Parallel synthesis of potent dopaminergic N-phenyltriazole carboxamides applying a novel click chemistry based phenol linker

Article abstract of DOI:10.1016/j.bmc.2009.06.041

Taking advantage of our click chemistry based methodology to construct novel SPOS (solid phase organic synthesis) resins, the triazolylmethyl linked catechol 6a was discovered, which is readily available via copper(I)-catalyzed azide-alkyne cycloaddition

Full text of DOI:10.1016/j.bmc.2009.06.041

Bioorganic & Medicinal Chemistry 17 (2009) 5482–5487
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Parallel synthesis of potent dopaminergic N-phenyltriazole carboxamides
applying a novel click chemistry based phenol linker
*
Pilar Rodriguez Loaiza, Stefan Löber, Harald Hübner, Peter Gmeiner
Department of Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander University, Schuhstrasse 19, D-91052 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Taking advantage of our click chemistry based methodology to construct novel SPOS (solid phase organic
synthesis) resins, the triazolylmethyl linked catechol 6a was discovered, which is readily available via
copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) of azidomethyl substituted polystyrene with
O-propargylcatechol and can be applied for the parallel synthesis of N-phenyltriazole carboxamides. As
a proof-of-concept, a ‘catch-and-release’ strategy could be successfully applied for a parallel synthesis
of dopaminergic phenyltriazoles of type 2. A focused model library of 20 test compounds revealing three
points of diversity was generated by a three-step SPOS approach. Product purification was performed
employing a solid-supported carboxylic acid anhydride as a scavenger. GPCR-ligand binding screening
revealed dopamine D3 receptor ligands with Ki values in the single digit nanomolar range.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 15 April 2009
Revised 15 June 2009
Accepted 18 June 2009
Available online 25 June 2009
Keywords:
Dopamine receptor
D3
Click chemistry
Parallel synthesis
Catch and release
Triazole
1. Introduction
drug abuse.8–10 Extensive structure–activity relationship studies
revealed that aryl carboxamides bearing a N-alkyl-N0-arylpiper-
The ‘click chemistry’ concept developed by Sharpless and co-
workers focuses on the utilization of highly efficient organic
reactions aiming to maximize the synthetic efficiency of selective
compound modifications.1 Within the ‘click chemistry’ toolbox,
the copper catalyzed azide–alkyne [3+2] cycloaddition (CuAAC)
proved to be a highly versatile and useful reaction leading to the
1,2,3-triazole heterocycle. Click chemistry has become a powerful
method within combinatorial chemistry, and it has found increas-
ing applications in drug discovery and lead optimization pro-
grams.2,3 Click chemistry has been accepted as a highly beneficial
tool for parallel synthesis. Because an appropriate linker displaying
optimal reactivity and swelling is crucial for the success, we
prepared novel, functionalized resins, taking advantage of the con-
cept of click chemistry. Thus, CuAAC of alkynyl-substituted handles
with azidomethyl polystyrene led to a new family of highly effi-
cient BAL (backbone amide linker),4 REM (regenerative Michael
acceptor),5 SPAn (solid/solution-phase annulation)6 and scavenger
resins.7 ‘Click resins’ enable solid phase supported reactions to
work under nearly perfect conditions fulfilling the requirements
of click chemistry.
azine side chain exhibit preferential D3 recognition.11–15 Taking
advantage of our click chemistry based BAL resins, we reported
on a parallel synthesis of biphenyl carboxamides of type 1a16 and
N-benzyl-1,2,3-triazole carboxamides of type 1b.17 To further opti-
mize the receptor binding profiles, we intended to approach struc-
tural hybrids of type 2 modifying the described N-benzyltriazole
based ligands towards N-phenyl substituted derivatives (Fig. 1).
Unfortunately, the elaborated backbone amide linker strategy
including a [3+2]-cycloaddition of a solid-supported propynoic
acid amide with benzyl azide could not be applied for the construc-
tion of phenyltriazoles since the employed arylazides decomposed
at the required temperature of 150 °C. Thus, we planned to com-
pass an alternative strategy for a parallel synthesis of N-phenyl-
1,2,3-triazole-4-carboxamides of type 2 taking advantage of our
click chemistry based methodology to construct novel SPOS (solid
phase organic synthesis) resins. Since solid-supported phenol han-
dles proved to be efficient tools for the SPOS of carboxamides via a
‘catch-and-release’ approach,18–20 we intended to construct novel
triazole linked phenols by applying our highly efficient, reliable
and robust click chemistry based concept utilizing the CuAAC.21,22
The presented methodology should allow an efficient parallel syn-
thesis of N-phenyl-1,2,3-triazole carboxamides. This structural fea-
ture can be found in a variety of pharmacological relevant
compounds like tyrosine phosphatase inhibitors, allosteric mGluR1
antagonists and anti-platelet active agents.23–25 As a proof-of-con-
cept we intended to construct a focused library of putative dopa-
mine receptor ligands.
