2
2
2
4.5.5. Cleavage. Products were cleaved from the resins
by agitation with 2% TFA in CH Cl (10 mL) at room
bined filtrates were collected and washed with NaH-
CO (saturated aqueous solution), the organic phase
2
2
3
temperature for 2 h. Each resin was filtered and rinsed
with CH Cl , the combined filtrates were collected and
washed with a saturated solution of NaHCO . The or-
was separated, dried with Na SO and the solvent
2
4
evaporated under vacuum. The residues were dried
under vacuum overnight to afford the crude products
2a–t. The products were analyzed by LC/MS for the
determination of the purity. For representative NMR
data, see below.
2
2
3
ganic layer was separated, dried with Na SO and the
2
4
solvent was evaporated under reduced pressure. The
resulting residues were dried under high vacuum over-
night to afford the crude products which were analyzed
by LC/MS for the determination of the purity. For rep-
resentative analytical data, see below.
4.6.1. N-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}-4-
ethynylbenzamide (2e, A2B1C1). H NMR: (CDCl3,
1
360 MHz) d (ppm) = 1.72–1.83 (m, 4H), 2.63–2.73
4.5.6. N-(1-Benzylpiperidin-4-yl)-4-phenylethynylbenza-
mide 9b.
(
(m, 2H), 2.81–2.93 (m, 4H), 3.10–3.19 (m, 4H), 3.21
(s, 1H), 3.48–3.57 (m, 2H), 3.88 (s, 3H), 6.88–6.99
(m, 3H), 7.02–7.08 (m, 1H), 7.57 (d, J = 8.4 Hz, 2H),
7.79 (d, J = 8.4 Hz, 2H). APCI-MS (m/z): 392.3
1
H
ppm) = 1.81–1.96 (m, 2H), 2.07–2.18 (m, 2H), 2.43–
NMR: (CDCl3, 360 MHz)
d
2
4
8
.56 (m, 2H), 3.15–3.27 (m, 2H), 3.85 (s, 2H), 4.07–
.20 (m, 1H), 6.34 (d, J = 7.9 Hz, 1H), 7.35–7.45 (m,
H), 7.55–7.63 (m, 4H), 7.77 (d, J = 8.2 Hz, 2H).
+
(M+1) .
+
APCI-MS (m/z): 395.2 (M+1) .
4.6.2. N-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}-4-
phenylethynylbenzamide (2f, A2B1C2).
1
H
NMR:
4
.5.7. N-(1-Benzylpiperidin-4-yl)-3-ethynylbenzamide 9c.
(CDCl , 360 MHz) d (ppm) = 1.73–1.85 (m, 4H),
3
1
H NMR: (CDCl , 600 MHz) d (ppm) = 1.86–1.98 (m,
2
1
1
J = 7.2 Hz, 1H), 7.74–7.81 (d, J = 7.2 Hz, 1H), 7.89 (s,
1
2.65–2.73 (m, 2H), 2.82–2.95 (m, 4H), 3.14–3.25 (m,
4H), 3.50–3.59 (m, 2H), 3.89 (s, 3H), 6.89 (d,
J = 8.1 Hz, 1H), 6.93–6.97 (m, 2H), 6.98–7.08 (m,
3H), 7.37–7.42 (m, 3H), 7.55–7.59 (m, 2H), 7.61 (d,
J = 8.6 Hz, 2H), 7.83 (d, J = 8.2 Hz, 2H). APCI-MS
3
H), 2.05–2.17 (m, 2H), 2.48–2.60 (m, 2H), 3.14 (s,
H), 3.20–3.32 (m, 2H), 3.90 (s, 2H), 4.08–4.19 (m,
H), 6.55 (br s, 1H), 7.30–7.46 (m, 6H), 7.61 (d,
+
H). APCI-MS (m/z): 319.2 (M+1) .
+
(m/z): 468.3 (M+1) .