The dopamine D3 receptor has attracted enormous attention as
a potential drug target, since selective D3 ligands are promising
agents for the therapy of schizophrenia, Parkinson’s disease and
* Corresponding author. Tel.: +49 9131 852 9383; fax: +49 9131 852 2585.
E-mail address: peter.gmeiner@medchem.uni-erlangen.de (P. Gmeiner).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.041

P. R. Loaiza et al. / Bioorg. Med. Chem. 17 (2009) 5482–5487
5483
Table 1
Determination of linker loading
O
O
N
Cl
3,5-dichloro-
R
Pr
N
H
N
benzoyl chloride,
Et3N, CH2Cl2
( ) n
N
H
NH2-Pr, CH2Cl2
or
6a
6b
7
Cl
1a
O
Theoretical loadingb
(mmol/g)
N
Resin Yield of 7 (mmol) per Triazole loading (mmol)
R
N
g resin
per g resina
N
( )n
N
N
H
6a
6b
0.98
0.99
0.99
0.96
0.85–1.03
0.85–1.03
N
CHO
R'
1b
a
Calculated by elementary analysis of nitrogen.
Calculated on the loading of Merrifield resin (1.0–1.2 mmol/g).
b
BAL
O
N
raphy, the yields of 7 indicated a loading of 0.98 and 0.99 mmol/g
for catechol and hydroquinone, respectively (Table 1). This data is
in high accordance to the loading calculated by elementary analy-
sis of nitrogen. The theoretical loading which was based on the
amount of chloromethyl groups in the starting material (1.0–
1.2 mmol/g), indicated nearly quantitative conversion.
R
N
1,3
N
( )
N
N
OH
H
N
2
R'
catch and release
To investigate the reliability and the scope of our solid phase
supported method, we elaborated the synthesis of a model com-
pound. An objective of this step was to identify the more efficient
one of the two resins 6a,b to optimize suitable reaction conditions
for the cycloaddition reaction and to validate the use of a scavenger
resin for the removal of the excess of amine reactant after the
product cleavage.
In detail, diisopropyl carbodiimide (DIC) promoted acylation of
the phenol resins 6a,b with propynoic acid furnished the respec-
tive esters 8a,b (Scheme 2). Product formation could be easily
monitored by IR spectroscopy indicating the presence of both the
ester C@O and C–C triple bond. Cycloaddition with p-nitrophenyl
azide was performed either in the presence or absence of CuI at
Figure 1. Phenyltriazole carboxamides as a potential novel class of dopamine D3
receptor ligands.
2. Results and discussion
2.1. Chemistry
In contrast to previously used phenylthioethers for the linkage
of catch and release functionalities, we tried to take advantage of
putatively chemically more stable phenylether based oxa-analogs
of type 6 to be functionalized and immobilized via click chemistry.
Thus, azidomethyl substituted polystyrene 5, which is readily
available from Merrifield resin, was planned to be used as a start-
ing material. Catechol and hydroquinone (3a,b) were mono-func-
tionalized by O-propargylation to give the terminal alkynes 4a
and 4b, respectively. Using CuI as a catalyst, [3+2]-cycloaddition
of the immobilized azide 5 and 4a,b resulted in formation of the
triazole-resins 6a,b (Scheme 1). To determine the amount of phe-
nol residues available for the aspired ‘catch-and-release’ strategy,
the functional resins 6a,b were acylated with 3,5-dichlorobenzoyl
chloride. Subsequent release by aminolysis with n-propylamine
afforded the carboxamide 7. After purification by flash chromatog-
O
O
O
a
6a,b
N
N
N
8a,b
NO2
b or c
N
OH
OH
a
N
O
N
O
O
HO
O
3a: ortho
3b: para
4a: ortho
4b: para
9a,b
N
N
N
N3
b
Cl
d
(e)
= polystyrene
(1% crosslinked)
N
Cl
5
N
OH
O
N
( )3
N
H
O
N
N
2m
N
N
O2N
N
6a: ortho
6b: para
Scheme 2. (a) (1) Propynoic acid, DIC, CH2Cl2, 7 h, (2) propynoic acid, DIC, CH2Cl2,
14 h; (b) p-nitrophenyl azide, CuI, DMF, 120 °C, 24 h; (c) p-nitrophenyl azide, DMF,
120 °C, 48 h; (d) N-(4-aminobutyl)-N0-(dichlorophenyl)piperazine, CH2Cl2, 20 °C,
18 h; (e) MP anhydride resin, CH2Cl2, 20 h, 24 h.