4
2
.6. 3D library: synthesis of ethynylphenyl carboxamides
a–t
4.6.3. N-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}-3-
ethynylbenzamide (2g, A2B2C1). H NMR: (CDCl3,
1
360 MHz) d (ppm) = 1.70–1.80 (m, 4H), 2.61–2.69 (m,
Resin 6 (20· 100 mg, 20· 0.1 mmol) was distributed
into 20 Teflon vessels followed by a solution of the
2H), 2.78–2.89 (m, 4H), 3.11 (s, 1H), 3.13–3.21 (m,
4H), 3.49–3.56 (m, 2H), 3.85 (s, 3H), 6.89 (d,

7256
P. Rodriguez Loaiza et al. / Bioorg. Med. Chem. 15 (2007) 7248–7257
3
responding receptor and [ H]spiperone at a final
J = 7.2Hz, 1H), 6.93–6.97 (m, 2H), 7.0–7.07 (m, 1H),
.41 (dd, J = 7.9, 7.5 Hz, 1H), 7.60–7.64 (m, 1H),
7
concentration of 0.1 nM. The assays were carried out
with a protein concentration of 3–10 lg/assay tube and
Kd values of 0.06–0.07 nM for D2long, 0.09–0.15 nM
for D2short, 0.08–0.25 nM for D3 and 0.22–0.28 nM
for D4.4.
7
(
.80–7.84 (m, 1H), 7.91 (t, J = 1.4 Hz, 1H). APCI-MS
m/z): 392.3 (M+1) .
+
4
3
.6.4. N-{5-[4-(2-Methoxyphenyl)piperazin-1-yl]pentyl}-
-phenylethynylbenzamide (2h, A2B2C2). H NMR:
1
(
CDCl , 360 MHz) d (ppm) = 1.43–1.53 (m, 2H), 1.59–
The investigation of serotonin 5-HT1A and 5-HT2 as
well as adrenergic a1 binding was performed as
described in the literature. In brief, porcine cortical
3
1
4
2
1
.76 (m, 4H), 2.48 (t, J = 7.5 Hz, 2H), 2.65–2.77 (m,
H), 3.08–3.22 (m, 4H), 3.51 (dd, J = 12.9, 6.8 Hz,
H), 3.89 (s, 3H), 6.17 (br s, 1H), 6.88 (d, J = 8.6 Hz,
H), 6.92–7.05 (m, 3H), 7.36–7.42 (m, 3H), 7.45 (dd,
1
5
membranes were subjected to the binding assay at a con-
centration of 80 lg/assay tube for determination of
3
5-HT1 and 5-HT2 binding utilizing [ H]WAY100635
J = 7.9, 7.5 Hz, 1H), 7.54–7.59 (m, 2H), 7.65–7.69 (m,
H), 7.74–7.81 (m, 1H), 7.93 (t, J = 1.4 Hz, 1H).
APCI-MS (m/z): 482.3 (M+1) .
A
3
and [ H]ketanserin each at a final concentration of 0.1
1
+
and 0.5 nM with KD values of 0.03–0.04 nM (for
5
-HT1 ) and 1.5–2.1 nM (for 5-HT2). Cortical mem-
A
3
4
4
6
2
1
.6.5. N-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butyl}-
-ethynylbenzamide (2i, A3B1C1). H NMR: (CDCl ,
branes at 55 lg/assay tube and the radioligand [ H]pra-
zosin at a final concentration of 0.1 nM were applied to
determine adrenergic a1 binding with KD values of
0.06 nM.
1
3
00 MHz) d (ppm) 0 1.70–1.84 (m, 4H), 2.69–2.78 (m,
H), 2.81–2.99 (m, 4H), 3.10–3.19 (m, 4H), 3.21 (s,
H), 3.47–3.56 (m, 2H), 6.92–7.0 (m, 1H), 7.15–7.24
(
2
m, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.4 Hz,
Screening studies were established when using test com-
pounds at a final concentration of 10 lM, 100 nM and
+
H). APCI-MS (m/z): 430.5 (M+1) .
1 nM as triplicates and the assay conditions as described
above (5-HT1 screening was done with the radioligand
4
3
6
.6.6. N-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butyl}-
A
1
-ethynylbenzamide (2k, A3B2C1). H NMR: (CDCl ,
3
[ H]8-OH-DPAT at a concentration of 0.5 nM).
3
00 MHz) d (ppm) = 1.67–1.78 (m, 4H), 2.53 (dd,
J = 7.2, 6 Hz, 2H), 2.63–2.78 (m, 4H), 2.99–3.09 (m,
H), 3.14 (s, 1H), 3.47–3.55 (m, 2H), 6.91 (d,
J = 6.6 Hz, 1H), 7.13–7.21 (m, 2H), 7.41 (dd, J = 7.8,
.2 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.79 (d,
J = 7.8 Hz, 1H), 7.87 (s, 1H). APCI-MS (m/z): 430.4
Protein concentration was established by the method of
2
Lowry using bovine serum albumin as standard.