Scheme 1. Reagents and conditions: (a) propargyl bromide, K2CO3, acetone, reflux,
18 h; (b) Cu(I)I, THF/DIPEA, 35 °C, 36 h.

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P. R. Loaiza et al. / Bioorg. Med. Chem. 17 (2009) 5482–5487
Table 2
The results of this SPOS validation and optimization are sum-
marized in Table 2. A couple of clear conclusions can be drawn
from the obtained results:
Optimization of SPOS condition
Resin
6a
6b
Catalyst
Method Ac
CuIa (%)
Noneb (%)
CuIa (%)
Noneb (%)
(A) Giving comparable yields, the use of CuI as a catalyst allows
choosing shorter reaction times. Exclusive formation of the
1,4-disubstituted triazole isomer was observed.
(B) The catechol-based resin 6a works more efficiently regard-
ing to the obtained product yields and purities.
Yielde
Purityf
Yielde
Purityf
50
>95
79
54
>95
46
43
>95
63
40
>95
91
Method Bd
98
97
87
86
a
Reaction time 24 h.
Reaction time 48 h.
Purification by flash chromatography.
Purification by scavenger resin.
Determined by 1H NMR.
b
c
d
e
f
(C) Application of an amine scavenger resin revealed to be a
highly efficient alternative to chromatographic purification.
Using the elaborated solid phase supported methodology de-
scribed above, five aminoalkylpiperazines, two alkynyl carboxylic
acids and two aromatic azides were chosen to construct a three
dimensional focused library of putative D3 receptor ligands of type
2. Following the described procedure including DIC-mediated acyl-
ation of the triazolylmethylcatechol (TMC) resin 6a, CuI-catalyzed
cycloaddition and aminolysis followed by removal of the excessive
amine, the aryltriazoles 2a–t were obtained. LC–MS derived puri-
ties and yields are displayed in Table 3. In general, the obtained
Determined by LC–MS (254 nm).
120 °C to give the triazoles 9a,b. Finally, the cleavage with 3 equiv
of N-(4-aminobutyl)-N’-(dichlorophenyl)piperazine afforded the
triazole carboxamide 2m, when product purification was done
alternatively by addition of a macroporous anhydride26 as a scav-
enger resin or by flash chromatography.
Table 3
Purities, yields and receptor screening results for SPOS products 2a–t
A1-2
B1-2
C1-5
6a
2a-t
a
b
c, d
Azides:
Acids:
COOH
COOH
N3
R
B1: R= NO2
B2: R= OMe
A1
A2
Amines:
C1: R= OMe, R'= H, n= 1
C2: R= Cl, R'= Cl, n= 1
C3: R= OMe, R'= H, n= 3
N
N
H2N
( )n
R
R'
C4: R= Cl, R'= Cl, n= 3
C5: R= Cl, R'= H, n= 3
a: 1) A1-2, DIC, CH2,Cl2, 7h, 2) A1-2, DIC, CH2,Cl2, 14h; b: B1-2, CuI,
DMF, 120°C, 24h; c: C1-5, CH2Cl2, 20°C, 18h; d: MP anhydride resin,
CH2Cl2, 20° C, 24h.
a
Determined by LC–MS (254 nm).

P. R. Loaiza et al. / Bioorg. Med. Chem. 17 (2009) 5482–5487
5485
Table 4
digit nanomolar dopamine D3 receptor binding with a superior Ki
Receptor binding data of selected compounds at human D2long, D2short, D3 and D4.4
receptors as well as porcine D1 and
value of 1.0 nM for the dichlorophenylpiperazine 2m and
tor preference for the methoxyphenylpiperazine 2a.
a1 recep-
a
1 receptors
Compound
Ki valuesa (nM)
4. Experimental
4.1. General
pD1
hD2long
hD2short
hD3
hD4.4
pa1
2a
4100
810
1700
2200
1800
750
49
44
83
120
470
53
68
47
42
180
1.0
4.9
5.7
9.3
140
280
320
240
200
6.5
17
46
28
42
2m
2n
2o
2p
Polystyrene resins were purchased from NovaBiochem. SPOS
were performed manually in a Heidolph Synthesis I equipped with
PFAvessels. Absolutesolventswere purchased fromAcros. Commer-
cially available starting material was used without further purifica-
tion. IR spectra were registered on JASCO model FTIR 410 instrument
via KBr pellet. 1H NMR (360 MHz or 600 MHz) spectra were deter-
minedon a BrukerAM360 or a BrukerAVANCEspectrometer insolu-
tion. LC–MS analyses were conducted in an Agilent Binary Gradient
System (MeOH/0.1 N aq HCO2H 10/90–90/10) in combination with
ChemStation Software and UV detection at 254 nm using a Zorbax
a
Ki values in nM are based on the means of 2–4 experiments each done in
triplicate.
purities of 73–95% allowed biological screening of all 20 library
members without further purification steps.
2.2. Biological investigations
SB-C8 (4.6 mm ꢀ 150 mm, 5
lm) with a flow rate of 0.5 mL/min.
A GPCR (G-protein coupled receptor) screening focused on bio-
genic amine receptors was performed to characterize all library
members to determine their ability to bind to the cloned human
D2long, D2short, D3 and D4 dopamine receptor subtypes, and
Mass detection was pointed out with a Bruker Esquire 2000 ion-trap
mass spectrometer using an APC ionization source. EI-MS spectra
were recorded on FINNIGAN MAT TSQ 70 spectrometer. Flash chro-
matography was done using silica gel (40–63 lm) as stationary
phase. TLCanalyseswere done on Merck60 F254 glass plates and ana-
lyzed by UV light (254 nm) or by iodine vapor.
the porcine D1 dopamine and
a
1 adrenergic receptors.16,27 This
was accomplished in a screening system by measuring the dis-
placement of the following radioligands: [3H]spiperone for D2,
D3, D4, [3H]SCH23390 for D1 receptors and [3H]prazosin for
a1,
4.2. Synthesis of linker precursors
using 100 nM concentration of the test compounds. The results
in Table 3 reflect the percentage of radioligand displacement from
the six different GPCRs. None of the compounds was able to bind
significantly to the D1 receptor. The affinities to the D2long,
D2short and D4 subtypes were weak to moderate. In contrast,
the majority of the test compounds showed excellent binding to
the D3 receptor. Interestingly, the most potent D3 ligands
2m,n,o,p feature the piperazinylbutylamine moiety in combination
Propargyloxyphenols 4a and 4b were synthesized starting from
catechol (3a) and hydroquinone (3b), respectively, according to a
procedure described in the literature.28
4.2.1. 2-Propargyloxyphenol 4a
1H NMR: (CDCl3, 360 MHz) d (ppm) = 2.58 (t, J = 2.3 Hz, 1H),
4.79 (d, J = 2.4 Hz, 2H), 6.85–6.91 (m, 1H), 6.95 (dd, J = 7.9 Hz,
1.5 Hz, 1H), 6.97–7.05 (m, 2H). EI-MS (m/z): 148 (M+).
with the dichlorophenyl substituent. For the a1 adreno-receptor, a
different structure activity relationship could be observed. Here,
the incorporation of a 2-(methoxyphenyl)piperazine moiety leads
to highest affinities (compounds 2i,k,l).