3
4
7
Data analysis of the resulting competition curves was
accomplished by non-linear regression analysis using
the algorithms in PRISM (GraphPad Software, San
+
(
M+1) .
Diego, CA). K values were derived from the corre-
i
4
4
6
4
.6.7. N-{5-[4-(2,3-Dichlorophenyl)piperazin-1-yl]pentyl}-
sponding EC data utilizing the equation of Cheng
5
and Prusoff.
0
1
-ethynylbenzamide (2q, A5B1C1). H NMR: (CDCl ,
31
3
00 MHz) d (ppm) = 1.44–1.51 (m, 2H), 1.63–1.73 (m,
H), 2.50–2.61 (m, 2H), 2.67–2.92 (m, 4H), 3.09–3.18
4.8. Functional experiments
(
6
(
m, 4H), 3.21 (s, 1H), 3.5 (dd, J = 13.2, 6.6 Hz, 2H),
.97 (dd, J = 7.8, 1.8 Hz, 1H), 7.15–7.21 (m, 2H), 7.57
d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H). APCI-
Determination of the stimulating effect of the test com-
pounds on mitogenesis as a functional assay was done
with a CHO dhfr cell line stably transfected with the
+
À
MS (m/z): 444.5 (M+1) .
human dopamine D3 receptor and with a CHO 10001
cell line stably expressing the human dopamine D4.2
4
3
6
4
.6.8. N-{5-[4-(2,3-Dichlorophenyl)piperazin-1-yl]pentyl}-
1
-ethynylbenzamide (2s, A5B2C1). H NMR: (CDCl ,
15
receptor according to literature. The concentration of
3
3
00 MHz) d (ppm) = 1.44–1.52 (m, 2H), 1.62–1.73 (m,
H), 2.49–2.62 (m, 2H), 2.65–2.90 (m, 4H), 3.08–3.20
the radiotracer was 0.02 lCi/well of [ H]thymidine (spe-
cific activity 25 Ci/mmol, Amersham Biosciences) and
the incubation was 4 or 2 h for D3 and D4 expressing
cells, respectively.
(
m, 5H), 3.50 (dd, J = 13.2, 6.6 Hz, 2H), 6.23 (br s,
1H), 6.98 (dd, J = 7.2, 1.8 Hz, 1H), 7.15–7.21 (m, 2H),
7.43 (t, J = 7.8 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.8
(
4
d, J = 7.8 Hz, 1 H), 7.88–7.90 (s, 1H). APCI-MS (m/z):
Experimental data resulting from the mitogenesis assay
were each normalized and then combined to get a mean
curve. Nonlinear regression analysis of this curve pro-
vided the EC50 value as a measure of potency. The top
value of the curve represented the maximal intrinsic
activity which was correlated to the effect of the full ago-
nist quinpirole (100%).
+
44.5 (M+1) .
4
.7. Binding studies
Receptor binding studies were carried out as described
2
9
in the literature. In brief, the dopamine D1 receptor
assay was done with porcine striatal membranes at a fi-
nal protein concentration of 60 lg/assay tube and the
3
radioligand [ H]SCH 23390 at 0.3 nM (K = 0.95 nM).
Acknowledgment
d
Competition experiments with the human D2long
,
D2short, D3 and D4.4 receptors were run with prepara-
tions of membranes from CHO cells expressing the cor-
This work was supported by the Deutsche Forschungs-
gemeinschaft (DFG).

P. Rodriguez Loaiza et al. / Bioorg. Med. Chem. 15 (2007) 7248–7257
7257
Supplementary data
13. Hocke, C.; Prante, O.; L o¨ ber, S.; H u¨ bner, H.; Gmeiner,
P.; Kuwert, T. Bioorg. Med. Chem. Lett. 2005, 15, 4819–
4
823.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.08.038.
1
1
1
4. Bettinetti, L.; Schlotter, K.; H u¨ bner, H.; Gmeiner, P.
J. Med. Chem. 2002, 45, 4594–4597.
5. Schlotter, K.; B o¨ ckler, F.; H u¨ bner, H.; Gmeiner, P.
J. Med. Chem. 2005, 48, 3696–3699.
6. Salama, I.; Hocke, C.; Utz, W.; Prante, O.; Boeckler, F.;
H u¨ bner, H.; Kuwert, T.; Gmeiner, P. J. Med. Chem. 2007,
References and notes
5
0, 489–500.
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