Equilibrium binding constants (Ki-values) were determined of
those compounds showing a percentage displacement greater than
70% at the D3 receptor as well as for 2a, for which the screening
4.2.2. 4-Propargyloxyphenol 4b
Analytical data are identical to those reported in the
literature.29
4.3. Synthesis of phenol resins 6a and 6b
results indicated strong
a1 selectivity. The affinity constant (Ki)
Merrifield resin (6.0 g, 1.1 mmol/g) was reacted with NaN3
(1.3 g, 20 mmol) in DMSO (70 mL) at 70 °C for 48 h. After being
cooled to room temperature the resin was sequentially washed
with DMSO (3 ꢀ 50 mL), H2O (3 ꢀ 50 mL), MeOH (5 ꢀ 50 mL),
CH2Cl2 (3 ꢀ 50 mL) and Et2O (3 ꢀ 50 mL), dried under vacuum
and analyzed by IR spectroscopy showing a strong absorption at
2090 cmꢁ1. The resulting azidomethyl polystyrene 5 (1.5 g) was
shaken together with 2-propargyloxyphenol 4a or 4-propargyloxy-
phenol 4b (0.6 g, 4.1 mmol) and CuI (30 mg, 0.17 mmol) in a mix-
ture of THF/DIPEA 2:1 (20 mL) at 35 °C for 36 h whereas a
disappearance of the IR-absorption at 2090 cmꢁ1 could be ob-
served. 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), CH2Cl2
(5 ꢀ 50 mL) and Et2O (3 ꢀ 50 mL) followed by drying of the resin
under vacuum affording the resins 6a and 6b.
values are shown in Table 4. The N-piperazinylethyl substituted
triazole carboxamide 2a displays low nanomolar affinity
(Ki = 6.5 nM) for the a1 receptor in combination with a high selec-
tivity over all tested dopamine receptor subtypes. On the other
hand, the N-(dichlorophenyl)piperazine with the four-carbon
spacer proved to be a scaffold leading to Ki values in the single digit
nanomolar range. Within this group, the (p-nitrophenyl)triazole
carboxamide 2m (A1B1C4) turned out to be the most potent li-
brary member with a Ki(D3) value of 1.0 nM and a 50-fold selectiv-
ity over the D2 subtypes. Taking this compound as a starting point
for further lead optimization projects, additional structural modifi-
cations—preferably at the aryltriazole moiety—should most proba-
bly yield subnanomolar D3 ligands.
3. Conclusion
In conclusion, utilizing the triazolylmethylcatechol (TMC) resin
6a, which is readily available via [3+2]azide–alkyne cycloaddition,
a ‘catch-and-release’ strategy could be successfullyapplied for a par-
allel synthesis of dopaminergic phenyltriazoles. A focused library of
20 test compounds revealing three points of diversity was generated
by a three-step SPOS approach followed by treatment with macro-
porous anhydride resin. GPCR-ligandbinding assays indicated single
4.4. Determination of resin capacity
Resins 6a or 6b (100 mg) were distributed in teflon vessels and
each reacted with 3,5-dichlorobenzoyl chloride (120 mg,
0.55 mmol) in a mixture of Et3N and CH2Cl2 (1/10, 10 mL) at room
temperature for 48 h. The resin were thoroughly washed with

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P. R. Loaiza et al. / Bioorg. Med. Chem. 17 (2009) 5482–5487
DMF (3 ꢀ 25 mL), CH2Cl2 (3 ꢀ 25 mL), Et2O (2 ꢀ 25 mL) and dried
by suction. Subsequent addition of 1-propylamine (20 mg,
0.55 mmol) in CH2Cl2 (5 mL) was followed by shacking for 18 h at
ambient temperature. The cleavage solution was collected by filtra-
tion and evaporated. The residue was dried under vacuum and puri-
fied by flash chromatography using hexane/ethyl acetate (1/1) to
afford 22.5 mg (from 6a) and 23 mg (from 6b) of 7, respectively.
7.2 Hz, 2H), 3.05–3.18 (m, 4H), 3.41–3.50 (m, 2H), 3.90 (s, 3H),
6.97 (ddd, J = 7.3 Hz, 2.1 Hz, 1.8 Hz, 1H), 7.00–7.10 (m, 2H), 7.14–
7.22 (m, 2H), 7.59–7.70 (m, 2H), 7.73 (s, 1H). APCI-MS (m/z):
532.8 (M+1)+.
4.11. N-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}-4-[1-(4-
nitrophenyl)-1H-[1,2,3]triazol-4-yl]butyramide (2k, A2B1C3)
4.5. Synthesis of phenylazide B1
1H NMR: (CDCl3, 360 MHz) d (ppm) = 1.60–1.72 (m, 2H), 1.80–
2.05 (m, 4H), 2.14 (t, J = 7.2 Hz, 2H), 2.40 (t, J = 7.2 Hz, 2H), 2.80–
3.00 (m, 6H), 3.34–3.50 (m, 6H), 3.88 (s, 3H), 6.85–6.98 (m, 2H),
7.01–7.11 (m, 2H), 8.07–8.18 (m, 2H), 8.33 (s, 1H), 8.33–8.43 (m,
2H). APCI-MS (m/z): 522.3 (M+1)+.
1-Azido-4-methoxybenzene was synthesized from 1-bromo-4-
iodobenzene in the presence of CuI and
L-proline according to a
method described in the literature.30
4.6. Synthesis of phenylazide B2
4.12. N-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}-4-[1-(4-
methoxyphenyl)-1H-[1,2,3]triazol-4-yl]butyramide (2l, A2B2C3)
1-Azido-4-nitrobenzene was synthesized from 4-nitroaniline
according to a method described in the literature.31
1H NMR: (CDCl3, 360 MHz) d (ppm) = 1.58–1.75 (m, 4H), 2.06–
2.17 (m, 2H), 2.33 (dd, J = 7.2 Hz, 6.8 Hz, 2H), 2.52–2.60 (m, 2H),
2.72–2.82 (m, 4H), 2.86–2.89 (m, 2H), 3.10–3.22 (m, 4H), 3.27–
3.35 (m, 2H), 3.87 (s, 3H), 3.88 (s, 3H), 6.88 (d, J = 7.5 Hz, 1H),
6.92–6.97 (m, 3H), 6.96–7.08 (m, 2H), 7.59–7.70 (m, 2H), 8.03 (s,
1H). APCI-MS (m/z): 507.4 (M+1)+.
4.7. 3D library. Synthesis of N-phenyltriazoles 2a–t
TMC resin 6a (20 ꢀ 100 mg, 20 ꢀ 0.1 mmol) was distributed into
20 teflon vessels followed by a solution of DIC (each 69 mg,
0.55 mmol) and the corresponding carboxylic acids 10 ꢀ A1 and
10 ꢀ A2 (each 0.5 mmol) in CH2Cl2 (5 mL). After agitation for 7 h at
room temperature the resins were washed with CH2Cl2 and the en-
tire acylation process was repeated with a prolonged reaction time
of 14 h. The resins were washed with CH2Cl2 (3 ꢀ 20 mL) and Et2O
(3 ꢀ 20 mL) and subsequently treated with a solution of the corre-
sponding arylazides 10 ꢀ B1 and 10 ꢀ B2 (each 1.0 mmol) and CuI
(2 mg, 0.01 mmol) in DMF (1 mL). After agitation at 120 °C for 24 h
the resins were washed extensively with hot DMF (10 ꢀ 20 mL),
CH2Cl2 (8 ꢀ 25 mL) and Et2O (2 ꢀ 20 mL). A solution of the primary
amines C1–C5 (each 0.4 mmol) in CH2Cl2 (3 mL) was added and agi-
tation was continued for 18 h at room temperature. Anhydride MP
scavenger resin (20 ꢀ 130 mg, 20 ꢀ 1.0 mmol) was added and the
mixtures were shaken for another 24 h. The cleavage solutions were
collected by filtration and the resins were washed with CH2Cl2 (each
20 mL). After evaporation of the combined solutions, the residues
were dried under vacuum over night 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.
4.13. N-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butyl}-1-(4-
nitrophenyl)-1H-[1,2,3]triazol-4-carboxamide (2m, A1B1C4)
1H NMR: (CDCl3, 600 MHz) d (ppm) = 1.68–1.83 (m, 4H), 2.55 (t,
J = 6.8 Hz, 2H), 2.65–2.79 (m, 4H), 3.10–3.20 (m, 4H), 3.58 (dd,
J = 12.4 Hz, 6.3 Hz, 2H), 7.00 (dd, J = 6.5 Hz, 2.9 Hz, 1H), 7.13–7.21
(m, 2H), 7.57 (br s, 1H), 8.00–8.10 (m, 2H), 8.43–8.50 (m, 2H),
8.70 (s, 1H). APCI-MS (m/z): 518.3 (M+1)+.
4.14. N-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butyl}-1-(4-
methoxyphenyl)-1H-[1,2,3]triazol-4-carboxamide (2n, A1B2C4)
1H NMR: (CDCl3, 600 MHz) d (ppm) = 1.71–1.84 (m, 4H), 2.68–
2.75 (m, 2H), 2.81–2.94 (m, 4H), 3.18–3.24 (m, 4H), 3.53 (dd,
J = 13.2 Hz, 6.6 Hz, 2H), 3.87 (s, 3H), 6.97 (ddd, J = 7.5 Hz, 1.8 Hz,
1.2 Hz, 1H), 7.00–7.09 (m, 2H), 7.12–7.21 (m, 2H), 7.59–7.68 (m,
2H), 8.50 (s, 1H). APCI-MS (m/z): 503.3 (M+1)+.
4.15. Binding studies
4.8. N-{2-[4-(2,3-Dichlorophenyl)piperazin-1-yl]ethyl}-1-(4-
nitrophenyl)-1H-[1,2,3]triazole-4-carboxamide (2e, A1B1C2)
Receptor binding studies were carried out as described in liter-
ature.24 In brief, the dopamine D1 receptor assay was done with
porcine striatal membranes at a final protein concentration of
1H NMR: (CDCl3, 600 MHz) d (ppm) = 2.71–2.82 (m, 6H), 3.09–
3.18 (m, 4H), 3.65–3.71 (m, 2H), 7.00 (d, J = 6.6 Hz, 1H), 7.15–
7.21 (m, 2H), 7.74 (br s, 1H), 8.00–8.09 (m, 2H), 8.44–8.53 (m,
2H), 8.67 (s, 1H). APCI-MS (m/z): 490.1 (M+1)+.
60
(Kd = 0.95 nM). Competition experiments with the human D2long
l
g/assay tube and the radioligand [3H]SCH 23390 at 0.3 nM
,
D2short, D3 and D4.4 receptors were run with preparations of mem-
branes from CHO cells expressing the corresponding receptor and
[3H]spiperone at a final concentration of 0.5 nM. The assays were
4.9. N-{2-[4-(2,3-Dichlorophenyl)piperazin-1-yl]ethyl}-4-[1-(4-
nitrophenyl)-1H-[1,2,3]triazol-4-yl]butyramide (2g, A2B1C2)
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
0.08–0.23 nM for D3 and 0.22–0.28 nM for D4.4.
,
1H NMR: (CDCl3, 360 MHz) d (ppm) = 2.10–2.19 (m, 2H), 2.37 (t,
J = 7.2 Hz, 2H), 2.58–2.66 (m, 2H), 2.67–2.78 (m, 4H), 2.94 (t, 7.2 Hz,
2H), 3.05–3.18 (m, 4H), 3.40–3.50 (m, 2H), 6.98 (ddd, J = 7.3 Hz,
2.1 Hz, 1.8 Hz, 1H), 7.14–7.22 (m, 2H), 7.95 (s, 1H), 7.93–8.05 (m,
2H), 8.29–8.40 (m, 2H). APCI-MS (m/z): 532.8 (M+1)+.
The investigation of adrenergic a1 binding was performed as
described in literature.15 In brief, porcine cortical membranes
were subjected to the binding assay at a concentration of 55
l
g/
assay tube for determination of adrenergic
a1 binding utilizing
[3H]prazosin at a final concentration of 0.4 with a KD value of
0.06 nM.
4.10. N-{2-[4-(2,3-Dichlorophenyl)piperazin-1-yl]ethyl}-4-[1-
(4-methoxyphenyl)-1H-[1,2,3]triazol-4-yl]butyramide (2h,
A2B2C2)
Screening studies were established when using test compounds
at a final concentration of 10 lM, 100 nM and 1 nM as triplicates
and the assay conditions as described above.
1H NMR: (CDCl3, 360 MHz) d (ppm) = 2.07–2.18 (m, 2H), 2.37 (t,
J = 7.2 Hz, 2H), 2.63 (t, J = 5.7 Hz, 2H), 2.67–2.78 (m, 4H), 2.91 (t,
Protein concentration was established by the method of Lowry
using bovine serum albumin as standard.32

P. R. Loaiza et al. / Bioorg. Med. Chem. 17 (2009) 5482–5487
5487
9. Schwartz, J. C.; Diaz, J.; Pilon, C.; Sokoloff, P. Brain Res. Brain Res. Rev. 2000, 31,
277.
10. Pilla, M.; Perachon, S.; Sautel, F.; Garrido, F.; Mann, A.; Wermuth, C. G.;
Schwartz, J. C.; Sokoloff, P. Nature 1999, 400, 371.
11. Boeckler, F.; Gmeiner, P. Pharmacol. Therap. 2006, 112, 281.
12. Leopoldo, M.; Berardi, F.; Colabufo, N. A.; De Giorgio, P.; Lacivita, E.; Perrone, R.;
Tortorella, V. J. Med. Chem. 2002, 45, 5727.
Data analysis of the resulting competition curves was accom-
plished by non-linear regression analysis using the algorithms in
PRISM (GraphPad Software, San Diego, CA). Ki values were derived
from the corresponding EC50 data utilizing the equation of Cheng
and Prusoff.33
13. Bettinetti, L.; Schlotter, K.; Hübner, H.; Gmeiner, P. J. Med. Chem. 2002, 45, 4594.
14. Newman, A. H.; Cao, J.; Bennett, C. J.; Robarge, M. J.; Freeman, R. A.; Luedtke, R.
R. Bioorg. Med. Chem. Lett., 2003. 2179.
Acknowledgments
15. Schlotter, K.; Böckler, F.; Hübner, H.; Gmeiner, P. J. Med. Chem. 2005, 48, 3696.
16. Bettinetti, L.; Löber, S.; Hübner, H.; Gmeiner, P. J. Comb. Chem. 2005, 7, 309.
17. Rodriguez Loaiza, P.; Löber, S.; Hübner, H.; Gmeiner, P. J. Comb. Chem. 2006, 8,
252.
18. Marshall, D. L. J. Org. Chem. 1970, 35, 867.
19. Breitenbucher, J. G.; Johnson, C. R.; Haight, M.; Phelan, J. C. Tetrahedron Lett.
1998, 39, 1295.
20. Fang, L.; Demee, M.; Sierra, T.; Kshirsagar, T.; Celebi, A. A.; Yan, B. J. Comb. Chem.
2002, 4, 362.
21. Tornoe, C.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
22. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.
23. Xie, J.; Seto, C. T. Bioorg. Med. Chem. 2007, 15, 458.
24. Noeske, T.; Jirgensons, A.; Starchenkovs, I.; Renner, S.; Jaunzeme, I.; Trifanova,
D.; Hechenberger, M.; Bauer, T.; Kauss, V.; Parsons, C. G.; Schneider, G.; Weil, T.
ChemMedChem 2007, 2, 1763.
25. Cunha, A. C.; Figueiredo, J. M.; Tributino, J. L. M.; Miranda, A. L. P.; Castro, H. C.;
Zingali, R. B.; Fraga, C. A. M.; de Souza, M. C. B. V.; Ferreira, V. F.; Barreiro, E. J.
Bioorg. Med. Chem. 2003, 11, 2051.
The Fonds der Chemischen Industrie is acknowledged for finan-
cial support. We thank Dr. J.-C. Schwartz and Dr. P. Sokoloff (IN-
SERM, Paris), Dr. H. H. M. Van Tol (Clarke Institute of Psychiatry,
Toronto) and Dr. J. Shine (The Garvan Institute of Medical Research,
Sydney) for providing D3, D4.4 and D2 receptor expressing cell
lines. This work was supported by the Deutsche Forschungsgeme-
inschaft (DFG).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.06.041.
References and notes
26. Purchased from Novabiochem, Merck Biosciences.
27. Hübner, H.; Haubmann, C.; Utz, W.; Gmeiner, P. J. Med. Chem. 2000, 43, 756.
28. Manivannan, S.; Sankararaman, S.; Hopo, H.; Dix, I.; Jones, P. G. J. Org. Chem.
2001, 66, 4299.
29. Venugopalan, B.; Balasubbramanian, K. K. Heterocycles 1985, 23, 81.
30. Zhu, W.; Ma, D. Chem. Commun. 2004, 888.
31. Tanno, M.; Sueyoshi, S.; Kamiya, S. Chem. Pharm. Bull. 1982, 30, 3125.
32. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193,
265.
1. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
2. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128.
3. Tron, G. C.; Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba, G.; Genazzani, A. A.
Med. Res. Rev. 2007, 28, 278.
4. Löber, S.; Rodríguez Loaiza, P.; Gmeiner, P. Org. Lett. 2003, 5, 1753.
5. Löber, S.; Gmeiner, P. Tetrahedron 2004, 60, 8699.
6. Dolle, R. E.; MacLeod, C.; Martinez-Teipel, B.; Barker, W.; Seida, P. R.; Herbertz,
T. Angew. Chem., Int. Ed. 2005, 44, 5830.
7. Leeb, L.; Gmeiner, P.; Löber, S. QSAR Comb. Sci. 2007, 26, 1145.
8. Joyce, J. N. Pharmacol. Therap. 2001, 90, 231.
33. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.